WO1997035008A1 - Procedes et composition de polypeptides chimeres pour des vaccins antigeniques tumoraux - Google Patents
Procedes et composition de polypeptides chimeres pour des vaccins antigeniques tumoraux Download PDFInfo
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- WO1997035008A1 WO1997035008A1 PCT/US1997/004656 US9704656W WO9735008A1 WO 1997035008 A1 WO1997035008 A1 WO 1997035008A1 US 9704656 W US9704656 W US 9704656W WO 9735008 A1 WO9735008 A1 WO 9735008A1
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- region
- chimeric polypeptide
- polypeptide
- tumor
- hbcag
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Classifications
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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
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4727—Mucins, e.g. human intestinal mucin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/82—Translation products from oncogenes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2730/00—Reverse transcribing DNA viruses
- C12N2730/00011—Details
- C12N2730/10011—Hepadnaviridae
- C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
- C12N2730/10123—Virus like particles [VLP]
Definitions
- the present invention relates to a vaccine that elicits an immune response against tumors.
- the present invention provides a chimeric polypeptide comprising an epitope of hepatitis B virus core antigen (HBcAg) and an epitope of a tumor antigen, as well as methods for administering the chimeric polypeptide to a subject to inhibit tumor growth.
- HBcAg hepatitis B virus core antigen
- Tumor cells are known to express tumor-specific antigens on the cell surface. These antigens are believed to be poorly immunogenic, largely because they represent gene products of oncogenes or other cellular genes which are normally present in the host and are therefore not clearly recognized as nonself. Although numerous investigators have tried to target immune responses against epitopes from various tumor specific antigens, none have been successful in eliciting adequate tumor immunity in vivo (for review, see, for example, Finn et al., 1995. Immunological Reviews 145:61-88; "Partially purified tumor antigen vaccines" (section 23.4), In: Biologic Therapy of Cancer: Principles and Practice, 2nd ed. Edited by DeVita et al., J.B. Lippincott Co., 1995).
- Nucleocapsid or core antigen from hepatitis B virus is known to be highly immunogenic and to have very efficient T-cell helper function
- the protein subunit spontaneously assembles into 27 nm core particles when expressed in Escherichia coh HBcAg has been used as a earner of peptide epitopes from viral and bacterial proteins linked by genetic engineering to yield a specific immune response to these epitopes
- a chimeric polypeptide comprising several human papillomavirus (HPV) B-epitopes and a "universal" T-helper epitope linked to HBcAg was found to elicit antibody and T-helper responses in mice (Tindle et al 1994 Virology 200 547-557)
- HPV human papillomavirus
- the present invention provides a chimeric polypeptide comprising a hepatitis B virus core antigen (HBcAg) region and a region comprising an epitope of a tumor antigen.
- the present invention further provides a nucleic acid encoding the chimeric polypeptide, a vector comprising the nucleic acid and a cell comprising the vector.
- Also provided is a method of inhibiting the growth of tumor cells in a subject comprising administering to the subject a tumor cell growth-inhibiting amount of a chimeric polypeptide comprising a HBcAg region and a region comprising an epitope of a tumor antigen corresponding to a tumor antigen in the subject.
- the present invention further provides a chimeric polypeptide comprising a HBcAg region and an antigenic polypeptide region comprising greater than 40 amino acids. Furthermore, the present invention provides a chimeric polypeptide comprising a HBcAg region and a antigenic polypeptide region comprising greater than 40 amino acids, which assembles into core particles.
- HBcAg region and an antigenic polypeptide region comprising greater than 40 amino acids, which assembles into core particles, comprising cloning into an expression vector a first DNA fragment encoding a HBcAg region and a second DNA fragment encoding an antigenic polypeptide region greater than 40 amino acids; and expressing the DNA of the expression vector in an expression system whereby the chimeric polypeptide assembles into core particles.
- the present invention provides a chimeric polypeptide comprising a HBcAg region and a region comprising an epitope of a tumor antigen.
- the chimeric polypeptide can be present in a purified form and can induce an immune response against the epitope ofthe tumor antigen and inhibit the growth of tumor cells expressing the epitope ofthe tumor antigen.
- "Purified” as used herein means the polypeptide is sufficiently free of contaminants or cell components with which proteins normally occur to allow the peptide to be used therapeutically.
- chimeric polypeptide means a polypeptide made up of two or more amino acid sequences representing peptides or polypeptides from different sources.
- epipe refers to a specific amino acid sequence of limited length which, when present in the proper conformation, provides a reactive site for an antibody or T cell receptor.
- tumor antigen describes a polypeptide expressed on the cell surfaces of specific tumor cells and which can serve to identify the type of tumor.
- the epitope ofthe tumor antigen can be any site on the antigen that is reactive with an antibody or T cell receptor.
- the tumor antigen can be from any type of tumor now known or identified in the future.
- the tumor antigen can be, but is not limited to human epithelial cell mucin (MUCl), the Ha-ras oncogene product, p53, carcino-embryonic antigen (CEA), the raf oncogene product, GD2, GD3, GM2, TF, sTn, MAGE-1, MAGE-3, tyrosinase, gp75, Melan-A/Mart-1, gplOO, HER2/neu, EBV- LMP 1 & 2, HPV-F4, 6, 7, prostatic serum antigen (PSA), alpha-fetoprotein (AFP), CO 17-1 A, GA733, gp72 and any other tumor antigens now known or identified in the future.
