WO2004069206A2 - Immunogenic cd91 ligand-antigenic molecule complexes and fusion proteins - Google Patents
Immunogenic cd91 ligand-antigenic molecule complexes and fusion proteins Download PDFInfo
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- WO2004069206A2 WO2004069206A2 PCT/US2004/003864 US2004003864W WO2004069206A2 WO 2004069206 A2 WO2004069206 A2 WO 2004069206A2 US 2004003864 W US2004003864 W US 2004003864W WO 2004069206 A2 WO2004069206 A2 WO 2004069206A2
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/62—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
Definitions
- the present invention relates to complexes and fusion proteins comprising a CD91 ligand and an antigenic molecule, for use in the treatment or prevention of a disease.
- CD91 also known as LDL receptor related protein, is structurally related to other receptors in the LDL receptor gene family. Like other members of the LDL receptor gene family, CD91 has various functions besides lipid metabolism, including homeostasis of proteinases and proteinase inhibitors, cellular entry of toxins and viruses, lysosomal enzyme activation, cellular signal transduction and neurotransmission. Herz and Srtickland, 2001, J. Clin. Invest. 108(6):779-84.
- CD91 is primarily expressed in liver, brain and placenta.
- the extracellular domain of the human receptor comprises six 50-amino acid EGF repeats and 31 complement repeats of approximately 40-42 amino acids.
- the complement repeats are organized, from the amino to the carboxy-terminus, into clusters of 2, 8, 10 and 11 repeats, called Cluster I, II, III and IV (Herz et al, 1988, EMBO J. 7:4119-4127).
- Cluster I, II, III and IV Cluster I, II, III and IV
- Cluster I, II, III and IV Cluster I, II, III and IV
- CD91 binds to at least 30 different ligands with high affinity, including ApoE, lipoprotein lipase, hepatic lipase, factor IXa, factor Villa, factor VIIa/TFPI, MMP-13 (matrix metaloproteinase-13), MMP-9 (matrix metaloproteinase-9), spingolipid activator protein, pregnancy zone protein, PAI-1 (plasminogen activator inhibitor- 1), antithrombin III, tissue factor pathway inhibitor (TFPI), Pseudomonas exotoxin A, heparin cofactor ⁇ , APP (amyloid precursor protein), thrombospondin- 1 , thrombospondin-2, lactoferrin, rhinovirus, RAP (alpha-2 macroglobulin receptor associated protein) and HIV-Tat protein.
- ApoE lipoprotein lipase, hepatic lipase, factor IXa, factor Villa, factor VIIa/TFPI, MMP-13 (matrix metaloproteinase
- the present invention relates to novel complexes and fusion proteins comprising a CD91 ligand and an antigenic molecule.
- the complexes and fusion proteins of the invention can induce an immune response against the antigenic molecule and are useful in the treatment or prevention of a disease.
- the invention also relates to methods of producing the complexes and fusion proteins, as well as compositions of complexes or fusion proteins.
- the present invention also provides methods of treating or preventing a disease in a subject using said complexes and fusion proteins and methods of inducing an immune response in a subject using said complexes and fusion proteins.
- CD91 ligands listed by way of example and not limitation, are suitable for use in the invention: ApoE, lipoprotein lipase, hepatic lipase, factor IXa, factor Villa, factor VIIa/TFPI, MMP-13, MMP-9, spingolipid activator protein, pregnancy zone protein, PAI-1, antithrombin El, Pseudomonas exotoxin A, tissue factor pathway inhibitor (TFPI), heparin cofactor II, APP, thrombospondin-1, thrombospondin-2, lactoferrin, rhinovirus, RAP and HIV-Tat protein.
- a purified complex is provided comprising a CD91 ligand and an antigenic molecule.
- the complex is immunogenic, i.e., when the complex is introduced into a suitable host, the host develops an immune response against the antigenic molecule of the complex.
- the antigenic molecule is not an antibody or a derivative thereof comprising a binding region thereof, hi one embodiment, the CD91 ligand is noncovalently associated with the antigenic molecule.
- the CD91 ligand is cross-linked to the antigenic molecule, hi yet another embodiment, the complex is the product of a method comprising complexing said CD91 ligand and said antigenic molecule in vitro, hi another embodiment, the antigenic molecule is a protein.
- the antigenic molecule is derived from an antigenic cell.
- the complex is free of biological cells and vesicles.
- the CD91 ligand is derived from a mammal.
- the CD91 ligand is derived from a human.
- the antigenic molecule is derived from a mammal.
- the antigenic molecule is derived from a human.
- the CD91 ligand and the antigenic molecule are derived from the same subject.
- the CD91 ligand and the antigenic molecule are derived from different subjects.
- a purified complex comprising a fragment of a CD91 ligand and an antigenic molecule.
- the complex is immunogenic.
- the fragment comprises a receptor binding domain.
- a purified fusion protein comprising an antigenic protein fused via a peptide bond to a CD91 ligand.
- the fusion protein is immunogenic.
- the complex is free of biological cells and vesicles.
- a purified fusion protein comprising an antigenic protein fused via a peptide bond to a fragment of a CD91 ligand.
- the fusion protein is immunogenic.
- the antigenic protein is not an antibody or a part thereof.
- the fragment comprises a receptor binding domain.
- the invention provides a composition comprising a plurality of purified complexes.
- each complex comprises a different antigenic molecule.
- the population of complexes of CD91 ligands bound to antigenic molecules is purified to apparent homogeneity, as viewed on an SDS- PAGE gel.
- a pharmaceutical composition comprising an amount of purified complex effective for treatment or prevention of a disease or a disorder.
- the complex of the pharmaceutical composition is immunogenic.
- the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
- Another embodiment of the invention provides a method for preparing immunogenic, in vitro complexes of a CD91 ligand associated with one or more antigenic molecules.
- the method comprises a) incubating a CD91 ligand or fragment thereof and one or more antigenic molecules under conditions and for a length of time sufficient for formation of complexes of the CD91 ligand non-covalently bound to the antigenic molecules; and b) isolating said complexes.
- the complexes are free of biological cells and vesicles, hi yet a further embodiment, the CD91 ligand is purified.
- a method of treating or preventing a disease in a subject is provided.
- the method comprises administering to the subject an amount of a purified complex effective to treat or prevent a disease.
- the CD91 ligand is noncovalently associated with the antigenic molecule.
- the CD91 ligand is cross-linked to the antigenic molecule.
- the complex is free of biological cells and vesicles.
- the antigenic molecule displays the antigenicity of a cancer-associated or cancer-specific antigen, or of an antigen that causes or is associated with a disease, respectively.
- Another embodiment of the invention provides a method of treating or preventing a disease in a subject comprising administering to the subject an amount of a purified fusion protein comprising an antigenic protein fused via a peptide bond to a CD91 ligand effective to treat or prevent a disease.
- the fusion protein is free of biological cells and vesicles.
- the antigenic molecule displays the antigenicity of a cancer-associated or cancer-specific antigen, or of an antigen that causes or is associated with a disease, respectively.
- the invention further provides a recombinant cell transformed with a) a first nucleic acid comprising a first nucleotide sequence that is operably linked to a first promoter and that encodes a CD91 ligand, and b) a second nucleic acid comprising a second nucleotide sequence that is operably linked to a second promoter and encodes an antigenic molecule, such that the CD91 ligand and the antigenic molecule are expressed within the cell and noncovalently associate with each other to form a complex that in sufficient amount is capable of eliciting an immune response to the antigenic molecule.
- the cell is a human cell.
- a recombinant cell in another aspect of the invention, is provided that is transformed with a nucleic acid comprising a nucleotide sequence that is operably linked to a promoter, wherein said nucleotide sequence encodes a fusion protein comprising an antigenic protein fused via a peptide bond to a CD91 ligand.
- the fusion protein is immunogenic.
- the invention provides pharmaceutical compositions comprising recombinant cells and pharmaceutically acceptable carriers.
- a method for preparing in vitro complexes of a CD91 ligand associated with one or more antigenic molecules comprising cross-linking the CD91 ligand to one or more antigenic molecules using a cross-linking agent is provided.
- the complex is immunogenic.
- Another aspect of the invention provides a method for preparing a fusion protein comprising a) culturing a recombinant cell transformed with a nucleic acid comprising a nucleotide sequence that is operably linked to a promoter, wherein said nucleotide sequence encodes a fusion protein comprising an antigenic protein fused via a peptide bond to a CD91 ligand, under conditions such that the fusion protein is expressed by the cell and b) recovering the fusion protein from the cells.
- the fusion protein is immunogenic.
- the complexes of the invention can be used in combination with other therapies in the treatment or prevention of a disease or disorder.
- Another aspect of the invention provides a method of inducing an immune response against an antigenic molecule in a subject comprising administering to the subject an effective amount of a purified complex comprising said antigenic molecule and a CD91 ligand.
- the CD91 ligand and antigenic molecule are non-covalently bound to each other.
- the CD91 ligand and antigenic molecule are cross-linked to each other.
- the complex is free of biological cells and vesicles.
- the immune response to the antigen is induced by enhancing uptake of said antigenic molecule by antigen presenting cells.
- Another aspect of the invention provides a method of inducing an immune response against an antigenic protein in a subject comprising administering to the subject an effective amount of purified fusion protein comprising said antigenic protein fused via a peptide bond to a CD91 ligand.
- the fusion protein is free of biological cells and vesicles.
- the immune response is induced by enhancing uptake of said antigenic protein by antigen presenting cells.
- the present invention provides complexes and fusion proteins of CD91 ligands and antigenic molecules, compositions of said fusion proteins and complexes, methods of preparing said fusion proteins and complexes, methods of treating or preventing a disease in a subject by immunizing with said fusion proteins and complexes, and methods of inducing an immune response to an antigenic molecule using the complexes and fusion proteins of the invention.
- the compositions and formulations of the present invention are administered to a subject to prevent a disease, including inhibiting the onset or development of the disease in a subject not having the disease, and inhibiting the progression of a disease in an asymptomatic subject.
- the complexes and or fusion proteins of the invention in such compositions and formulations are purified.
- disease means any abnormal physical or mental condition, or a condition of a living animal or plant body or of one of its parts that impairs normal functioning.
- Types of disease include, but are not limited to cancer, infectious diseases, neurodegenerative diseases, endocrine/metabolic diseases and vascular diseases.
- the compositions and formulations of the present invention are administered to a subject that has been diagnosed with a disease or is suspected of having a disease.
- treatment of a disease encompasses the treatment of subjects already diagnosed as having a disease; the delay of the onset or evolution or aggravation or deterioration of the symptoms or signs of a disease; and/or promoting regression of a disease in symptomatic subjects.
- CD91 ligand means a molecule that binds to CD91.
- antigenic molecule means any molecule containing an antigenic determinant, i.e., capable of being bound by an antibody or recognized by a T cell in the context of MHC Class I, Class II molecules, or CD1 molecules.
- Antigenic molecules can be, but not limited to, carbohydrates, lipids, polypeptides and peptides (polypeptides and peptides collectively referred to as "proteins").
- the term “antigenic molecule” does not encompass an antibody or a derivative thereof comprising a binding region thereof.
- an antigenic molecule comprises one or more antigenic determinants (epitopes) of a single protein.
- an antigenic molecule comprises multiple antigenic determinants, at least one antigenic determinant from each of two or more proteins.
- an antigenic molecule comprises one or more antigenic determinants (epitopes) of a single naturally occurring protein.
- an antigenic molecule comprises multiple antigenic determinants, at least one antigenic determinant from each of two or more naturally occuring proteins.
- the antigenic molecule comprises one or more antigenic determinants from a first naturally occurring protein and one or more antigenic determinants from a second naturally occurring protein, i.e., it can be a fusion protein of at least a portion of two different naturally occurring proteins.
- immunogenic in reference to a substance means that the substance, when introduced into a suitable host, can cause the production by the host of an immune response (antibody or cell-mediated) against the substance.
- CD91 ligands and antigenic molecules can be produced by any of a number of methods.
- antigenic molecules associated with a disease can be obtained by recombinant or synthetic methods, or can be isolated and purified from recombinant cells.
- the antigenic molecules can also be isolated from cells removed from a subject or cells in culture.
- Complexes between antigenic molecules and CD91 ligands can be formed by covalent or non-covalent association of antigenic molecules with CD91 ligands.
- the complexes of the invention are used in purified form, preferably to apparent homogeneity as viewed on an SDS-PAGE gel, or to at least 60%, 70%, 80%, or 90% of total protein.
- complexes may be formed in vitro using a variety of methods, described herein. Methods for preparing such CD91 ligand-antigenic molecule complexes are described in detail in Sections 4.3 to 4.5, below.
- CD91 ligand-antigenic peptide complexes and fusion proteins may be used as vaccines against various diseases. Without being bound by any particular theory, such complexes or fusion proteins may act by eliciting a B-cell and/or T-cell response in patients with such disorders. Methods for the use of such CD91 ligand-antigenic molecule complexes and fusion proteins as vaccines against various diseases are described in Section 4.6 in detail herein.
- the complexes comprise antigenic molecules complexed to CD91 ligands.
- the antigenic molecule is prepared by synthetic means.
- the antigenic molecule is prepared by recombinant methods, hi yet another embodiment, the antigenic molecules are from a preparation of proteins from an antigenic cell of interest.
- the antigenic molecules are prepared from tissue which displays the pathologic or histologic changes associated with a disease.
- the antigenic molecules are prepared from body fluid or any other biological source.
- compositions of the invention also comprise complexes of CD91 ligands and antigenic peptides that are prepared by first, generating a population of peptides from a preparation of proteins of the antigenic cells of interest, and then complexing the peptides to CD91 ligands.
- the methods used for preparing a protein preparation of antigenic cells do not selectively remove or retain any particular protein or peptide from other proteins and peptides in the antigenic cell. Even in certain embodiments when cytosolic proteins or membrane-derived proteins are used, the methods used to make the preparations do not selectively remove or retain any particular protein of the cytosol or of the membranes. Therefore, the majority of the proteins present in the cytosol or in the membranes are also present in the respective preparations of antigenic proteins and peptides from antigenic cells.
- substantially the entire repertoire of antigenic proteins and peptides of the antigenic cells, and substantially all the antigenic proteins and peptides in the cytosol or in the membranes are present in the complexing reaction and form complexes with CD91 ligands.
- Antigenic proteins or antigenic fragments thereof may be used as antigenic molecules.
- the proteins or fragments may be purified.
- Antigenic epitopes of proteins may optionally be screened using any method known in the art. Such techniques include, but are not limited to, methods that are based on algorithmic identification, peptide elution and cell-based binding assay techniques. Protein sequences may be analyzed to identify antigen-specific epitopes that meet criteria such as conservancy, binding, population coverage and immunogenicity. Potential epitopes can be identified by, e.g. a hydrophilicity analysis (see e.g., Hopp and Woods, 1981, Proc. Natl. Acad. Sci. USA 78:3824). Another such process for epitope identification is described in Sette et al., 2002,
- Curr Opin Investig Drugs 3:132-9 Briefly, computer algorithms analyze the amino acid sequence of all known antigens associated with the target indication for the presence of peptides which contain epitope motifs and which meet sequence conservancy requirements (epitopes from variable regions of the antigen are avoided). Peptides meeting the requirements of the computer screen are synthesized and in vitro HLA binding assays are performed. Peptides are evaluated for superfamily binding (assessing their ability to bind broadly within a family of HLA molecules). Identifying epitopes which bind broadly within superfamilies ensures broad population coverage for the ultimate vaccine. Peptides are then tested for immunogenicity, both in vivo in mice which express human MHC and in vitro against infected or transfected cells.
