WO2000038708A1 - Traitement et prevention des reactions de rejets immunitaires - Google Patents

Traitement et prevention des reactions de rejets immunitaires Download PDF

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Publication number
WO2000038708A1
WO2000038708A1 PCT/US1999/030818 US9930818W WO0038708A1 WO 2000038708 A1 WO2000038708 A1 WO 2000038708A1 US 9930818 W US9930818 W US 9930818W WO 0038708 A1 WO0038708 A1 WO 0038708A1
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cells
hydrolase
hydrolases
preventing
ameliorating
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PCT/US1999/030818
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WO2000038708A9 (fr
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Richard L. Franklin
Yves St. Pierre
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Phairson Medical Inc.
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Priority to AU28454/00A priority Critical patent/AU2845400A/en
Publication of WO2000038708A1 publication Critical patent/WO2000038708A1/fr
Publication of WO2000038708A9 publication Critical patent/WO2000038708A9/fr

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    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/46434—Antigens related to induction of tolerance to non-self
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • 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)
    • G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)

Definitions

  • the present invention relates to a treatments to prevent immune rejection reactions, such as graft vs host disease (GVHD), with a hydrolase effective to remove cell surface adhesion molecules involved in triggering such immune reactions
  • GVHD graft vs host disease
  • One embodiment uses a krill-derived multifunctional enzyme and a family of crustacean and fish derived enzymes having substantial structural or functional similarity to the multifunctional enzyme derived from antarctic krill
  • Another particularly preferred enzyme is an Atlantic cod ⁇ Gadus morhua) trypsin, particularly that described European J Biochem.
  • aquatic or other enzymes that are substantially or functional structurally similar to the krill-derived multifunctional enzyme have the same utility as the krill enzyme In particular, these enzymes are useful for treating viral infections and other disorders, as outlined for example in U S Patent Application Nos 08/486,820, 08/338,501 (filed November 22, 1994) and U S Patents 5,945,102 and 5,958,406
  • Do ⁇ ng et al , J. Immunol. 154 4842-4850, 1995 article (“Do ⁇ ng”) describes the effects of two enzymes found in sputum from cystic fibrosis patients on CD4 and CD8
  • the enzymes are polymorphonuclear leukocyte-derived proteinase elastase and cathepsin G Exposure to the polymorphonuclear leukocyte-derived proteinase elastase apparently reduced the cytotoxic response of a T-cell clone
  • the invention provides a method of preventing or ameliorating transplantation rejection reactions comprising treating the donor tissue with a rejection reaction preventing or ameliorating effective amount of a hydrolase (which can be a mixture of hydrolases), such as a protease
  • a hydrolase which can be a mixture of hydrolases
  • the invention can comprise selecting an hydrolase that is effective to induce tolerance in an immune cell to an antigen or cell to which the immune cell was previously reactive
  • the invention can comprise selecting a hydrolase (or an appropriate mix of hydrolases) that disrupts signal 2 mediators of an immune cell or signal 1 mediators (or both)
  • the invention can comprise selecting a hydrolase (or an appropriate mixture of hydrolases) that disrupts signal 2 mediators, while leaving in place signal 1
  • the invention can comprise selecting a hydrolase that removes, destroys, inactivates or
  • the hydrolase employed is more effective than papain, or bromelain, or mammalian trypsin
  • the hydrolase employed is more effective in removing one or more of CD28, ICAM-1 (CD54), GP39 (CD154), an lnteg ⁇ n, CD40 and CD80 than is the krill multifunctional enzyme
  • the hydrolase employed is more effective in removing CD28 than is one or more of the krill multifunctional enzyme
  • the hydrolase employed is more effective in removing ICAM-1 (CD54) than is one or more of the krill multifunctional enzyme
  • the hydrolase employed is more effective in removing an lnteg ⁇ n than is one or more of the k ⁇ ll multifunctional enzyme
  • the hydrolase employed is more effective
  • the invention also provides a method of preventing or ameliorating transplantation rejection reactions comprising treating a donor source of immune cell (e g , lymphocyte) precursor cells (such as from bone marrow) with a rejection preventing or ameliorating effective amount of a hydrolase, and administering the treated lymphocyte precursor cells to a recipient
  • a donor source of immune cell e g , lymphocyte
  • lymphocyte precursor cells such as from bone marrow
  • the invention further provides a method of preventing or ameliorating transplantation rejection reactions comprising isolating from a source of immune cells taken from a donor (a) a fraction enriched in mature T-cells and (b) a fraction containing immune cell precursor cells, treating the mature T-cells of fraction (a) with a rejection preventing or ameliorating effective amount of a hydrolase, and administering the mature T-cells of fraction (a) and fraction (b) to a recipient.
  • the hydrolase treated mature T-cells are contacted with cells of fraction (b) prior to administration to the recipient.
  • the invention still further provides a method of preventing or ameliorating transplantation rejection reactions comprising treating a source of immune cells taken from a recipient or donor (for example where the recipient does not have an immune system) with a rejection preventing or ameliorating effective amount of a hydrolase; incubating the treated source of immune cells with a donor organ, tissue or cell type; transplanting the donor organ, tissue or cell type into the recipient; and administering the treated cells into the recipient.
  • the treated cells include mature T- cells.
  • the invention also provides a method of preventing or ameliorating allergic or autoimmune reactions comprising: treating a source of immune cells taken from a treatment subject or donor (for example where the recipient does not have an immune system) with an allergic or autoimmune reaction preventing or ameliorating effective amount of a hydrolase; exposing the immune cells to an antigen that induces the allergic reaction or which contains autoimmune epitopes, and restoring the treated and exposed immune cells cells to the treatment subject.
  • the invention provides a method of preventing or ameliorating allergic, autoimmune or transplantation rejection reactions with a hydrolase, comprising: identifying the hydrolase or mixture of hydrolases as a hydrolase or mixture of hydrolases with a relative selective preference for disabling signal 2 and/or signal 1, or effective for inducing tolerance in immune cells to a substance or to a cell; treating immune cells with the hydrolase or mixture of hydrolases; and administering the treated cells to a mammal.