- the effectiveness ofthe chimeric protein in eliciting an immune response against a particular tumor antigen can be determined following the methods set forth in the Examples.
- Tumor antigens can be obtained following known procedures or are commercially available. See generally, The Oncogene Handbook. T. Curran, E P. Reddy, and A. Salka (ed ), Elsevier Science Publishers, The Netherlands (1 88).
- the HBcAg region ofthe chimeric polypeptide can comprise the HBcAg protein in its entirety or a portion ofthe HBcAg protein.
- sections ofthe HBcAg gene containing the amino terminus, sections ofthe HBcAg gene containing the carboxy terminus and/or sections ofthe HBcAg gene encoding internal amino acid sequences can be cloned separately or in any combination into the construct ofthe chimeric polypeptide, according to the teachings herein.
- a section of interest can initially be chosen on the basis of its potential for immunogenicity as indicated by computer models and mutation analysis and its suitability as part ofthe chimeric polypeptide ofthe present invention can be determined by measuring the immunogenic and therapeutic response of the section of interest, linked to an epitope of a tumor antigen, according to standard techniques in the art, such as, for example, as taught herein in the Examples.
- the invention also includes peptides and polypeptides having slight variations in amino acid sequences or other properties. Such variations may arise naturally as allelic variations (e.g., due to genetic polymorphism) or may be produced by human intervention (e.g., by mutagenesis of cloned DNA sequences), such as induced point, deletion, insertion and substitution mutants. Minor changes in amino acid sequence are generally preferred, such as conservative amino acid replacements, small intemal deletions or insertions, and additions or deletions at the ends ofthe molecules. Substitutions may be designed based on, for example, the model of Dayhoff, et al. (in Atlas of Protein Sequence and Structure 1978, Nat'l Biomed. Res.
- the polypeptides can comprise one or more selected epitopes on the same tumor antigen, one or more selected epitopes on different tumor antigens, as well as repeats ofthe same epitope, either in tandem or interspersed along the amino acid sequence ofthe HBcAg polypeptide.
- the epitope of the tumor antigen can be positioned in the chimeric polypeptide at the carboxy terminus of HBcAg, the amino terminus of HBcAg and/or at one or more intemal sites within the HBcAg amino acid sequence.
- compositions comprising a chimeric polypeptide comprising a HBcAg region and a region comprising an epitope of a tumor antigen, and a pharmaceutically acceptable carrier.
- the composition can include an effective amount ofthe chimeric polypeptide in combination with a pharmaceutically acceptable carrier and in addition, can include other medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents, etc.
- pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected antigen without causing substantial deleterious biological effects or interacting in a deleterious manner with any ofthe other components ofthe composition in which it is contained. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington's Pharmaceutical Sciences (Martin, E.W., ed., latest edition, Mack Publishing Co., Easton, PA).
- the composition can comprise a suitable adjuvant.
- suitable adjuvant describes an adjuvant capable of being combined with the chimeric polypeptide to enhance an immune response in a subject without deleterious effect on the subject.
- a suitable adjuvant can be, but is not limited to, for example, SYNTEX adjuvant formulation 1 (SAF-1) composed of 5 percent (wt/vol) squalene (DASF, Parsippany, N J ), 2.5 percent Pluronic, L121 polymer (Aldrich Chemical, Milwaukee), and 0.2 percent polysorbate (Tween 80, Sigma) in phosphate-buffered saline.
- Suitable adjuvants include Freund's adjuvant, alum, aluminum phosphate, aluminum hydroxide, N-acetyl- muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D- isoglutamine (CGP 11637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D- isoglutaminyl-L-alanine-2-( 1 '-2'-dipalmitoyl-sn-glycero-3 -hydroxyphosphoryloxy)- ethylamine (CGP 19835 A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trealose dimycolate and cell wall skeleton (MPL+TDM+CWS) in 2% squalene Tween 80
- isolated nucleic acid is meant nucleic acid molecules that are substantially free ofthe other nucleic acids and other components commonly found in association with nucleic acid in a cellular environment. Separation techniques for isolating nucleic acids from cells are well known in the art and include phenol extraction followed by ethanol precipitation and rapid solubilization of cells by organic solvent or detergents (See, e.g., Sambrook et al., 1989. Molecular Cloning: A Laboratory Manual, 2d. edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
- the nucleic acid encoding the chimeric polypeptide can be any nucleic acid that functionally encodes the chimeric polypeptide.
- the nucleic acid can include, for example, expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites and transcriptional terminator sequences.
- expression control sequences are promoters derived from metallothionine genes, actin genes, immunoglobulin genes, CMV, SV40, adenovirus, bovine papilloma virus, etc.
- a nucleic acid encoding a selected chimeric polypeptide can readily be determined based upon the genetic code for the amino acid sequence ofthe selected chimeric polypeptide and many nucleic acids will encode any selected chimeric polypeptide. Modifications in the nucleic acid sequence encoding the chimeric polypeptide are also contemplated. Modifications that can be useful are modifications to the sequences controlling expression ofthe chimeric polypeptide to make production of the chimeric polypeptide inducible or repressible as controlled by the appropriate inducer or repressor. Such means are standard in the art (see, e.g., .