- Using such screening techniques allows identification of the specific amino acid sequence or sequences of a protein which elicit an immune response.
- Disease relevant epitopes may be tested and validated via functional T-cell assays, ensuring their clinical relevance, e.g., screened using T-cells from humans, confronted with the disease and who developed a protective natural immune response.
- Epitopes may be modified, e.g., by changing one or more amino acid residues, to enhance/optimize immunogenicity. See e.g., U.S. Patent 6,037,135, herein expressly incorporated by reference in its entirety.
- the invention provides complexes comprising these antigenic epitopes and CD91 ligands.
- a tumor specific or tumor associated antigen may be used as the antigenic molecule in the complexes or fusion proteins of the invention.
- Any such antigens known in the art may be used in the complexes and fusion proteins of the invention, in addition to the following tumor antigens, listed by way of example and not limitation: 707- AP (707 alanine proline), AFP (alpha ( ⁇ )-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T cells 4), BAGE (B antigen; /3-catenin/m, jS-catenin/mutated), Bcr-abl (breakpoint cluster region- Abelson), CAMEL (CTL-recognized antigen on melanoma) , CAP-1 (carcinoembryonic antigen peptide - 1), CASP-8 (caspase-8), CDC27m (cell-division cycle 27 mutated), CDK4/m
- a molecule comprising one or more epitopes of an infectious agent (e.g., viral antigen, bacterial antigen, etc.) that is the causative agent of the disease is used.
- an infectious agent e.g., viral antigen, bacterial antigen, etc.
- a molecule comprising epitope(s) of a virus is used; where it is desired to treat or prevent a bacterial infection, a molecule comprising epitope(s) of bacteria is used; where it is desired to treat or prevent protozoal infection, a molecule comprising epitope(s) of protozoa is used; and where it is desired to treat or prevent a parasitic infection, a molecule comprising epitope(s) of parasites is used.
- a molecule comprising epitope(s) of an antigenic molecule associated with a neurodegenerative disease, or epitope(s) of an antigenic molecule associated with an amyloid disease, including but not limited to a fibril peptide or protein, is used as the antigenic molecule of the invention.
- such a neurodegenerative disease- associated antigenic molecules may be a molecule associated with Alzheimer's Disease, age-related loss of cognitive function, senile dementia, Parkinson's disease, amyotrophic lateral sclerosis, Wilson's Disease, cerebral palsy, progressive supranuclear palsy, Guam disease, Lewy body dementia, prion diseases, spongiform encephalopathies, Creutzfeldt- Jakob disease, polyglutamine diseases, Huntington's disease, myotonic dystrophy, Freidrich's ataxia, ataxia, Gilles de la Tourette's syndrome, seizure disorders, epilepsy, chronic seizure disorder, stroke, brain trauma, spinal cord trauma, AIDS dementia, alcoholism, autism, retinal ischemia, glaucoma, autonomic function disorder, hypertension, neuropsychiatric disorder, schizophrenia, or schizoaffective disorder.
- Antigenic molecules that are suitable for in vitro complexing methods are disclosed in PCT publication no.
- WO 01/52890 dated July 26, 2001, which is incorporated by reference herein in its entirety, and include, but are not limited to, /3-amyloid or a fragment thereof, an oligomeric A/3 complex or a fragment thereof, an ApoE4-A/3 complex, tau protein or an fragment thereof, amyloid precursor protein or a fragment thereof, a mutant amyloid precursor protein or a fragment thereof, presenillin or a fragment thereof, a mutant of presenillin or a fragment thereof, ⁇ -synuclein or a fragment thereof, or a prion protein or a fragment thereof, and the antigenic derivatives of any of the foregoing proteins or fragments thereof.
- Amyloid disease associated antigenic molecules may be molecules associated with diseases characterized by the extracellular deposition of protein and/or peptide fibrils which form amyloid deposits or plaques, including but not limited to type II diabetes and amyloidoses associated with chronic inflammatory or infectious disease states and malignant neoplasms, e.g., myeloma.
- Certain amyloid disease such as Alzheimer's disease and prion diseases, e.g., Creutzfeldt Jacob disease, are neurodegenerative diseases.
- the antigenic molecule of the invention is related to an autoimmune disease.
- autoimmune diseases include, but are not limited to, insulin-dependent diabetes mellitus (i.e., IDDM, or autoimmune diabetes), multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, polymyositis, chronic active hepatitis, mixed connective tissue disease, primary biliary cirrhosis, pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease, vitiligo, gluten-sensitive enteropathy, Graves' disease, myasthenia gravis, autoimmune neutropenia, idiopathic thrombocytopenia purpura, rheumatoid arthritis, cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture's disease, bullous pemphigoid, discoid l
- IDDM insulin-dependent diabetes mellitus
- cholesteryl ester transfer protein can be an antigenic molecule.
- vascular diseases e.g., cardiovascular disease
- molecules comprising epitopes of antigenic molecules associated with vascular diseases are used as the antigenic molecules of the invention.
- angiotensin II can be an antigenic molecule in the treatment or prevention of vascular diseases.
- Antigenic proteins used in the complexes of the invention can be obtained from antigenic cells of interest. Since whole cancer cells, infected cells or other antigenic cells are used in one embodiment of the present methods, in such embodiment, it is not necessary to isolate or characterize or even know the identities of these antigenic proteins in advance of using such methods.
- the source of the antigenic cells may be selected, depending on the nature of the disease with which the antigens are associated.
- any tissues, or cells isolated from a cancer, including cancer that has metastasized to multiple sites can be used as an antigenic cell in the present method. For example, leukemic cells circulating in blood, lymph or other body fluids can also be used, solid tumor tissue (e.g., primary tissue from a biopsy) can be used.
- antigenic cells can be cancer cells or preneoplastic cells.
- the transition from non-neoplastic cell growth to neoplasia commonly consists of hyperplasia, metaplasia, and dysplasia (for review of such abnormal growth conditions (See Robbins and Angell, 1976, Basic Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-79).
- a non-limiting list of cancers, the cells of which can be used herein is provided in Section 4.13.
- Cancer cells derived from cancer tissues, cancer cells, infected cells, or cells that displays the pathologic or histologic changes associated with a disease can also be used as antigenic cells.
- Cancer or infected tissues, cells, or cell lines of human origin are preferred.
- Cancer cells, infected cells, or antigenic cells can be identified and isolated by any method known in the art. For example, cancer cells or infected cells can be identified by morphology, enzyme assays, proliferation assays, or the presence of pathogens or cancer-causing viruses. If the characteristics of the antigenic molecule of interest are known, antigenic cells can also be identified or isolated by any biochemical or immunological methods known in the art.
- cancer cells or infected cells can be isolated by surgery, endoscopy, other biopsy techniques, affinity chromatography, and fluorescence activated cell sorting (e.g., with fluorescently tagged antibody against an antigen express by the cells).
- Antigenic cells that display similar antigenicity have one or more antigenic determinants in common against which an immune response in a subject is desired (e.g., for therapeutic or prophylactic purposes).
- the cells may be cultured in vitro by standard methods to expand the number of cells prior to use in the present methods. There is no requirement that a clonal or homogeneous or purified population of antigenic cells be used. A mixture of cells can be used provided that a substantial number of cells in the mixture contain the antigens of interest. In a specific embodiment, the antigenic cells and/or immune cells are purified.
- the methods of the invention provide compositions of a CD91 ligand complexed to an antigenic molecule displaying the antigenicity of a tumor or cancer antigen of the same type of cancer, e.g., an antigen overexpressed in tumor or cancer cells relative to nontumor or noncancerous cells ("tumor associated antigens"), or an antigen expressed in tumor or cancer cells and not expressed in nontumor or noncancerous cells ("tumor specific antigen").
- tumor associated antigens an antigen overexpressed in tumor or cancer cells relative to nontumor or noncancerous cells
- tumor specific antigen an antigen expressed in tumor or cancer cells and not expressed in nontumor or noncancerous cells
- the antigenic molecules are antigenic peptides derived from cancer cells, preferably human cancers, e.g., tumor specific antigens and tumor associated antigens.
- the peptides can be generated by proteolytic digestion of proteins (e.g., cytosolic and/or membrane-derived proteins) from cancer cells, or antigenic cells that share antigenic determinants with or display similar antigenicity as the cancer cells.
- the antigenic molecules are antigenic peptides derived from cells infected by a pathogen or infectious agent that causes the infectious disease, or the pathogen which includes but is not limited to, a virus, bacterium, fungus, protozoan, parasite, etc.
- the pathogen is one that infects humans.
- the methods of the invention provide compositions of CD91 ligands complexed to antigenic molecules that display the antigenicity of an antigen of an infectious agent that causes an infectious disease or of an antigen that is associated with or causes an infectious disease.
- the antigenic peptides are generated by proteolytic digestion of (e.g., cytosolic and/or membrane-derived) proteins obtained from infected cells, antigenic cells that share antigenic determinants with or display similar antigenicity as the infected cells, or the pathogens including viral particles.
- the antigenic peptides can also be generated from antigenic cells that display the antigenicity of an agent (pathogen) that causes the infectious disease, or a variant of such agent. Infectious agents that can infect cells which can be used herein is provided in
- any pathogen or infectious agent that can cause an infectious disease can be used to infect a cell, and the infected cell used as an antigenic cell for the preparation of antigenic peptides.
- Variants of a pathogen or infectious agent such as but limited to replication-defective variants, non-pathogenic or attenuated variants, non- infectious variants, can also be used as an antigenic cell for this purpose.
- many viruses, bacteria, fungi, parasites and protozoans that can be cultured in vitro or isolated from infected materials can serve as a source of antigenic cells. Methods known in the art for propagating such pathogens including viral particles can be used.
- uninfected cells of a cell type susceptible to infection by the pathogen or infectious agent that causes the disease can be infected in vitro.
- standard techniques can be used to facilitate infection by the pathogen or infectious agent, and propagation of the infected cells.
- influenza viruses may be used to infect normal human fibroblasts; and mycobacteria may be used to infect normal human Schwann cells.
- variants of an infectious agent such as replication-defective viruses, non-pathogenic or attenuated mutants, or temperature- sensitive mutants can also be used to infect or transform cells to generate antigenic cells for the preparation of antigenic peptides.
- any method known in the art can be used to propagate and grow the pathogens. Such methods will depend on the pathogen, and may not involve infecting a host. For example, many techniques are known in the art for growing pathogenic bacteria, fungi and other non- viral microorganisms in culture, including large scale fermentation.
- any cell or tissue that displays the pathologic or histologic changes associated with a disease can be used as antigenic cell for the preparation of antigenic peptides.
- neurons that display the pathologic changes associated with Alzheimer's disease, such as plaque formation are suitable as antigenic cells.
- the methods of the invention provide compositions of CD91 ligands complexed to antigenic molecules displaying the antigenicity of an antigen that causes or is associated with the disease.
- the antigenic molecules are from cells which display the pathologic or histologic changes associated with the disease.
- normal cells of the appropriate cell type from the intended recipient maybe transformed or transfected in vitro with an expression construct comprising a nucleic acid molecule encoding such antigen, such that the antigen is expressed in the recipient's cells.
- an expression construct comprising a nucleic acid molecule encoding such antigen, such that the antigen is expressed in the recipient's cells.
- more than one such antigen may be expressed in the recipient's cell in this fashion, as will be appreciated by those skilled in the art, any techniques known, such as those described in Ausubel et al, 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, New York, may be used to perform the transformation or transfection and subsequent recombinant expression of the antigen gene in recipient's cells.
- a protein preparation which is derived from a pathogen, a cancer cell, an infected cell or a cell that displays the pathologic or histologic changes associated with a disease.
- the protein preparations are prepared, postoperatively, from tumor cells obtained from a cancer patient, hi another embodiment of the present invention, one or more antigenic proteins of interest are synthesized in cell lines modified by the introduction of recombinant expression systems that encode such antigens, and such cells are used to prepare the proteins.
- the proteins can be obtained from one or more cellular fraction(s), for example, the cytosol of the antigenic cells, or they can be extracted or solubilized from the membranes or cell walls of the antigenic cells.
- a cellular fraction comprises at least 20, 50, 100, 500, 1,000, 5,000, 10,000, or 20,000 different proteins.
- the term "protein preparation” refers to a mixture of proteins obtained from antigenic cells, a cellular fraction of antigenic cells, or virus particles.
- the proteins can be obtained from a cellular fraction, such as the cytosol.
- the proteins can also be non-cytosolic proteins (e.g., those from cell walls, cell membranes or organelles), or both.
- Cellular fractions may include but are not limited to cytosolic fractions, membrane fractions, and organelle fractions, such as nuclear, mitochondrial, lysosomal, and endoplasmic reticulum-derived fractions.
- the protein preparations can be obtained from non-recombinant or recombinant cells.
- antigenic proteins as used herein also encompasses antigenic polypeptides and antigenic peptides that may be present in the preparation.
- the protein preparation obtained from the antigenic cells or cellular fractions thereof or virus particles can optionally be purified from other non-proteinaceous materials to various degrees by techniques known in the art.
- the protein preparation may comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% 97%, 98%, 99% of the different proteins and peptides present in the antigenic cells or virus particles or a fraction of the antigenic cells.
- the protein preparations have not been subjected to any method of preparation that selectively removes or retains one or more particular protein(s) from the other proteins in the antigenic cells.
- the protein preparation is the total cell lysate which is not fractionated and/or purified, and may contain other non-proteinaceous materials of the cells
- the protein preparation is total protein in a cellular fraction, which has not been subjected to further fractionation or purification, and may contain other non-proteinaceous materials of the cells.
- the protein preparation is the total protein in a preparation of viral particles.
- the protein preparation comprises total cellular protein, total cytosolic proteins, or total membrane-bound proteins of antigenic cell(s).
- the protein preparation comprises at least 20, 50, 100, 500, 1,000, 5,000, 10,000, or 20,000 different proteins.
- a plurality of different antigenic proteins are present in a protein preparation of antigenic cells.
- the proteins in the protein preparation may be subjected to a step of protease digestion prior to in vitro complexing to CD91 ligands.
- the proteins in the protein preparation are not subjected to a step of protease digestion prior to in vitro complexing to CD91 ligands.
- the lysing of antigenic cells or disruption of cell walls, cell membranes, or viral particle structure can be performed using standard protocols known in the art.
- the antigenic cells can be lysed, for example, by mechanical shearing, sonication, freezing and thawing, adjusting the osmolarity of the medium surrounding the cells, or a combination of techniques.
- the antigenic cells can be lysed by chemicals, such as detergents.
- cellular debris materials that are non-proteinaceous or materials that do not contain cytosolic, and/or membrane-derived proteins (including proteins in the membranes of organelles). Removal of these components can be accomplished by techniques such as low speed centrifugation or filtration. After removing cellular debris and intact cells, a high speed centrifugation step can be used to separate the cytosolic proteins which are in the supernatant, and the membrane-derived proteins which are collected in the pellet. Standard procedures commonly known in the art allows the further isolation of the membrane-derived proteins from the pellet. Standard techniques commonly known in the art can be used to extract viral proteins from viral particles.