  • the hydrolase can be selected on the basis of specificity for cell surface adhesion molecules
  • the invention further provides (a) methods relating to certain conditions using effective amounts of hydrolase, (b) compositions for use in such methods, (c) pharmaceutical compositions containing effective amounts of hydrolase for use in such methods, and (d) uses of the hydrolase composition for manufacturing a medicament for use in such methods.
  • the methods are include • treating a tissue, body fluid or composition of cells to remove or inactivate a cell adhesion component comprising, wherein the enzyme is administered to the tissue, body fluid or composition of cells, preferably a cell-adhesion component removing or inactivating effective amount or an immune rejection inhibiting amount of the enzyme is administered, wherein preferably the tissue, body fluid or composition of cells is treated e tra- corporeally, although they may also be treated in silii in an animal; or
  • the method comprises administering a composition comprising a hydrolase described above.
  • the invention fiirther provides (a) methods for treating or prophylactically preventing a cell-cell or cell-virus adhesion syndrome comprising administering an anti- adhesion effective amount of a hydrolase effective to remove or inactivate a cellular or viral acceptor or receptor adhesion component that is involved in the cell-cell or cell- virus adhesion, (b) compositions or substances for use in such methods, (c) pharmaceutical compositions containing effect i ⁇ e amounts of enzyme for use in such methods, and (d) uses of the enzyme composition for manufacturing a medicament for use in such methods.
  • the syndrome comprises inflammation, shock, tumor metastases. autoimmune disease, transplantation rejection reactions or microbial infections.
  • the syndrome is selected from the group consisting of graft versus host disease, organ or tissue transplantation rejection, autoimmune disease and associated conditions, microbial infection, immune disorder, cystic fibrosis, COPD, atherosclerosis, cancer, asthma, septic shock, toxic shock syndrome, conjunctivitis, reperfusion injury and pain, and (b) a cell surface adhesion molecule, associated with the cell-cell or cell-virus adhesion syndrome, is removed or inactivated by the administered hydrolase.
  • the cell surface adhesion molecule can be selected from the group consisting of ICAM-1 (also know as CD54), ICAM-2 (also known as CD102), VCAM-1. CD3, CD4, CD8, CD 1 1 , CDl 8.
  • CD44. CD 49, CD62L, CD 102, GP39 (CDl 54), integrins (e g., of ⁇ - 1 subfamily >e g,. ⁇ - 1 (CD29) with ⁇ l (CD49a), ⁇ 2 (CD49b), ⁇ 3 (CD49c).
  • ⁇ -2 subfamily e.g., ⁇ -2 (CDl l a) with ⁇ L (CD l lb), ⁇ VI (CD ) o ⁇ X (CD l l c), or of ⁇ -3 subfa ilv
  • the invention further provides a pharmaceutical composition for removing or inactivating a cell-surface adhesion molecule comprising a cell-surface adhesion molecule removing or inactivating effective amount of a hydrolase
  • hydrolases include a number of enzymes such as cod trypsin and other hydrolases.
  • proteases with multiple classes of proteolytic activity such as the multifunctional enzyme having: activity comprising at least one of a chymotrypsin, trypsin.
  • compositions typicalh include a pharmaceutically acceptable diluent or earner.
  • the invention still further provides a pharmaceutical composition for treating or prophylactically preventing a cell-cell or cell-virus adhesion syndrome comprising a cell- cell or cell-vi ⁇ is adhesion syndrome treating or preventing effective amount of a composition comprising a hydrolase
  • the hydrolase is multifunctional enzyme having activity comprising at least one of a chymotrypsin, trypsin. collagenase, elastase or exo peptidase activity; a molecular Height between about 20 kd and about 40 kd as determined by SDS PAGE, and substantial homology to the krill-derived multifunctional hydrolase
  • Such compositions typically include a pharmaceutically acceptable diluent or carrier.
  • HlV-infected patients are treated to slow the progression of the associated diseases by the process of ( i ) isolating ' I -cells from the patient. (2) treating the T-cells with a hydrolase effective to remove CD4. and (3 ) injecting the T-cells into the patient.
  • the method of extra-corporeally treating a tissue bodv fluid or composition of cells to remove cell adhesion components reduces the immune rejection of a tissue, body fluid or composition of cells that is transplanted from one individual to another in another aspect, such treatments remove or inactivate the cell adhesion components found in the treated tissue, bodv fluid or composition of cells involved in a microbial infection
  • the invention relates to a hydrolase having multifunctional activity comprising at least one of a chymotrypsin, trypsin, collagenase, elastase or exo peptidase activity, a molecular weight between about 20 kd and about 40 kd as determined by SDS PAGE, and substantial homology to krill-derived multifunctional hydrolase
  • the enzyme has a molecular weight of from about 26 kd to about 32 kd as determined by SDS (sodium dodecyl sulfate) polvacrylamide gel electrophoresis ("sodium dodecyl
  • the enzvme has an N-terminal sequence comprising: I-V-G-G-X-E/D-B-X-X-X-Z/B -P-Z/H-Q-B-X- B7Z, wherein X is any amino acid, Z is an aromatic amino acid, B is an amino acid having a C I to C6 alkyl side chain, and B ' is leucine or isoleucine More preferably, all amino acids represented by X, Z or B are natural amino acids.
  • the enzyme has an N-terminal sequence comprising: I-V-G-G-X-E/D-B wherein X is any amino acid, B is an amino acid having a CI to C6 alkyl side chain.
  • the enzx e is the krill-derived multifunctional hydrolase
  • the ⁇ -terminal sequence is I-V-G-G-X-E-V-T-P-H-A-Y-P-W-Q-V-G-L-F-I-D-D-M-Y-F (SEQ ID NO 20).
  • the enzyme has the N-terminal sequence: I-V-G-G-N/M-E-V-T-P-H-A- Y-P-W-Q-V-G-L-F-I-D-D-M-Y-F (SEQ ID NO 1).
