EP1218030A1 - Procedes et compositions pour le traitement et la prevention des rejets de greffes utilisant des proteines de choc thermique - Google Patents

Procedes et compositions pour le traitement et la prevention des rejets de greffes utilisant des proteines de choc thermique

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Publication number
EP1218030A1
EP1218030A1 EP00961701A EP00961701A EP1218030A1 EP 1218030 A1 EP1218030 A1 EP 1218030A1 EP 00961701 A EP00961701 A EP 00961701A EP 00961701 A EP00961701 A EP 00961701A EP 1218030 A1 EP1218030 A1 EP 1218030A1
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EP
European Patent Office
Prior art keywords
heat shock
tissue
shock protein
organ
grafted
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP00961701A
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German (de)
English (en)
Other versions
EP1218030A4 (fr
Inventor
Pramod K. Srivastava
Rajiv Y. Chandawarkar
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Fordham University
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Fordham University
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Publication date
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Publication of EP1218030A1 publication Critical patent/EP1218030A1/fr
Publication of EP1218030A4 publication Critical patent/EP1218030A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/001—Preparations to induce tolerance to non-self, e.g. prior to transplantation
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6043—Heat shock proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/622—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier non-covalent binding

Definitions

  • the present invention relates to methods for treatment and prevention of graft rejection, e . g . , in response to tissue or organ transplantation.
  • compositions of complexes of heat shock/stress protein including, but not limited to, hsp70, hsp90, and gp96, either alone or in combination with each other, noncovalently bound to antigenic molecules, are used to suppress the immune response to the grafted tissue or organ.
  • compositions containing un-complexed stress proteins i.e., free of antigenic molecules
  • the invention encompasses administration of heat shock proteins before, after, or both before and after transplantation or grafting.
  • the invention encompasses administration of donor tissue sample prior to administration of heat shock protein and subsequent transplantation or grafting.
  • the Immunology of Transplant and Graft Rejection Organs are transplanted clinically to rectify an irreversible functional deficit but, unless donor and recipient are genetically identical, graft antigens will trigger a rejection response by the recipient.
  • the study of skin graft rejection in mice led to the discovery of the major histocompatibility complex (MHC) antigens, the function of which is to bind processed antigens and present them to T lymphocytes.
  • MHC major histocompatibility complex
  • T lymphocytes are pivotal in transplant rejection.
  • the sensitization phase of rejection is due mainly to passenger leucocytes in the graft being recognized as foreign by the recipient's CD4+ T cells.
  • the effector phase of rejection involves these activated recipient T cells entering the graft and locally producing cytokines.
  • Organ transplantation is now the treatment of choice for end stage organ failure.
  • the ultimate goal in transplantation has been the development of strategies to induce specific tolerance to the allograft.
  • the MHC antigens are the principal targets of the immune response to allografts and T cell recognition of allo-MHC is the initial event which initiates allograft rejection.
  • the availability of sequences of MHC genes in mice, rats, and humans has made it possible to prepare synthetic peptides for the study of the role of MHC peptides in allorecognition and tolerance induction.
  • MHC molecules contain an array of endogenous peptides bound in their antigen presentation groove.
  • T cells recognize specific processed alloantigen presented as peptides in the context of self MHC by antigen-presenting cells (APCs) .
  • APCs antigen-presenting cells
  • synthetic MHC peptides can immunomodulate the alloimmune response both in vitro and vivo, and that allo-tolerance can be induced with synthetic MHC peptides.
  • Two types of effects mediated by synthetic MHC peptides have been reported: (1) suppression of the alloimmune response by relatively non-polymorphic peptides and (2) antigen-specific unresponsiveness induced by polymorphic peptides.
  • Hsps in grafted tissue have been suggested to be alloantigenic targets of heart graft rejecting immune responses.
  • Qian et al., 1995, Transplant Immunology 3: 114- 123 reported elevated hsp expression in cardiac allografts in mice.
  • Qian et al. also reported the presence of infiltrating lymphocytes reactive with mycobacterial hsp60 and hsp70 and with murine grp78 in cardiac allografts undergoing rejection.
  • Moliterno et al., 1995, J. Heart Lung Transplant. 14: 329-337 also reported that anti-hsp60 autoimmune T cells accumulate at sites of inflammation in transplanted heart.
  • Chaperonin 10 has also been referred to as early pregnancy factor (EPF) .
  • CpnlO is homologous to the heat shock protein groES.
  • Administration of cpnlO following skin grafts was reported to significantly prolong the viability of allogenic skin grafts in rats (International Publication Nos. WO 95/15338 and WO 95/15339).
  • the present invention relates to compositions and methods for the treatment and prevention of graft rejection.
  • Treatment regimens include the administration of heat shock proteins (hsps) . Because the protection is based on the immunoregulatory role of the hsp itself (and not its antigenicity) , the effectiveness of the treatment is general — unlike free peptide or other specific graft alloantigen approaches (including where the hsp itself is an alloantigen) , the treatment is not limited to a specific target alloantigen of the rejection process. The effectiveness of the hsp administration is not dependent on identity between the organ or tissue from which the hsp was obtained and the tissue or organ which is being transplanted.
  • hsps heat shock proteins
  • the hsp-mediated suppression of graft rejection may be dependent on the pre-existing development of the graft- specific autoimmune attack.
  • the source of hsp does not require tissue specificity in order to effect suppression because its suppressive activity may attain specificity by acting against a previously activated T cell response, which is specific.
  • the treatment regimens disclosed are useful for the treatment and prevention rejection of a variety of grafted tissues and organs.
  • the example in Section 6, below, demonstrates in detail the effectiveness of prevention of skin graft rejection using the heat shock protein gp96.
  • the treatment methods of the invention are more specific than common cytokine approaches to induction of suppression which are excessively systemic.
  • the hsps used in accordance with the invention exert a more local and targeted immunosuppressive effect at the site of immune cellular activity.
  • Hsps may be administered, in accordance with the invention, before, after, or both before and after transplantation or grafting.
  • the invention encompasses administration of donor tissue sample prior to administration of heat shock protein and subsequent transplantation or grafting.
  • the invention provides methods for determining doses of hsp administered for treatment and prevention of graft rejection. In general, the dosages required for suppressing the immune response are higher than those typically used for generating an immune response.
  • the invention provides pharmaceutical formulations for administration of the compositions in appropriate dosages. The invention also provides routes of administration of the compositions used for treatment and prevention of graft rejection.
  • the invention encompasses a method of preventing or treating rejection of a grafted cell, tissue, or organ in a mammal comprising administering to the mammal a composition comprising a purified complex consisting essentially of a heat shock protein noncovalently bound to an antigenic molecule, wherein the composition does not comprise a heat shock protein that is an alloantigen of the grafted cells, tissue, or organ.
  • the invention encompasses a method of preventing or treating rejection of a grafted cell, tissue, or organ in a mammal comprising administering to the mammal a composition comprising a purified complex consisting essentially of a heat shock protein noncovalently bound to an antigenic molecule, wherein the composition does not comprise any of said complex wherein said antigenic molecule is an alloantigen of the grafted cells, tissue, or organ.
  • compositions comprising gp96 in the prevention of skin graft rejection.