- the methods used do not or are not designed to selectively remove or retain any one or more particular protein(s) from other proteins that are present in the antigenic cell, in the cytosol or in the membranes, hi other embodiments, optionally, the proteins from the antigenic cells can be separated by their general biochemical and/or biophysical properties, such as size, charge, or combinations thereof. Any techniques known in the art can be used to perform the separation.
- Selected fractions of the proteins/peptides that comprise at least 20, 50, 100, 500, 1,000, 5,000, 10,000, or 20,000 different proteins or that comprise at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% 97%, 98%, 99% of the different proteins present in the antigenic cells or a cellular fraction thereof, or virus particles, are used to form complexes to a CD91 ligand.
- An exemplary, but not limiting, method that may be used to make a protein preparation comprising cytosolic proteins is as follows:
- Cells which may be tumor cells derived from a biopsy of the patient or tumor cells cultivated in vitro, or cell infected with a pathogenic agent, are suspended in 3 volumes of IX Lysis buffer comprising 30mM sodium bicarbonate pH 7.5, lmM PMSF, incubated on ice for 20 minutes and then the hypotonically-swollen cells are homogenized in a dounce homogenizer until >95% cells are lysed.
- IX Lysis buffer comprising 30mM sodium bicarbonate pH 7.5, lmM PMSF
- hypotonically-swollen cells are homogenized in a dounce homogenizer until >95% cells are lysed.
- cells can be sonicated, on ice, until >99% cells are lysed as determined by microscopic examination.
- a buffer such as phosphate buffered saline (PBS) which may comprises 1 mM PMSF, before sonication.
- PBS phosphate buffered sa
- the lysate is centrifuged at 1,000 x g for 10 minutes to remove intact cells, nuclei and other cellular debris.
- the resulting supernatant is recentrifuged at about 100,000 x g for about one hour, and the supernatant recovered.
- the 100,000 x g supernatant may be dialyzed for 36 hours at 4°C
- Cells which may be tumor cells derived from a biopsy of the patient or tumor cells cultivated in vitro, or cells infected with a pathogenic agent, are suspended in 3 volumes of IX Lysis buffer comprising 30mM sodium bicarbonate pH 7.5, lmM PMSF, incubated on ice for 20 minutes and then the hypotonically-swollen cells are homogenized in a dounce homogenizer until >95% cells are lysed.
- IX Lysis buffer comprising 30mM sodium bicarbonate pH 7.5, lmM PMSF
- hypotonically-swollen cells are homogenized in a dounce homogenizer until >95% cells are lysed.
- cells can be sonicated, on ice, until >99% cells are lysed as determined by microscopic examination.
- a buffer such as phosphate buffered saline (PBS) which may comprises 1 mM PMSF, before sonication.
- PBS phosphate buffered sa
- the lysate is then centrifuged at 100,000 x g for 10 minutes to collect the cell membranes.
- Membrane-derived proteins can be dislodged from the lipid bilayer and isolated from the 100,000g pellet (where the membrane- derived proteins are located) by resuspending the pellet in 5 volumes of PBS containing 1% sodium deoxycholate (without Ca 2+ and Mg 2+ ) and incubated on ice for 1 h.
- the resulting suspension is centrifuged for 30 min at 20,000g and the resulting supernatant harvested and dialyzed against several changes of PBS (without Ca 2+ and Mg 2+ ) to remove the detergent.
- the resulting dialysate is centrifuged for 90 min at 100,000g and the supernatant purified further.
- the population of cytosolic and/or membrane-derived proteins obtained from antigenic cells can be complexed to a CD91 ligand directly without protease treatment or any further extraction or selection processes. Alternatively, the proteins can be subjected to protease treatment prior to complexing.
- the cytosolic and membrane-derived proteins obtained from antigenic cells can be optionally digested to generate antigenic peptides.
- either the cytosolic or the membrane-derived proteins are used in the digestion.
- the cytosolic and membrane-derived proteins are combined in the digestion reaction to generate antigenic peptides.
- the protein preparations that are used in the protease digestion have not been subjected to any method(s) of preparation that selectively remove or retain one or more particular protein(s) from the other proteins in the antigenic cells, or the cytosol or membranes of the antigenic cells.
- proteases or proteolytic enzymes can be used in the invention to produce from a protein preparation of antigenic cells a population of peptides which comprises antigenic peptides.
- the enzymatic digestions can be performed either individually or in suitable combinations with any of the proteolytic enzymes that are well known in the art including, but not limited to, trypsin, Staphylococcal peptidase I (also known as protease V8), chymotrypsin, pepsin, cathepsin G, thermolysin, elastase, and papain.
- Trypsin is a highly specific serine proteinase that cleaves on the carboxyl-terminal side of lysines and arginines.
- Staphylococcal peptidase I a serine proteinase
- a digestion can be carried out with a single protease or a mixture of proteases.
- the proteases or proteolytic enzymes used are incubated under conditions suitable for the particular enzyme. Preferably, the enzyme is purified.
- Non-enzymatic methods such as cyanogen bromide cleavage, can also be used for generating peptides.
- the protein preparation to be digested can be aliquoted into a plurality of reactions each using a different enzyme, and the resulting peptides may optionally be pooled together for use. It may not be necessary to completely digest the proteins in the enzymatic reactions. These reactions results in the generation of a diverse and different set of peptides for each protein that is present in the protein preparation.
- the production of different peptide sets allows for a greater probability of generating antigenic peptides that are capable of inducing an immune response to the antigens in the protein preparation when they are complexed to a CD91 ligand.
- the protein preparation to be digested is aliquoted into two separate reactions and two different proteolytic enzymes are used to produce two different sets of peptides of the proteins present in the protein preparation.
- trypsin and Staphylococcal peptidase I are used separately to digest the protein preparation.
- the proteolytic enzymes used in the invention exhibit similar activities as the proteolytic activities that are found in the proteasome. The proteasome is responsible for the extralysosomal, endocatalytic degradation of cytosolic and nuclear proteins which are mis-folded or damaged in a cell.
- the proteasome can degrade proteins completely to yield single amino acids, can generate optimal major histocompatibility complex class I (MHC I)-binding epitopes, and can generate longer peptide precursors which could potentially undergo further trimming elsewhere in the cell to yield cytotoxic T cell epitopes.
- Cleavage preferences of the proteasome is on the carboxyl (COOH)-side of basic, acidic, and hydrophobic amino acids.
- Three known proteolytic enzymatic activities that are present in the proteasome are chymotrypsin-like activity, trypsin-like activity, and peptidylglutamylpeptide-hydrolyzing activity (Uebel and Tampe, 1999, Curr. Opin. Immunol. 11:203-208).
- enzymes having such activities and specificities can be used separately or in combination to digest the protein preparation.
- trypsin, chymotrypsin, and/or peptidylglutamylpeptide-hydrolase are used.
- the resulting peptide digestions comprise antigenic peptides, non-antigenic peptides, and single amino acid residues.
- the reactions may also comprise undigested or incompletely digested antigenic proteins.
- the proteolytic enzymatic digestions of the invention are monitored in order to generate peptides that fall within a desirable range of lengths. In a preferred embodiment, the peptides generated are from about 7 to about 20 amino acid residues. Most antigenic peptides that are presented to T cells by MHC class I and class II fall within this range.
- the population of peptides comprises peptides having a size range of 6 to 21, 8 to 19, 10 to 20, or at least 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50, amino acid residues.
- the antigenic peptides have 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 35 amino acid residues.
- a test reaction can be performed where small aliquots of a protein digestion are taken out of the reaction and monitored for the progression of digestion through either tricine-polyacrylamide gel electrophoresis ("tricine-PAGE"), high performance liquid chromatography (“HPLC”), or mass spectrometry, or any other method known in the art to determine the size of peptides.
- Other variables of the reaction that can be manipulated include the amount of protein in the reaction, the temperature, the duration of incubation, the presence of cofactors, etc.
- Enzymatic inhibitors that can be used in the invention include, but are not limited to, PMSF, bestatin, amastatin, leupeptin, and cystatin, depending on which enzymes are used in the protein digestion. Inhibitors for most proteases are well known in the art.
- another method of terminating an enzymatic digestion is by physical removal of the enzyme from the reaction. This can be done by attaching the enzyme of choice to a solid phase, such as a resin or a material that can easily be removed from the reaction by well known methods such as centrifugation or filtration. The protein preparation is allowed to contact or flow across the solid phase for a period of time.
- Such immobilized enzymes can be purchased commercially or can be produced by procedures for immobilizing enzymes that are well known in the art.
- the peptides can optionally be separated from low molecular weight materials, such as dipeptides, or single amino acid residues, in the preparation.
- the peptides can be isolated by centrifugation through a membrane, such as the Centriprep-3.
- the peptides can be separated by their general biochemical and/or biophysical properties, such as size, charge, or combinations thereof. Any techniques known in the art can be used to perform the separation resulting in digested protein preparation comprising at least 50, 100, 500, 1,000, 5,000, 10,000, 20,000, 50,000, or 100,000 different peptides.
- peptides that are endogenously present in antigenic cells can be used in the invention either alone or in combination with the peptides generated by the proteolytic digestion of the cytosolic and membrane-derived proteins.
- such peptides that are isolated directly from a protein preparation of antigenic cells can be complexed to CD91 ligands.
- the cytosolic or the membrane-derived proteins are used in the isolation process.
- the cytosolic and membrane- derived proteins are combined in the isolation process.
- the protein preparations that are used in the isolation have not been subjected to any method(s) of preparation that selectively remove or retain one or more particular protein(s) from the other proteins in the antigenic cells, or the cytosol or membranes of the antigenic cells.
- the protein preparation comprises comprise at least 20, 50, 100, 500,
- the method comprise treating the protein preparation to ATP, guanidium hydrochloride, and/or exposing the protein preparation to acidic conditions such that antigenic peptides in the protein preparation can be eluted.
- acidic conditions such that antigenic peptides in the protein preparation can be eluted.
- acids can be used, including but not limited to, trifluoroacetic acid. Methods known in the art such as those described in Marston and Hartley, 1990, Meth. Enzymol. 182:264-276 for dissociating protein aggregates can be used.
- the isolation process comprises exposing a protein preparation of antigenic cells with ATP, for example, at room temperature for one hour, and/or treating a protein preparation of antigenic cells with trifluoroacetic acid (TFA), for example 0.1% TFA.
- TFA trifluoroacetic acid
- the treatment preferably comprises sonicating the protein preparation in 0.1% TFA.
- a protein preparation is first exposed to ATP, followed by sonication in 0.1% TFA.
- Various protease inhibitors can be used in the invention prior to cell lysis and the isolation process.
- a mixture of 14 protease inhibitors can be used: phenylmethylsulfonyl fluoride (PMSF) 2 mM, ethylenediaminetetreacedic acid (EDTA) 1 mM, ethylene glycolbis(P-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA) 1 mM, (all obtained from Sigma, St.
- PMSF phenylmethylsulfonyl fluoride
- EDTA ethylenediaminetetreacedic acid
- EGTA ethylene glycolbis(P-aminoethyl ether)N,N,N',N'-tetraacetic acid
- the peptides resulting from the protein preparation comprise antigenic peptides and non-antigenic peptides of a variety of sizes ranging from at least 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50, amino acid residues.
- the peptides have 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 35 amino acid residues.
- the peptides are preferably recovered by separating from the proteins in the preparation prior to complexing with a CD91 ligand.
- the peptides can be recovered by centrifugation through a membrane, such as the Centriprep-3, by drying under vacuum, or by reverse phase chromatography, e.g., fractionation in a BioCad20 microanalytiocal HPLC Poros RH2 column (Perseptive Biosystems, Cambridge, MA), equilibrated with 0.1 % TFA in water and elution by acetonitrile.
- antigenic peptides that are endogenously present in antigenic cells and that are isolated directly from a protein preparation can be complexed to a CD91 ligand.
- a mixed population of peptides comprising peptides that are endogenously present in antigenic cells and peptides from digested cytosolic and membrane-derived proteins, can be complexed to a CD91 ligand.
- the peptide can be produced, either by recombinant techniques or by synthetic methods.
- the antigenic peptide may be synthesized using conventional peptide synthesis or any of a number of other protocols well known in the art.
- a peptide can be synthesized by use of a peptide synthesizer. Either the entire protein can be synthesized, or an antigenic determinant thereof, preferably the portion of the protein that contains the mutant or variant amino acid(s).
- Peptides may be synthesized by solid-phase peptide synthesis using procedures similar to those described by Merrifield, 1963, J. Am. Chem. Soc. 85:2149.
- N- ⁇ -protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to an insoluble polymeric support i.e., polystyrene beads.
- the peptides are synthesized by linking an amino group of an N-cu- deprotected amino acid to an ⁇ -carboxyl group of an N- ⁇ -protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide.
- N- ⁇ -protecting groups include Boc which is acid labile and Fmoc which is base labile. Details of appropriate chemistries, resins, protecting groups, protected amino acids and reagents are well known in the art (see Atherton et al, 1989, Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, and Bodanszky, 1993, Peptide Chemistry, A Practical Textbook, 2nd Ed., Springer- Verlag).
- nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the sequence of the antigenic proteins.
- Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, ⁇ -amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, 7-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, /3-alanine, fluoro-amino acids, designer amino acids such as /3
- the synthetic peptide prepared is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% ,96%, 97%, 98%, 99% or 100% pure.
- antigenic protein can produced by recombinant means. Once the nucleotide sequence encoding an antigenic protein has been identified, the nucleotide sequence, or a fragment thereof, can be obtained and cloned into an expression vector for recombinant expression. The expression vector can then be introduced into a host cell for propagation of the antigen. Methods for recombinant production of antigenic proteins are described in detail herein.
- Any recombinant antigenic protein useful in the complexes of the invention is preferably 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% ,96%, 97%, 98%, 99% or 100% pure.
- the DNA may be obtained by DNA amplification or molecular cloning directly from a tissue, cell culture, or cloned DNA (e.g., a DNA "library") using standard molecular biology techniques (see, e.g., Methods in Enzymology, 1987, volume 154, Academic Press; Sambrook et al. 1989, Molecular Cloning - A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, New York; and Current Protocols in Molecular Biology, Ausubel et al.
- Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will contain only exon sequences. Whatever the source, the antigen gene should be cloned into a suitable vector for propagation of the gene.
- DNA can be amplified from genomic or cDNA by polymerase chain reaction (PCR) amplification using primers designed from the known sequence of a related or homologous antigen.
- PCR is used to amplify the desired sequence in DNA clone or a genomic or cDNA library, prior to selection.
- PCR can be carried out, e.g., by use of a thermal cycler and Taq polymerase (Gene Amp®).
- the polymerase chain reaction (PCR) is commonly used for obtaining genes or gene fragments of interest.
- a nucleotide sequence encoding an antigenic protein of any desired length can be generated using PCR primers that flank the nucleotide sequence encoding the peptide- binding domain.
- an antigenic protein gene sequence can be cleaved at appropriate sites with restriction endonuclease(s) if such sites are available, releasing a fragment of DNA encoding the antigenic protein gene, or an antigenic derivative thereof. If convenient restriction sites are not available, they may be created in the appropriate positions by site-directed mutagenesis and/or DNA amplification methods known in the art (see, for example, Shankarappa et al, 1992, PCR Method Appl. 1 :277-278). The DNA fragment that encodes the antigenic protein is then isolated, and ligated into an appropriate expression vector, care being taken to ensure that the proper translation reading frame is maintained.