  • the multifunctional enzyme of the invention has at least two of the identified proteolytic activities, more preferably at least three, still more preferably at least four Yet more preferably, the enzyme has all of the identified proteolytic activities
  • the multifunctional enzyme has substantial anti cell-cell and cell-viius adhesion activity
  • the multifunctional me has substantial homology with the krill-derived multifunctional hydrolase
  • the multifunctional enzyme shall include an amino acid sequence having at least about 70% identity with a "reference sequence" described below, more preferably at least about 80% identity, still more preferably at least about 90% identity, yet still more preferably at least about 95% identity.
  • the krill-derived multifunctional hydrolase can be the multifunctional enzyme
  • the reference sequence is (i) the amino acid 64-300 sequence of SEQ ID NO 2 I . or (i) a sequence which is that of the amino acid 64-300 sequence of SEQ ID NO 2 1 except that it has one or more of the amino acid substitutions found in the amino acid 1 - 185 sequence of SEQ ID NO 22, one or more of the amino acid substitutions found in the amino acid 72-
  • the hydrolase is selectively reactive with cell-surface receptors such as proteins or glycolipids.
  • the hydrolase is substantially purified.
  • the hydrolase has a purity with respect to macromolecules of at least about 90%, more preferably least about 95%, more preferably about 97%, still more preferabh about 99%, yet more preferably 9 ⁇ .7% with respect to macromolecules.
  • substantially pure shall mean about 60% purity.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the multifunctional enzyme of claim I and a pharmaceutically acceptable diluent or carrier
  • Figures 1A and IB survival of (C57BL/6 x DBA/2)BDFI recipients of semi-allogeneic C57BL/6 bone marrow cells mixed with PHM-treated C57BL/6 splenocytes.
  • Figure 2 Survival of (C57BL/6 x DBA/2)BDFI recipients of semi-allogeneic C57BL/6 bone marrow cells mixed with protease-treated C57BL/6 splenocytes.
  • Figure 3 (3A-3C) shows the effects of cod trypsin of PHM incubations on surface markers.
  • Figures 4 and 5 show the effects of various hydrolases on a number of cell surface adhesion molecules.
  • the multifunctional enzyme and other hydrolases effectively remove or inactivate certain cell-surface adhesion molecules, such as ICAM-1 (CD54), ICAM-2 (CD 102), VCAM-1, CD4, CD8, CD28, CD31, CD 11 a, CD49d, other integrin component chains, CD44, the asialo GMl ceramide, CD40 and CD80 without affecting cell viability
  • This adhesion site removal or inactivation phenomenon is believed to provide at least a partial explanation for effectiveness against many, though probably not all, of the indications against which, for example, the multifunctional enzyme is effective
  • the ant ⁇ -CD4 cell surface adhesion molecule activity of the multifunctional enzyme is believed to be responsible, at least in part, for the enzyme's HIV-transmission inhibitory activity
  • the HIV infective pathway utilizes the CD4 cell-surface molecule See, Lentz, "Molecular Interaction of Viruses with Host-Cell Receptors," in Adhesion Molecule
  • CD3 and CD90 show little or no change, CD28, CD49, CD29D, CD 18 and CDl 1 are significantly destroyed or inactivated, about 25%> to about 40% reduction detectable antigen, ICAM-1 (CD54), ICAM-2 (CD 102), CD44, CD31, CD62L
  • L-selectin CD4, and CD8 are substantially destroyed or inactivated, generally about 70%) to about 100% reduction in detectable antigen
  • antibodies against asialo GM-1 have indicated reductions in the immunologically detectable amount of this ceramide in the membranes of lung epithelial cells following exposure to the multifunctional enzyme of the invention
  • such treatment of lung epithelial cells with the krill hydrolase reduces the level attachment ⁇ Pseudomonas bacteria to the lung epithelial cells
  • hydrolases of interest remove certain cell surface molecules that are believed to contribute to the signal 2 pathway for activation of T-cells, these cell surface molecules include one or more of CD4, CD8, CD28, and CDl 54, while having substantially less effect on the T-cell receptor (TcR), which is involved in the signal 1 pathway for activation of T-cells
  • TcR T-cell receptor
  • hydrolases that substantially interfere with the signal 2 pathway or another accessory pathway e g , remove, destroy, inactivate or disable at least 60% of at least one CD4, CD8, CD28, or CDl 54
  • do not substantially interfere with the signal 1 pathway e g , no more than 50% of TcR is removed, destroyed, inactivated or disabled
  • TcR T-cell receptor
  • adhesion molecule a molecule found on the surface of a cell involved, directly or indirectly, in transmitting signals to the cell
  • cell-cell or cell-virus adhesion syndrome a disease in which a receptor or acceptor cell adhesion component plays a role in the etiology of the disease, for instance by playing a role in the development, transmission, growth or course of the disease
  • hydrolase an enzyme that degrades bonds formed by dehydration reactions such as amide, ester, or ether bonds
  • the term encompasses, but is not limited to, proteases such as trypsin and chymotrypsin
  • identity is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as the case may be, as determined by comparing the sequences
  • identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences "Identity" can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A M , ed , Oxford University Press, New York, 1988, Biocomputing- Informatics and Genome Projects, Smith, D W , ed , Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin,
  • test sequence includes a contiguous segment that is the reference amino acid sequence described above , or is identical with the reference sequence except that, over the entire length corresponding to the reference sequence, the amino acid sequence has an average of up to five substitutions, deletions or insertions for every 100 amino acids of the reference sequence
  • methods to determine identity are codified in publicly available computer programs
  • Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J , et al , Nucleic Acids Research 12(1) 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S F et al .
  • BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S , et al , NCBI NLM NIH Bethesda, MD 20894, Altschul, S , et al , J Mol Biol 215 403-410 (1990)
  • the method of Needleman and Wunch, using the parameters set forth in Version 2 of DNASIS can also be used Additionally, the well known Smith Waterman algorithm can be used to determine identity Alternatively, Parameters for polypeptide sequence comparison include the following
  • a polypeptide sequence of the present invention may be identical to the reference sequence, that is it may be 100% identical, or it may include up to a certain integer number of amino acid alterations as compared to the reference sequence such that the percent identity is less than 100%> identity
  • Such alterations are selected from the group consisting of at least one amino acid deletion, substitution, including conservative and non-conservative substitution, or insertion, and wherein said alterations may occur at the amino- or carboxy-terminal positions of the reference polypeptide sequence or anywhere between those terminal positions, interspersed either individually among the amino acids in the reference sequence or in one or more contiguous groups within the reference sequence
  • immune disorder any disorder caused by an immune reaction to foreign substances, tissues or cells or to autologous or transplanted tissue The term encompasses autoimmune diseases
  • immune cell a lymphocyte, such as a B-cell or T-cell, or a precursor cell to a lymphocyte
  • krill-derived multifunctional hydrolase a multifunctional enzyme having the same sequence as the enzyme isolated from krill having the properties of the protein described in Examples I B, 1 C and ID This enzyme is also referred to as the "krill multifunctional hydrolase” or the “krill multifunctional enzyme” or the “krill-derived multifunctional enzyme "
  • macromolecule for determining purity, this means a biological polymer such as a protein, nucleic acid or carbohydrate of molecular weight greater than about 1000
  • multifunctional enzyme an enzyme having activity comprising at least one of a chymotrypsin, trypsin, collagenase, elastase or exo peptidase activity, a molecular weight between about 20 kd and about 40 kd, and substantial homology to k ⁇ ll-de ⁇ ved multifunctional hydrolase
  • multifunctional enzyme derived from fish or crustacean refers to an enzyme having the same sequence as an enzyme isolated from fish or crustacean
  • protein for the purpose of determining purity, this means a polypeptide of molecular weight greater than about 1000
  • reactive with a cell-surface protein or glycolipid means removes, destroys, inactivates or disables the detectable presence of the cell-surface molecule, by whatever mechanism
  • reactive with a cellular or viral acceptor or receptor adhesion component means removes, destroys, inactivates or disables a cell's or a virus' ability to interact with a cell, virus, ligand, group or molecule, regardless of the mechanism •
  • SDS-PAGE means polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate
  • systemic administration an administration of a biological agent, such as the multifunctional enzyme, designed deliver the agent to the blood or other circulatory system (such as the lymphatic system) of an animal
  • Hydrolases have unit activity according to a recognized assay for the particular type of hydrolase, and is typically defined as the amount of enzyme that catalyzes the hydrolysis of 1 ⁇ mol of substrate per minute at 25°C
  • succ ⁇ nyl-ala-ala-pro-phe- ?-n ⁇ troan ⁇ l ⁇ de Sigma Chemical Co , St Louis, MO
  • the extinction coefficient, ⁇ , of/j-n ⁇ troan ⁇ l ⁇ de is 8800 M 'cm ', thus the multiplication factor to convert dA/minute into U/minute of sample is 5 68, when 20 ⁇ l of sample is used
  • the substrate is CBZ-GRPpNA
  • HL60 cells promyelocyte cells believed to give rise to T-cells, derived from an acute progranulocytic leukemia
  • their binding to TNF ⁇ stimulated endothehal cells is inhibited by more than about 60%
  • treatment of HL60 or endothehal cells with the multifunctional enzyme of the invention will inhibit HL60 cell binding to TNF ⁇ stimulated endothehal cells by at least about 20%, more preferably at least about 40%>, still more preferably at least about 60%o, yet more preferably at least about 80%>
  • the multifunctional enzyme will preferably have at least about 30% of the adhesion-inhibiting activity of the k ⁇ ll-de ⁇ ved multifunctional hydrolase
  • the multifunctional enzyme shall have at least about 60% of the adhesion inhibiting activity of the k ⁇ ll-de ⁇ ved multifunctional hydrolase, still more preferably at least about 80%>, yet more preferably at least about 100% Transplantation, Autoimmune
  • GVHD graft-vs -host disease
  • BMT bone marrow transplantation
  • Acute and chronic GVHD develops in a significant proportion of transplant recipients and represents a major cause of morbidity and mortality after bone marrow transplantation between imperfectly matched individuals (I e , allogeneic transplantation)
  • Efforts to prevent GVHD should reduce morbidity and mortality of transplantation, and enhance the long term outcome of a transplant GVHD is a T-cell mediated disease affecting multiple organ systems The risk of death due to GV
  • T-cells must receive two signals one through the T cell receptor (TcR), and a second signal delivered by accessory molecules, such as CD28, which bind to their counter receptors expressed at the surface of antigen presenting cells (APC)
  • TcR T cell receptor
  • APC antigen presenting cells
  • immune cells When immune cells are treated and contacted with other immune reaction mediating cells prior to administration to a patient such contacting is, for example, conducted under appropriate conditions for maintaining metabohcally active immune reaction mediating cells for, for example, from a tew minutes to a few hours, preferably from about 1 hour to about 4 hours
  • immune reaction mediating cells are treated with hydrolase, exposed to a preparation which would trigger the immune response sought to be avoided, and reintroduced into a treatment subject
  • hydrolase treatment is typically ex vivo, and the exposure is preferably conducted ex vivo
  • ex vivo exposing (l e , contacting) is, for example, conducted under appropriate conditions for maintaining metabohcally active immune reaction mediating cells for, for example, from a few minutes to a few hours, preferably from about 1 hour to about 4 hours
  • Examples of autoimmune-associated antigen preparations include, without limitation, myelin sheath preparations, myelin basic protein and preparations of one or more types of collagen.
  • Antigen preparations can be used, for example, in the treatment of multiple sclerosis, irritable bowel disease (including Crohn's Disease and ulcerative colitis), pernicious anemia, juvenile onset diabetes, thyroiditis, systemic lupus erythematosis (SLE), scleroderma, polyarteritis nodosa and other vasculitides, myasthenia gravis, motor neuron disease, encephlomyelitis, subacute sclerosing pan- encephalitis, Goodpasture's Syndrome, haemolytic anemia, thrombocytopenia, pemphigus vulgaris and bullous pemphigoid.
  • Other examples of autoimmune diseases and examples of allergies can be found in standard texts on allergies or immunology, such as Roitt, Essential Immunology, Eighth Edition, Blackwell Scientific Publications, Oxford, 1994.
  • Transplantation or tolerizing protocols according to the invention include:
  • first cells which are immune cells from a recipient animal with second cells or immunogens to which one seeks to induce tolerance, where the first cells or the second cells are immune cells that are treated with hydrolase, administering the first cells to the recipient animal, and, if appropriate, transplanting tissue from the animal source of the second cells to the recipient;
  • first cells which are immune cells from a donor animal with second cells wherein the first cells or second cells comprise antigens to which one seeks to induce tolerance, where the first cells or the second cells are immune cells that are treated with hydrolase, administering the first cells to a recipient animal, and, if appropriate, transplanting tissue form the animal source of the second cells to the recipient.
  • the cells contacted with the treated immune cells can be the tissue to be transplanted.
  • the immune cells are administered some time before transplantation, such as 12, 24, 48, 72 hours.
  • the second cells can also be treated with the hydrolase.
  • the tolerized cells are T-cells.
  • the T-cells are tolerized by contact with T-cell depleted cells, preferably immune cells.
  • the T-cells can then be isolated from the tolerizing mixture of cells by an affinity binding protocol or cell sorting with appropriate cell-specific antibody reagents
  • Thy-1 (CD90) antibodies tagged with a magnetically susceptible material can be used to isolate T-cells by by magnetic separation
  • hydrolases include metalloproteinases (such as matrix metalloproteinases, including human fibroblast collagenase, interstitial collagenase, stromelysin, gelatinase A, gelatinase B, adamalysins, microbial metalloproteinases and the like), elastases, trypsins, chymotrypsins, other serine proteinases, and the like
  • Such hydrolases include hydrolases of aquatic origin, as described herein
  • Other applicable hydrolases are believed to include, for example, mammalian and non-mammalian trypsins, mammalian and non-mammalian chymotrypsins, mammalian and non-mammalian elastases, papains, bromelains, mammalian and non-mammalian collagenases, subtihsins and mammalian and non-mammalian cathe
  • a hydrolase exposed to immune cells removes, destroys, inactivates or disables at least 60%, 75%, 85%, 90%, 95%, 98% or 99% of at least one of CD4, CD8, a CDl 1, CD25, CD28, a CD49, CDl 52 and CD 154. These molecules are involved in the signal 2 pathway.
  • the hydrolases removes, destroys, inactivates or disables a significant portion of (a) CD4 and CD8, (b) CD4 and CD28, (c) CD4 and CD 154, (d) CD4, CD8 and CD28, (e) CD4, CD8 and CDl 54, (f) CD4, CD28 and CD 154, (g) CD4, CD8, CD28 and CD 154, (h) CD8 and CD28, (i) CD8 and CD 154, (j) CD8, CD28 and CD 154, or (k) CD28 and CD 154.
  • the hydrolases removes, destroys, inactivates or disables a significant portion of (a) CD4 and CD8, (d) CD4, CD8 and CD28, (e) CD4, CD8 and CD 154 or (g) CD4, CD8, CD28 and CD 154.
  • a hydrolase with the immune cells Preferably, no more than 50%, 35%, 20% or 10% of TcR, which is associated with signal 1 , is removed, destroyed, inactivated or disabled by contacting a hydrolase with the immune cells.
  • no more than 50%, 35%, 20% or 10% of CD3, which is associated with signal 1, is removed, destroyed, inactivated or disabled by contacting a hydrolase with the immune cells.
  • a mixture of two or more hydrolases is used to provide the removing, destroying, inactivating or disabling activity, i.e., induce tolerance, or the signal 2 disrupting activity.
  • the mixture can be selected on the basis of mixing a second (or third, etc.) hydrolase that is more effective against given cell surface adhesion molecule than another hydrolase in the mixture.
  • the effects of hydrolase treatment is supplemented with the use of antibodies to specific cell adhesion molecules.
  • This approach can be used for example to alter the kinetics of cell surface effects or supplement effects against certain cell adhesion molecules.
  • the hydrolase selected could be very effective against certain of the targeted cell surface adhesion molecules, but less effective against others.
  • the antibodies, which are preferably monoclonal are used to target the cell surface adhesion molecules against which the hydrolase is less effective.
  • the selected hydrolase can be effective against targeted cell surface adhesion molecules, but a further effect can be achieved with the antibodies.
  • the antibodies can monovalent (e.g., fab fragments), especially as to a given cell surface target (such that an antibody monovalent as to a cell surface adhesion molecule has another binding pocket with another specificity)
  • Antibody sources for use in this aspect of the invention include Boeh ⁇ nger
  • a suitable dose of hydrolase per application ranges from about 0 1 ⁇ g/cm ⁇ to about 1 mg/cm ⁇ , preferably from about 1 ⁇ g/cm ⁇ (for example using about 10 ⁇ g/ml) to about 1 mg/cm ⁇ (for example, using about 10 mg/ml) more preferably from about 5 ⁇ g/cm ⁇ (for example, using about 50 ⁇ g/ml) to about 100 ⁇ g/cm ⁇ (for example using about 1 mg/ml), yet more preferably from about 10 ⁇ g/cm ⁇ to about 250 ⁇ g/cm ⁇ , still yet more preferably from about 10 ⁇ g/cm ⁇ (for example using about 100 ⁇ g/ml) to about 50 ⁇ g/cm ⁇ (for example, about 500 ⁇ g/ml)
  • dosages will generally be selected to maintain a serum level of hydrolase between about 0 1 ⁇ g/l OOcc and about 5 ⁇ g/lOOcc, preferably between
  • the airways of the patient preferably from about 0.5 ml to about 1.0 ml.
  • hydrolase for scar and keloid treatments, generally between about 0.1 mg and about 5 mg of hydrolase will be injected into each cm ⁇ of the lesion, preferably from about 0.5 mg to about 3 mg.
  • hydrolase for treating adhered connective tissue or joints, generally between about 0.5 mg and about 10 mg of hydrolase will be injected interstitially at the adhesion, preferably -between about 1 mg and about 5 mg.
  • the enzyme composition will generally be applied from about 1 to about 10 times per day, preferably from about 2 to about 5 times per day.
  • the composition may be applied systemically or in a manner adapted to target the affected tissue or cells, or a tissue or cells implicated in the disorder can be treated extra-corporeally.