  • FIG. 1 Summary of results of skin graft Experiment 1. Results from Day 14 (4 days after engraftment) ; Day 15, (5 days after engraftment; Day 17 (7 days after engraftment) ; Day 18 (8 days after engraftment) ; Day 19 (9 days after engraftment) ; and Day 20 (10 days after engraftment) are each shown as five rows of five ovals. Each oval represents the skin graft of a different mouse. For each day's results, the rows correspond to five different mice each treated as follows: l sc Row: Buffer (phosphate buffered saline) , alone;
  • FIG. 2. Summary of results of skin graft Experiment 2. Results from Day 18 (8 days after engraftment); Day 22, (12 days after engraftment; Day 24 (14 days after engraftment); Day 18 (8 days after engraftment) ; Day 26 (16 days after engraftment); and Day 29 (19 days after engraftment) are each shown as five row of two ovals. Each oval represents the skin graft of a different mouse. For each day's results, the rows correspond to two different mice each treated as follows: 1 st R Rooww:: No treatment;
  • FIGS. 3A-B Results of Day 18 (8 days after engraftment) from skin graft Experiment 2, presented as described for FIG. 2 , above.
  • FIGS. 4A-B Results of Day 22 (12 days after engraftment) from skin graft Experiment 2, presented as described for FIG. 2, above.
  • FIGS. 5A-B Results of Day 24 (14 days after engraftment) from skin graft Experiment 2, presented as described for FIG. 2, above.
  • FIGS. 6A-B Results of Day 26 (16 days after engraftment) from skin graft Experiment 2, presented as described for FIG. 2, above.
  • FIGS. 7A-B Results of Day 29 (19 days after engraftment) from skin graft Experiment 2, presented as described for FIG. 2, above.
  • the invention is based, in part, on newly discovered immunotherapeutic and immunoprophylactic treatment regimens for graft rejection.
  • hsps in accordance with the present invention are not dependent on administration of any particular target antigen of the rejection process.
  • “Graft” and “Transplant” are used interchangeably herein and each encompass the transfer of cells, tissues, or organs from one location to another, including from one individual to another individual.
  • Antigenic molecule refers to any molecule noncovalently bound to a heat shock protein, including, but not limited to, the peptides with which the hsps are endogenously associated in vivo as well as exogenous antigens/ immunogens (i.e., with which the hsps are not complexed in vivo) or antigenic/ immunogenic fragments and derivatives thereof.
  • the hsps of the present invention that can be used include but are not limited to, gp96, hsp90, and hsp70, either alone or in combination with each other.
  • the hsps are mammalian hsps. More preferably, for the treatment or prevention of graft rejection in humans, the hsps are human hsps.
  • the hsp is not cpnlO. In yet another particular embodiment, the hsp is not hsp60.
  • Heat shock proteins which are also referred to interchangeably herein as stress proteins, useful in the practice of the instant invention can be selected from among any cellular protein that satisfies any one of the following criteria.
  • a heat shock protein is characterized by having its intracellular concentration increase when a cell is exposed to a stressful stimulus, by being capable of binding other proteins or peptides, and by being capable of releasing the bound proteins or peptides in the presence of adenosine triphosphate (ATP) or low pH, or by having at least 35% homology with any cellular protein having any of the above properties.
  • ATP adenosine triphosphate
  • the first stress proteins to be identified were the heat shock proteins (hsps) .
  • hsps heat shock proteins
  • hsps are synthesized by a cell in response to heat shock.
  • three major families of hsp have been identified based on molecular weight. The families have been called hsp60, hsp70 and hsp90 where the numbers reflect the approximate molecular weight of the stress proteins in kilodaltons.
  • Mammalian hsp90 and gp96 each are members of the hsp90 family. Many members of these families were found subsequently to be induced in response to other stressful stimuli including, but not limited to, nutrient deprivation, metabolic disruption, oxygen radicals, and infection with intracellular pathogens. (See Welch, May 1993, Scientific American 56-64; Young, 1990,
  • hsps/ stress proteins belonging to all of these three families can be used in the practice of the instant invention.
  • the major hsps can accumulate to very high levels in stressed cells, but they occur at low to moderate levels in cells that have not been stressed.
  • the highly inducible mammalian hsp70 is hardly detectable at normal temperatures but becomes one of the most actively synthesized proteins in the cell upon heat shock (Welch, et al., 1985, J.
  • hsp90 and hsp60 proteins are abundant at normal temperatures in most, but not all, mammalian cells and are further induced by heat (Lai, et al., 1984, Mol . Cell . Biol . 4:2802-10; van Bergen en Henegouwen, et al., 1987, Genes Dev . 1:525-31).
  • Heat shock proteins are among the most highly conserved proteins in existence.
  • DnaK the hsp70 from E. coli has about 50% amino acid sequence identity with hsp70 proteins from excoriates (Bardwell, et al., 1984, Proc . Natl . Acad . Sci . 81:848-852).
  • the hsp60 and hsp90 families also show similarly high levels of intra families conservation (Hickey, et al., 1989, Mol . Cell . Biol . 9:2615-2626; Jindal,
  • hsp60, hsp70 and hsp90 families are composed of proteins that are related to the stress proteins in sequence, for example, having greater than 35% amino acid identity, but whose expression levels are not altered by stress. Therefore it is contemplated that the definition of stress protein, as used herein, embraces other proteins, muteins, analogs, and variants thereof having at least 35% to 55%, preferably 55% to 75%, and most preferably 75% to 85% amino acid identity with members of the three families whose expression levels in a cell are enhanced in response to a stressful stimulus. The purification of stress proteins belonging to these three families is described below.
  • immunogenic hsp-peptide complexes of the invention include any complex containing an hsp and a peptide that is
  • TJ TJ i-l CD tr 0 ⁇ tn rt 0 ⁇ c rt i ⁇ ft Oi ⁇ rt TJ 3 ⁇ TJ 3 3 • TJ TJ TJ TJ TJ TJ rt rt ft rt O _3 ⁇ TJ — ⁇ r ⁇ H, 3 ft H- H- ft P- tn ⁇ ft rt TJ ⁇ ⁇ ⁇ O • rt i
  • additional molecules such as antibodies, including monoclonal antibodies, or soluble receptors or soluble receptor analogues, that may contact and/or effectively modify the functional capabilities of immune system cells, such as antigen presenting cells, with which the hsp may come into contact.
  • additional molecules such as antibodies, including monoclonal antibodies, or soluble receptors or soluble receptor analogues, that may contact and/or effectively modify the functional capabilities of immune system cells, such as antigen presenting cells, with which the hsp may come into contact.
  • the hsps and/or antigenic molecules can be purified from natural sources, chemically synthesized, or recombinantly produced.
  • the invention provides methods for determining doses for treatment and prevention of graft rejection by evaluating the optimal dose of hsp, both unbound and noncovalently bound to peptide, in experimental animal models and extrapolating the data.
  • the graft or transplant is allogeneic to the individual recipient. In another specific embodiment, the graft or transplant is xenogeneic to the recipient.
  • human recipients may receive grafts or transplants from non-human mammalian donors, including but not limited to pigs and sheep.
  • the methods disclosed herein encompass the prevention and treatment of graft rejection in human and non-human transplant recipients, including but not limited to non-human mammals such as dogs, cats, and horses.
  • the therapeutic regimens and pharmaceutical compositions of the invention can be used with additional immune response enhancers or biological response modifiers including, but not limited to, the cytokines IFN- ⁇ , IFN- ⁇ , IL-2, IL-4 , IL-6, TNF, or other cytokine affecting immune cells.
  • the hsp either uncomplexed or complexed with antigenic molecule is administered in combination therapy with one or more of these cytokines.
  • the therapeutic regimens and pharmaceutical compositions of the invention can be used with additional immunosuppressants or biological response modifiers including, but not limited to, cyclosporine, azathioprine, mycophenolate mofetil, tacrolimus, corticosteroids, prednisone, cyclophosphamide, and antilymphocytes such as antilymphocyte globulin (ALG) , antithymocyte globulin (ATG) , and orthoclone OKT3.
  • ALG antilymphocyte globulin
  • ATG antithymocyte globulin
  • orthoclone OKT3 orthoclone OKT3.
  • the hsp either uncomplexed or complexed with antigenic molecule is administered in combination therapy with one or more of these immunosuppressants . Accordingly, the invention provides methods of preventing and treating graft rejection in an individual comprising administering a composition which elicits specific immunotolerance to the target host cells or tissue.
  • Grafted cells, tissues, and organs whose rejection by recipient can be treated and prevented by the methods of the present invention include, but are not limited to, skin, liver, kidney, heart, bone marrow, pancreas, lung, cornea, and cartilage, and cells obtained from these tissues and organs, including but not limited to pancreatic islet cells.
  • the hsps used in accordance with the invention can be complexed with antigenic molecules (e.g., peptides), or uncomplexed. Whether complexed or not, the hsps can be native (non-recombinant) or recombinant.
  • the antigenic molecules can be endogenous, i.e., naturally associated with hsp intracellularly. Alternatively, the antigenic molecules can be exogenous, i.e., not naturally occurring in a noncovalent complex with hsps, or eluted from a cellularly derived noncovalent complex with hsps and reconstituted with other hsps in vitro.
  • the hsp, or complex is used in purified form, preferably to homogeneity as viewed on a polyacrylamide gel, or to at least 60%, 70%, 80%, or 90% of total protein.
  • the hsp-peptide complexes can be isolated as such from cells wherein the hsp and antigenic molecule are produced.
  • Hsps or exogenous antigenic molecules can be produced in the cell by recombinant expression of a gene encoding that component (either hsp or antigenic molecule) , or can be isolated from native sources.
  • the hsps and exogenous antigenic molecule components can be produced and isolated independently and complexed in vitro .
  • complexes of hsps and endogenous peptides can be isolated from cells.
  • the hsp component is first isolated from cells as a complex, and then purified away from the noncovalently bound endogenous peptide with which it is complexed, prior to complexing in vitro with the exogenous antigenic molecule of interest.
  • the hsp component is first isolated from cells as a complex, and then the noncovalently bound endogenous peptide with which it is complexed is exchanged in vitro with the exogenous antigenic molecule of interest.
  • protocols described herein can be used to isolate and produce purified hsps or purified complexes of hsps and antigenic molecules.
  • Uncomplexed endogenous hsps and endogenous hsps complexed with antigenic molecules can be isolated from any eukaryotic cells, including but not limited to, tissues, isolated cells, and immortalized eukaryotic cell lines.
  • the tissue source need not be the same as the tissue which is targeted by the subject graft. Suitable source tissues include, but are not limited to liver, pancreas, or any other organ of mammalian or non-mammalian origin.
  • the hsps can be produced by recombinant
  • Peptides derived from either a naturally expressed protein (i.e., native peptide) or from a recombinantly expressed protein can be isolated by first isolating the corresponding hsp-peptide complex and then eluting the peptide. Methods for eluting noncovalently bound peptide from the hsp-peptide complex are described in Section 5.2.4, below.
  • Peptides can also be produced synthetically and subsequently complexed with hsps in vitro .
  • the hsps to be used therapeutically, alone or complexed, can but need not be isolated from a sample from the patient to which they are then to be administered to treat or prevent graft rejection, i.e., the hsps (and antigenic molecules) can be autologous or non-autologous.
  • Hsp-Peptide Complexes The methods described in Sections 5.2.1.1-5.2.1.3, below, can be used to isolate hsps complexed with antigenic molecules from cells, preferably from cells expressing non- recombinant hsps, although cells expressing recombinant hsps may also be used. A population of purified hsp-peptide complexes, comprising different peptides, can thus be obtained. These same methods may also be used to prepare purified hsp, by removing the endogenous antigenic molecules from the isolated complexes by methods described in Section 5.2.3, below.
  • a procedure that can be used is as follows: A pellet of eukaryotic cells (e . g . , from liver, pancreas, or any other convenient organ) is resuspended in 3 volumes of buffer consisting of 30mM sodium bicarbonate buffer (pH 7.5) and lmM PMSF and the cells allowed to swell on ice 20 minutes. The cell pellet then is homogenized in a Dounce homogenizer (the appropriate clearance of the homogenizer will vary according to each cells type) on ice until >95% cells are lysed.
  • a Dounce homogenizer the appropriate clearance of the homogenizer will vary according to each cells type
  • the lysate is centrifuged at l,000Xg for 10 minutes to remove unbroken cells, nuclei and other debris.
  • the supernatant from this centrifugation step then is recentrifuged at 100,000Xg for 90 minutes.
  • the gp96-peptide complex can be purified either from the 100,000Xg pellet or from the supernatant.
  • the supernatant is diluted with equal volume of 2X lysis buffer and the supernatant mixed for 2-3 hours at 4°C with Con A-Sepharose® (Pharmacia, Inc., Sweden) equilibrated with PBS containing 2mM Ca : ⁇ and 2mM Mg -* .
  • the slurry is packed into a column and washed with IX lysis buffer until the OD : , : drops to baseline. Then, the column is washed with 1/3 column bed volume of 10% ⁇ -methyl mannoside ( ⁇ -MM) dissolved in PBS containing 2mM Ca " and 2mM Mg * , the column sealed with a piece of parafilm, and incubated at 37 °C for 15 minutes. Then the column is cooled to room temperature and the parafilm removed from the bottom of the column. Five column volumes of the ⁇ -MM buffer are applied to the column and the eluate analyzed by SDS-PAGE.
  • ⁇ -MM ⁇ -methyl mannoside
  • the resulting material is about 60-95% pure, however this depends upon the cell type and the tissue-to-lysis buffer ratio used.
  • the sample is applied to a Mono Q® FPLC ion -exchange chromatographic column (Pharmacia, Inc. , Piscataway, NJ) equilibrated with a buffer containing 5mM sodium phosphate, pH 7.
  • the proteins then are eluted from the column with a 0- 1M NaCl gradient and the gp96 fraction elutes between 400mM and 550mM NaCl.
  • One optional step involves an ammonium sulfate precipitation prior to the Con A purification step and the other optional step involves DEAE-Sepharose® purification after the Con A purification step but before the Mono Q® FPLC step.
  • the supernatant resulting from the l00,000Xg centrifugation step is brought to a final concentration of 50% ammonium sulfate by the addition of ammonium sulfate.
  • the ammonium sulfate is added slowly while gently stirring the solution in a beaker placed in a tray of ice water.
  • the solution is stirred from about % to 12 hours at 4°C and the resulting solution centrifuged at 6,000 rpm (Sorvall SS34 rotor) .
  • the supernatant resulting from this step is removed, brought to 70% ammonium sulfate saturation by the addition of ammonium sulfate solution, and centrifuged at 6,000 rpm (Sorvall SS34 rotor).
  • the resulting pellet from this step is harvested and suspended in PBS containing 70% ammonium sulfate in order to rinse the pellet. This mixture is centrifuged at 6,000 rpm (Sorvall SS34 rotor) and the pellet dissolved in PBS containing 2mM Ca :* and Mg :* . Undissolved material is removed by a brief centrifugation at 15,000 rpm (Sorvall SS34 rotor).
  • the solution is mixed with Con A Sepharose® and the procedure followed as before.
  • the gp96 containing fractions eluted from the Con A column are pooled and the buffer exchanged for 5mM sodium phosphate buffer, pH 7, 300mM NaCl by dialysis, or preferably by buffer exchange on a Sephadex® G25 column (Pharmacia, Inc. , Sweden) .
  • the solution is mixed with DEAE-Sepharose® previously equilibrated with 5mM sodium phosphate buffer, pH 7, 300mM NaCl.
  • the protein solution and the beads are mixed gently for 1 hour and poured into a column.
  • the column is washed with 5mM sodium phosphate buffer, pH 7, 300mM NaCl, until the absorbance at 280nM drops to baseline. Then, the bound protein is eluted from the column with five volumes of 5mM sodium phosphate buffer, pH 7, 700mM NaCl. Protein containing fractions are pooled and diluted with 5mM sodium phosphate buffer, pH 7 in order to lower the salt concentration to 175mM. The resulting material then is applied to the Mono Q® FPLC column (Pharmacia) equilibrated with 5mM sodium phosphate buffer, pH 7 and the protein that binds to the Mono Q® FPLC column (Pharmacia) is eluted as described before.
  • the pellet When the gp96 fraction is isolated from the 100,000Xg pellet, the pellet is suspended in 5 volumes of PBS containing either 1% sodium deoxycholate or 1% octyl glucopyranoside (but without the Mg ⁇ * and Ca " ) and incubated on ice for 1 hour. The suspension is centrifuged at 20,000Xg for 30 minutes and the resulting supernatant dialyzed against several changes of PBS (also without the Mg " and Ca " ) to remove the detergent. The dialysate is centrifuged at 100,000Xg for 90 minutes, the supernatant harvested, and calcium and magnesium are added to the supernatant to give final concentrations of 2mM, respectively. Then the sample is purified by either the unmodified or the modified method for isolating gp96-peptide complex from the 100,000Xg supernatant, see above.
  • the gp96-peptide complexes can be purified to apparent homogeneity using this procedure. About 10-20 ⁇ g of gp96- peptide complex can be isolated from lg cells/tissue.
  • hsp70-peptide complexes The purification of hsp70-peptide complexes has been described previously, see, for example, Udono et al., 1993, J. Exp . Med . 178:1391-1396.
  • a procedure that can be used, presented by way of example but not limitation, is as follows: Initially, cells (e . g . , from liver, pancreas, or any other convenient organ) are suspended in 3 volumes of IX lysis buffer consisting of 5mM sodium phosphate buffer, pH 7, 150mM NaCl, 2mM CaCl , 2mM MgCl and lmM phenyl methyl sulfonyl fluoride (PMSF) .
  • IX lysis buffer consisting of 5mM sodium phosphate buffer, pH 7, 150mM NaCl, 2mM CaCl , 2mM MgCl and lmM phenyl methyl sulfonyl fluoride (PM
  • the pellet is sonicated, on ice, until >99% cells are lysed as determined by microscopic examination.
  • the cells can be lysed by mechanical shearing and in this approach the cells typically are resuspended in 30mM sodium bicarbonate pH 7.5, lmM PMSF, incubated on ice for 20 minutes and then homogenized in a dounce homogenizer until >95% cells are lysed.
  • the lysate is centrifuged at l,000Xg for 10 minutes to remove unbroken cells, nuclei and other cellular debris.
  • the resulting supernatant is recentrifuged at 100,000Xg for
  • the material that fails to bind is harvested and dialyzed for 36 hours (three times, 100 volumes each time) against lOmM Tris-Acetate pH 7.5, O.lmM EDTA, lOmM NaCl, lmM PMSF. Then the dialyzate is centrifuged at 17,000 rpm (Sorvall SS34 rotor) for 20 minutes. Then the resulting supernatant is harvested and applied to a Mono Q® FPLC column equilibrated in 20mM Tris- Acetate pH 7.5, 20mM NaCl, O.lmM EDTA and 15mM 2- mercaptoethanol.
  • the column is then developed with a 20mM to 500mM NaCl gradient and then eluted fractions fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and characterized by immunoblotting using an appropriate anti-hsp70 antibody (such as from clone N27F3-4, from StressGen, Victoria, British Columbia, Canada) .
  • SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
  • Fractions strongly immunoreactive with the anti-hsp70 antibody are pooled and the hsp70-peptide complexes precipitated with ammonium sulfate; specifically with a 50%- 70% ammonium sulfate cut.
  • the resulting precipitate is then harvested by centrifugation at 17,000 rpm (SS34 Sorvall rotor) and washed with 70% ammonium sulfate.
  • the washed precipitate is then solubilized and any residual ammonium sulfate removed by gel filtration on a Sephadex® G25 column (Pharmacia) . If necessary the hsp70 preparation thus obtained can be repurified through the Mono Q® FPLC column as described above.
  • the hsp70-peptide complex can be purified to apparent homogeneity using this method. Typically lmg of hsp70- peptide complex can be purified from lg of cells/tissue.
  • the present invention further describes a rapid method for purification of hsp70-peptide complexes.
  • This improved method comprises contacting cellular proteins with ADP or a nonhydrolyzable analog of ATP affixed to a solid substrate, such that hsp70 in the lysate can bind to the ADP or nonhydrolyzable ATP analog, and eluting the bound hsp70.
  • a preferred method uses column chromatography with ADP affixed to a solid substratum (e.g., ADP-agarose) .
  • the resulting hsp70 preparations are higher in purity and devoid of contaminating peptides.
  • the hsp70 yields are also increased significantly by about more than 10 fold.
  • chromatography with nonhydrolyzable analogs of ATP instead of ADP, can be used for purification of hsp70-peptide complexes.
  • Hsp 90-peptide Complexes 500 million cells (e.g., from liver, pancreas, or any other convenient organ) are homogenized in hypotonic buffer and the lysate is centrifuged at 100,000Xg for 90 minutes at 4°C. The supernatant is applied to an ADP-agarose column. The column is washed in buffer and is eluted with 5 column volumes of 3 mM ADP. The hsp70-peptide complexes elute in fractions 2 through 10 of the total 15 fractions which elute. The eluted fractions are analyzed by SDS-PAGE. The hsp70- peptide complexes can be purified to apparent homogeneity using this procedure. 5.2.1.3. Preparation and Purification of Hsp 90-peptide Complexes
  • cells e.g., from liver, pancreas, or any other convenient organ
  • IX Lysis buffer consisting of 5mM sodium phosphate buffer (pH7) , 150mM NaCl, 2mM CaCl , 2mM MgCl : and lmM phenyl methyl sulfonyl fluoride (PMSF) .
  • PMSF phenyl methyl sulfonyl fluoride
  • the cells can be lysed by mechanical shearing and in this approach the cells typically are resuspended in 30mM sodium bicarbonate pH 7.5, lmM PMSF, incubated on ice for 20 minutes and then homogenized in a dounce homogenizer until >95% cells are lysed.
  • the lysate is centrifuged at l,000Xg for 10 minutes to remove unbroken cells, nuclei and other cellular debris.
  • the resulting supernatant is recentrifuged at l00,000Xg for 10 minutes.
  • Hsp90-peptide complexes can be purified to apparent homogeneity using this procedure. Typically, 150-200 ⁇ g of hsp90-peptide complex can be purified from lg of cells/tissue.