- DNA fragments are generated to form a genomic library. Since some of the sequences encoding related protein antigens are available and can be purified and labeled, the cloned DNA fragments in the genomic DNA library may be screened by nucleic acid hybridization to a labeled probe (Benton and Davis, 1977, Science 196:180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Those DNA fragments with substantial homology to the probe will hybridize. It is also possible to identify an appropriate fragment by restriction enzyme digestion(s) and comparison of fragment sizes with those expected according to a known restriction map.
- RNA for cDNA cloning of the antigenic protein gene can be isolated from cells which express the antigenic protein.
- a cDNA library may be generated by methods known in the art and screened by methods, such as those disclosed for screening a genomic DNA library. If an antibody to the antigenic protein is available,the antigenic protein may be identified by binding of a labeled antibody to the antigenic protein synthesizing clones.
- Other specific embodiments for the cloning of a nucleotide sequence encoding an antigenic protein are presented as examples but not by way of limitation, as follows:
- nucleotide sequences encoding an antigenic protein can be identified and obtained by hybridization with a probe comprising a nucleotide sequence encoding the antigenic protein under conditions of low to medium stringency.
- procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:6789-6792).
- Filters containing DNA are pretreated for 6 h at 40°C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1%
- Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ⁇ g/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 X 10 6 cpm 32 P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 h at 40°C, and then washed for 1.5 h at 55°C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS.
- the wash solution is replaced with fresh solution and incubated an additional 1.5 h at 60°C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68°C and reexposed to film.
- Other conditions of low stringency which may be used are well known in the art (e.g., as employed for cross-species hybridizations).
- Any technique for mutagenesis known in the art can be used to modify individual nucleotides in a DNA sequence, for purpose of making amino acid substitution(s) in the expressed peptide sequence, or for creating/deleting restriction sites to facilitate further manipulations.
- Such techmques include but are not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson et al, 1978, J. Biol. Chem. 253:6551), oligonucleotide-directed mutagenesis (Smith, 1985, Ann. Rev. Genet. 19:423-463; Hill et al., 1987, Methods Enzymol. 155:558-568), PCR-based overlap extension (Ho et al.
- Nucleotide sequences encoding an antigenic protein can be inserted into the expression vector for propagation and expression in recombinant cells.
- An expression construct refers to a nucleotide sequence encoding an antigenic protein operably associated with one or more regulatory regions which allows expression of the antigenic protein in an appropriate host cell. "Operably-associated” refers to an association in which the regulatory regions and the antigenic protein sequence to be expressed are joined and positioned in such a way as to permit transcription, and ultimately, translation of the protein sequence.
- a variety of expression vectors may be used for the expression of proteins, including, but not limited to, plasmids, cosmids, phage, phagemids, or modified viruses. Examples include bacteriophages such as lambda derivatives, or plasmids such as pBR322 or pUC plasmid derivatives or the Bluescript vector (Stratagene). Typically, such expression vectors comprise a functional origin of replication for propagation of the vector in an appropriate host cell, one or more restriction endonuclease sites for insertion of the antigenic protein gene sequence, and one or more selection markers. Vectors based on E. coli are the most popular and versatile systems for high level expression of foreign proteins (Makrides, 1996, Microbiol. Rev.
- Non-limiting examples of prokaryotic expression vectors may include the ⁇ gt vector series such as ⁇ gtl 1 (Huynh et al, 1984 in "DNA Cloning Techniques", Vol. I: A Practical Approach (D. Glover, ed.), pp. 49-78, IRL Press, Oxford), and the p ⁇ T vector series (Studier et al, 1990, Methods Enzymol., 185:60-89).
- Non-limiting examples of regulatory regions that can be used for expression in E. coli may include but not limited to lac, trp, Ipp, phoA, recA, tac, ⁇ P L, and phage T3 and T7 promoters (Makrides, 1996, supra).
- a potential drawback of a prokaryotic host- vector system is the inability to perform many of the post-translational processing events of mammalian cells.
- a eukaryotic host-vector system is preferred, a mammalian host-vector system is more preferred, and a human host-vector system is the most preferred.
- the regulatory regions necessary for transcription of an antigenic protein or polypeptide can be provided by the expression vector.
- a translation initiation codon (ATG) may also be provided to express a nucleotide sequence encoding an antigenic protein that lacks an initiation codon.
- RNA polymerase and transcription factors will bind to the regulatory regions on the expression construct to effect transcription of the antigenic protein sequence in the host organism.
- the precise nature of the regulatory regions needed for gene expression may vary from host cell to host cell.
- a promoter is required which is capable of binding RNA polymerase to initiate the transcription of an operably-associated nucleic acid sequence.
- Such regulatory regions may include those 5 '-non-coding sequences involved with initiation of transcription and translation, such as a TATA box, cap site, a CAAT box, transcription factor binding sites, enhancer elements, and the like.
- the non-coding region 3' to the coding sequence may contain transcriptional termination regulatory sequences, such as terminators and polyadenylation sites.
- Both constitutive and inducible regulatory regions may be used for expression of the antigenic protein. It may be desirable to use inducible promoters when the conditions optimal for growth of the recombinant cells and the conditions for high level expression of the antigenic protein are different. Examples of useful regulatory regions are provided in the next section below.
- regulatory regions For expression of antigenic proteins in mammalian host cells, a variety of regulatory regions can be used, for example, the SV40 early and late promoters, the cytomegalovirus
- CMV Rous sarcoma virus long terminal repeat
- RSV- LTR Rous sarcoma virus long terminal repeat
- Inducible promoters that may be useful in mammalian cells include but are not limited to those associated with the metallothionein II gene, mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV-LTR), the /3-interferon gene, and the Hs ⁇ 70 gene (Williams et al, 1989, Cancer Res. 49:2735-42 ; Taylor et al, 1990, Mol. Cell. Biol. 10:165-75).
- elastase I gene control region which is active in pancreatic acinar cells (Swift et al, 1984, Cell 38:639-646; Ornitz et al, 1986, Cold Spring Harbor Symp. Quant. Biol.
- mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al, 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al, 1987, Genes Dev. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al, 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al, 1987, Science 235:53-58; alpha 1-antirrypsin gene control region which is active in the liver (Kelsey et al, 1987, Genes Dev.
- beta-globin gene control region which is active in myeloid cells (Mogram et al, 1985, Nature 315:338-340; Kollias et al, 1986, Cell 46: 89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al, 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283- 286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al, 1986, Science 234: 1372-1378).
- the efficiency of expression of the antigenic protein in a host cell may be enhanced by the inclusion of appropriate transcription enhancer elements in the expression vector, such as those found in SV40 virus, Hepatitis B virus, cytomegalovirus, immunoglobulin genes, metallothionein, /3-actin (see Bittner et al, 1987, Methods in Enzymol. 153:516-544; Gorman, 1990, Curr. Op. Biotechnol. 1:6-47).
- the expression vector may also contain sequences that permit maintenance and replication of the vector in more than one type of host cell, or integration of the vector into the host chromosome.
- sequences may include but are not limited to replication origins, autonomously replicating sequences (ARS), centromere DNA, and telomere DNA. It may also be advantageous to use shuttle vectors that can be replicated and maintained in at least two types of host cells.
- the expression vector may contain selectable or screenable marker genes for initially isolating or identifying host cells that contain DNA encoding an antigenic protein. For long term, high yield production of antigenic proteins, stable expression in mammalian cells is preferred.
- selectable or screenable marker genes for initially isolating or identifying host cells that contain DNA encoding an antigenic protein. For long term, high yield production of antigenic proteins, stable expression in mammalian cells is preferred.
- a number of selection systems may be used for mammalian cells, including, but not limited, to the Herpes simplex virus thymidine kinase (Wigler et al, 1977, Cell 11:223), hypoxanthine-guanine phosphoribosylfransferase (Szybalski and Szybalski, 1962, Proc. Natl. Acad. Sci. U.S.A.
- adenine phosphoribosylfransferase (Lowy et al, 1980, Cell 22:817) genes can be employed in tk ' , hgprf or aprf cells, respectively.
- antimetabolite resistance can be used as the basis of selection for dihydrofolate reductase (dhfr), which confers resistance to methotrexate (Wigler et al, 1980, Natl. Acad. Sci. U.S.A. 77:3567; O'Hare et al, 1981, Proc. Natl. Acad. Sci. U.S.A.
- DNA sequences with regulatory functions such as promoters
- linkers or adapters providing the appropriate compatible restriction sites may be ligated to the ends of cDNA or synthetic DNA encoding an antigenic protein, by techniques well known in the art (Wu et al, 1987, Methods Enzymol. 152:343-349). Cleavage with a restriction enzyme can be followed by modification to create blunt ends by digesting back or filling in single-stranded DNA termini before ligation.
- a desired restriction enzyme site can be introduced into a fragment of DNA by amplification of the DNA by use of PCR with primers containing the desired restriction enzyme site.
- the expression construct comprising an antigenic protein sequence operably associated with regulatory regions can be directly introduced into appropriate host cells for expression and production of antigenic protein-CD91 ligand complexes without further cloning (see, for example, U.S. Patent No. 5,580,859).
- the expression constructs may also contain DNA sequences that facilitate integration of the antigenic protein nucleotide sequence into the genome of the host cell, e.g., via homologous recombination.
- an expression vector comprising a replication origin suitable for appropriate host cells in order to propagate and express the antigenic protein in the host cells.
- Expression constructs containing cloned nucleotide sequence encoding antigenic polypeptides can be introduced into the host cell by a variety of techniques known in the art, including but not limited to, for prokaryotic cells, bacterial transformation (Hanahan, 1985, in DNA Cloning, A Practical Approach, 1:109-136), and for eukaryotic cells, calcium phosphate mediated transfection (Wigler et al, 1977, Cell 11:223-232), liposome-mediated transfection (Schaefer-Ridder et al.
- Cell lines that stably express antigenic proteins or antigenic protein-CD91 ligand complexes may be engineered by using a vector that contains a selectable marker.
- engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
- the selectable marker in the expression construct confers resistance to the selection and optimally allows cells to stably integrate the expression construct into their chromosomes and to grow in culture and to be expanded into cell lines. Such cells can be cultured for a long period of time while antigenic protein is expressed continuously.
- any of the cloning and expression vectors described herein may be synthesized and assembled from known DNA sequences by techniques well known in the art.
- the regulatory regions and enhancer elements can be of a variety of origins, both natural and synthetic.
- Some vectors and host cells may be obtained commercially. Non-limiting examples of useful vectors are described in Appendix 5 of Current Protocols in Molecular Biology, 1988, ed. Ausubel et al, Greene Publish. Assoc. & Wiley Interscience, which is incorporated herein by reference; and the catalogs of commercial suppliers such as Clontech
- a number of viral-based expression systems may also be utilized with mammalian cells to produce antigenic proteins.
- Vectors using DNA virus backbones have been derived from simian virus 40 (SV40) (Hamer et al, 1979, Cell 17:725), adenovirus (Van Doren et al, 1984, Mol. Cell Biol. 4:1653), adeno-associated virus (McLaughlin et al, 1988, J. Virol. 62:1963), and bovine papillomas virus (Zinn et al, 1982, Proc. Natl. Acad. Sci. 79:4897).
- SV40 simian virus 40
- adenovirus Van Doren et al, 1984, Mol. Cell Biol. 4:1653
- adeno-associated virus McLaughlin et al, 1988, J. Virol. 62:1963
- bovine papillomas virus Zainn et al, 1982, Proc.
- the donor DNA sequence may be ligated to an adenovirus transcription translation control region, e.g., the late promoter and tripartite leader sequence.
- This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing heterologous products in infected hosts (see, e.g, Logan and Shenk, 1984, Proc. Natl. Acad. Sci. U.S.A. 81:3655-3659).
- Bovine papillomavirus can infect many higher vertebrates, including man, and its DNA replicates as an episome.
- a number of shuttle vectors have been developed for recombinant gene expression which exist as stable, multicopy (20-300 copies/cell) extrachromosomal elements in mammalian cells.
- these vectors typically contain a segment of BPV DNA (the entire genome or a 69% transforming fragment), a promoter with a broad host range, a polyadenylation signal, splice signals, a selectable marker, and "poisonless" plasmid sequences that allow the vector to be propagated in E. coli.
- the expression gene construct is transfected into cultured mammalian cells, for example, by the techniques of calcium phosphate coprecipitation or elecfroporation.
- selection of transformants is achieved by use of a dominant selectable marker, such as histidinol and G418 resistance.
- BPV vectors such as pBCMGSNeo and pBCMGHis may be used to express antigenic peptide sequences (Karasuyama et al, Eur. J. Immunol.
- the vaccinia 7.5K promoter may be used (see, e.g., Mackett et al, 1982, Proc. Natl. Acad. Sci. U.S.A. 79:7415-7419; Mackett et al, 1984, J. Virol. 49:857- 864; Panicali et al, 1982, Proc. Natl. Acad. Sci. U.S.A.
- vectors based on the Epstein-Barr virus (EBV) origin (OriP) and EBV nuclear antigen 1 (EBNA-1 ; a trans-acting replication factor) may be used.
- EBV Epstein-Barr virus
- EBNA-1 EBV nuclear antigen 1
- Such vectors can be used with a broad range of human host cells, e.g., EBO-pCD (Spickofsky et al, 1990, DNA Prot. Eng. Tech. 2:14-18), pDR2 and XDR2 (available from Clontech Laboratories).
- Antigenic proteins may also be made with a retrovirus-based expression system.
- retroviruses can efficiently infect and transfer genes to a wide range of cell types including, for example, primary hematopoietic cells.
- retroviruses such as Moloney murine leukemia virus
- most of the viral gene sequences can be removed and replaced with nucleic acid sequences encoding the antigenic protein, while the missing viral functions can be supplied in trans.
- the host range for infection by a retroviral vector can also be manipulated by the choice of envelope used for vector packaging.
- a retroviral vector can comprise a 5' long terminal repeat (LTR), a 3' LTR, a packaging signal, a bacterial origin of replication, and a selectable marker.
- the antigenic peptide DNA is inserted into a position between the 5 ' LTR and 3 ' LTR, such that transcription from the 5' LTR promoter transcribes the cloned DNA.
- the 5' LTR comprises a promoter, including but not limited to an LTR promoter, an R region, a U5 region and a primer binding site, in that order. Nucleotide sequences of these LTR elements are well known in the art.
- a heterologous promoter as well as multiple drug selection markers may also be included in the expression vector to facilitate selection of infected cells (see McLauchlin et al, 1990, Prog. Nucleic Acid Res. and Molec. Biol. 38:91-135; Morgenstern et al, 1990, Nucleic Acid Res. 18: 3587-3596; Choulika et al, 1996, J. Virol 70:1792-1798; Boesen et al, 1994, Biotherapy 6: 291-302; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114).
- yeast a number of vectors containing constitutive or inducible promoters may be used with Saccharomyces cerevisiae (baker's yeast), Schizosaccharomyces pombe (fission yeast), Pichia pastoris, and Hansenula polymorpha (methylotropic yeasts).
- Saccharomyces cerevisiae bakeer's yeast
- Schizosaccharomyces pombe Schizosaccharomyces pombe
- Pichia pastoris Pichia pastoris
- Hansenula polymorpha methylotropic yeasts
- AcNPV can be used as a vector to express an antigenic peptide in Spodopterafrugiperda cells.