- nucleic acids can be generated by means standard in the art, such as by recombinant nucleic acid techniques, as exemplified in the examples herein, and by synthetic nucleic acid synthesis or in vitro enzymatic synthesis.
- a vector comprising the nucleic acids ofthe present invention and a cell comprising the vector ofthe present invention is also provided.
- the vectors ofthe invention can be in a host (e.g., cell line or transgenic animal) that can express the chimeric polypeptide contemplated by the present invention.
- E. coli Esscherichia coli
- Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteria, such as Salmonella, Serratia, as well as various Pseudomonas species.
- These prokaryotic hosts can support expression vectors which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).
- any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
- the promoters will typically control expression, optionally with an operator sequence and have ribosome binding site sequences for example, for initiating and completing transcription and translation.
- an amino terminal methionine can be provided by insertion of a Met codon 5' and in-frame with the protein.
- the carboxy-terminal extension ofthe protein can be removed using standard oligonucleotide mutagenesis procedures.
- yeast expression can be used. There are several advantages to yeast expression systems. First, evidence exists that proteins produced in a yeast secretion systems exhibit correct disulfide pairing. Second, post-translational glycosylation is efficiently carried out by yeast secretory systems.
- the Saccharomyces cerevisiae pre-pro-alpha-factor leader region (encoded by the MFa-1 gene) is routinely used to direct protein secretion from yeast (Brake et al., 1984. "Alpha-factor-directed synthesis and secretion of mature foreign proteins in Saccharomyces cerevisiae, " PNAS 82:4642-4646 ).
- the leader region of pre-pro-alpha-factor contains a signal peptide and a pro-segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene.
- This enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage-signal sequence.
- the polypeptide coding sequence can be fused in-frame to the pre-pro-alpha-factor leader region. This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter.
- the protein coding sequence is followed by a translation termination codon which is followed by transcription termination signals.
- polypeptide coding sequence of interest can be fused to a second protein coding sequence, such as Sj26 or ⁇ -galactosidase, used to facilitate purification ofthe fusion protein by affinity chromatography.
- a second protein coding sequence such as Sj26 or ⁇ -galactosidase
- the insertion of protease cleavage sites to separate the components ofthe fusion protein is applicable to constructs used for expression in yeast.
- Efficient post-translational glycosylation and expression of recombinant proteins can also be achieved in Baculovirus systems in insect cells.
- Mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures and secretion of active protein.
- Vectors useful for the expression of proteins in mammalian cells are characterized by insertion ofthe protein coding sequence between a strong viral promoter and a polyadenylation signal.
- the vectors can contain genes conferring either gentamicin or methotrexate resistance for use as selectable markers.
- the antigen and immunoreactive fragment coding sequence can be introduced into a Chinese hamster ovary (CHO) cell line using a methotrexate resistance-encoding vector.
- Presence ofthe vector RNA in transformed cells can be confirmed by Northern blot analysis and production of a cDNA or opposite strand RNA corresponding to the protein coding sequence can be confirmed by Southern and Northern blot analysis, respectively.
- suitable host cell lines capable of secreting intact proteins include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, and the like.
- Expression vectors for these cells can include expression control sequences, as described above.
- the vectors containing the nucleic acid sequences of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cell host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cell hosts.
- vectors for the expression of protein in mammalian cells similar to those developed for the expression of human gamma-interferon, tissue plasminogen activator, clotting Factor VIII, hepatitis B virus surface antigen, protease Nexinl, and eosinophil major basic protein, can be employed.
- the vector can include CMV promoter sequences and a polyadenylation signal available for expression of inserted nucleic acid in mammalian cells (such as COS7).
- the nucleic acid sequences can be expressed in hosts after the sequences have been positioned to ensure the functioning of an expression control sequence.
- These expression vectors are typically replicable in the host organisms either as epi somes or as an integral part ofthe host chromosomal DNA.
- expression vectors can contain selection markers, e.g., tetracycline resistance or hygromycin resistance, to permit detection and/or selection of those cells transformed with the desired nucleic acid sequences (see, e.g., U.S. Patent 4,704,362).
- the present invention also provides a chimeric polypeptide comprising a HBcAg region and an antigenic polypeptide region comprising greater than 40 amino acids.
- the invention provides a chimeric polypeptide having an antigenic polypeptide region comprising, for example, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113,
- the present invention also contemplates a chimeric polypeptide comprising an HBcAg region and an antigenic polypeptide region comprising greater than 300 amino acids, which can function as a carrier for epitopes, in a conformation other than an assembled core particle of HBV.
- the antigenic polypeptide region can comprise an immunoglobulin variable heavy chain, an immunoglobulin variable light chain, either of which can comprise about 100 to 130 amino acids, or the antigenic polypeptide region can comprise a single chain antibody which can comprise about 280 amino acids.
- the chimeric polypeptide comprising an antigenic polypeptide region comprising greater than 40 amino acids can be in a composition comprising a pharmaceutically acceptable carrier and a suitable adjuvant, as described previously for the chimeric polypeptide comprising a tumor epitope.
- the present invention contemplates a nucleic acid encoding the chimeric polypeptide comprising a HBcAg region and an antigenic polypeptide region comprising greater than 40 amino acids, as well as a vector comprising the nucleic acid and a cell comprising the vector, as described previously.
- the chimeric polypeptide comprising a HBcAg region and an antigenic polypeptide region can have the structure: HBcAg-X, wherein X is an antigenic polypeptide region comprising greater than 40 amino acids.
- the invention provides a chimeric polypeptide having an antigenic polypeptide region wherein X comprises, for example, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,
- X can be an immunoglobulin variable heavy chain, an immunoglobulin variable light chain, either of which can comprise about 100 to 130 amino acids, or a single chain antibody, which can comprise about 280 amino acids.
- a chimeric polypeptide comprising an epitope of a hepatitis B virus core antigen and an antigenic polypeptide region having the structure.
- HBcAg-X wherein X is an antigenic polypeptide region comprising greater than 300 amino acids, which can assemble into core particles or can function as a carrier of various epitopes in a conformation which is not an assembled core particle.
- a chimeric polypeptide comprising a HBcAg region and a antigenic polypeptide region comprising greater than 40 amino acids, which assembles into core particles, is also provide in the present invention.
- the invention provides a chimeric polypeptide having an antigenic polypeptide region comprising, for example, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109
- the antigenic polypeptide region can comprise an immunoglobulin variable heavy chain, an immunoglobulin variable light chain, either of which can comprise about 100 to 130 amino acids, or the antigenic polypeptide region can comprise a single chain antibody, which can comprise about 280 amino acids and which can assemble into core particles.
- the present invention also contemplates a chimeric polypeptide comprising an HBcAg region and an antigenic polypeptide region comprising greater than 300 amino acids, which can assemble into core particles.
- the chimeric polypeptide comprising an antigenic polypeptide region comprising greater than 40 amino acids, which can assemble into core particles can be in a composition comprising a pharmaceutically acceptable carrier and a suitable adjuvant, as described previously for the chimeric polypeptide comprising a tumor epitope.
- the present invention contemplates a nucleic acid encoding the chimeric polypeptide comprising a HBcAg region and an antigenic polypeptide region comprising greater than 40 amino acids, which can assemble into 40 nm like particles, as well as a vector comprising the nucleic acid and a cell comprising the vector, as described previously.
- the present invention also provides a method for inducing an immune response in a subject capable of inducing an immune response and preferably human, comprising administering to the subject an immune response inducing amount of a chimeric polypeptide comprising a HBcAg region and an antigenic polypeptide region comprising greater than 40 amino acids.
- an immune response inducing amount is that amount of chimeric polypeptide which is capable of producing in a subject a humoral and/or cellular immune response capable of being detected by standard methods of measurement, such as, for example, as described herein.
- the antigenic polypeptide region can induce an antibody response.
- the antibodies can treat or prevent a pathological or harmful condition in the subject in which the antibodies are produced or the antibodies can be removed from the subject and administered to another subject to treat or prevent a pathological or harmful condition.
- the immune response can be the production in the subject of anti-idiotype antibodies, which represent the image ofthe original antigen and can function in a vaccine preparation to induce an immune response to a pathogenic antigen, thereby avoiding immunization with the antigen itself (Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1988).
- the anti-idiotype antibodies can treat or prevent a pathological or harmful condition in the subject in which the anti- idiotype antibodies are produced or the anti-idiotype antibodies can be removed from the subject and administered to another subject to treat or prevent a pathological or harmful condition.
- a method for inhibiting the growth of tumor cells in a subject comprising administering to the subject a tumor cell growth- inhibiting amount of a chimeric polypeptide comprising a HBcAg region and an antigenic polypeptide region comprising greater than 40 amino acids.
- the chimeric polypeptide comprising an HBcAg region and an antigenic polypeptide region comprising greater than 40 amino acids can be used in in vitro diagnostic assays, as well as in screening assays for identifying unknown tumor epitopes and fine mapping of tumor epitopes.
- HBcAg region and an antigenic polypeptide region comprising greater than 40 amino acids which assembles into core particles, comprising cloning into an expression vector a first DNA fragment encoding a HBcAg region and a second DNA fragment encoding an antigenic polypeptide region greater than 40 amino acids; and expressing the DNA of the expression vector in an expression system under conditions whereby the chimeric polypeptide assembles into core particles.
- the expression vector and expression system can be of any ofthe types as described herein.
- the cloning ofthe first and second DNA segments into the expression vector and expression ofthe DNA under conditions which allow for the formation of core particles can be carried out as described in the Examples section included herein.
- the phrase "assembles into core particles” means that the chimeric polypeptide self assembles by a mechanism similar to the self assembly mechanism of wild type virus core protein in mammalian cells.
- the present invention provides a method of inhibiting the growth of tumor cells in any animal subject capable of eliciting an immune response to a tumor, preferably human, comprising administering to the subject a tumor cell growth-inhibiting amount of a chimeric polypeptide comprising a HBcAg region and a region comprising an epitope of a tumor antigen corresponding to a tumor antigen in the subject.
- inhibiting the growth of tumor cells means that following administration ofthe chimeric polypeptide, a measurable immune response against the tumor cell epitope is elicited in the subject, resulting in the inhibition of growth of tumor cells present in the subject.
- the humoral immune response can be measured by detection, in the serum ofthe subject, of antibodies reactive with the epitope ofthe tumor antigen present on the chimeric polypeptide, according to protocols standard in the art, such as enzyme linked immunosorbent immunoassay (ELISA) and Western blotting protocols.
- ELISA enzyme linked immunosorbent immunoassay
- the cellular immune response can be measured by, for example, footpad swelling in laboratory animals, peripheral blood lymphocyte (PBL) proliferation assays and PBL cytotoxicity assays, as would be known to one of ordinary skill in the art of immunology and particularly as set forth in the available handbooks and texts of immunology protocols (see, for example,” Immunologic Studies in Humans,” In Current Protocols in Immunology, J. E. Coligan et al., eds. John Wiley & Sons, New York (1991).
- PBL peripheral blood lymphocyte
- the animals can either be pre ⁇ treated with the chimeric polypeptide and then challenged with a lethal dose of tumor cells, or the lethal dose of tumor cells can be administered to the animal prior to receipt ofthe chimeric polypeptide and survival times documented.
- standard clinical response parameters can be analyzed. These parameters, as would be known to one of ordinary skill in the art of oncology and tumor biology, can include physical examination ofthe subject, measurements of tumor size, X-ray studies and biopsies.
- the chimeric polypeptide can be administered to the subject orally or parenterally, as for example, by intramuscular injection, by intraperitoneal injection, topically, transdermally, injection directly into the tumor, or the like, although subcutaneous injection is typically preferred.
- Immunogenic and tumor cell growth inhibiting amounts ofthe chimeric polypeptide can be determined using standard procedures, as described. Briefly, various doses ofthe chimeric polypeptide are prepared, administered to a subject and the immunological response to each dose is determined. (Arnon, R. (Ed.) Synthetic Vaccines 1:83-92, CRC Press, Inc., Boca Raton, Florida, 1987).
- the exact dosage ofthe chimeric polypeptide will vary from subject to subject, depending on the species, age, weight and general condition ofthe subject, the severity ofthe cancer that is being treated, the particular tumor antigen being used, the mode of administration, and the like. Thus, it is not possible to specify an exact amount. However, an appropriate amount may be determined by one of ordinary skill in the art using only routine screening given the teachings herein.
- the dosage will approximate that which is typical for the administration of vaccines, and typically, the dosage will be in the range of about 1 to 500 ⁇ g ofthe chimeric polypeptide per dose, and preferably in the range of 50 to 250 ⁇ g ofthe chimeric polypeptide per dose.
- This amount can be administered to the subject once every other week for about eight weeks or once every other month for about six months.
- the effects ofthe administration ofthe chimeric polypeptide can be determined starting within the first month following the initial administration and continued thereafter at regular intervals, as needed, for an indefinite period of time.
- Parenteral administration if used, is generally characterized by injection.
- Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
- a more recently revised approach for parenteral administration involves use of a slow release or sustained release system, such that a constant level of dosage is maintained. See, e.g., U.S. Patent No. 3,710,795, which is incorporated by reference herein.
- conventional nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, and the like.
- Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc. an active compound as described herein and optional pharmaceutical adjuvants in an excipient, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
- the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.
- auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc.
- fine powders or granules may contain diluting, dispersing, and/or surface active agents, and may be presented in water or in a syrup, in capsules or sachets in the dry state, or in a nonaqueous solution or suspension wherein suspending agents may be included, in tablets wherein binders and lubricants may be included, or in a suspension in water or a syrup. Where desirable or necessary, flavoring, preserving, suspending, thickening, or emulsifying agents may be included. Tablets and granules are preferred oral administration forms, and these may be coated.
- the present invention also provides a method for producing single chain antibodies against tumor epitopes comprising producing an chimeric polypeptide comprising a HBcAg region and a region comprising an epitope of a tumor antigen; immunizing animals with an amount ofthe chimeric polypeptide sufficient to produce a humoral immune response to the chimeric polypeptide; isolating spleen cells expressing immunoglobulin specific for the chimeric polypeptide; isolating the immunoglobulin variable genes from the spleen cells; cloning the immunoglobulin variable genes into an expression vector; expressing the immunoglobulin variable genes in a bacteriophage; infecting E. coli cells with the bacteriophage; isolating bacteriophage from the E. coli cells which express the immunoglobulin variable genes and isolating the immunoglobulin variable gene products for use as single chain antibodies.
- tumor epitopes in HBcAg particles would be repeated 180 to 240 times per core particle and substantial amounts of epitope would be exposed on the surface ofthe chimeric core particles.
- These particles such as Muccore, would be better targets than tumor cells or purified tumor antigen peptides for antibody selection approaches such as phage displayed scFv production.
- the specific phage producing scFv are selected by several rounds of binding elution and infection in E. coli, using biotin labeled HBcAg-tumor epitope particles (e.g., Muccore).
- biotin labeled HBcAg-tumor epitope particles e.g., Muccore
- the biotin enables selection of high affinity scFv-phage through binding to streptavidin conjugated magnetic beads. This approach provides simple, fast and efficient production of specific anti-tumor epitope scFv.
- the present invention is more particularly described in the following examples which are intended as illustrative only as numerous modifications and variations therein will be apparent to those skilled in the art.
- HBcAg Cloning of HBcAg.
- the gene coding for HBcAg was amplified by the polymerase chain reaction (PCR) (30 cycles of 30 sec. at 94°C, followed by 30 sec. at 56°C, followed by 30 sec at 72°C) from the plasmid pHBV320, which contains the entire genome of hepatitis B vims (HBV) (subtype ayw, gift of Dr. P.
- HBcAg DNA fragment was cloned using Neo I and Hind III sites into the NlpBtaccore707AB expression vector.
- the expression vector NlpBtaccore70'/AB was constructed by inserting a PCR fragment with a Shine Delgarno sequence, optimized 5 '-UTR, a portion ofthe amino-terminus of HBcAg (70 bp) and unique restriction enzyme sites into the pBtac2 plasmid (Boehringer Mannheim).
- a tumor epitope was inserted into HBcAg either through insertion between positions 78 and 79 in the amino acid sequence or insertion after either position 149 or 176 ofthe amino acid sequence near the carboxy terminus of HBcAg.
- unique restriction sites Xho I and Sma I
- GGATCCGGACACTCGCCCAGCACCGGGTTCTACTGCTCCGCCGGCA CACGGTGTAACTTCTGCCCGGG (SEQ ID NO:4) using primers PRMUC-1 (GGATCCGGACACTCGCCCA) (SEQ ID NO: 5) and PRMUC-R1 (CCCGGGCAGAAGTTACACCGTG (SEQ ID NO:6) and cloned into the HBcAg gene utilizing Mbo I and Sma I sites incorporated between the nucleotide positions in the HBcAg gene corresponding to positions 78 and 79 in the amino acid sequence.
- a modified MUCl epitope GVTSAPDTRPAPGSTAPPAH (SEQ ID NO:7) containing the immunogenic sequence
- PDTRP SEQ ID NO:8
- MUCl epitopes were called Muccore.
- DNA representing epitopes from other tumor antigens can be isolated or synthesized by methods known in the art, as described above, amplified by PCR and cloned into a constmct containing all or a portion ofthe HBcAg gene
- These amplified DNA sequences can be inserted internally into the nucleotide sequence ofthe HBcAg gene or at either the carboxy terminus or amino terminus ofthe HBcAg gene
- the DNA can be inserted either by splicing into restriction sites that are created or occur naturally in the HBcAg gene sequence or the DNA can be ligated at the desired insertion site with DNA ligase, for example, on the end of an oligonucleotide
- the chimeric constmct can be inserted into an expression vector by standard cloning techniques, e g , as described above All of these procedures are standard molecular biology techniques (as set forth, for example, in Sambrook et al.
- HBcAg fused to the lymphoma specific variable fragments of immunoglobulin molecule or scFv.
- a plasmid was constmcted consisting of three units (1) DNA fragment (fragment- 1, core fragment) coding for HBcAg (from amino acid residues 1 to 176), (2) fragment-2, coding for the entire portion of either heavy or light variable chains of murine lymphoma 38c 13 specific immunoglobulin, and (3) an expression vector for E.col ⁇ , such as the two commercially available vectors with pTac or T7 promoters, pBTac2 (Boehringer Mannhein,
- the core fragment was amplified by PCR (30 cycles of 30 seconds each at 94, 58 and 72°C) from the plasmid containing the entire HBV genome, subtype ayw (Dr Pauls Pumpa, Riga, Norway) using specific oligonucleotide primers: PRCORE5 -1 : GAATTCGGAGGAAAAAAACCATGGACATTGATCCTTATAAAGAA (SEQ ID NO: 19) (Eco RI and Neo I sites underlined and SD and ATG codon in bold); PRCORE3'Rl : AGATCTTCTGCGACGCGG (SEQ ID NO:20) (Bgl II site underlined)
- the fragment- 1 purified from an agarose gel, was digested with Bgl II restriction endonuclease in the manufacturer's recommended buffer containing 50 mM Na-salt (Life Technologies, Gaithersburg, MD) for one hour at 37°C.
- the fragment ends were filled with dNTP (100 mM) using two u of Klenow fragment for ten minutes at room temperature (RT).
- the fragment was extracted with phenol-chloroform and precipitated in ethanol containing 0.3 M Na-acetate.
- the fragment- 1 was digested with Eco RI restriction endonuclease and extracted and precipitated as described.
- the resulting fragment was resuspended in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).
- the plasmid N3scFv38MHpBtac/AB (Arya Biragyn) was digested with Eco RI and Bam HI endonucleases. About 50 ng ofthe vector was mixed with 200 ng each ofthe fragments and ligated for 1-2 hours at 16°C in the ligation mixture containing 2mM ATP and 1-5 u of T4 DNA ligase. XLl blue competent E coli.
- N 17coreL- Vh38MHpTac/AB The expression vector N28coreL- Vh38MHpETl 1/AB was constructed similarly by cloning fragment- 1 and fragment-2 under T7 promoter control in the modified plasmid, pETl ld/AB.
- the DNA fragment for the variable light (VI) chain of 38c 13 IgM (Vk38) was removed from the plasmid NlscFv38CM/AB (Arya Biragyn) by Sal I and Bam HI digestion and inserted into the N28coreL-Vh38pETl 1/AB plasmid by replacing the Vh38 fragment, Xho I and Bam HI.
- the resulting plasmid was verified by DNA sequencing analysis and called N5coreL-Vk38MHpETl 1/AB.
- the DNA fragment for the 38cl 3 lymphoma specific single chain antibody is the DNA fragment for the 38cl 3 lymphoma specific single chain antibody
- E.coli expression and purification of core fusion proteins were grown overnight in 150-200 ml Superbroth medium (SB, Digene Diagnostics Inc., Beltsville, MD) containing 150-200 ⁇ g/ml Amp and 1% dextrose at 30°C, at a shaker speed of about 180-200 ⁇ m.
- Cell cultures were diluted 2X in the same medium in a 2 L flask and grown for an additional two hours. The cell culture was then diluted with 1 L of SB containing 150 ⁇ g/ml AMP and 0.5 mM IPTG (powdered IPTG was added directly to the medium) and cells were incubated further at 30°C on the shaker at a speed of 200- 250 ⁇ m for six hours.
- the bacterial DNA was digested for ten minutes at RT by adding 3 ⁇ l of DNase I (100,00 u/ml, Life Technologies, Gaithersburg, MD) and 200 ⁇ l of IM MgCl 2 .
- the cell debris was removed by centrifuging at 14,000 ⁇ m for 20 minutes at 4°C (Sorvall RC-5B, rotor SS-34)
- Recombinant HBcAg (core) was precipitated in 30% saturated ammonium sulfate (SAS)
- SAS saturated ammonium sulfate
- the core-V proteins were enriched by Hydroxy-apatite column chromatography (Bio-Rad Labs, Hercules, CA).
- the 02 M Na-phosphate fractions were pooled and precipitated with an equal volume of SAS.
- the pellet was resuspended in 3 ml of PBS containing 0 3% Triton X-100, 5% glycerol and the mixture was applied onto a C16/100 column with Sepharose 4CLB (Pharmacia Biotech, Uppsala, Sweden)
- the recombinant HBcAg particles came out in the fractions corresponding to the first pick just after the void volume HBcAg positive fractions were pooled together and precipitated by adding equal volumes of SAS
- the precipitated pellet of HBcAg particles was resuspended in 2 ml PBS and dialyzed against 4 L of PBS at 4°C overnight in 50,000 kDa cut-off dialysis membrane.
- the protein concentration
- the Nl 2core- scFv38MHpTac/AB plasmid, encoding HBcAg (176 amino acid residues) fused to a single chain antibody fragment (scFv38, about 280 amino acid residues) was constmcted to examine the size limits ofthe fusion protein on the particle formation ability of HBcAg N12core-scFv38MHpTac/AB plasmid was expressed in the XL1 blue strain of E.
- HBcAg fused with Vh or Vk immunoglobulin fragments from B cell lymphoma 38c 13 Two types of plasmids coding for HBcAg fused with Vh or Vk immunoglobulin fragments from B cell lymphoma 38c 13 were constructed N28coreL-Vh38MHpETl l/AB and N5coreL-Vk38MHpETl l/AB, respectively
- the size ofthe immunoglobulin fragments fused to HBcAg (176 amino acid residues), including spacer regions and c-myc His-tags, was approximately 160 amino acids
- the plasmids were expressed in the BL21(DE3) strain of E. coli and the proteins produced were tested by Western blot hybridization using 9E10 and anti-
- WHBc antibodies (described above) Both antibodies detected a protein band of about 32-36 KDa
- the fusion core-Vh and core-Vk proteins migrated in Sepaharose 4CLB size exclusion chromatography very close to the void volume and were eluted from the first major pick, which corresponds to the partculated HBcAg
- This fraction contained a positive band ofthe expected size for both the purified core- Vh and core-Vk samples with reducing PAGE.
- Non-reducing PAGE did not reveal a significant positive signal, which indicated that the majority of purified core-V proteins were assembled into particles
- These data correlated well with the electron micrographs ofthe samples, which showed that the fusion proteins of core-Vh and core- Vk were assembled into HBcAG-like particles.
- Sandwich ELISA of these samples revealed that variable fragments ofthe immunoglobulin molecule were exposed on the surface ofthe HBcAg particles.
- HBcAg retained the ability to efficiently self-assemble when expressed as a fusion protein with immunoglobulin variable region fragments.
- HBcAg-f ⁇ sed immunoglobulin variable fragments could be used to recreate immunoglobulin idiotype on the surface of HBcAg particles, either by co-expressing both core-Vh and core- VI in a single host, or by chemically induced particulation of denatured core-Vh and core- VI proteins
- HBcAg particles expressing tumor epitopes for production of tumor epitope specific antibodies are useful tools for generating anti-tumor epitope antibodies in vivo.
- Tumor epitopes in HBcAg particles are repeated about 180 to 240 times per core particle, exposing substantial amounts of epitope on the surface ofthe chimeric core particles.
- These particles like Muccore, are better targets than tumor cells or purified tumor antigen peptides for antibody selection protocols such as phage displayed scFv production.
- the spleen cells from mice immunized with chimeric core particles e.g., Muccore
- phage displayed scFv technology The specific phage producing scFv is selected through several rounds of binding, elution andE. coli.
- Example II In vivo studies on immunity and tumor suppression induced by administration of chimeric polypeptides.
- hu-PBL-SCID mice Immunity and tumor suppression in hu-PBL-SCID mice against human pancreatic tumor cells.
- Two Muccore fusion proteins (differing by the position ofthe immunogenic sequence in the tumor antigen epitope, as described above), containing HBc antigen and a single tandem repeat of mucin antigen were constructed as described herein and studied as vaccines for inducing mucin-specific immune responses and tumor inhibition in hu-PBL-SCID mice.
- the hu-PBL-SCID mice received HBc-MUCl as the active immunization.
- the vaccine was administered intraperitoneally at 60 ⁇ g/mouse for a total of three immunizations.
- the control animals received phosphate buffered saline as mock immunization.
- the anti-mucin antibody responses were measured by ELISA.
- the mucin-specific delayed type hypersensitivity was studied by determining mucin- induced footpad swelling and human lymphocytic infiltration.
- PANC-1 human pancreatic cancer
- the tumor growth kinetics were studied in both immunized and control animals. Both fusion proteins were shown to induce anti-mucin humoral and cellular immunities in hu-PBL-SCID mice.
- One preparation was shown to be effective in the induction of antibody response while the other one was found slightly more effective in inducing cellular immunity.
- HBc- MUCl fusion proteins were demonstrated to be immunogenic and tumor-suppressive in SCID mice reconstituted with human PBL. Immunity and tumor suppression in BALB/c mice against lethal challenge with tumor cells harboring a Ha-ras Arg-12 mutation.
- a chimeric fusion HBcAg gene bearing a mutated amino-terminal portion ofthe Ha-ras oncogene was constmcted as described herein and expressed in E. coli.
- the fusion protein, HBcAg/Arg-12 ras efficiently self-assembles into core particles, as verified by electron microscopy.
- mice were immunized with either 80 ⁇ g of purified HBcAg/Arg-12 ras or Muccore (as a control) and subsequently challenged with a lethal dose of Ha-Balb tumor cells containing arg-12-mutated ras (Dr. Larry Kwak, NCI, Frederick, MD).
- the control mice immunized with Muccore died by day 25 of post tumor challenge, with the median at day 20.
- HBcAg/Arg-12 immunized mice had significantly suppressed tumor growth and prolonged survival with median at day 35.
- Example DI Administration of chimeric polypeptides to human subjects
- a chimeric polypeptide comprising an epitope of HBcAg and an epitope of a tumor antigen present in the human subject is administered to the subject subcutaneously in a dose ranging from 1 to 500 ⁇ g ofthe polypeptide once weekly for about eight weeks or once monthly for about six months.
- blood samples can be taken from the subject and analyzed to determine the effects of administration ofthe chimeric polypeptide.
- the presence in the subject's semm, of antibodies reactive with the epitope ofthe tumor antigen on the chimeric protein can be determined by ELISA, Western blotting or radioimmunoprecipitation, or other methods for detecting the formation of antigen/antibody complexes as would be standard practice for one of ordinary skill in the art of immunology.
- a cellular immune response to the epitope ofthe tumor antigen in the chimeric polypeptide can be detected by PBL proliferation assays, PBL cytotoxicity assays, cytokine measurements, or other methods for detecting delayed type hypersensitivity and cellular immune response, as would be standard practice for one of ordinary skill in the art of immunology.
- the kinetics of tumor growth and inhibition of tumor cell growth can be determined by monitoring the subject's clinical response, through physical examination, tumor measurement, x-ray analysis and biopsy.
- the exact dosage can be determined for a given subject by following the teachings as set forth herein, as would be standard practice for one of ordinary skill in the art of vaccine development.
- TITLE OF INVENTION METHODS AND COMPOSITIONS OF CHIMERIC POLYPEPTIDES FOR TUMOR ANTIGEN VACCINES
- MOLECULE TYPE DNA (genomic) (XI) SEQUENCE DESCRIPTION: SEQ ID NO:l: CCATGGACAT TGATCCTTAT AAAGAA 26
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- MOLECULE TYPE DNA (genomic)
- XI SEQUENCE DESCRIPTION: SEQ ID NO:18: GGATCCGCAG AAGAACAGAA ACTGATCTCA GAAGAGGATC TGGCCCACCA CCATCACCAT 60 CACTAACCCG GG 72
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Cited By (7)
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WO1999046392A1 (fr) * | 1998-03-12 | 1999-09-16 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Methodes et compositions utilisant des proteines de fusion chemokine-antigene tumoral comme vaccins anti-cancer |
WO2001077158A1 (fr) * | 2000-04-07 | 2001-10-18 | University Of Leeds Innovations Limited | Proteines hybrides de l'antigene capsidique de l'hepatite b |
US6911204B2 (en) | 2000-08-11 | 2005-06-28 | Favrille, Inc. | Method and composition for altering a B cell mediated pathology |
US7754676B2 (en) | 2000-09-15 | 2010-07-13 | The United States Of America As Represented By The Department Of Health And Human Services | Defensin-antigen fusion proteins |
US7897152B2 (en) | 2000-09-18 | 2011-03-01 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Viral chemokine-antigen fusion proteins |
EP2366709A1 (fr) * | 2010-03-16 | 2011-09-21 | BioNTech AG | Vaccination anti-tumeur impliquant une réponse immunitaire humorale contre les protéines endogènes |
WO2011113546A1 (fr) * | 2010-03-16 | 2011-09-22 | Biontech Ag | Vaccination antitumorale suscitant une réponse immunitaire humorale dirigée contre les autoprotéines |
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