  • hsps genes encoding hsps have been cloned and sequenced, including, for example, human hsp70 (GenBank Accession Nos. M11717 and M15432; see also Hunt and Morimoto, 1985, Proc. Natl. Acad. Sci. USA 82: 6455-6459), human hsp90 (GenBank Accession No. X15183; see also Ya azaki et al., 1989, Nucleic Acids Res. 17: 7108), and human gp96 (GenBank Accession No. M33716; see also Maki et al., 1990, Proc. Natl. Acad. Sci. USA 87: 5658-5662) .
  • the hsps can be produced by recombinant DNA technology using techniques well known in the art. Methods which are well known to those skilled in the art can be used to construct expression vectors containing hsp coding sequences and appropriate transcriptional/translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, for example, the techniques described in Sambrook et al., 1989, supra , and
  • host-expression vector systems can be utilized to express the hsp genes. These include but are not limited to microorganisms such as bacteria (e.g., E . coli , B . subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing the hsp coding sequence; yeast (e.g.
  • Sac char omyces , Pichia transformed with recombinant yeast expression vectors containing the hsp coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the hsp coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing the hsp coding sequence; or mammalian cell systems (e.g.
  • COS COS, CHO, BHK, 293, 3T3 harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) .
  • promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter) .
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not limited, to the E . coli expression vector pUR278
  • pGEX vectors can also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned hsp gene protein can be released from the GST moiety.
  • AcNPV Autographa calif ornica nuclear polyhedrosis virus
  • the virus grows in Spodoptera frugiperda cells.
  • the hsp gene can be cloned individually into non- essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter) .
  • Successful insertion of the hsp coding sequence will result in inactivation of the polyhedrin gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene) .
  • non-occluded recombinant virus i.e., virus lacking the proteinaceous coat coded for by the polyhedrin gene
  • These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed.
  • a number of viral-based expression systems can be utilized.
  • the hsp coding sequence can be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene can then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing hsps in infected hosts.
  • a non-essential region of the viral genome e.g., region El or E3
  • Specific initiation signals may also be required for efficient translation of inserted hsp coding sequence. These signals include the ATG initiation codon and adjacent sequences. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al. , 1987, Methods in Enzymol. 153:516-544).
  • a host cell strain can be chosen which modulates the expression of the inserted sequences, or modifies and processes the hsp in the specific fashion desired. For example, choosing a system that allows for appropriate glycosylation is especially important in the case of gp96.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins such as glycosylation.
  • Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.
  • the histidine-nickel (his-Ni) tag system is used (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA 88: 8972-8976) .
  • the his-Ni system the his-Ni system, the hsp is expressed in human cell lines as a fusion protein which can be readily purified in a non-denatured form.
  • the gene of interest i.e., the hsp gene
  • the gene of interest is subcloned into a vaccinia recombination plasmid such that the gene's open reading frame is translationally fused to an amino-terminal tag consisting of six histidine residues.
  • Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni ⁇ * ⁇ nitriloacetic acid-agarose columns and histidine-tagged proteins are selectively eluted with imidazole-containing buffers.
  • Kits for expressing an isolating proteins using the his- Ni system are commercially available from Invitrogen®, San Diego, California.
  • recombinant hsps produced in eukaryotic hosts cells as described in this section, above can be purified according to the respective methods detailed in Section 5.2.1, above.
  • Uncomplexed hsps The following methods can be used to obtain uncomplexed hsps, i.e., hsps that are substantially free of noncovalently bound antigenic molecules such as peptides.
  • the hsps can be administered in their uncomplexed form in accordance with the invention for the treatment and prevention of graft rejection.
  • the uncomplexed hsps can be used to design hsp-antigenic molecule complexes by complexing them in vitro with antigenic molecules of interest, as described in
  • acetic acid or trifluoroacetic acid is added to the purified hsp- antigenic molecule complex to give a final concentration of 10% (vol/vol) and the mixture incubated at room temperature or in a boiling water bath or any temperature in between, for 10 minutes ⁇ See , Van Bleek, et al., 1990, Nature 348:213-216; and Li, et al., 1993, EMBO Journal 12:3143-3151).
  • the resulting samples are centrifuged through a Centricon® 10 assembly.
  • the high and low molecular weight fractions are recovered.
  • the remaining large molecular weight hsp70— peptide complexes can be reincubated in low pH to remove any remaining peptides.
  • the resulting higher molecular weight fractions containing hsp are pooled and concentrated.
  • the hsp70-peptide complex is purified as described above in Section 5.2.1.2.
  • the peptide is eluted from the hsp70 by either of the following two preferred methods. More preferably, the hsp70-peptide complex is incubated in the presence of ATP. Alternatively, the hsp70-peptide complex is incubated in a low pH buffer, as described in Section 5.2.2, above .
  • the complex is centrifuged through a Centricon® 10 assembly (Millipore) to remove any low molecular weight material loosely associated with the complex.
  • the large molecular weight fraction can be removed and analyzed by SDS- PAGE while the low molecular weight can be analyzed by HPLC as described below.
  • the stress protein-peptide complex in the large molecular weight fraction is incubated with lOmM ATP for 30 minutes at room temperature.
  • the resulting samples are centrifuged through a Centricon® 10 assembly as mentioned previously.
  • the high and low molecular weight fractions are recovered.
  • the remaining large molecular weight hsp70-peptide complexes can be reincubated with ATP to remove any remaining peptides.
  • the resulting higher molecular weight fractions containing hsp70 are pooled and concentrated.
  • immunogenic or antigenic peptides can be isolated from either stress protein-peptide complexes or MHC-peptide complexes for use subsequently as antigenic molecules, by complexing in vitro to hsps.
  • Exemplary protocols for isolating peptides and/or antigenic components from either of the these complexes are set forth below in Sections 5.2.4.1 and 5.2.4.2.
  • the methods detailed in Section 5.2.3, above, can be used to elute the peptide from a stress protein-peptide complex.
  • One approach involves incubating the stress protein-peptide complex in the presence of ATP.
  • the other approach involves incubating the complexes in a low pH buffer.
  • the complex of interest is centrifuged through a Centricon® 10 assembly (Millipore) to remove any low molecular weight material loosely associated with the complex.
  • the large molecular weight fraction can be removed and analyzed by SDS-PAGE while the low molecular weight can be analyzed by HPLC as described below.
  • the stress protein-peptide complex in the large molecular weight fraction is incubated with lOmM ATP for 30 minutes at room temperature.
  • acetic acid or trifluoroacetic acid is added to the stress protein-peptide complex to give a final concentration of 10% (vol/vol) and the mixture incubated at room temperature or in a boiling water bath or any temperature in between, for 10 minutes (See , Van Bleek, et al., 1990, Nature 348:213-216; and Li, et al., 1993, EMBO
  • Centricon® 10 assembly as mentioned previously.
  • the high and low molecular weight fractions are recovered.
  • the remaining large molecular weight stress protein-peptide complexes can be reincubated with ATP or low pH to remove any remaining peptides.
  • MHC molecules is well known in the art and so is not described in detail herein (See , Falk, et al., 1990, Nature
  • MHC-peptide complexes can be isolated by a conventional immunoaffinity procedure.
  • the peptides then can be eluted from the MHC-peptide complex by incubating the complexes in the presence of about 0.1% TFA in acetonitrile.
  • the eluted peptides can be fractionated and purified by reverse phase HPLC, as before.
  • amino acid sequences of the eluted peptides can be determined either by manual or automated amino acid sequencing techniques well known in the art. Once the amino acid sequence of a potentially protective peptide has been determined the peptide can be synthesized in any desired amount using conventional peptide synthesis or other protocols well known in the art.
  • Peptides having the same amino acid sequence as those isolated above can be synthesized by solid-phase peptide synthesis using procedures similar to those described by Merrifield, 1963, J . Am . Chem . Soc , 85:2149. During synthesis, N- ⁇ -protected amino acids having protected side chains are added stepwise to a growing polypeptide chain linked by its C-terminal and to an insoluble polymeric support i.e., polystyrene beads.
  • the peptides are synthesized by linking an amino group of an N- ⁇ -deprotected amino acid to an ⁇ -carboxy group of an N- ⁇ -protected amino acid that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide.
  • a reagent such as dicyclohexylcarbodiimide.
  • the attachment of a free amino group to the activated carboxyl leads to peptide bond formation.
  • the most commonly used N- ⁇ -protecting groups include Boc which is acid labile and Fmoc which is base labile. Briefly, the C-terminal N- ⁇ -protected amino acid is first attached to the polystyrene beads. The N- ⁇ -protecting group is then removed.
  • the deprotected ⁇ -amino group is coupled to the activated ⁇ -carboxylate group of the next N- ⁇ - protected amino acid.
  • the process is repeated until the desired peptide is synthesized.
  • the resulting peptides are then cleaved from the insoluble polymer support and the amino acid side chains deprotected. Longer peptides can be derived by condensation of protected peptide fragments. Details of appropriate chemistries, resins, protecting groups, protected amino acids and reagents are well known in the art and so are not discussed in detail herein (See, Atherton, et al., 1989,
  • complexes of hsps and the peptides with which they are endogenously associated in vivo are not employed, and it is desired to use hsp-antigenic molecule complexes, complexes of hsps to antigenic molecules are produced in vitro .
  • the peptides either isolated by the aforementioned procedures or chemically synthesized or recombinantly produced can be reconstituted with a variety of purified natural or recombinant stress proteins in vitro to generate immunogenic noncovalent stress protein-antigenic molecule complexes.
  • exogenous antigens or antigenic/ immunogenic fragments or derivatives thereof can be noncovalently complexed to stress proteins for use in the immunotherapeutic or prophylactic vaccines of the invention.
  • a preferred, exemplary protocol for noncovalently complexing a stress protein and an antigenic molecule in vitro is discussed below.
  • the hsps Prior to complexing, the hsps are pretreated with ATP or low pH to remove any peptides that may be associated with the hsp of interest.
  • ATP ATP
  • excess ATP is removed from the preparation by the addition of apyranase as described by Levy, et al., 1991, Cell 67 : 265-274.
  • the buffer is readjusted to neutral pH by the addition of pH modifying reagents.
  • the antigenic molecules (l ⁇ g) and the pretreated hsp (9 ⁇ g) are admixed to give an approximately 5 antigenic molecule: 1 stress protein molar ratio.
  • the mixture is incubated for 15 minutes to 3 hours at 4° to 45°C in a suitable binding buffer such as one containing 20mM sodium phosphate, pH 7.2, 350mM NaCl, 3mM MgCl ; and lmM phenyl methyl sulfonyl fluoride (PMSF) .
  • a suitable binding buffer such as one containing 20mM sodium phosphate, pH 7.2, 350mM NaCl, 3mM MgCl ; and lmM phenyl methyl sulfonyl fluoride (PMSF) .
  • the preparations are centrifuged through a Centricon® 10 assembly (Millipore) to remove any unbound peptide.
  • the association of the peptides with the stress proteins can be assayed by SDS-PAGE. This is the preferred method for in vitro complexing of peptides isolated from MHC-peptide complexes of peptides disassociated from endogenous hsp
  • hsp70 preferred for producing complexes of hsp70 to exogenous antigenic molecules such as peptides
  • 5-10 micrograms of purified hsp is incubated with equimolar quantities of the antigenic molecule in 20mM sodium phosphate buffer pH 7.5, 0.5M NaCl, 3mM MgCl and lmM ADP in a volume of 100 microliter at 37°C for 1 hr. This incubation mixture is further diluted to 1ml in phosphate-buffered saline.
  • a suitable buffer such as one containing 20mM sodium phosphate buffer pH 7.5 , 0.5M NaCl, 3nM MgCl at 60-65°C for 5-20 min.
  • equimolar or excess quantities of peptide e.g., exogenous peptide
  • the incubation mixture is allowed to cool to room temperature and centrifuged one or more times if necessary, through a Centricon® 10 assembly (Millipore) to remove any unbound peptide.
  • 100-300nM purified peptide is added to lOOnM purified gp96.
  • 100-300nM peptide e.g., exogenous peptide
  • purified gp96-peptide (endogenous) complex such that the exogenous peptide is exchanged for the endogenous peptide.
  • the mixture is incubated in a binding buffer consisting of 20mM HEPES, pH 7.2 , 20 mM NaCl, and 2mM MgCl ; at 60°C for 10 min. and allowed to cool to room temperature for an additional 10 min. After centrifugation, the sample is incubated for 30 min. at room temperature. Free peptide is removed completely using a microcon 50 (Amicon, Inc. ) .
  • hsp covalently linked i.e., covalently coupled or joined
  • peptide may be used in accordance with the invention to inhibit graft rejection.
  • hsp and peptide can be prepared separately according to the methods described in Sections 5.2.3-5.2.4, above. Free peptide can then be covalently linked to hsp by mixing each component in the presence of a cross-linking agent, including but not limited to glutaraldehyde.
  • Such covalently linked hsp-peptide complexes can be made using, for example, the method of Lussow et al., 1991, Eur. J. Immunol. 21:2297-2302 and Barrios et al., 1992, Eur. J. Immunol. 22:1365-1372, each of which is hereby incorporated by reference in its entirety.
  • a peptide can be covalently linked to an hsp by genetically engineering an hsp-peptide fusion protein, using recombinant DNA techniques well known in the art. More specifically, the coding sequence of an hsp can be obtained as described in Section 5.2.2, above, for example, and then fused to a DNA sequence coding for a peptide. This construct can then be expressed in a host cell and purified as an intact fusion protein, using the methods described in Section 5.2.2, above, for example. Preferably, the peptide is fused to the peptide binding domain of the hsp.
  • Hsps and hsp-antigenic molecule complexes are administered to mammalian subjects, e.g., primates, dogs, cats, mice, rats, horses, cows, pigs, etc., preferably humans, in doses in a range of about 5 ⁇ g to about 5000 ⁇ g, alternatively in a range of about 5 ⁇ g to about 1500 ⁇ g.
  • mammals a range of about 50 ⁇ g to about 500 ⁇ g, either intradermally or subcutaneously may be used.
  • a range of about 50 ⁇ g to about 200 ⁇ g subcutaneously and about 5 ⁇ g to about 100 ⁇ g intradermally may be used.
  • intradermal injections typically require a lower dosage and are, therefore, preferred with respect to economy of materials.
  • an effective dose for prevention of graft rejection in mice is 100 ⁇ g and 200 ⁇ g gp96 subcutaneously for mice of average mass of 20-25 g. These amounts of hsp (100-200 ⁇ g range) are high compared to the relatively small amounts of hsp-peptide complex that are required to elicit an effective immune response against an antigenic peptide, such as a complexed tumor antigen. Similar high dosages of 100-200 ⁇ g, or more than 200 ⁇ g, of hsp may also be effective in treatment of larger mammals, including humans.
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the hsps or complexes may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents.
  • Administration can be systemic or local; this may be achieved, for example and not by way of limitation, by topical application, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • the hsp compositions are administered, either intradermally or subcutaneously, with sites of administration varied sequentially.
  • sites of administration varied sequentially.
  • the doses recited above are given once weekly for a period of about 4 to 6 weeks, and the mode of administration is varied with each administration.
  • Each site of administration may be varied sequentially.
  • the first injection can be given, either intradermally or subcutaneously, on the left arm, the second on the right arm, the third on the left belly, the fourth on the right belly, the fifth on the left thigh, the sixth on the right thigh, etc.
  • the same site can be repeated after a gap of one or more injections.
  • split injections can be given.
  • half the dose can be given in one site and the other half in another site on the same day.
  • further injections are preferably given at two-week intervals over a period of time of one month.
  • Later injections can be given monthly.
  • the pace of later injections can be modified, depending upon the patient's clinical progress and responsiveness to the immunotherapy.
  • the mode of administration is sequentially varied, e.g., weekly administrations are given in sequence intradermally or subcutaneously.
  • the uncomplexed hsps or hsps complexed with antigenic molecules in accordance with the invention, can be formulated into pharmaceutical preparations for administration to mammals, preferably humans, for treatment or prevention of graft rejection.
  • immunosuppressive agents as described in Section 5, above, can be formulated separately from or in combination with the hsps and hsp-antigenic molecule complexes described herein for use in accordance with the invention.
  • Compositions comprising a compound of the invention formulated in a compatible pharmaceutical carrier can be prepared, packaged, and labeled for treatment and prevention of rejection of grafted tissues and organs, such as skin, liver, kidney, heart, bone marrow, pancreas, lung, cornea, and cartilage.
  • the complex is water-soluble, then it can be formulated in an appropriate buffer, for example, phosphate buffered saline or other physiologically compatible solutions. Alternatively, if the resulting complex has poor solubility in aqueous solvents, then it can be formulated with a non-ionic surfactant such as Tween, or polyethylene glycol.
  • a non-ionic surfactant such as Tween, or polyethylene glycol.
  • the compounds and their physiologically acceptable solvates can be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral, rectal administration.
  • the pharmaceutical preparation can be in liquid form, for example, solutions, syrups or suspensions, or can be presented as a drug product for reconstitution with water or other suitable vehicle before use.
  • Such liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats) ; emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid) .
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, or fractionated vegetable oils
  • preservatives e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid
  • the pharmaceutical compositions can take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose) ; fillers (e.g., lactose, macrocrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate) ; or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinized maize starch, polyvinyl pyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, macrocrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.
  • Preparations for oral administration can be suitably formulated to give controlled release of the active compound.
  • the compositions can take the form of tablets or lozenges formulated in conventional manner .
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator
  • the compounds can be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multi- dose containers, with an added preservative.
  • the compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and can contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds can also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds can also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds can be formulated with suitable polymeric or hydrophobic materials (for example, as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • suitable polymeric or hydrophobic materials for example, as an emulsion in an acceptable oil
  • ion exchange resins for example, as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • Liposomes and emulsions are well known examples of delivery vehicles or carriers for hydrophilic drugs.
  • compositions can, if desired, be presented in a pack or dispenser device which can contain one or more unit dosage forms containing the active ingredient.
  • the pack can for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device can be accompanied by instructions for administration.
  • kits for carrying out the therapeutic regimens of the invention comprise in one or more containers therapeutically or prophylactically effective amounts of the hsp or hsp-antigenic molecule complexes in pharmaceutically acceptable form.
  • the hsp or hsp-antigenic molecule complex in a vial of a kit of the invention can be in the form of a pharmaceutically acceptable solution, e.g., in combination with sterile saline, dextrose solution, or buffered solution, or other pharmaceutically acceptable sterile fluid.
  • the complex can be lyophilized or desiccated; in this instance, the kit optionally further comprises in a container a pharmaceutically acceptable solution (e.g., saline, dextrose solution, etc.), preferably sterile, to reconstitute the complex to form a solution for injection purposes.
  • a kit of the invention further comprises a needle or syringe, preferably packaged in sterile form, for injecting the complex, and/or a packaged alcohol pad. Instructions are optionally included for administration of hsp or hsp-antigenic molecule complexes by a clinician or by the patient.
  • the hsp-based compositions and formulations described above in Sections 5.2 and 5.4 can be used to treat or prevent graft rejection of cells, tissues, and organs, including but not limited to skin, liver, kidney, heart, bone marrow, pancreas, lung, cornea, and cartilage and cells derived therefrom.
  • Suppression of a rejection response may also be enhanced by administration of the hsp after transplantation.
  • Transplantation may trigger an incipient graft rejection response.
  • Administration of hsp after transplantation may specifically suppress such an activated rejection response.
  • the treatment regimens provided herein comprise administration of the hsps after the onset of the graft rejection response; i.e., after the specific immune response has already developed.
  • Hsp administration results in regulation of the activity of the relevant, pathologically active effector cells.
  • the treatment methods of the present invention exploit not only the general properties of hsps but also the specificity of the naturally arisen pathological immune response.
  • the treatment methods of the invention are more specific than common cytokine approaches to induction of suppression which are excessively systemic.
  • the hsps used in accordance with the invention exert a more local and targeted immunosuppressive effect at the site of autoimmune cellular activity.
  • the invention encompasses administration of hsp before, after, or both before and after grafting or transplantation.
  • Pre-treatment of the recipient with a sample of donor tissue and hsp may be used, in accordance with the invention, to exploit the ability of hsps to specifically suppress an activated immune response.
  • the recipient may be pre-treated, prior to transplantation, with a tissue sample obtained from the donor organ.
  • this tissue is a dispensable sample which would not jeopardize the health of the recipient if rejected.
  • Examples of such pre-treatment tissue include, but are not limited to, small portions of the actual tissue or organ to be transplanted.
  • the tissue used in the pre- treatment need not be the same as the tissue to be transplanted.
  • the pre-treatment tissue can be different than the tissue or organ to be transplanted.
  • the key aspect of the pre-treatment tissue is that it expresses alloantigens of the donor. These alloantigens elicit a T cell response against the tissue of the donor. This response could potentially damage the eventual transplanted donor tissue or organ.
  • hsp is administered to the recipient after exposure to the tissue sample, but prior to actual transplantation. In this manner, the hsp specifically suppresses this response against the sample donor tissue.
  • administration of hsp prior to transplantation may further comprise, in accordance with the invention, treatment of the recipient with a sample of donor tissue prior to administration of hsp.
  • Donor tissues and organs for engraftment and transplantation can be selected using standard screening and tissue typing methods well known in the art, so as to minimize the likelihood of rejection. These methods include, but are not limited to, matching HLA phenotypes of donor and recipient which is well known in the art and described in detail in Valente et al., 1998 "Immunobiology of Renal Transplantation", in The Surgical Clinics of America, Venkateswara, K.R. , ed. , Vol. 78, No. 1 (W.B. Saunders Company: Philadelphia) at pages 1-26, which is hereby incorporated by reference in its entirety.
  • donors and recipients may be screened for suitability of transplantation to determine the extent of contraindications using the criteria detailed in Kasiske, 1998 , "The Evaluation of Prospective Renal Transplant Recipients and Living Donors," in The Surgical Clinics of America, Venkateswara, K.R. , ed. , Vol. 78, No. 1 (W.B. Saunders Company: Philadelphia) at pages 27-39, which is hereby incorporated by reference in its entirety.
  • Experiment 1 demonstrates the effectiveness of the heat shock protein gp96 in inhibiting graft rejection.
  • gp96 was isolated as a non-covalent gp96-peptide complex from liver and from skin of BALB/cJ (H-2 1 ) mice (i.e., syngeneic with graft donor BALB/cJ (H-2 ') mice) , according to the method described Srivastava et al., 1986, Proc. Natl. Acad. Sci. USA 83: 3407-3411.
  • Purified gp96 (complexed with peptide) was suspended in phosphate buffered saline (PBS) .
  • Donor skin cell and liver cell lysates were obtained as a 100,000Xg supernatant prepared as described in Srivastava et al. , 1986, supra .
  • each C57BL/6 (H-2 b ) recipient received a full thickness skin graft as follows.
  • Recipient C57BL/6 (H-2 C ) mice were wounded by creating a wound of 1.2 cm in diameter, which expanded to 1.6 cm in diameter.
  • An appropriately sized disk was marked with a pen, and full-thickness skin was excised using a scalpel.
  • Skin grafts were obtained from BALB/cJ (H-2 ⁇ ) donor mice by excising a 1.6 cm diameter patch of skin. Grafts were sewn onto the wound of recipient mice using 4.0 silk interrupted sutures. Grafts were meshed by making random incisions on the surface to allow seepage and prevent tenting of the graft.
  • Grafts were analyzed 4, 5, 7, 8, 9, and 10 days after engraftment (on Day 14, Day 15, Day 17, Day 18, Day 19, and Day 20, respectively) .
  • Experiment 2 also demonstrates the effectiveness of the heat shock protein gp96 in inhibiting graft rejection.
  • gp96 was isolated as a non-covalent gp96-peptide complex from liver of BALB/cJ (H-2 d ) mice, (i.e., syngeneic with graft donor BALB/cJ (H-2 ⁇ ) mice) or liver of Lewis rats, according to the method described in Srivastava et al., 1986, supra .
  • Skin grafts were obtained from BALB/cJ (H-2 J ) donor mice as described in Section 6.1, above. Grafts were analyzed 8, 12, 14, 16, and 19 days after engraftment (Day 18, Day 22, Day 24, Day 26, and Day 29, respectively) .
  • Results The results for all of Days 18, 22, 24, 26, and 29 are summarized in FIG. 2.
  • the results for Day 18 are depicted in FIGS. 3A-B.
  • the results for Day 22 are depicted in FIGS. 4A- B.
  • the results for Day 24 are depicted in FIGS. 5A-B.
  • the results for Day 26 are depicted in FIGS. 6A-B.
  • the results for Day 29 are depicted in FIGS. 7A-B.
  • mice 1 in group 8 and mouse 1 in group 9 each had a healthy graft on Day 18 and Day 22.
  • mouse 2 in group 8 and mouse 2 in group 9 each had a grafts that were much healthier than the grafts of all the mice from the other groups on Day 22.
  • the present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

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Abstract

L'invention porte sur un procédé de traitement et de prévention des rejets de greffes suite par exemple à une transplantation d'organe. Ledit procédé consiste à administrer une composition de complexes de protéines de choc thermique dont non exclusivement des hsp70, hsp90, et gp96, soit seules, soit combinées, et non liées par covalence à des molécules d'antigènes pour supprimer la réponse immunitaire vis-à-vis du tissu ou de l'organe greffé. L'invention porte également sur l'administration de compositions contenant des protéines de choc non complexées (c.-à-d. exemptes de molécules d'antigène) pour supprimer la réponse immunitaire vis-à-vis du tissu ou de l'organe greffé. L'invention préconise l'administration des protéines de choc thermique avant ou après ou avant et après la transplantation et l'administration d'échantillons de tissus du donneur avant celle des protéines de choc thermique et la transplantation ou la greffe.
EP00961701A 1999-09-10 2000-09-08 Procedes et compositions pour le traitement et la prevention des rejets de greffes utilisant des proteines de choc thermique Withdrawn EP1218030A4 (fr)

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PCT/US2000/024711 WO2001017554A1 (fr) 1999-09-10 2000-09-08 Procedes et compositions pour le traitement et la prevention des rejets de greffes utilisant des proteines de choc thermique

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EP1296711B1 (fr) 2000-06-26 2006-05-17 Stressgen Biotechnologies Corporation Hpv-e7 pour le traitement des infections par le papillomavirus
US6921534B2 (en) 2001-02-05 2005-07-26 Stressgen Biotechnologies Corporation Hepatitis B virus treatment
ES2433915T3 (es) 2002-05-21 2013-12-13 Irun R. Cohen Vacunas de ADN que codifican proteínas de choque térmico
AU2002952492A0 (en) * 2002-11-06 2002-11-21 Cbio Limited Chaperonin 10 immunosuppression
JP2007530420A (ja) * 2003-07-07 2007-11-01 ザ・ジェネラル・ホスピタル・コーポレイション ｆｕｇｅｔａｃｔｉｃタンパク質、組成物および使用方法
BRPI0708865A8 (pt) 2006-03-14 2019-01-22 Univ Oregon Health & Science métodos para produzir uma resposta imune à tuberculose
US8188214B2 (en) 2007-02-28 2012-05-29 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Brachyury polypeptides and methods for use
EP3578195B1 (fr) 2008-06-26 2023-08-09 Zevra Denmark A/S Utilisation du hsp70 en tant que régulateur de l'activité enzymatique
US8664183B2 (en) 2009-02-27 2014-03-04 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services SPANX-B polypeptides and their use
CN102462841A (zh) * 2010-11-09 2012-05-23 中国科学院微生物研究所 一种促进gp96蛋白的免疫活性的方法及其应用
RU2013125923A (ru) 2010-11-30 2015-01-10 Орфазиме Апс СПОСОБЫ УВЕЛИЧЕНИЯ ВНУТРИКЛЕТОЧНОЙ АКТИВНОСТИ Hsp70
EP2895191B1 (fr) 2012-09-14 2019-06-05 The U.S.A. as represented by the Secretary, Department of Health and Human Services Protéine brachyury, vecteurs adénoviraux codant pour la protéine brachyury, et leur utilisation
BR102014002362A8 (pt) 2014-01-30 2018-04-17 Uniao Brasileira De Educacao E Assistencia Mantenedora Da Puc Rs método de imunomodulação e/ou preservação de órgãos ex-vivo, composições, processo e usos
HUE054957T2 (hu) 2014-09-15 2021-10-28 Orphazyme As Arimoklomol készítése
WO2017024000A1 (fr) 2015-08-03 2017-02-09 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Mutants de délétion de la protéine brachyury, vecteurs sans levure codant pour les mutants de délétion de la protéine brachyury, et leur utilisation
WO2017178029A1 (fr) 2016-04-13 2017-10-19 Orphazyme Aps Protéines de choc thermique et homéostasie du cholestérol
HUE052158T2 (hu) 2016-04-29 2021-04-28 Orphazyme As Arimoklomol a glükocerebroszidázzal társult rendellenességek kezeléséhez
IL303026A (en) 2020-11-19 2023-07-01 Zevra Denmark As Processes for preparing arimoclomol citrate and its intermediates

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CA2383213A1 (fr) 2001-03-15

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