- the antigenic protein DNA may be cloned into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter). These recombinant viruses are then used to infect host cells in which the inserted DNA is expressed (see, e.g., Smith et al, 1983, J. Virol. 46:584; Smith, U.S. Patent No. 4,215,051).
- the expression vector must be used with a compatible host cell which may be derived from a prokaryotic or an eukaryotic organism, including, but not limited to bacteria, yeasts, insects, mammals, and humans. Any cell type that is compatible with the expression vector may be used, including those that have been cultured in vitro or genetically engineered. Host cells may be obtained from normal or affected subjects, including healthy humans, patients with cancer or an infectious disease, private laboratory deposits, public culture collections such as the American Type Culture Collection, or from commercial suppliers.
- Preferred mammalian host cells include but are not limited to those derived from humans, monkeys and rodents, (see, for example, Kriegler M. in “Gene Transfer and Expression: A Laboratory Manual", New York, Freeman & Co. 1990), such as monkey kidney cell line transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line (293, 293-EBNA), or 293 cells subcloned for growth in suspension culture (Graham et al, 1977, J. Gen. Virol. 36:59), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary-cells-DHFR (CHO, Uriaub and Chasin, 1980, Proc. Natl. Acad. Sci.
- mice sertoli cells (Mather, 1980, Biol. Reprod. 23:243-251), mouse fibroblast cells (NIH-3T3), monkey kidney cells (CVI ATCC CCL 70), african green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75), human liver cells (Hep G2, KB 8065), and mouse mammary tumor cells (MMT 060562, ATCC CCL51).
- Exemplary cancer cell types used for demonstrating the utility of recombinant cells as a cancer vaccine are provided as follows: mouse fibroblast cell line, NTH3T3, mouse Lewis lung carcinoma cell line, LLC, mouse mastocytoma cell line, P815, mouse lymphoma cell line, EL4 and its ovalbumin transfectant, E.G7, mouse melanoma cell line,
- B16F10 mouse fibrosarcoma cell line, MC57, and human small cell lung carcinoma cell lines, SCLC#2 and SCLC#7.
- the recombinant cells may be cultured under standard conditions of temperature, incubation time, optical density, and media composition.
- a cells may be cultured under conditions emulating the nutritional and physiological requirements of a cell in which the antigenic protein is endogenously expressed.
- the antigenic protein or an antigenic portion thereof, can be purified by any methods appropriate for the protein, and then used to form complexes with CD91 ligands as described in section 4.4, below.
- the recombinant antigenic proteins of the invention can be recovered and purified from recombinant cell cultures by known methods, including ammonium sulfate precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, immunoaffinity chromatography, hydroxyapatite chromatography, and lectin chromatography.
- the invention provides methods for purification of recombinant antigenic proteins by affinity purification.
- affinity chromatography well known in the art. One approach is based on specific molecular interactions between an affinity label present on the antigenic protein and its binding partner. A second approach, immunoaffinity chromatography, relies on the immunospecific binding of an antibody to an epitope present on the tag.
- Protein A affinity chromatography a method that is generally applicable to purifying recombinant antigenic proteins that are fused to the constant regions of immunoglobulin, is a well known technique in the art.
- Staphylococcus protein A is a 42 kD polypeptide that binds specifically to a region located between the second and third constant regions of heavy chain immunoglobulins. Because of the Fc domains of different classes, subclasses and species of immunoglobulins, affinity of protein A for human Fc regions is strong, but may vary with other species. Subclasses that are less preferred include human IgG-3, and most rat subclasses.
- protein G (of Streptococci) may be used in place of protein A in the purification.
- Protein-A sepharose (Pharmacia or Biorad) is a commonly used solid phase for affinity purification of antibodies, and can be used essentially in the same manner for the purification of an antigenic protein of interest fused to an immunoglobulin Fc fragment.
- Bound antigenic protein of interest can be eluted by various buffer systems known in the art, including a succession of citrate, acetate and glycine-HCl buffers which gradually lowers the pH. This method is less preferred if the recombinant cells also produce antibodies which will be copurified with the antigenic protein. See, for example, Langone, 1982, J. Immunol. Meth. 51:3; Wilchek et al, 1982, Biochem. itl. 4: 629; Sjobring et al, 1991, J. Biol. Chem. 26:399; page 617-618, in Antibodies A Laboratory Manual, edited by Harlow and Lane, Cold Spring Harbor laboratory, 1988.
- a polyhistidine tag may be used, in which case, the antigenic protein can be purified by metal chelate chromatography.
- the polyhistidine tag usually a sequence of six histidines, has a high affinity for divalent metal ions, such as nickel ions (Ni 2+ ), which can be immobilized on a solid phase, such as nitrilotriacetic acid matrices.
- the purification method comprises loading the cell culture supernatant onto
- Ni 2+ -NTA-agarose can be obtained from commercial suppliers such as Sigma (St. Louis) and Qiagen.
- Antibodies that recognize the polyhistidine tag are also available which can be used to detect and quantify the antigenic protein.
- GST glutathione-S-transferase
- an antigenic protein-GST fusion expressed in a prokaryotic host cell such as E. coli
- GST glutathione-S-transferase
- an antigenic protein-GST fusion expressed in a prokaryotic host cell such as E. coli
- E. coli can be purified from the cell culture supernatant by absorption with glutathione agarose beads, followed by elution in the presence of free reduced glutathione at neutral pH. Denaturing conditions are not required at any stage during purification, and therefore, it may be desirable co-purification of CD91 ligands and antigenic proteins, for use in the loading of immobilized CD91 ligands with antigenic proteins.
- GST is known to form dimers under certain conditions, dimeric antigenic proteins may be obtained. See, Smith, 1993, Methods Mol. Cell Bio. 4:220-229.
- MBP maltose binding protein
- E. coli Another useful affinity label that can be used is the maltose binding protein (MBP) of E. coli, which is encoded by the malE gene.
- MBP maltose binding protein
- the secreted protein-MBP present in the cell supernatant binds to amylose resin while contaminants are washed away.
- the bound antigenic protein-MBP is eluted from the amylose resin by maltose. See, e.g., Guan et al, 1987, Gene 67:21-30.
- the second approach for purifying antigenic proteins is applicable to affinity labels that contain an epitope for which polyclonal or monoclonal antibodies are available.
- Various methods known in the art for purification of protein by immunospecific binding such as immunoaffinity chromatography, and immunoprecipitation, can be used. See, e.g., Chapter 13 in Antibodies A Laboratory Manual, edited by Harlow and Lane, Cold Spring Harbor laboratory, 1988; and Chapter 8, Sections I and II, in Current Protocols in Immunology, ed. by Coligan et al, John Wiley, 1991; the disclosure of which are both incorporated by reference herein.
- CD91 ligands suitable for the complexes and fusion proteins of the invention can be isolated from the cells and/or serum of a subject by various methods described in the art (see, e.g., Bensadoun et al, Methods in Molecular Biology, Vol. 109, Humana Press Inc., Totowa, NJ, (Doolittle and Reue eds.), pages 145-50).
- Amino acid sequences and nucleotide sequences of many CD91 ligands are generally available in sequence databases, such as GenBank.
- Computer programs, such as Entrez can be used to browse the database, and retrieve any amino acid sequence and genetic sequence data of interest by accession number. These databases can also be searched to identify sequences with various degrees of similarities to a query sequence using programs, such as FASTA and BLAST, which rank the similar sequences by alignment scores and statistics.
- GenBank accession numbers for some CD91 ligands ApoE (GenBank Accession Number: LPHUE), Lipoprotein lipase (GenBank Accession Number: LIHUL), Hepatic lipase (GenBank Accession Number: AAC34206), Factor IXa (GenBank Accession Number: KFHU), Factor Villa (GenBank Accession Number: P00451, GenBank Accession Number: EZHU), Factor VIIa TFPI (GenBank Accession Number: KFHU7, GenBank Accession Number: PI 0646), MMP-13 (GenBank Accession Number: P45452), MMP-9 (GenBank Accession Number: CAC07541), Pregnancy Zone Protein (GenBank Accession Number: S29738), PAI- 1 , Antithrombin HI (GenBank Accession Number: AAB40025), Tissue factor pathway inhibitor (TFPi)(GenBank Accession Number: KFHU7), Heparin cofactor II (GenBank Accession Number: NP_
- CD91 ligands can be produced by any method known in the art, such as the recombinant methods described above (Section 4.1.3) for the production of antigenic proteins.
- CD91 ligand- encoding cDNA or genomic DNA may be obtained from any species, for example, by PCR amplification.
- Oligonucleotide primers representing known nucleic acid sequences of related CD91 ligands can be used as PCR primers. One can choose to synthesize several different degenerate primers for use in the PCR reactions.
- the size of the coding region of the CD91 ligand gene being amplified is too large to be amplified in a single PCR, several PCR covering the entire gene, preferably with overlapping regions, may be carried out, and the products of the PCR ligated together to form the entire coding sequence.
- a segment of a CD91 ligand gene is amplified, that segment may be cloned, and utilized as a probe to isolate a complete cDNA or genomic clone.
- the CD91 ligand gene may then be cloned into an appropriate expression vector, and be produced, propogated, isolated and purified according to methods for recombinant production of proteins, such as those described for recombinant production of antigenic proteins described in Section 4.1.3, above.
- a CD91 ligand, or a fragment thereof can be synthesized using methods known in the art, such as those described for the synthesis of antigenic proteins, in Section 4.1.2, above.
- conventional peptide synthesis may be used, or other synthetic protocols well known in the art.
- CD91 ligand Purification of the resulting CD91 ligand, or fragment thereof, may then be accomplished using conventional procedures, such as preparative HPLC using gel permeation, partition and/or ion exchange chromatography.
- preparative HPLC using gel permeation, partition and/or ion exchange chromatography.
- matrices and buffers are well known in the art.
- the invention contemplates complexes and fusion proteins comprising a CD91 ligand fragment and an antigenic molecule.
- the CD91 ligand fragment contains a receptor binding domain.
- Receptor binding domains have been identified for various CD91 ligands (see, e.g., Swertfeger and Hui, 2001, J. Biol. Chem. 276:25043 and Weisgraber et al, 1983, J. Biol. Chem. 258:12348 (receptor binding domain for Apo E at amino acid residues 141-155); Williams et al, 1994, J. Biol. Chem. 269:8653 and Krapp et al, 1995, J. Lipid Res.
- Chem. 274:37685-92 (receptor binding domain for factor Villa, A2 domain at amino acid residues 484-509); Lenting et al, 1999, J. Biol. Chem. 274:23734-39 (receptor binding domain for factor Villa, light chain-C2 domain at amino acid residues 2173- 2332); Iakhiaev et al, 1999, J. Biol. Chem. 274:36995-37003 and Hamik et al, 1999, J. Biol. Chem. 274:4962-69 (receptor binding domain for TFPI at C terminal domain); Steffansson et al, 1998, J. Biol. Chem.
- One such method for identifying a binding domain of a CD91 ligand comprises constructing mutants of the CD91 ligand (for example, the mutants may contain a deletion, truncation or point mutation). The mutants are then assayed for their ability to interact with a receptor which is known to bind to the CD91 ligand. In this manner, the binding domain of the ligand can be identified.
- Another such method for identifying a binding domain of a CD91 ligand comprises assaying for the ability of a particular fragment or epitope of a CD91 ligand to modulate receptor-ligand binding events.
- Another example of a method for identifying a binding domain of a CD91 ligand comprises the use of an antibody that binds to a particular epitope on the CD91 ligand. The CD91 ligand is first exposed to the antibody, and then the CD91 ligand is assayed for its ability to bind a receptor of interest. Any method known in the art for detecting receptor binding may be used (see, e.g. ,
- cell free assays that may be used include assays in which an immobilized protein is tested for its ability to bind to a putative cognate protein (e.g., ELISA and affinity matrices), and two hybrid or three hybrid assays (see, e.g., U.S. Patent No. 5,283,317; Zervos et al, 1993, Cell 72:223-232; Madura et al, 1993, J. Biol. Chem.
- any cell based assay known in the art may be used to detect receptor- ligand interactions.
- a receptor protein or biologically active portion thereof is contacted with either 1) mutant ligand molecules, 2) fragments or epitopes of ligand, or 3) ligand molecules in the presence of an antibody, or fragments or epitopes of ligand.
- the receptor-ligand binding interaction is then monitored, for example, by cellular phenotype or a receptor mediated cellular response (e.g. , uptake of ligand or modulation of signal transduction).
- CD91 ligands with antigenic molecules are produced in vitro.
- Immunogenic CD91 ligand-antigenic molecule complexes can be generated in vitro by covalent or non-covalent coupling of a CD91 ligand with an antigenic molecule.
- antigenic molecules may be isolated from various sources, chemically synthesized, or produced recombinantly.
- CD91 ligands may also be prepared by a variety of methods, as described in Section 4.2, above. After isolation of CD91 ligands and antigenic molecules, complexes are produced in vitro.
- non-covalent immunogenic complexes of the invention Any method known in the art can be used for preparing non-covalent complexes of the invention.
- the formation of non-covalent immunogenic complexes of CD91 ligands and antigenic molecules can be achieved under conditions that favor complex formation.
- the following example describes a method for preparing non- covalent immunogenic complexes of CD91 ligands and antigenic molecules:
- CD91 ligands are prepared, as described in Section 4.2, above.
- the antigenic molecules and the CD91 ligand are mixed together.
- the mixture is then incubated in a suitable buffer.
- the preparations are then centrifuged through filter to remove any unbound peptide. If the CD91 ligand is bound to a solid phase, the CD91 ligand-antigenic molecule complexes formed can be washed free of unbound antigenic molecules prior to eluting the CD91 ligand-antigenic molecule complexes off the solid phase.
- the association of the antigenic molecules with the CD91 ligands can be assayed by any method known in the art.
- the following example describes a preferred method for preparing non- covalent immunogenic complexes of CD91 ligands and antigenic molecules:
- CD91 ligands are prepared, as described in Section 4.2, above.
- the antigenic molecules (lOO ⁇ g) and the pretreated CD91 ligand (l ⁇ g-lmg) are mixed together to give an approximately 5:1 antigenic molecule : CD91 ligand molar ratio.
- the mixture is then incubated for 15 minutes to 3 hours at 4° to 45°C in a suitable binding buffer such as one containing 20mM sodium phosphate, pH 7.2, 350mM NaCl, 3mM MgCl 2j lmM phenyl methyl sulfonyl fluoride (PMSF), and 1-10 niM ADP.
- a suitable binding buffer such as one containing 20mM sodium phosphate, pH 7.2, 350mM NaCl, 3mM MgCl 2j lmM phenyl methyl sulfonyl fluoride (PMSF), and 1-10 niM ADP.
- the preparations are centrifuged through a Centricon 10 assembly (Millipore) to remove any unbound peptide. If the CD91 ligand is bound to a solid phase, the CD91 ligand-antigenic molecule complexes formed can be washed free of unbound antigenic molecules prior to eluting the CD91 ligand-antigenic molecule complexes off the solid phase. The association of the antigenic molecules with the CD91 ligands can be assayed by SDS-PAGE. Following complexing, the immunogenic CD91 ligand-antigenic molecule complexes can optionally be assayed in vivo or in vitro using, for example, the methods described in Section 4.7, below.
