WO1998051341A1 - ANTIGEN-mPEG CONJUGATES SUPPRESS HUMORAL AND CELL MEDIATED IMMUNE RESPONSES - Google Patents

ANTIGEN-mPEG CONJUGATES SUPPRESS HUMORAL AND CELL MEDIATED IMMUNE RESPONSES Download PDF

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WO1998051341A1
WO1998051341A1 PCT/US1998/009786 US9809786W WO9851341A1 WO 1998051341 A1 WO1998051341 A1 WO 1998051341A1 US 9809786 W US9809786 W US 9809786W WO 9851341 A1 WO9851341 A1 WO 9851341A1
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mpeg
protein
conjugate
cells
ova
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PCT/US1998/009786
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French (fr)
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WO1998051341B1 (en
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Alec H. Sehon
Judith A. Kapp
Glen M. Lang
Yong Ke
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The University Of Manitoba
Emory University
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Priority to AU74853/98A priority Critical patent/AU7485398A/en
Publication of WO1998051341A1 publication Critical patent/WO1998051341A1/en
Publication of WO1998051341B1 publication Critical patent/WO1998051341B1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • 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
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a method of inducing tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses by administering an effective amount of an Ag conjugated with monomethoxypolyethylene glycol (mPEG) conjugate.
  • Methods of treating allergies, autoimmune diseases and preventing an immune rejection of organ transplants and rejection of genetically engineered cells in gene therapy, are also disclosed.
  • exogenous protein Ag can activate CD8 + T cells that suppress immune responses in an Ag-specific fashion (1-5) .
  • MHC major histocompatibility complex
  • nonphagocytic EL4 cells do not present soluble ovalbumin (OVA) to MHC class I-restricted CD8 + T cells after pulsing with native OVA (6) .
  • OVA ovalbumin
  • exogenous OVA could be presented via MHC class I pathway by these cells if delivered by other means, such as osmotic lysis (6) , electroporation (7) , liposomes (8) , mycobacterial infection (9) , nonionic triblock copolymers (10) or receptor-mediated uptake (11) .
  • the antigen-specific receptors expressed by T cells do not recognize native, intact proteins. Rather, they bind to protein fragments that are bound by proteins encoded by the Major Histocompatability Complex expressed on the surface of specialized antigen presenting cells.
  • the PCT publication WO 95/12413 does not propose that mPEG antigens would be used to inhibit T cells. This treatment would be non-antigen-specific. Such nonspecific inhibition would have little advantage over the immunosuppressive drugs that are currently in use.
  • mPEG modification of proteins renders them non-immunogenic in that they fail to stimulate antibody responses. More importantly, the mPEG modified proteins are tolerogenic rendering exposed individuals unable to respond to a subsequent challenge with the unmodified, protein. Thus, tolerance induced by mPEG modified proteins is due to induction of suppressor T cells that in turn down regulate in an Ag-specific manner the immune response of the recipient if administered at any time prior to challenge with the unmodified antigen. Sehon has obtained several patents on a procedure for preventing IgE mediated allergic responses, and favoring the tilting of the immune response to the production of IgG antibodies in allergic individuals. However, the effects of mPEG modified proteins on cell-mediated immune responses have only recently been studied.
  • U.S. Patent No. 4,296,097 discloses a process for the suppression of the formation of anti-BPO (benzylpenicilloyl) antibodies by administering a conjugate of penicillin and an amino-derivative of polyvinylalcohol polymers.
  • U.S. Patent No. 4,261,973 discloses a method of suppressing the induction of reaginic antibodies to an allergan by administering a covalent conjugate of the allergen and non-immunogenic water soluble polymers.
  • U.S. Patent No. 5,447,722 describes a method of suppression of an IgG immune response to an antigenic protein by administering a tolerogenic conjugate of monomethoxypolyethylene glycol and the antigenic protein one day prior to adminstration of the protein alone.
  • U.S. Patent No. 5,358,710 describes a method of suppressing an animals antibody-mediated immune response to a second antigenic polypeptide by selecting a mammal which is unsensitized to a first antigenic polypeptide, and administering a tolerogenic conjugate of the first antigenic peptide covalently bound to a water soluble polymer. Then an adduct of the first antigenic polypeptide bound to a second antigenic polypeptide is administered.
  • the administration of the tolerogenic conjugate suppresses the capacity of the mammal to mount a humoral antibody response to the first antigenic peptide so that when the first antigenic peptide is conjugated to the second antigenic peptide the anibody mediated immune response to the second antigenic peptide is also suppressed.
  • the goal of this invention is to provide a method for induction of suppression of both humoral (antibody) , and cellular (cell mediated) immune responses to an Ag, by the administration of the antigen in the form of a conjugate with mPEG, (i.e. Ag(mPEG) containing an optimal number of mPEG molecules coupled covalently onto the antigen) at any time prior to injection of the Ag by itself or in conjunction with an appropriate adjuvant, so as to induce humoral and cellular responses to the Ag in question.
  • mPEG conjugate with mPEG, (i.e. Ag(mPEG) containing an optimal number of mPEG molecules coupled covalently onto the antigen) at any time prior to injection of the Ag by itself or in conjunction with an appropriate adjuvant, so as to induce humoral and cellular responses to the Ag in question.
  • the immunosuppressive Ag(mPEG) n conjugate is also referred to as a tolerogenic conjugate.
  • Part I of the application generally enables methods of suppressing the humoral and cell mediated immune responses.
  • Part II of the application enables specific applications of the above method to gene therapies, organ transplantation and treatment of autoimmune conditions.
  • the invention provides a method of obtaining passive transfer of suppression of an immune response comprising treating an animal, preferably a syngeneic animal with Ag(mPEG) conjugate and transferring lymphocytes from said animal to a recipient animal, wherein said lymphocytes provide suppression of Ag-specific cytotoxic lymphocyte (CTL) activity in said recipient animal.
  • an animal preferably a syngeneic animal with Ag(mPEG) conjugate
  • lymphocytes provide suppression of Ag-specific cytotoxic lymphocyte (CTL) activity in said recipient animal.
  • CTL cytotoxic lymphocyte
  • the invention advantageously provides a method of treating a condition selected from the group consisting of allergies and autoimmune diseases by inducing tolerance to an antigen (Ag) in both humoral and cell mediated immune responses comprising administering an effective amount of Ag(mPEG) conjugate to induce tolerance to an antigen (Ag) .
  • the invention provides a method of preventing an immune rejection of organ transplants comprising administering an effective amount of Ag(mPEG) conjugate to induce tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses.
  • the invention provides a method of treating organ-specific autoimmune diseases in animal comprising administration of mPEG conjugates of autoantigens selected from the group consisting of collagen-induced arthritis by type II collagen, experimental autoimmune encephalomyelitis by myelin basic protein, and diabetes in NOD mice by insulin to induce tolerance to an antigen (Ag) in both humoral and cell mediated immune responses.
  • autoantigens selected from the group consisting of collagen-induced arthritis by type II collagen, experimental autoimmune encephalomyelitis by myelin basic protein, and diabetes in NOD mice by insulin to induce tolerance to an antigen (Ag) in both humoral and cell mediated immune responses.
  • the invention provides a method of conducting gene therapy including administering to a mammal an immunosuppressive effective amount of a tolerogenic conjugate consisting of a protein coupled to monomethoxypolyethylene glycol (mPEG) having a molecular weight of about 2,000-10,000 daltons, wherein administration of said tolerogenic conjugate is at least one day prior to administration of a gene therapy vector encoding a gene for a protein, wherein said protein is identical to said protein which is coupled to mPEG, and wherein said method results in the suppression of an immune response and in the development of tolerance to the protein expressed by said gene encoded by said gene therapy vector.
  • mPEG monomethoxypolyethylene glycol
  • Ab antibody; Ag, antigen, APC, antigen presenting cell; CFA, complete Freund's adjuvant; CTL, cytotoxic T lymphocyte; HIgG, human monoclonal (myeloma) immunoglobulin G; IFN- ⁇ interferon; IL, interleukin, LNL, lymph nodal lymphocyte; MHC, major histocompatibility complex; mPEG, monomethoxypolyethylene glycol; OVA, ovalbumin, PCA, passive cutaneous anaphylaxis; TCR, T cell receptor, Th, helper T cells; Ts suppressor T cell; TsF, Ts factor; f.p., foot pad.
  • FIG. 1 shows that OVA(mPEG) conjugate induces suppression of Ab responses.
  • Groups of C57BL/6 mice were injected i.p. with PBS, 2.5 mg OVA(mPEG) or 1 mg OVA. After one week, mice were primed via the f.p. with 200 ⁇ g OVA in CFA and bled two weeks later. Sera from each group were tested individually for OVA-specific IgG (upper panel ) or IgG isotypes (lower panel , at 1:100 dilution) by ELISA and absorbance was read at 405 nm. Normal mouse sera were used as negative control and results are shown as averages of three mice + SD.
  • FIG. 2 shows specificity of suppression of humoral and cell mediated immune responses induced by conjugates of protein Ag and mPEG.
  • C57BL/6 mice were either untreated, or injected i.p. with 2.5 mg OVA(mPEG) or 2.5 mg H ⁇ gG(mPEG). After one week, half of each group of mice were primed via f.p. with 200 ⁇ g OVA in CFA and the other half with 200 ⁇ g HIgG in CFA. Mice were bled two weeks later and sera (1:50 dilution) from each group were tested individually for IgG specific for OVA (left panel ) or HIgG (right panel ) by ELISA, and absorbance was read at 405 nm.
  • mice + SD Normal mouse serum was used as negative control and the results are shown averages of three mice + SD.
  • FIG. 3 shows that OVA(mPEG) conjugate induces suppression of lymphokine production by helper T cells.
  • C57BL/6 mice were pretreated and primed as described in Figure 1. Two weeks after immunization, draining lymph nodes were harvested and 2 x 10° of LNL were incubated at 37 °C with 2 x 10° of irradiated APC in the presence or absence of 200 ⁇ g/ml OVA. As control, LNL were incubated with 200 ⁇ g/ml ConA in the absence of APC. Supematants were harvested 24 h later and tested for lymphokine production using HT-2 cells. Results are shown as averages of triplicates + SD.
  • FIG. 4 shows that OVA(mPEG) conjugate induces suppression of lymphokine production by T cells.
  • C57BL/6 mice were pretreated and primed as described in Figure 1. Draining lymph nodes were harvested two weeks after immunization and LNL were incubated with APC in the presence or absence of OVA as described in Figure 2. Supematants were tested for IL-2 , IL-4 and IFN- by lymphokine ELISA.
  • FIG. 5 shows that OVA(mPEG) conjugate inhibits activation of T cells.
  • C57BL/6 mice were primed via f.p. with 200 ⁇ g OVA in CFA.
  • 3 x 10 6 /ml of spleen cells were incubated with medium alone, 200 ⁇ g/ml OVA or 500 ⁇ g/ml OVA(mPEG) conjugate.
  • the supematants were tested for IFN- lymphokine ELISA.
  • FIG. 6 shows that OVA(mPEG) conjugate induces suppression of cytolytic responses.
  • C57BL/6 mice were injected i.p. with (A) PBS, (B) 2.5 mg OVA(mPEG) or (C) 1 mg native OVA. After one week, mice were primed via f.p. with 200 ⁇ g OVA in CFA. Spleens were harvested two weeks later and spleen cells were stimulated with irradiated E.G7-OVA cells. After six days, cytolytic activity of cultured cells was measured using 51 Cr-labeled E.G7-OVA or EL4 targets. Results are shown as % specific lysis at various E:T ratios and represent averages of triplicates + SD.
  • FIG. 7 shows that immunosuppression could be achieved by transfer of spleen cells of syngeneic mice that had been tolerized by OVA(mPEG) .
  • Donor C57BL/6 mice were injected i.p. with (B) PBS, (C) 2.5 mg OVA(mPEG) or (D) 1 mg OVA.
  • B PBS
  • C 2.5 mg OVA(mPEG)
  • D 1 mg OVA.
  • 1 x 10 s of spleen cells were transferred i.v. to syngeneic naive mice.
  • control mice (A) and recipients (B to D) were primed via f.p. with 200 ⁇ g OVA in CFA.
  • spleen cells from recipients were stimulated with irradiated E.G7-OVA cells for six days and their cytolytic activity was tested in a 4-h standard 51 Cr-release assay as described in Figure 6.
  • FIGS. 8, 9 and 10 show diagrams illustrating the efficiency of the invention.
  • the percentages in brackets of Figs. 8 and 10 represent the degree of suppression with respect to the control in animals receiving phosphate buffered saline (PBS) in lieu of the conjugates.
  • PBS phosphate buffered saline
  • the present invention provides a method for inducing specific suppression to a given antigen (Ag) of both humoral and cell-mediated immune responses comprising administering an effective amount of a tolerogenic Ag(mPEG) conjugate.
  • a tolerogenic Ag(mPEG) conjugate In one embodiment the tolerance induced by Ag(mPEG) conjugates is Ag specific and the suppression of the humoral response is induced in an isotype-nonspecific manner.
  • the tolerance is mediated by Ag-specific CD8 + suppressor T (Ts) cells.
  • the conjugate in another embodiment, suppresses IL-2 production by lymph node lymphocytes (LNL) . In yet another embodiment the conjugate suppresses IL-2 , IFN- and IL-4 lymphokine production.
  • the method does not skew CD4 + T cells toward Thl or Th2 phenotype.
  • the Ag(mPEG) conjugate inhibits in vitro lymphokine production by in vivo primed CD4 + Th cells.
  • the Ag(mPEG) conjugate advantageously inhibits both arms of cell-mediated immune responses in vivo .
  • the invention also provides a method for obtaining passive transfer of suppression of an immune response comprising treating an animal with Ag(mPEG) conjugate and transferring lymphocytes from said animal to a recipient animal, wherein said lymphocytes provide suppression of Ag-specific cytotoxic lymphocyte (CTL) activity in said recipient animal.
  • CTL cytotoxic lymphocyte
  • the transfer of Ts cells inhibited cytolytic responses in recipients.
  • the transfer of splenic Ts cells from an Ag (mPEG) -tolerized animal leads to downregulation of primary IgE and IgG responses in recipient animals.
  • This ability to suppress the immune response provides a method of treating a condition preferably selected from the group consisting of allergies and autoimmune diseases by inducing tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses comprising administering an effective amount of Ag(mPEG) conjugate to induce tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses.
  • the ability to suppress the immune response also provides for a method of preventing an immune rejection of organ transplants, or rejection of DNA transfected cells and their expressed protein product, comprising administering an effective amount of Ag(mPEG) conjugate to induce tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses.
  • an antigen Ag
  • antibodies of all IgG subclasses are suppressed.
  • IgG isotopes dependent upon Thl and Th2 lymphokines are both inhibited by said Ag(mPEG) conjugates.
  • lymphokines produced by CD4 + Th cells are inhibited by said Ag(mPEG) conjugate. These lymphokines are selected from the group consisting of IL-2 , IL-4 and IFN- .
  • the invention provides a method of treating organ-specific autoimmune diseases in animal comprising administration of mPEG conjugates of autoantigens selected from the group consisting of collagen-induced arthritis by type II collagen, experimental autoimmune encephalomyelitis by myelin basic protein (58) , and diabetes in NOD mice by insulin to induce tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses.
  • autoantigens selected from the group consisting of collagen-induced arthritis by type II collagen, experimental autoimmune encephalomyelitis by myelin basic protein (58) , and diabetes in NOD mice by insulin to induce tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses.
  • the inventors demonstrated the induction of CD8 + T cells activated by exogenous protein antigens (Ag) in immune regulation. It has been demonstrated that exogenous ovalbumin (OVA) primed murine CD8 + cytotoxic T lymphocytes (CTL) precursors, if administered with complete Freund's adjuvant (CFA) , these CD8 + CTL suppressed Ag-specific humoral and cell-mediated immune responses in recipients after transfer. Thus, CD8 + CTL are immunosuppressive and can be accounted as regulatory cells in some situations.
  • OVA ovalbumin
  • CTL cytotoxic T lymphocytes
  • CFA complete Freund's adjuvant
  • the inventors have shown that oral administration of protein Ag induced tolerance of both humoral and cell-mediated immune responses.
  • This tolerance was mediated by Ag-specific CD8 + suppressor T (Ts) cells that were phenotypically distinguished from CD8 + CTL by reactivity with a monoclonal antibody (mAb) specific for the murine Ts cells.
  • mPEG modified ovalbumin OVA
  • This effect was antigen-specific in that responses to unrelated antigens were not inhibited by mPEG modified OVA (and vice versa) .
  • the inventors investigate the mechanisms of immune regulation by using the tolerogenic conjugates of protein Ag and mPEG.
  • the results show that a single injection of OVA(mPEG) conjugate but not unconjugated OVA suppressed subsequent Ab responses, lymphokine production by CD4 + T cells and inhibited priming of CD8 + CTL precursors to OVA.
  • the tolerance induction by Ag(mPEG) conjugates was Ag specific, and suppression could be transferred by splenic T cells from tolerized mice to syngeneic mice.
  • the inventors confirmed that Ag(mPEG) conjugates not only suppress Ab (i.e. humoral) responses suppressing different, i.e., Ig classes but also inhibit cell-mediated immune responses in vivo .
  • Ag(mPEG) conjugates Covalent coupling of diverse protein antigens and mPEG resulted in Ag(mPEG) conjugates that have been found to be tolerogenic rather than immunogenic (18-22) .
  • Administration of Ag(mPEG) conjugates is a well-established method to induce long-lasting tolerance (20,23) and has been used to prevent the induction of Ag specific allergic responses which are mediated by IgE Abs (19,24).
  • Ag(mPEG) conjugates stimulated the induction of Ag-specific, non cytotoxic Ts cells (25,26), which expressed Thy-1 and CD8 + markers (26,27), and reacted with a mAb specific for the activated murine Ts cells (17) .
  • TsF soluble Ts factor
  • CD8 + Ts cells produced a ThO-like pattern of cytokines (IL-2, IL-4 , IFN ⁇ , TGF-?, TNF- ⁇ and lymphotoxin) that were found not to be directly responsible for the observed downregulation of Ab responses (30) .
  • cytokines IL-2, IL-4 , IFN ⁇ , TGF-?, TNF- ⁇ and lymphotoxin
  • the inventors investigated the tolerance induction of cell-mediated immune responses by Ag(mPEG) conjugates. Indeed, in addition to inducing suppression of humoral responses, OVA(mPEG) conjugate but not the unmodified OVA, inhibited lymphokine production by CD4 + Th cells and priming of CD8 + CTL precursors upon challenge with OVA in CFA. Moreover, tolerance induced by OVA(mPEG) conjugate was mediated by Ts cells that induced also Ag-specific suppression on transfer to naive syngeneic recipients.
  • mice Eight- to 12-week-old female C57BL/6, mice (H-2 ) were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and used exclusively in this study. Outbred, 250 to 350 g male Sprague-Dawley or Long Evans hooded rats were obtained from the Central Animal Care Services of the University of Manitoba.
  • Purified chicken egg OVA (grade VI) was purchased from Sigma Chemical Co. (St. Louis, MO) .
  • Human monoclonal IgG (HIgG) was isolated from serum of a myeloma patient by ammonium sulfate precipitation and ion exchange chromatography on DEAE (20) .
  • the tolerogenic conjugates used in this study OVA(mPEG) 10 and HgG(mPEG) 25 , were prepared as described earlier (23,25,33 incorporated by reference herein) and dissolved in phosphate-buffered saline (PBS, pH 7.4).
  • PBS phosphate-buffered saline
  • any antigen may be used in conjunction with mPEG having a molecular weight in the range of about 3000-25,000, preferably 5000-20,000.
  • the subscript n in the formula Ag(mPEG) n represents the degree of conjugation, i.e. the average number of mPEG molecules coupled per molecule of protein Ag. (Although the example presented herein uses OVA Ag as a model antigen, any antigen to which tolerance is desired can be converted to an immunosuppressive mPEG conjugate and be used according to the method of the present invention. )
  • the tolerogenic conjugates 0VA(mPEG) u and HIgG (mPEG) 25 were synthesized by a modification of the procedure previously reported (21) . Aggregate free protein is concentrated by ultrafiltration iva an Amicon filter to 5-10 mg/ml and then reacted with a large excess, an 8-molar excess of the electrophilically activated mPEG intermediate e.g., mPEG para-nitrophenol carbonate (which reacts with alpha and epsilon amino groups) , commercially available from Shearwater Polymers, Huntsville, Alabama, with respect to the total lysine content of the protein. This procedure is applicable to any protein.
  • mPEG para-nitrophenol carbonate which reacts with alpha and epsilon amino groups
  • the content of the dialysis bag was applied to a Pharmacia BloPilot gel filtration column (Superdex 60/600) equilibrated with DDW for the isolation of the conjugate, which elutes in the void volume, from the hydrolyzed mPEG and from the para-nitrophenol which elutes with the buffer salts, and to place the conjugate in DDW for lyophilization.
  • the conjugate is stored lyophilized at -20°C.
  • mice were pretreated by intraperitoneal (i.p.) injection with 2.5 mg OVA(mPEG) or 1 mg native OVA.
  • the control mice received 0.5 ml PBS in lieu of Ag.
  • the OVA(mPEG) conjugates used in this study contained -40% (wt/wt) protein, i.e. 2.5 mg OVA(mPEG) conjugates contained about 1 mg native OVA within the complex.
  • mice were immunized in the hindfoot pads (f.p.) with 200 ⁇ g of OVA emulsified in CFA containing M . tuberculosis H37Ra (Difco Labs, Detroit, MI) .
  • mice Two weeks after immunization, mice were bled and sacrificed. Draining lymph nodes and spleens were harvested for testing lymphokine production and cytolytic activity, respectively (4,13). In some control experiments, mice were pretreated with 2.5 mg H ⁇ gG(mPEG) and then immunized with 200 ⁇ g HIgG in CFA.
  • PCA Passive cutaneous anaphylaxis
  • the PCA titer was expressed as the reciprocal of the highest dilution of the serum giving a blue area of at least 5 mm in diameter.
  • the Student's t-test was used to compare the PCA titer of each group with that of the control. The difference was considered statistically significant only when the p value was smaller than 0.01.
  • Enzyme-linked immunosorbent assay A solid-phase ELISA was used to determine Ag-specific IgG production in primed mice (13). Microliter plates were coated with OVA or HIgG at a concentration of 10 ⁇ g/ml in borate-buffered saline (BBS, pH 8.2) overnight at 4°C and then blocked with PBS containing 1% bovine serum albumin (BSA) and 0.1% sodium azide. The test sera were added in three-fold serial dilutions and incubated at 37 °C for 1 h. Plates were washed extensively with PBS between each step.
  • BBS borate-buffered saline
  • Alkaline phosphatase (AP) -conjugated goat anti-mouse IgG (Cappel, Durham, NC) was then added and incubated at 37 °C for 1 h.
  • rabbit anti-mouse IgG heavy chains ( ⁇ , ⁇ Za , ⁇ zb and ