Classifications

• • 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
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
  • C12N9/6467—Granzymes, e.g. granzyme A (3.4.21.78); granzyme B (3.4.21.79)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
  • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
  • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
  • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
  • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
  • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
  • G01N2333/96433—Serine endopeptidases (3.4.21)

Definitions

• Apoptosis is a genetically determined cellular program. It consists of a stereotypic sequence of events characterized by nuclear condensation, cellular membrane blebbing, and DNA degradation. Apoptosis plays a vital role in normal development and tissue remodeling, escape from cellular transformation, and the immune response. Apoptosis can be initiated by a variety of exogenous factors, e.g., radiation, oxidative stress, or chemotherapeutic agents, or by natural endogenous processes, e.g., cytotoxic T lymphocytes, corticosteroids, or engagement of cell surface molecules such as fas or TNF.
• exogenous factors e.g., radiation, oxidative stress, or chemotherapeutic agents
• Apoptosis is particularly important for protection against intracellular pathogens and in tumor immunosurveillance. Unwanted apoptosis is associated with certain autoimmune diseases and transplant graft rejection. There is a need for therapeutic treatments which activate apoptosis so as to inhibit diseases associated with unwanted cell or infectious particle proliferation, as well as a need for therapeutic treatments which inhibit apoptosis so as to treat certain autoimmune diseases and transplant graft rejections.
• serine proteases e.g., granzymes
• serine protease binding molecules to aid in the treatment, diagnosis and/or identification of therapeutic agents for diseases characterized by abnormal apoptosis.
• the invention features a method for determining if an animal is at risk for a disease resulting in abnormal apoptosis.
• An animal is provided.
• An aspect of the metabolism or structure of a serine protease e.g., a granzyme, e.g., granzyme A, or a serine protease binding molecule, e.g., a granzyme binding molecule, e.g., a granzyme A binding molecule, e.g., PHAP I, PHAP II, a complex comprising PHAP II, or heat shock protein 27, is evaluated in the animal.
• Another aspect of the invention is the agent so identified.
• Another aspect of the invention is a method for effecting apoptosis in a cell.
• a cell which is deficient in effecting apoptosis is provided.
• An effective amount of an active endogenous DNase capable of degrading genomic DNA in the cell is provided.
• the DNase is administered to the cell so as to effect apoptosis.
• Another aspect of the invention is a method for providing an animal having cancer with a therapeutic level of a polypeptide, e.g., granzyme A, a binding molecule for granzyme A, e.g., PHAP I, PHAP II, a complex comprising PHAP II, heat shock protein 27, or a biologically active analog or fragment thereof.
• the polypeptide is provided to the animal by administering to the animal a nucleic acid encoding the polypeptide.
• Another aspect of the invention is a method for treating an animal at risk for unwanted cell or infectious particle proliferation.
• An animal at risk for unwanted cell or infectious particle proliferation is provided.
• An agent capable of activating apoptosis is provided.
• the agent is administered to the animal in a therapeutically effective amount such that treatment of the unwanted cell or infectious particle proliferation occurs.
• Another aspect of the invention is a pharmaceutical composition for treating unwanted cell or infectious particle proliferation in an animal comprising a therapeutically effective amount of an agent capable of effecting apoptosis, e.g., a serine protease, a binding molecule for a serine protease, or a biologically active analog or fragment thereof, and a pharmaceutically acceptable carrier.
• an agent capable of effecting apoptosis e.g., a serine protease, a binding molecule for a serine protease, or a biologically active analog or fragment thereof.
• Another aspect of the invention is a pharmaceutical composition for treating unwanted cell or infectious particle proliferation in an animal comprising a therapeutically effective amount of a recombinant nucleic acid encoding a polypeptide capable of effecting apoptosis, e.g., a serine protease, a binding molecule for a serine protease, or a biologically active analog or fragment thereof, and a pharmaceutically acceptable carrier.
• a recombinant nucleic acid encoding a polypeptide capable of effecting apoptosis, e.g., a serine protease, a binding molecule for a serine protease, or a biologically active analog or fragment thereof, and a pharmaceutically acceptable carrier.
• Another aspect of the invention is a method for treating an autoimmune disease or a transplant graft rejection in an animal.
• An animal in need of treatment for an autoimmune disease or a transplant graft rejection is provided.
• An agent which inhibits apoptosis is provided.
• the agent is administered to the animal in a therapeutically effective amount such that treatment of the autoimmune disease or transplant graft rejection occurs.
• Another aspect of the invention is a method for treating an autoimmune disease or a transplant graft rejection in an animal.
• a therapeutically effective amount of an agent capable of inhibiting DNase activity of a complex comprising PHAP II or a component of the complex, or capable of inhibiting granzyme A cleavage of PHAP II, is administered to an animal having an autoimmune disease or a transplant graft rejection.
• Another aspect of the invention is a method for treating an animal at risk for an autoimmune disease or a transplant graft rejection.
• An animal at risk for an autoimmune disease or a transplant graft rejection is provided.
• An agent capable of inhibiting apoptosis is provided. The agent is administered to the animal in a therapeutically effective amount such that treatment of the autoimmune disease or transplant graft rejection occurs.
• Another aspect of the invention is a pharmaceutical composition for treating an autoimmune disease or a tissue graft rejection in an animal comprising a therapeutically effective amount of an agent capable of inhibiting apoptosis by inhibiting a serine protease, a binding molecule for a serine protease, or a biologically active analog or fragment thereof, and a pharmaceutically acceptable carrier.
• Another aspect of the invention is a pharmaceutical composition for treating an autoimmune disease or a transplant graft rejection in an animal comprising a therapeutically effective amount of a recombinant nucleic acid encoding a polypeptide capable of inhibiting apoptosis by inhibiting a serine protease, a binding molecule for a serine protease, or a biologically active analog or fragment thereof, and a pharmaceutically acceptable carrier.
• Yet another aspect of the invention is a method for producing granzyme A comprising culturing a cell having a recombinant expression vector described above under conditions that permit expression of the granzyme A.
• Fig. 1 depicts a recombinant plasmid for Pro-rGranA and a recombinant plasmid for S->rGranA.
• a serine protease binding molecule is meant any molecule which binds to a serine protease, e.g., a substrate, a regulatory element, a structural component, a chaperone (transport) molecule, or any other type of ligand.
• a serine protease binding molecule can be, e.g., a polypeptide or a nucleic acid, e.g., RNA or DNA.
• Preferred serine protease binding molecules are granzyme binding molecules.
• a preferred granzyme binding molecule is a granzyme A binding molecule. Examples of serine protease binding molecules that have been identified by this invention include PHAP I, PHAP II and heat shock protein 27.
• PHAP II or a complex comprising PHAP II migrates from the cytoplasm into the nucleus of the cell where PHAP II is cleaved. See Example 8.
• the term "a complex comprising PHAP II" as used herein means the complex as described in Example 9.
• Levels of protein, mRNA or modifications of the serine protease or serine protease binding molecule can be measured, e.g., in a sample, e.g., a tissue sample, by standard methods known to those skilled in the art.
• the invention also includes a method for evaluating an agent for use in modulating apoptosis.
• a cell is provided.
• An agent is provided.
• the agent is administered to the cell in a therapeutically effective amount.
• the effect of the agent on an aspect of the metabolism or structure of a serine protease or a serine protease binding molecule is evaluated.
• a change in the aspect of the metabolism or structure is indicative of the usefulness of the agent in modulating apoptosis.
• Fragments of a serine protease or the serine protease binding molecule can be generated by methods known to those skilled in the art.
• the fragment is biologically active.
• the ability of a candidate fragment to exhibit a biological activity of the serine protease or serine protease binding molecule can be assessed by methods known to those skilled in the art, e.g., as described herein.
• fragments containing residues that are not required for biological activity of the fragment or that result from alternative mRNA splicing or alternative protein processing events are also included.
• Antibodies are meant to include antibodies against any moiety that directly or indirectly affects the metabolism of a serine protease or a serine protease binding molecule, preferably granzyme A, PHAP I, PHAP II, a complex comprising PHAP II, or heat shock protein 27.
• the antibodies can be directed against, e.g., a serine protease or a serine protease binding molecule, or a complex, subunit, fragment or analog thereof, or a biologically active fragment or analog thereof.
• antibodies include anti-granzyme A, anti-PHAP I, anti-PHAP II, anti- PHAP Il-containing complex, and anti-heat shock protein 27 antibodies.
• expression vectors can be used for in vivo transfection and expression of a polypeptide in particular cell types so as to reconstitute the function of, or alternatively, abrogate the function of, the polypeptide in a cell in which non-wild type polypeptide is expressed.
• Expression constructs of the polypeptide, and mutants thereof can be administered in any biologically effective carrier, e.g. any formulation or composition capable of effectively delivering the gene for the agent to cells in vivo.
• An animal at risk for unwanted cell or infectious particle proliferation is provided.
• An agent capable of activating apoptosis is provided.
• the agent is administered to the animal in a therapeutically effective amount such that treatment of the unwanted cell or infectious particle proliferation occurs.
• Being at risk for the disease can result from, e.g., a family history of the disease, a genotype which predisposes to the disease, or phenotypic symptoms which predispose to the disease.
• the serine protease is a granzyme, e.g., granzyme A
• the binding molecule for a serine protease is a granzyme binding molecule, e.g., a granzyme A binding molecule, e.g., PHAP I, PHAP II, a complex comprising PHAP II or heat shock protein 27.
• the granzyme insertion was excised and modified by PCR amplification with primers encoding an enterokinase site 5' of the predicted first amino acid of the active enzyme and BamHl and Xhol restriction sites for insertion into pet26b (Novagen, Madison, WI). The sequence was verified via dideoxy sequencing (Sequenase; USB/Amersham, Cleveland, OH). Plasmid expression of transfected colonies of BL21-DE3 (Novagen, Madison, WI) was induced with 1 mM IPTG.
• Mutation of the catalytic site Ser 184 to Ala was carried out via PCR mutagenesis. (Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)). The mutated sequence was reamplified using a 5' primer containing the bacterial pel B leader sequence 5' of the coding sequence and the previously used 3' primer. The sequence was confirmed by dideoxy sequencing. The mutant protein (S->ArGranA) was expressed and purified as above without enterokinase cleavage. The mutant enzyme was designed so that the bacterial signal peptidase cleavage produced a protein whose N terminus coincided with that of the active enzyme. See Fig. 1. The mutant enzyme migrated with the same mobility as the enterokinase-cleaved rGranA. The final yield of the mutant protein was approximately 1 mg/4L.
• mice have been immunized four times with rPHAP II (with sera ELISA titers >1 :8000), so as to fuse spleens from immunized mice to produce PHAP II mAb.
• rPHAP II serum-binding protein
• Commercial monoclonal antibodies to hsp27 are readily available from Stressgen, Victoria, BC.

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• 1998
  • 1998-08-17 CA CA002298867A patent/CA2298867A1/en not_active Abandoned
  • 1998-08-17 EP EP98942081A patent/EP1007723A4/de not_active Withdrawn
  • 1998-08-17 WO PCT/US1998/017022 patent/WO1999009206A1/en not_active Application Discontinuation

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