- a linker molecule may be used to noncovalently tether a CD91 ligand to an antigenic molecule.
- a linker molecule is a bispecific molecule in which one moiety of the linker molecule binds a CD91 ligand, while another moiety of the linker molecule binds an antigenic molecule.
- Such linker molecules are described in PCT WO 01/78772, which is incorporated by reference herein, hi a particular embodiment, the linker molecule is covalently linked to an antigenic molecule and noncovalently linked to a CD91 ligand.
- the linker molecule is noncovalently linked to an antigenic molecule and non-covalently linked to a CD91 ligand.
- the linker molecule is non-covalently linked to an antigenic molecule and covalently linked to a CD91 ligand.
- antigenic molecules covalently attached to CD91 ligands may be used as vaccines to elicit an immune response.
- CD91 ligands and antigenic molecules are prepared, as described in Sections 4.2 and 4.1, respectively.
- the CD91 ligand and the antigenic molecule are then covalently coupled using any method known in the art.
- CD91 ligands may be covalently coupled to antigenic molecules by chemical cross-linking.
- Chemical cross-linking agents such as aldehydes, ketones and glyoxals, and their use are well known in the art and are suitable for use in the invention.
- Cross-linking agents including but not limited to, protein A, glutaraldehyde, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl
- sSMCC 4-(N-maleimidomethyl) cyclohexane-1-carboxylate
- glutaraldehyde may be used to cross-link a CD91 ligand and an antigenic molecule.
- other methods for covalent attachment of proteins known in the art can be used in the invention, such as photo cross-linking (See Current Protocols in Molecular Biology, Ausubel et al. (eds.), Greene Publishing Associates and Wiley Interscience, New York and Binder et al, 2000, Nature Immunol. 1:151-155, which are incorporated by reference herein).
- recombinant fusion proteins comprising a CD91 ligand fused via a peptide bond to an antigenic protein
- recombinant fusion proteins may be used to treat or prevent cancer or an infectious disease.
- an expression vector is constructed using nucleic acid sequences encoding a CD91 ligand fused to sequences encoding an antigenic protein, using recombinant methods known in the art, such as those described in Section 4.1.3, above.
- CD91 ligand-antigenic molecule fusions are then expressed and isolated.
- the N-terminal portion of a CD91 ligand or CD91 ligand fragment is fused to the C-terminal portion of an antigenic molecule.
- the C-terminal portion of a CD91 ligand or CD91 ligand fragment is fused to the N-terminal portion of an antigenic molecule.
- Such fusion proteins can be used to elicit an immune response (Suzue et al, 1997, Proc. Natl. Acad. Sci. U.S.A. 94:13146-51).
- fusion proteins can be used to induce an immune response and in immunotherapy against target diseases or disorders.
- a nucleotide sequence encoding a CD91 ligand or a CD91 ligand fragment and an antigenic molecule can be introduced into cells including, but not limited to, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
- the choice of cell type depends on the type of antigenic protein being expressed, and can be determined by one of skill in the art.
- the cells used in the methods of the invention are of mammalian origin. Mammals contemplated by this aspect of the invention include humans, companion animals (e.g., dogs and cats), livestock animals (e.g., sheep, cattle, goats, pigs and horses), laboratory animals (e.g., mice, rats and rabbits), and captive or free wild animals.
- any cells, preferably human cells can be used in the present methods for producing CD91 ligand-antigenic protein fusion proteins.
- gene constructs encoding the CD91 ligand and the antigenic molecule can be carried out by any method known in the art, including gene therapy art, such as but not limited to transfection, elecfroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequence encoding the CD91 ligand and antigenic protein, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et al, 1993, Meth. Enzymol.
- the present invention encompasses methods for treatment and prevention of cancer and other diseases using complexes or fusion proteins of CD91 ligands and antigenic molecules.
- the present invention also provides methods of inducing an immune response to an antigenic molecule using the complexes and fusion proteins of the invention.
- an immunogenic amount of complex or fusion protein of a CD91 ligand and an antigenic molecule, wherein the antigenic molecule displays the antigenicity of an antigen that causes or is associated with a disease can be used in the treatment or prevention of the disease.
- the CD91 ligand and the antigenic molecule of the complex are noncovalently linked.
- the CD91 ligand and the antigenic molecule of the complex are chemically cross-linked.
- the purified CD91 ligand-antigenic molecule complex or fusion protein vaccines may have particular utility in the treatment of human cancer or other diseases. It is appreciated, however, that the vaccines developed using the principles described herein will be useful in treating diseases of other mammals, for example, farm animals including cattle, horses, sheep, goats, and pigs, and household pets including cats and dogs.
- the invention also provides a method of treating a disease or disorder amenable to treatment by induction of an immune response against an antigenic molecule comprising administering to a subject having the disease or disorder an immunogenic amount of a purified complex comprising the antigenic molecule noncovalently bound to, or cross- linked to, a CD91 ligand.
- the invention also provides a method of treating a disease or disorder amenable to treatment by induction of an immune response against an antigenic molecule comprising administering to a subject having the disease or disorder an immunogenic amount of a purified fusion protein comprising the antigenic molecule fused via a peptide bond to a CD91 ligand.
- the complexes and fusion proteins of the invention can be used to induce an immune response to an antigenic molecule in a subject. Without being bound by any particular theory, it is believed that an immune response to an antigenic molecule is induced by the complexes and fusion proteins of the invention by enhancing the uptake of the antigenic molecule by antigen presenting cells, including macrophages, dendritic cells and B cells.
- CD91 ligand-antigenic molecule complexes and fusion proteins can also be used for passive immunotherapy against cancer or other diseases.
- Passive immunity is the short- term protection of a host, achieved by the administration of pre-formed antibody (e.g., in purified form or by administering serum containing a pre-formed antibody(ies)), directed against an antigen or pathogenic organism of interest, e.g., a tumor or viral antigen.
- pre-formed antibody e.g., in purified form or by administering serum containing a pre-formed antibody(ies)
- an antigen or pathogenic organism of interest e.g., a tumor or viral antigen.
- CD91 ligand-antigenic molecule complexes or fusion proteins may be used to elicit an immune response in a subject, the sera removed from the subject and used for treatment or prevention of a disease in a subject having a disease caused by the presence of a common antigen.
- CD91 ligand-antigenic molecule complexes and fusion proteins can be assayed for immunogenicity using any method known in the art. By way of example but not limitation, one of the following three procedures can be used.
- mice are injected with the CD91 ligand-antigenic molecule complex or fusion protein, using any convenient route of administration.
- As a negative control other mice are injected with CD91 ligand-antigenic molecule complexes or fusion proteins not associated with the antigen of interest, or cells containing CD91 ligand-antigenic molecule complexes or fusion proteins not associated with the antigen of interest. Cells containing the antigen of interest may act as a positive control for the assay.
- the mice are injected twice, 7-10 days apart. Ten days after the last immunization, the spleens are removed and the lymphocytes released. The released lymphocytes may be re-stimulated subsequently in vitro by the addition of dead cells that expressed the antigen of interest.
- 8xl0 6 immune spleen cells may be stimulated with 4xl0 4 mitomycin C treated or ⁇ -irradiated (5-10,000 rads) cells containing the antigen of interest (or cells transfected with an appropriate gene, as the case may be) in 3ml RPMI medium containing 10% fetal calf serum.
- 33% secondary mixed lymphocyte culture supernatant may be included in the culture medium as a source of T cell growth factors (See, Glasebrook, et al., 1980, J. Exp. Med. 151:876).
- spleen cells may be cultured without stimulation, hi some experiments spleen cells of the immunized mice may also be re-stimulated with antigenically distinct cells, to determine the specificity of the cytotoxic T cell response.
- the mixed lymphocyte culture is added to a target cell suspension to give different effecto ⁇ target (E:T) ratios (usually 1 : 1 to 40: 1).
- E:T effecto ⁇ target
- the target cells are prelabelled by incubating lxl 0 6 target cells in culture medium containing 20 mCi 51 Cr/ml for one hour at 37°C. The cells are washed three times following labeling.
- Each assay point (E:T ratio) is performed in triplicate and the appropriate controls incorporated to measure spontaneous 51 Cr release (no lymphocytes added to assay) and 100% release (cells lysed with detergent). After incubating the cell mixtures for 4 hours, the cells are pelletted by centrifugation at 200g for 5 minutes. The amount of 51 Cr released into the supernatant is measured by a gamma counter. The percent cytotoxicity is measured as cpm in the test sample minus spontaneously released cpm divided by the total detergent released cpm minus spontaneously released cpm.
- a concentrated hybridoma supernatant derived from K-44 hybridoma cells (an anti-MHC class I hybridoma) is added to the test samples to a final concentration of 12.5%.
- Primary T cells are obtained from spleen, fresh blood, or CSF and purified by centrifugation using FICOLL-PAQUE PLUS (Pharmacia, Upsalla, Sweden) essentially as described by Kruse and Sebald, 1992, EMBO J. 11 : 3237-3244.
- the peripheral blood mononuclear cells are incubated for 7-10 days with a lysate of cells expressing an antigen of interest.
- Antigen presenting cells may, optionally be added to the culture 24 to 48 hours prior to the assay, in order to process and present the antigen in the lysate.
- the cells are then harvested by centrifugation, and washed in RPMI 1640 media (GibcoBRL, Gaithersburg, Md.).
- 5xl0 4 activated T cells/well are in RPMI 1640 media containing 10% fetal bovine serum, 10 mM HEPES, pH 7.5, 2 mM L-glutamine, 100 units/ml penicillin G, and 100 ⁇ g/ml streptomycin sulphate in 96 well plates for 72 hrs at 37°C, pulsed with 1 ⁇ Ci 3 H-thymidine (DuPont NEN, Boston, Mass.)/well for 6 hrs, harvested, and radioactivity measured in a TOPCOUNT scintillation counter (Packard Instrument Co., Meriden, Conn.).
- the immunogenicity of a CD91 ligand- antigenic molecule complex or fusion protein is determined by measuring antibodies produced in response to the vaccination with the complex or fusion protein, by an antibody response assay, such as an enzyme-linked immunosorbent assay (ELISA) assay.
- an antibody response assay such as an enzyme-linked immunosorbent assay (ELISA) assay.
- ELISA enzyme-linked immunosorbent assay
- microtitre plates (96-well Immuno Plate II, Nunc) are coated with 50 ⁇ l/well of a 0.75 ⁇ g/ml solution of a purified, non-CD91 ligand-complexed form of the antigen used in the vaccine (e.g. A/342) in PBS at 4°C for 16 hours and at 20°C for 1 hour.
- the wells are emptied and blocked with 200 ⁇ l PBS-T-BSA (PBS containing 0.05% (v/v) TWEEN 20 and 1% (w/v) bovine serum albumin) per well at 20°C for 1 hour, then washed 3 times with PBS-T.
- PBS-T-BSA PBS containing 0.05% (v/v) TWEEN 20 and 1% (w/v) bovine serum albumin
- Fifty ⁇ l/well of plasma or CSF from a vaccinated animal (such as a model mouse or a human patient) is applied at 20°C for 1 hour, and the plates are washed 3 times with PBS-T.
- the anti-antigenic molecule antibody activity is then measured calorimetrically after incubating at 20°C for 1 hour with 50 ⁇ l/well of sheep anti-mouse or anti-human immunoglobulin, as appropriate, conjugated with horseradish peroxidase (Amersham) diluted 1 : 1 ,500 in PBS-T-BSA and (after 3 further PBS-T washes as above) with 50 ⁇ l of an o-phenylene diamine (OPD)-H 2 O substrate solution.
- OPD o-phenylene diamine
- the reaction is stopped with 150 ⁇ l of 2M H 2 SO 4 after 5 minutes and absorbance is determined in a Kontron SLT-210 photometer (SLT Lab-instr., Zurich, Switzerland) at 492 nm (ref. 620 nm).
- dosages of CD91 ligand-antigenic molecule complexes and fusion proteins may be extrapolated from prior art methods established in experimental tumor models (Blachere et al, 1993, J. Immunotherapy 14:352-356).
- dosages may be extrapolated from prior art methods established in an appropriate experimental disease model.
- Extrapolation to human dosages is based on body weight and surface area.
- the effective dose of CD91 ligand-antigenic molecule complexes or fusion proteins in humans weighing 70kg should be lmg/kg ⁇ 12 x 70, i.e., about 6mg (5.8mg).
- Drug doses are also given in milligrams per square meter of body surface area because this method rather than body weight achieves a good correlation to certain metabolic and excretionary functions (Shirkey, 1965, JAMA 193:443).
- body surface area can be used as a common denominator for drug dosage in adults and children as well as in different animal species as indicated below in Table 1 (Freireich et al, 1966, Cancer Chemotherap. Rep. 50:219-244). TABLE 1
- Example:To express a mg/kg dose in any given species as the equivalent mg/sq m dose, multiply the dose by the appropriate km factor. In adult human, lOOmg/kg is equivalent to 100 mg kg x 37 kg/sq m 3700 mg/sq m.
- the dosage range for the complexes and fusion proteins of the invention is l ⁇ g to 5 mg, 1 ⁇ g to 1 mg, or 5 ⁇ g to 500 ⁇ g, with a preferred range being 10 ⁇ g to 250 ⁇ g.
- Representative dosages include 10 ⁇ g, 25 ⁇ g, 50 ⁇ g, 75 ⁇ g, 100 ⁇ g, 150 ⁇ g, 200 ⁇ g and 250 ⁇ g.
- the doses recited above are preferably given once weekly for a period of about 4-6 weeks, and the mode or site of administration is preferably varied with each administration. In a preferred example, intradermal administrations are given, with each site of administration varied sequentially.
- the first injection may be given subcutaneously on the left arm, the second on the right arm, the third on the left belly, the fourth on the right belly, the fifth on the left thigh, the sixth on the right thigh, etc.
- the same site may be repeated after a gap of one or more injections.
- split injections may be given.
- half the dose may be given in one site and the other half on an other site on the same day.
- the mode of administration is sequentially varied, e.g., weekly injections are given in sequence intradermally, intramuscularly, intravenously or intraperitoneally.
- injections are preferably given at two-week intervals over a period of time of one month. Later injections may be given monthly. The pace of later injections may be modified, depending upon the patient's clinical progress and responsiveness to the immunotherapy.
- the subject is immunized by administration of the vaccine formulation, in at least one dose, and preferably two or more doses.
- the subject may be administered as many doses as is required to maintain a state of immunity.
- boosters given at regular intervals, i.e., at six months or yearly, may be desirable in order to sustain immunity at an effective level.
- CD91 ligand-antigenic molecule complexes of the invention may be administered using any desired route of administration, including but not limited to, e.g., intradermally, subcutaneously, intravenously or intramuscularly, although intradermally is preferred. Advantages of intradermal administration include use of lower doses and rapid absorption, respectively. Mucosal routes of administration include, but are not limited to, oral, rectal and nasal administration.
- Preparations for mucosal administrations are suitable in various formulations as described below.
- the route of administration can be varied during a course of treatment.
- the invention also contemplates pharmaceutical compositions (vaccines) comprising a CD91 ligand-antigenic molecule complex or fusion protein and an adjuvant.
- the complex or fusion protein is preferably purified. More than one adjuvant can be used in pharmaceutical compositions of the invention.
- Some adjuvants that may be used in the invention include, but are not limited to: heat shock proteins, saponin adjuvants (e.g., QS-21), alpha 2 macroglobulin, lipopolysaccharide (LPS), immunostimulatory oligonucleotides which include CpG oligonucleotides, and complexes of heat shock proteins and antigenic molecules, such as peptides, or the like.
- saponin adjuvants e.g., QS-21
- LPS lipopolysaccharide
- immunostimulatory oligonucleotides which include CpG oligonucleotides, and complexes of heat shock proteins and antigenic molecules, such as peptides, or the like.
- the following patents and printed publications disclose immunostimulatory oligonucleotides which include CpG oligonucleotides that can be used in the compositions of the invention: U.S. Patents 6,207,646; 6,3
- a combination of saponin adjuvants e.g., QS-21
- immunostimulatory oligonucleotides e.g., a CpG oligonucleotide
- Other suitable adjuvants that can be used in the invention can be found in A
- a complex or fusion protein of the invention is administered separately from one or more adjuvants.
- a complex or fusion protein of the invention and an adjuvant are administered in a sequence and within a time interval such that the complex or fusion protein and an adjuvant can act together to provide an increased benefit that is greater than either administered alone.
- the complex or fusion protein and an adjuvant are admimstered sufficiently close in time so as to provide the desired therapeutic or prophylactic outcome. Each can be admimstered simultaneously and/or separately, in any appropriate form and by any suitable route.
- the complex or fusion protein and an adjuvant are administered by different routes of administration.
- each is administered by the same route of admimstration.
- the complex or fusion protein and an adjuvant can be administered at the same or different sites, e.g. arm and leg.
- a complex or fusion protein of the invention and an adjuvant are administered less than 1 hour apart, at about 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, no more than 24 hours apart or no more than 48 hours apart.
- a complex or fusion protein of the invention and an adjuvant are administered 2 to 4 days apart, 4 to 6 days apart, 1 week a part, 1 to 2 weeks apart, 2 to 4 weeks apart, one moth apart, 1 to 2 months apart, or 2 or more months apart.
- a complex or fusion protein of the invention and an adjuvant are administered in a time frame where both are still active.
- One skilled in the art would be able to determine such a time frame by determining the half life of each administered component.
- compositions comprising CD91 ligand-antigenic molecule complexes or fusion proteins formulated in a compatible pharmaceutical carrier may be prepared, packaged, and labeled for treatment of the indicated cancer or infectious disease.
- an amount of CD91 ligand-antigenic molecule complex or fusion protein is admimstered to a human that is in the range of about 2 to 150 ⁇ g, preferably 20 to 200 ⁇ g, most preferably about 5 ⁇ g, given once weekly for about 4-6 weeks, intradermally with the site of administration varied sequentially.
- the complex or fusion protein is water-soluble, then it may be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions.
- the resulting complex may be formulated with a non-ionic surfactant such as Tween, or polyethylene glycol.
- a non-ionic surfactant such as Tween, or polyethylene glycol.
- the CD91 ligand-antigenic molecule complexes and fusion proteins and their physiologically acceptable solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, rectal administration.
- the pharmaceutical preparation may be in liquid form, for example, solutions, syrups or suspensions, or may be presented as a drug product for reconstitution with water or other suitable vehicle before use.
- Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p- hydroxybenzoates or sorbic acid).
- suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
- emulsifying agents e.g., lecithin or acacia
- non-aqueous vehicles e.g., almond oil, oily esters, or fractionated vegetable oils
- preservatives e.
- compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
- binding agents e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose
- fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
- lubricants e.g., magnesium stearate, talc or silica
- disintegrants e.g., potato starch
- compositions for oral administration may be suitably formulated to give controlled release of the complexes.
- Such compositions may take the form of tablets or lozenges formulated in conventional manner.
- the complexes and compositions of the present invention are administered intrathecally by an implant be placed in or near the lesioned area.
- Suitable implants include, but are not limited to, gelfoam, wax, liposome or microparticle-based implants.
- Such compositions are preferably used when it is desired to achieve sustained release of the CD91 ligand-antigenic molecule complexes.
- the complexes and fusion proteins may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
- the dosage unit may be determined by providing a valve to deliver a metered amount.
- Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the complexes and a suitable powder base such as lactose or starch.
- the complexes and fusion proteins may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
- Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
- the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
- the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
- the complexes and fusion proteins may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
- the complexes and fusion proteins may also be formulated as a depot preparation.
- Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
- the complexes may be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
- suitable polymeric or hydrophobic materials for example, as an emulsion in an acceptable oil
- ion exchange resins for example, as sparingly soluble derivatives, for example, as a sparingly soluble salt.
- Liposomes and emulsions are well known examples of delivery vehicles
- compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
- the pack may for example comprise metal or plastic foil, such as a blister pack.
- the pack or dispenser device may be accompanied by instructions for administration.
- Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
- kits for carrying out the therapeutic regimens of the invention comprise in one or more containers therapeutically or prophylactically effective amounts of CD91 ligand-antigenic molecule complexes or fusion proteins in pharmaceutically acceptable form (preferably purified), optionally with a pharmaceutically acceptable carrier.
- the kit optionally further comprises in the same or different container an adjuvant, e.g., a saponin.
- the CD91 ligand-antigenic molecule complexes or fusion proteins in a vial of a kit of the invention may be in the form of a pharmaceutically acceptable solution, e.g., in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluid.
- the complex or fusion protein may be lyophilized or desiccated; in this instance, the kit optionally further comprises in a container a pharmaceutically acceptable solution (e.g., saline, dextrose solution, etc.), preferably sterile, to reconstitute the complex or fusion protein to form a solution for injection purposes.
- Kits directed to the treatment or prevention of cancer or infectious disease optionally further comprise in a separate container, antigen presenting cells (APCs), which may be sensitized. If the APCs are not sensitized, the kit may further comprise a purified antigenic molecule for sensitizing the APCs. The APCs are preferably purified.
- kits which, in addition to the components above, also include one or more therapeutic or preventive compositions known in the art in the treatment or prevention of a disease or disorder.
- therapeutic or preventive compositions known in the art in the treatment or prevention of a disease or disorder. Examples of such compositions, listed by way of example and not limitation are found in section 4.12, below.
- kits of the invention further comprises a needle or syringe, preferably packaged in sterile form, for injecting the complex or fusion protein, and/or a packaged alcohol pad. Instructions are optionally included for administration of CD91 ligand-antigenic molecule complexes or fusion proteins by a clinician or by the patient.
- the immunopotency of the complexes and fusion proteins of the invention can be determined by monitoring the immune response in test animals following immunization with the complexes and fusion proteins of the invention, or by use of any immunoassay known in the art.
- Generation of a humoral (antibody) response and/or cell-mediated immunity may be taken as an indication of an immune response.
- Test animals may include mice, hamsters, dogs, cats, monkeys, rabbits, chimpanzees, etc., and eventually human subjects.
- Methods of introducing the vaccine may include oral, intracerebral, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal or any other standard routes of immunization.
- the immune response of the test subjects can be analyzed by various approaches such as: the reactivity of the resultant immune serum to the antigen of interest, as assayed by known techniques, e.g., immunosorbant assay (ELISA), immunoblots, radioimmunoprecipitations, etc. , or by protection of the immunized host against cancer or the infectious disease.
- ELISA immunosorbant assay
- the vaccine of the invention may be tested in rabbits for the ability to induce an antibody response to the antigenic molecule.
- Male specific-pathogen-free (SPF) young adult New Zealand White rabbits may be used.
- the test group each receives a fixed concentration of the vaccine.
- a control group receives an injection of 1 mM Tris-HCl pH 9.0 without the antigen molecule.
- Blood samples may be drawn from the rabbits every one or two weeks, and serum analyzed for antibodies to the antigenic molecule.
- the presence of antibodies specific for the antigen may be assayed, e.g., using an ELISA assay.
- the effect of immunotherapy with CD91 ligand-antigenic molecule complexes or fusion proteins on progression of a disease can be monitored by any methods known to one skilled in the art.
- cellular immunity may optionally be monitored by methods including but not limited to measuring: a) delayed hypersensitivity as an assessment of cellular immunity; b) activity of cytolytic T-lymphocytes in vitro; c) levels of antigenic molecule.
- Delayed hypersensitivity skin tests are also of great value in the overall immunocompetence and cellular immunity to an antigen. Inability to react to a battery of common skin antigens is termed anergy (Sato et al., 1995, Clin. Immunol. Pathol. 74:35- 43).
- antigens Proper technique of skin testing requires that the antigens be stored sterile at 4°C, protected from light and reconstituted shortly before use.
- a 25- or 27-gauge needle ensures intradermal, rather than subcutaneous, administration of antigen. Twenty-four and 48 hours after intradermal administration of the antigen, the largest dimensions of both erythema and induration are measured with a ruler. Hypoactivity to any given antigen or group of antigens is confirmed by testing with higher concentrations of antigen or, in ambiguous circumstances, by a repeat test with an intermediate test.
- the activity of cytolytic T-lymphocytes can be assessed in vitro using the following method.
- 33% secondary mixed lymphocyte culture supernatant or IL-2 is included in the culture medium as a source of T cell growth factors.
- T cells are cultured without the stimulator cells. In other experiments, T cells are restimulated with antigenically distinct cells. After six days, the cultures are tested for cytotoxicity in a 4 hour 51 Cr-release assay. The spontaneous 51 Cr-release of the targets should reach a level less than 20%.
- a tenfold concentrated supernatant of W6/32 hybridoma is added to the test at a final concentration of 12.5% (Heike et al, J. Immunotherapy 15: 165-174).
- the amount of immunogen to be used and the immunization schedule will be determined by a physician skilled in the art and will be admimstered by reference to the immune response and antibody titers of the subject.
- the complexes and fusion proteins of the invention can be used in combination with other therapeutic compositions known in the art in the treatment or prevention of a disease or disorder, such as immunotherapeutic agents, antineoplastic agents, anti-viral agents, anti- fungal agents, antibiotics and anti-inflammatory agents.
- the complexes or fusion proteins may be coadministered with one or more therapeutic compositions known in the art or may be administered after or before one or more therapeutic compositions known in the art.
- the invention contemplates combinations of the complexes or fusion proteins of the invention and one or more therapeutic compositions know in the art to be useful in treating or preventing any of the diseases or disorders listed in sections 4.13-4.18 below.
- the administration of the complexes and fusion proteins of the invention can augment the effect of anti-cancer agents or anti-infectives, and vice versa.
- combination therapy additive potency or additive therapeutic effects can be observed.
- dosages that alone would be suboptimal can be employed.
- the use of combination therapy can also provide better therapeutic profiles than the administration of the treatment modality, or the complexes and fusion proteins of the invention alone.
- Additive or synergistic effects may allow the dosage and/or dosing frequency of either or both modalities to be adjusted to reduce or avoid unwanted or adverse effects.
- the invention also encompasses pharmaceutical compositions comprising a CD91 ligand-antigenic molecule complex or fusion protein and one or more therapeutic compositions know in the art to be useful in treating or preventing any of the diseases or disorders listed in sections 4.13-4.18 below.
- the invention also encompasses pharmaceutical compositions comprising an CD91 ligand-antigenic molecule complex or fusion protein and a reduced dose of one or more therapeutic compositions know in the art to be useful in treating or preventing any of the diseases or disorders listed in sections 4.13-4.18 below.
- the phrase "reduced dose” refers to a dose that is below the normally administered range, i.e., below the standard dose as suggested by the Physicians' Desk Reference, 54 th Edition (2000) or a similar reference.
- the complexes or fusion proteins of the invention may be administered in combination with one or more chemotherapeutic agent.
- chemotherapeutic agents include but are not limited to: methotrexate, taxol, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, etoposides, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel, doxorubicin, epirubicin, 5-fluorouracil, taxanes such as docetaxel, 5-fluorouraci
- suitable chemotherapeutics include, but are not limited to, methotrexate, taxol, L-asparaginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin, dacarbazine, procarbizine, topotecan, nitrogen mustards, cytoxan, etoposide, 5-fluorouracil, BCNU, irinotecan, camptothecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase, vinblastine, vincristine, vinorelbine, paclitaxel, and docetaxel.
- the anti-cancer agent can be, but is not limited to, methotrex
- Taxoids Paclitaxel
- DHFR inhibitors Methotrexate
- Cytosine analogs Cytarabine (ara C)
- Angiostatin plasmaogen fragment
- IP-10 lhterleukin-12
- Thrombos ⁇ ondin-1 (TSP-1)
- Vasostatin (calreticulin fragment)
- Antimitotic agents allocolchicine
- Halichondrin B colchicine colchicine derivative dolstatin 10 maytansine rhizoxin thiocolchicine trityl cysteine
- Dopaminergic neurotoxins l-methyl-4-phenylpyridinium ion
- Bleomycins Bleomycin A2
- Additional anti-cancer agents include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; alfretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzeles
- anti-cancer drugs that can be used include, but are not limited to: 20-epi-l,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; alfretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-C
- the complexes or fusion proteins of the invention may also be administered in combination with one or more cytokines or immunomodulatory agents.
- the cytokine is selected from the group consisting of IL-l ⁇ , IL- l ⁇ , IL-2, IL- 3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IFN ⁇ , IFN/3, IFN ⁇ , TNF ⁇ , TNF/3, G-CSF, GM-CSF, TGF-/3, JX-15, IL-18, GM-CSF, INF- ⁇ , INF- ⁇ , SLC, endothelial monocyte activating protein-2 (EMAP2), MIP-3o MIP-3/3, or an MHC gene, such as HLA- B7.
- EMF2 endothelial monocyte activating protein-2
- MIP-3o MIP-3/3 or an MHC gene, such as HLA- B7.
- cytokines include other members of the TNF family, including but not limited to TNF- ⁇ -related apoptosis-inducing ligand (TRAIL), TNF- ⁇ - related activation-induced cytokine (TRANCE), TNF- ⁇ -related weak inducer of apoptosis (TWEAK), CD40 ligand (CD40L), LT- ⁇ , LT-/3, OX4OL, CD4OL, FasL, CD27L, CD30L, 4-1BBL, APRIL, LIGHT, TL1, TNFSF16, TNFSF17, and AITR-L, or a functional portion thereof. See, e.g., Kwon et al, 1999, Curr. Opin. Immunol. 11:340-345 for a general review of the TNF family.
- the complexes or fusion proteins of the invention may also be administered in combination with one or more antifungal agents.
- Antifungal agents suitable for use in the invention include but are not limited to: polyenes (e.g., amphotericin b, candicidin, mepartricin, natamycin, and nystatin), allylarnines (e.g., butenafme, and naftifine), imidazoles (e.g., bifonazole, butoconazole, chlordantoin, flutrimazole, isoconazole, ketoconazole, and lanoconazole), thiocarbamates (e.g., tolciclate, tolindate, and tolnaftate), triazoles (e.g., fluconazole, itraconazole, saperconazole, and terconazole), bromosalicylchloranilide, buclosamide, calcium propionate, chlorphenesin, ciclopirox
- Antibiotic agents suitable for use in the invention include but are not limited to: aminoglycoside antibiotics (e.g., apramycin, arbekacin, bambermycins, butirosin, dibekacin, neomycin, neomycin, undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, and spectinomycin), amphenicol antibiotics (e.g., azidamfenicol, chloramphenicol, florfenicol, and thiamphenicol), ansamycin antibiotics (e.g., rifamide and rifampin), carbacephems (e.g., loracarbef), carbapenems (e.g., biapenem and imipenem), cephalosporins (e.g., cef
- antiviral agents include but are not limited to: ribavirin, rifampicin, AZT, ddl, ddC, acyclovir and ganciclovir.
- Immunoreactive reagents include antibodies, molecules or proteins engineered to include (i.e., comprise) the antigen binding portion of an antibody, molecules or proteins engineered to include an antigen binding domain that mediates antibody dependent immune responses, a peptide or domain that interacts specifically with the antigen of interest, or any antigen binding domain that interacts with an antigen/epitope of interest, and the domain of the constant region of an antibody that mediates antibody dependent immune effector cell responses or processes. Examples of such domains or regions within the Ab constant region that can be used in the present invention include those disclosed in Reddy et al, 2000, J.
- antibodies useful in the methods and compositions of the invention include, but are not limited to, MDX-010 (Medarex, NJ) which is a humanized anti-CTLA-4 antibody currently in clime for the treatment of prostate cancer; SYNAGIS® (Medhnmune, MD) which is a humanized anti-respiratory syncytial virus (RSV) monoclonal antibody for the treatment of patients with RSV infection; HERCEPTIN® (Trastuzumab) (Genentech, CA) which is a humanized anti-HER2 monoclonal antibody for the treatment of patients with metastatic breast cancer; REMICADE® (infiiximab) (Centocor, PA) which is a chimeric anti-TNFo! monoclonal antibody for the treatment of patients with Crone's disease; REOPRO® (abciximab)
- Ostavir which is a human anti Hepatitis B virus antibody (Protein Design Lab/Novartis);
- PROTOVIRTM which is a humanized anti-CMV IgGl antibody (Protein Design
- MAK-195 SEGARD
- IC14 which is an anti-CD14 antibody (ICOS Pharm)
- OVAREXTM which is a murine anti-CA 125 antibody (Altarex)
- PANOREXTM which is a murine anti-17-IA cell surface antigen IgG2a antibody (Glaxo Wellcome/Centocor)
- BEC2 which is a murine anti-idiotype (GD3 epitope) IgG antibody (ImClone System);
- C-C225 which is a chimeric anti-EGFR IgG antibody (ImClone System);
- VITAXINTM which is a humanized anti-0!Vj(33 integrin antibody (Applied Molecular Evolution/Medhnmune); Campath 1H/LDP-03 which is a humamze
- CDP870 is a humanized anti-TNF-o! Fab fragment (Celltech); IDEC-151 is a primatized anti-CD4 IgGl antibody (IDEC Pharm/SmithKline Beecham); MDX-CD4 is a human anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 is a humanized anti- TNF- ⁇ IgG4 antibody (Celltech); LDP-02 is a humamzed anti- ⁇ 4/37 antibody
- OrthoClone OKT4A is a humamzed anti-CD4 IgG antibody (Ortho Biotech);
- ANTOVATM is a humanized anti-CD40L IgG antibody (Biogen);
- ANTEGRENTM is a humanized anti-VLA-4 IgG antibody (Elan);
- MDX-33 is a human anti-CD64 (Fc R) antibody (Medarex/Centeon);
- SCH55700 is a humanized anti-IL-5 IgG4 antibody (Celltech/Schering); SB-240563 and SB-240683 are humanized anti-IL-5 and IL-4 antibodies, respectively, (SmithKline Beecham);
- rhuMab-E25 is a humanized anti-IgE IgGl antibody (Genentech/Norvartis/Tanox Biosystems);
- ABX-CBL is a murine anti CD-147
- IgM antibody (Abgenix); BTI-322 is a rat anti-CD2 IgG antibody (Medimmune/Bio
- Orthoclone/OKT3 is a murine anti-CD3 IgG2a antibody (ortho Biotech); S ULECTTM is a chimeric anti-CD25 IgGl antibody (Novartis Pharm); LDP-01 is a humanized anti-/3 2 -integrin IgG antibody (LeukoSite); Anti-LFA-1 is a murine anti CD 18 F(ab') 2 (Pasteur-Merieux/hnmunotech); CAT- 152 is a human anti-TGF-/3 2 antibody (Cambridge Ab Tech); and Corsevin M is a chimeric anti-Factor VII antibody (Centocor).
- Preferred antibodies for use include anti-CTLA-4 antibodies and anti-41BB antibodies.
- the above-listed immunoreactive reagents, as well as any other immunoreactive reagents, may be administered according to any regimen known to those of skill in the art, including the regimens recommended by the suppliers of the immunoreactive reagents.
- Types of cancers that can be treated or prevented by the compositions and methods of the present invention include, but are not limited to, human sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic
- the cancer is metastatic.
- the patient having a cancer is immunosuppressed by reason of having undergone anti-cancer therapy (e.g., chemotherapy radiation) prior to administration of the CD91 ligand-antigenic molecule complexes or fusion proteins.
- anti-cancer therapy e.g., chemotherapy radiation
- the preventive and therapeutic compositions and methods of the invention are directed at enhancing the immunocompetence of the cancer patient either before surgery, at or after surgery, and to induce tumor-specific immunity to cancer cells, with the objective being inhibition of cancer, and with the ultimate clinical objective being total cancer regression and eradication.
- the methods of the invention can also be used in individuals at enhanced risk of a particular type of cancer, e.g., due to familial history or environmental risk factors.
- Infectious diseases that can be treated or prevented by the compositions and methods of the present invention are caused by infectious agents including, but not limited to, viruses, bacteria, fungi protozoa and parasites.
- infectious agents including, but not limited to, viruses, bacteria, fungi protozoa and parasites.
- the invention is not limited to treating or preventing infectious diseases caused by intracellular pathogens.
- Viral diseases that can be treated or prevented by the methods of the present invention include, but are not limited to, those caused by hepatitis type A, hepatitis type B, hepatitis type C, influenza, varicella, adenovirus, he ⁇ es simplex type I (HSV-I), he ⁇ es simplex type II (HSV-II), rinde ⁇ est, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papova virus, cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, human immunodeficiency virus type I (HIV-I), and human immunodeficiency virus type II (HJTV- II).
- Bacterial diseases that can be treated or prevented by the methods of the present invention are caused by bacteria including, but not limited to, bacteria that have an intracellular stage in its life cycle, mycobacteria, rickettsia, mycoplasma, neisseria and legionella.
- Protozoal diseases that can be treated or prevented by the methods of the present invention are caused by protozoa including, but not limited to, leishmania, kokzidioa, and trypanosoma.
- Parasitic diseases that can be treated or prevented by the methods of the present invention are caused by parasites including, but not limited to, chlamydia and rickettsia.
- Neurodegenerative disorders that can be prevented or treated using the compositions and methods of the invention include disorders relating to the central nervous system and/or peripheral nervous system including, but not limited to, cognitive and neurodegenerative disorders such as Alzheimer's Disease, age-related loss of cognitive function and senile dementia, Parkinson's disease, amyotrophic lateral sclerosis, Wilson's Disease, cerebral and progressive supranuclear palsy, Guam disease, Lewy body dementia, prion diseases, such as spongiform encephalopathies, e.g., Creutzfeldt- Jakob disease, polyglutamine diseases, such as Huntington's disease, myotonic dystrophy, Freidrich's ataxia and other ataxias, well as Gilles de la Tourette's syndrome, seizure disorders such as epilepsy and chronic seizure disorder, stroke, brain or spinal cord trauma, AIDS dementia, alcoholism, autism, retinal ischemia, glaucoma, autonomic function disorders such as hypertension and sleep disorders,
- Endocrine/metabolic diseases that can be prevented or treated using the compositions and methods of the invention include, but are not limited to, hyperlipidemia, hypercholesterolemia and general disorders of lipid and/or cholesterol metabolism. 4.17 TARGET VASCULAR DISEASES
- Vascular diseases that can be treated or prevented by the compositions and methods of the invention include, but are not limited to, hypertensive heart disease, hypertensive renal disease, secondary hypertension and atherosclerosis.
- Autoimmune diseases that can be treated or prevented by administration of the antigenic molecules or fusion proteins of the invention include, but are not limited to, insulin-dependent diabetes mellitus (i.e., IDDM, or autoimmune diabetes), multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, polymyositis, chronic active hepatitis, mixed connective tissue disease, primary biliary cirrhosis, pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease, vitiligo, gluten- sensitive enteropathy, Graves' disease, myasthenia gravis, autoimmune neutropenia, idiopathic thrombocytopenia pu ⁇ ura, rheumatoid arthritis, cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture's disease, bullous pemphigoid, discoid lupus, ulcerative colitis, and
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Abstract
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002515122A CA2515122A1 (en) | 2003-02-04 | 2004-02-04 | Immunogenic cd91 ligand-antigenic molecule complexes and fusion proteins |
US10/544,663 US20060217298A1 (en) | 2003-02-04 | 2004-02-04 | Immunogenic cd91 ligand-antigenic molecule complexes and fusion proteins |
EP04708275A EP1594437A2 (en) | 2003-02-04 | 2004-02-04 | Immunogenic cd91 ligand-antigenic molecule complexes and fusion proteins |
AU2004208850A AU2004208850A1 (en) | 2003-02-04 | 2004-02-04 | Immunogenic CD91 ligand-antigenic molecule complexes and fusion proteins |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US44476603P | 2003-02-04 | 2003-02-04 | |
US60/444,766 | 2003-02-04 |
Publications (2)
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WO2004069206A2 true WO2004069206A2 (en) | 2004-08-19 |
WO2004069206A3 WO2004069206A3 (en) | 2008-01-24 |
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PCT/US2004/003864 WO2004069206A2 (en) | 2003-02-04 | 2004-02-04 | Immunogenic cd91 ligand-antigenic molecule complexes and fusion proteins |
Country Status (5)
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---|---|
US (1) | US20060217298A1 (en) |
EP (1) | EP1594437A2 (en) |
AU (1) | AU2004208850A1 (en) |
CA (1) | CA2515122A1 (en) |
WO (1) | WO2004069206A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2016504308A (en) * | 2012-12-05 | 2016-02-12 | ザヴァックス ジェネティクス ワクチン カンパニー リミテッドThevax Genetics Vaccine Co., Ltd. | Fusion proteins used as immunogen enhancers to induce antigen-specific T cell responses |
Families Citing this family (13)
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US7199102B2 (en) * | 2000-08-24 | 2007-04-03 | The Regents Of The University Of California | Orally administered peptides synergize statin activity |
US7723303B2 (en) * | 2000-08-24 | 2010-05-25 | The Regents Of The University Of California | Peptides and peptide mimetics to treat pathologies characterized by an inflammatory response |
US8568766B2 (en) | 2000-08-24 | 2013-10-29 | Gattadahalli M. Anantharamaiah | Peptides and peptide mimetics to treat pathologies associated with eye disease |
US7148197B2 (en) * | 2000-08-24 | 2006-12-12 | The Regents Of The University Of California | Orally administered small peptides synergize statin activity |
JP2008513479A (en) * | 2004-09-16 | 2008-05-01 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | G-type peptides and other agents for ameliorating atherosclerosis and other pathologies |
EP1827472A4 (en) | 2004-12-06 | 2012-09-05 | Univ California | Methods for improving the structure and function of arterioles |
WO2006089114A2 (en) * | 2005-02-17 | 2006-08-24 | The Board Of Trustees Of The Leland Stanford Junior University | Methods and compositions for treating an anthrax toxin mediated condition |
EA012440B1 (en) * | 2005-04-29 | 2009-10-30 | Зе Риджентс Оф Зи Юнивесити Оф Кэлифонье | Peptide, composition based thereon for treating pathologies associated with inflammatory processes |
US20080293639A1 (en) * | 2005-04-29 | 2008-11-27 | The Regents Of The University Of California | Peptides and peptide mimetics to treat pathologies characterized by an inflammatory response |
DK2195331T3 (en) | 2007-08-28 | 2014-02-03 | Uab Research Foundation | SYNTHETIC POLYPEPTIDS SIMILARING APOLIPOPROTEIN E AND METHODS OF USE |
CA2697957A1 (en) | 2007-08-28 | 2009-03-12 | Uab Research Foundation | Synthetic apolipoprotein e mimicking polypeptides and methods of use |
JP7081923B2 (en) | 2014-07-31 | 2022-06-07 | ユーエイビー リサーチ ファンデーション | Higher effectiveness for removing apoE mimetic peptides and plasma cholesterol |
CN107249629A (en) * | 2015-02-26 | 2017-10-13 | 生控基因疫苗股份有限公司 | Comprising immunogenic protein and combination adjuvant and to induce the vaccine combination that T cells with antigenic specificity reacts |
-
2004
- 2004-02-04 EP EP04708275A patent/EP1594437A2/en not_active Withdrawn
- 2004-02-04 US US10/544,663 patent/US20060217298A1/en not_active Abandoned
- 2004-02-04 CA CA002515122A patent/CA2515122A1/en not_active Abandoned
- 2004-02-04 AU AU2004208850A patent/AU2004208850A1/en not_active Abandoned
- 2004-02-04 WO PCT/US2004/003864 patent/WO2004069206A2/en not_active Application Discontinuation
Non-Patent Citations (3)
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BINDER R.J.: 'Adjuvanticity of Alpha-2-Macroglobulin, and Independent Ligand for the Heat Shock Protein Receptor CD91' JOURNAL OF IMMUNOLOGY vol. 166, 2001, pages 4968 - 4972 * |
CROY J.E.: 'All Three LDL Receptor Homology Regions of the LDL Receptor-Related Protein Bind Multiple Ligands' BIOCHEMISTRY vol. 42, no. 44, November 2003, pages 13049 - 13057 * |
NIELSEN M.S.: 'Analysis of Ligand Binding to the Alpha-2-Macroglobulin Receptor/Low Density Lipoprotein Receptor-related Protein' THE JOURNAL OF BIOLOGICAL CHEMISTRY vol. 270, no. 40, 1995, pages 23713 - 23719 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2016504308A (en) * | 2012-12-05 | 2016-02-12 | ザヴァックス ジェネティクス ワクチン カンパニー リミテッドThevax Genetics Vaccine Co., Ltd. | Fusion proteins used as immunogen enhancers to induce antigen-specific T cell responses |
JP2016504309A (en) * | 2012-12-05 | 2016-02-12 | ザヴァックス ジェネティクス ワクチン カンパニー リミテッドThevax Genetics Vaccine Co., Ltd. | Fusion proteins as immunogen enhancers for inducing antigen-specific T cell responses |
JP2017222673A (en) * | 2012-12-05 | 2017-12-21 | ザヴァックス ジェネティクス ワクチン カンパニー リミテッドThevax Genetics Vaccine Co., Ltd. | Fusion proteins for use as immunogenic enhancer for inducing antigen-specific t cell response |
JP2018030843A (en) * | 2012-12-05 | 2018-03-01 | ザヴァックス ジェネティクス ワクチン カンパニー リミテッドThevax Genetics Vaccine Co., Ltd. | Fusion proteins for use as immunogenic enhancers for inducing antigen-specific t cell responses |
Also Published As
Publication number | Publication date |
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WO2004069206A3 (en) | 2008-01-24 |
CA2515122A1 (en) | 2004-08-19 |
EP1594437A2 (en) | 2005-11-16 |
AU2004208850A1 (en) | 2004-08-19 |
US20060217298A1 (en) | 2006-09-28 |
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