  • the organ, tissue or cells to be transplanted will preferably be bathed in a solution of the hydrolase for between about 10 minutes and about 5 hours.
  • the enzyme solution will preferably contain between about 0.01 mg/ml or 0.5U/ml and about 25 mg/ml or l,250U/ml of the hydrolase, and in certain embodiment preferably, between about 0.5 mg/ml or 25U/ml and about 5 mg/ml and about 250U/ml.
  • the hydrolase can be administered systemically using the conditions described above.
  • the cells are preferably treated with an amount and time of treatment effective to reduce, remove or inactivate at least one cell surface protein by at least about 50%, more preferably by at least about 80%.
  • the hydrolase of the invention is administered orally, topically, rectally, vaginally, by instillation (for instance into the urinary tract or into fistulas), by pulmonary route by use of an aerosol, by application of drops to the eye, or systemically, such as parenterally, including, for example, intramuscularly, subcutaneously, intraperitoneally, intraarterially or intravenously.
  • the multifunctional enzyme is administered alone, or it is combined with a pharmaceutically-acceptable carrier or excipient according to standard pharmaceutical practice
  • the hydrolase is used in the form of tablets, capsules, lozenges, chewing gum, troches, powders, syrups, elixirs, aqueous solutions and suspensions, and the like
  • carriers that is used include lactose, sodium citrate and salts of phosphoric acid
  • disintegrants such as starch, and lubricating agents such as magnesium stearate and talc
  • useful diluents are lactose and high molecular weight polyethylene glycols
  • certain sweetening and/or flavoring agents are added
  • sterile solutions of the hydrolase are usually prepared, and the pHs of the solutions are suitably adjusted and buffered
  • the total concentration of solutes should be controlled to render the preparation isotonic
  • the hydrolase is usually prepared, and the pHs of the solutions are suitably adjusted and buffered
  • the total concentration of solutes should
  • the hydrolase is typically administered in aqueous form or in a hydrogel
  • a preferred hydrogel comprises an aqueous suspension of from about 1% (w/v) to about 10% of low molecular weight hydrolyzed starch
  • Suppository forms of the hydrolase are useful for vaginal, urethral and rectal administrations
  • Such suppositories will generally be constructed of a mixture of substances that is solid at room temperature but melts at body temperature
  • the substances commonly used to create such vehicles include theobroma oil, glycerinated gelatin, hydrogenated vegetable oils, mixtures of polyethylene glycols of various molecular weighty and fatty acid esters of polyethylene glycol See, Remington's Pharmaceutical Sciences, 16th Ed , Mack Publishing, Easton, PA, 1980, pp 1530-1533 for further discussion of suppository dosage forms
  • Analogous gels or cremes can be used for vaginal, urethral and rectal administrations
  • Numerous administration vehicles will be apparent to those of ordinary skill in the art, including without limitation slow release formulations, hposomal formulations and polymeric matrices
  • the cells or viruses involved can include, without limitation, endothehal cells, lymphocytes, including T-cells, tumor cells, microbial cells, viruses, including HIV and herpes
  • endothehal cells lymphocytes, including T-cells
  • tumor cells tumor cells
  • microbial cells viruses, including HIV and herpes
  • Adhesion processes are believed to be involved in tissue invasion, for instance, by immune cells, microbes, and tumor cells
  • hydrolases are proteases Particularly preferred is the multifunctional enzyme of the invention
  • the hydrolase will be administered in an effective amount
  • An effective amount is an amount effective to either (1) reduce the symptoms of the disease sought to be treated, (2) induce a pharmacological change relevant to treating the disease sought to be treated, (3) inhibit or prevent infection or re-infection by an infective agent, or (4) prevent or minimize the occurrence of a non-infectious disease (for instance a disease treatable by blocking a cell adhesion phenomenon)
  • hydrolases- can be used in many veterinary contexts to treat animals, preferably to treat mammals, as will be recognized by those of ordinary skill in light of the present disclosure
  • HL60 cells a hyman lymphocyte cell line
  • TNF tumor necrosis factor
  • E-selectin is a cell surface adhesion protein that appears to bind to a sialated carbohydrate See, Bevilacqua et al , Science
  • the sequence of the first 25 amino acids of the Krill derived multifunctional enzyme is I-V-G-G-N/M-E-V-T-P-H-A-Y-P-(W)-Q-V-G-L-F-I-D-D-M-Y-F (SEQ ID NO. 1).
  • the parentheses indicate a weak recovery of the 14th amino acid and "N/M" indicates heterogeneity at the 5th position.
  • a comparison of the N-terminal 20 to 25 amino acid sequences of various serine hydrolases is presented in Table 2, below
  • the enzyme can be manufactured by recombinant means.
  • sequences recited herein can be used as the basis of oligonucleotide probes for screening expression or genomic libraries to isolate the complete structural gene. See, e.g., Suggs et al., Proc. Nail. Acad. Sci. USA, 78 6613, 1981 or Berent et al., BioTechnic ⁇ ies, 3: 208, 1985.
  • known protein sequences can be used to design primers for use in PCR-based amplification of nucleic acid encoding a multifunctional enzyme.
  • the types of substitutions selected can be based on the analysis of the frequencies of amino acid substitutions between homologous proteins of different species developed by Schulz et al , Principles of Protein Structure, Sp ⁇ nger-Verlag, 1978, pp 14-16, on the analyses of structure-forming potentials developed by Chou and Fasman, Biochemistry 13, 21 1, 1974 or other such methods reviewed by Schulz et al, Principles in Protein Structure, Springer- Verlag, 1978, pp 108-130, and on the analysis of hydrophobicity patterns in proteins developed by Kyte and Doolittle, J Mo/ Biol 157 105-132, 1982 Krill, including without limitation krill of the genuses Euphausia (such as superba, crystallorphias, frigida, triacantha, vellantim, lougirostris, lucens, similis, spinifera, recurva and the like), Meganydiphanes (such as norvegica and the like) and Tysanoes
  • Example 1 In Vitro Binding of HL60 Cells to Endothelial Cells Endothelial cells Endothelial cells were first passaged onto 96 well plates at a given concentration
  • the endothelial cells used in the experiment are described in Edgell et al , Proc. Nat/. Acad. Sci. USA (1983) 80 3734 The cells were incubated at 37°C under a DMEM cell culture medium containing 10%> fetal calf serum and under a 5%> CO2 atmosphere
  • the medium was removed and replaced with 100 ⁇ l (microliter) of a suspension of 200,000 HL60 cells (a human lymphocyte cell line, available from the European Cell Culture Bank under ECACC Accession No 8501 143 1 ) in RPMI medium containing 10% fetal calf serum The cells were incubated for 30 minutes After this, the medium was removed and the adherent cells were washed two times with DMEM medium The relative adherence of the HL60 cells was measured by measuring the difference in optical density at 450nm between the plates on which the cells were co-incubated and plates having endothelial cells alone
  • TNF ⁇ was measured by adding TNF ⁇ at 1500 units/ml to the endothelial cells 4 hours before the incubation with HL60 cells
  • the effect of antibody to E-selectin was measured by adding 25 ⁇ g/ml of monoclonal antibody BBAZ (R&D Systems Europe, Oxford, England) to the HL60 cells
  • BBAZ monoclonal antibody
  • the amount of antibody binding was determined using a fluorescence-activated cell sorter From the results, it was determined that the order of sensitivity to inactivation or removal by the hydrolase was CD4, CD8 ⁇ ICAM-2 (CD 102) ⁇ CD44 ⁇ ICAM- 1 (CD54) ⁇ H-2K Using these same methods with appropriate cells, including endothehal cells, including the s-end- 1 endothehal cell line (Kinashi et al , J Leukocyte Biol 57 168, 1995) and T-cells isolated from the thymuses of C57BL/6 mice, it was determined that the VCAM-1, CD28, CD 31 and asialo GMl ceramide markers are sensitive to the hydrolase The antibodies used to make these determinations were
  • binding was detected with a labeled second antibody, for instance, binding of the asialo GMl antibody was detected with FITC-labeled Fab fragments that were specific for rabbit lgG (heavy and light chains), which was obtained from Caltag Laboratories, San Francisco, CA Example 3 - Timecourse of Cell Surface Recovery of Adhesion Molecules
  • O-l 1 10 T-cell hybrids (this cell line is described by Shimonkevitz et al , / Experimenta/ Med 158 303, 1983) were treated with 500 ⁇ g/ml of the k ⁇ ll-de ⁇ ved multifunctional hydrolase prepared as described in Example IB of WO 96/24371 (Phairson Medical) and tested for the CD4 marker as described in Example 2 Immediately after the treatment, well less than 1% of the amount of CD4 found in the controls was found on the hydrolase-treated cells 48 hours later, the levels in treated cells were the same as those in untreated cells
  • mice (abbreviated BDFl mice, H-2 b k ) were purchased from Charles River Laboratories (St- Constant, Quebec, Canada) Animals were housed microisolator cages at the Institut Armaud Frappier specific-pathogen free facility At the time of bone marrow transplantation, donors and recipients were 6 to 8 weeks of age in the first series of experiments, and 5 to 10 weeks of age in the second series of experiments Bone marrow transplantation (BMT)
  • Recipients were given a single dose of 700 r total body irradiation 2-4 hours before transplantation from a 6 °Co irradiator Irradiated recipients received, as a source of T-cells, a single intravenous injection via the tail vein of 5 x 10 6 bone marrow cells and 5 x 10 6 spleen cells Spleens, femurs, and tibias were aseptically collected from euthanized donors and placed in ice-cold Hank's balanced salt solution (HBSS).
  • HBSS Hank's balanced salt solution
  • Spleens were pressed through sterile wire mesh to obtain single cell suspensions which were further treated with Tris-buffered ammonium chloride or sterile distilled water (hypotonic lysis) to eliminate erythrocytes. Bone marrow cells were flushed with a needle and a syringe from femoral and tibial cavities of donor mice and collected. All cell suspensions were washed twice with HBSS before use. Treatment of spleen cells with proteases In some experiments, spleen cells were treated with proteases prior to injection in irradiated recipients. The purified krill-derived multifunctional enzyme ("PHM protease”) was obtained from Phairson Medical Ltd (Batch No.
  • PLM protease The purified krill-derived multifunctional enzyme
  • donor C57BL/6 spleen cells were treated with 50 ⁇ g/ml of PHM in serum-free RPMI medium for 2 h at 37°C, whereas DBA/2 cells were treated with 20 ⁇ g/ml of PHM in serum-free RPMI medium for 2 h at 37°C.
  • donor C57BL/6 spleen cells were treated with 20 ⁇ g/ml of protease (e.g. cod trypsin, papain, or PHM) in serum-free RPMI medium for 1 h at 37°C.
  • protease e.g. cod trypsin, papain, or PHM
  • Controls included spleen cells incubated without proteases in serum-free RPMI for the same period of time at 37°C. Cells were then washed twice in serum-free medium and counted using Trypan blue staining,
  • PE-labeled anti-CD4 (Pharmingen, San Diego, CA) and Red-613-labeled ant-CD8 antibodies (GIBCO-BRL, Mississaugua, Ont, Canada) obtained commercially.
  • the stained cells were analyzed on a Coulter XL-MCL laser flow cytometer (Hialeah, FL) Experimental design The experiments were designed to investigate the impact of protease treatment of splenocytes on the prevention of lethal GVHD.
  • Donor cells from C57BL/6 (H-2 ), or DBA 2 (H-2 k ) were injected in semi-allogeneic BDFl (H-2 b ) recipients. In this model.
  • GVHD is induced either upon allorecognition of H-2 k antigens expressed by the antigen presenting cells of the recipients following injection of C57BL/6 (H-2 ) donor T-cells, or upon allorecognition of H-2 b antigens on the antigen presenting cells of the recipients following injection of DBA/2 (H-2 k ) donor T-cells
  • H-2 k C57BL/6
  • H-2 k DBA/2
  • PHM can cleave several cell surface adhesion molecules from the surface of T lymphocyte cell lines in vitro, including CD4, CD8, CD62L, CD54, and others
  • PHM can cleave receptors from the surface of freshly isolated splenocytes of C57BL/6
  • the cleavage of the CD4 and CD8 molecules by PHM was dose-dependent
  • PHM is used at concentrations above 20 ⁇ g/ml
  • PHM completely removed the expression of CD4 or CD8 from the surface of splenocytes
  • Splenocytes from C57BL/6 mice were incubated with the indicated concentrations of PHM for 1 h at 37°C in serum- free RPMI medium
  • Cells were then stained with specific antibodies to CD4 or CD8, and analyzed by laser flow cytometry Since PHM can remove any expression of CD4 or CD8 at the surface of splenocytes, and since both CD4 and CD8 have been reported to play a key role in the induction of GVHD, the question of whether ex
  • GVHD is associated with severe weight loss BDFl recipients receiving C57BL/6 bone marrow mixed with splenocytes suffered irreversible and severe weight loss while recipients receiving histocompatible bone marrow inoculum occasionally lost some weight shortly after the transfer due to the irradiation, but subsequently showed signs of recovery as indicated by a continuous gain of weight BDFl recipients reconstituted with C57BL/6 bone marrow cells and the PHM-treated splenocytes also recovered from the initial weight loss associated with the irradiation then underwent a period of gradual weight loss between day 15 and they 30 After day 30, however, these BDFl recipients started to fully recover and most of these recipients survived and gained weight For these results, weights of individual mice were monitored twice a week for each BDFl recipient receiving C57BL/6 semi-allogeneic bone marrow cells mixed with (A) untreated splenocytes, (B) PHM-treated splenocytes, or (C) syngeneic, PHM-treated splenocytes
  • PBMCs Peripheral blood mononuclear cells
  • T-cell-depleted Balb/c stimulator cells containing mostly B cells and macrophages, and some dendritic cells, and few NK cells
  • RPMI Reactive Reassisted Magnetic Infiltration
  • fetal calf serum FCS - 10% final concentration
  • T-cell-depleted stimulator cells containing mostly B cells and macrophages, and some dendritic cells, and few NK cells
  • RPMI Reactive Rexin-free RPMI
  • FCS fetal calf serum
  • sequences recited in the Sequence Listing below as "DNA” or under some other apparently restrictive nomenclature, represent an exemplification of the invention, since those of ordinary skill will recognize that such sequences can be usefully employed in polynucleotides in general, including ribopolynucleotides.

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Abstract

Cette invention se rapporte notamment à un procédé servant à prévenir ou à réduire les réactions de rejets des transplantations, ce procédé consistant à traiter les tissus donneurs avec une quantité d'hydrolase ayant pour effet de prévenir ou de réduire la réaction de rejet et choisie de façon à réduire la quantité d'une ou plusieurs molécules d'adhésion des surfaces cellulaires.
PCT/US1999/030818 1998-12-24 1999-12-23 Traitement et prevention des reactions de rejets immunitaires WO2000038708A1 (fr)

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WO2000078332A2 (fr) * 1999-06-18 2000-12-28 Jon Bragi Bjarnason Proteinases de serine de poisson et leur utilisation pharmaceutique et cosmetique
EP1154686A1 (fr) * 1999-02-18 2001-11-21 FAUSTMAN, Denise L. Methode d'inhibition de rejet de greffe
US7485293B1 (en) 1999-02-18 2009-02-03 Faustman Denise L Method for inhibiting transplant rejection
US8372812B2 (en) 2009-02-26 2013-02-12 Aker Biomarine Asa Phospholipid and protein tablets
US8697138B2 (en) 2007-03-28 2014-04-15 Aker Biomarine As Methods of using krill oil to treat risk factors for cardiovascular, metabolic, and inflammatory disorders
US9644169B2 (en) 2007-03-28 2017-05-09 Aker Biomarine Antarctic As Bioeffective krill oil compositions
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EP1154686A1 (fr) * 1999-02-18 2001-11-21 FAUSTMAN, Denise L. Methode d'inhibition de rejet de greffe
EP1154686A4 (fr) * 1999-02-18 2003-07-02 Denise L Faustman Methode d'inhibition de rejet de greffe
US7485293B1 (en) 1999-02-18 2009-02-03 Faustman Denise L Method for inhibiting transplant rejection
WO2000078332A2 (fr) * 1999-06-18 2000-12-28 Jon Bragi Bjarnason Proteinases de serine de poisson et leur utilisation pharmaceutique et cosmetique
WO2000078332A3 (fr) * 1999-06-18 2001-07-05 Jon Bragi Bjarnason Proteinases de serine de poisson et leur utilisation pharmaceutique et cosmetique
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US9730966B2 (en) 2007-03-28 2017-08-15 Aker Biomarine Antartic As Method of reducing appetite in a human subject comprising administering krill oil composition
US8697138B2 (en) 2007-03-28 2014-04-15 Aker Biomarine As Methods of using krill oil to treat risk factors for cardiovascular, metabolic, and inflammatory disorders
US9816046B2 (en) 2007-03-28 2017-11-14 Aker Biomarine Antarctic As Bioeffective krill oil compositions
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US8372812B2 (en) 2009-02-26 2013-02-12 Aker Biomarine Asa Phospholipid and protein tablets
US11578289B2 (en) 2013-06-14 2023-02-14 Aker Biomarine Antarctic As Lipid extraction processes
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US9867856B2 (en) 2014-01-10 2018-01-16 Aker Biomarine Antarctic As Phospholipid compositions and their preparation
US10864223B2 (en) 2015-02-11 2020-12-15 Aker Biomarine Antarctic As Lipid compositions
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WO2017140735A1 (fr) * 2016-02-15 2017-08-24 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Prévention du rejet de greffe par utilisation préalable de greffons modifiés
US20190262396A1 (en) * 2016-02-15 2019-08-29 Fraunhofer-Gesellschaft Zur Fõrderung Der Angeewandten Forschung E.V. Prevention of graft rejection by prior use of modified grafts
JP2019509269A (ja) * 2016-02-15 2019-04-04 フラウンホッファー−ゲゼルシャフト ツァー フェーデルング デア アンゲバンテン フォルシュング エー ファー 修飾移植片を先に用いることによる移植拒絶反応の抑制
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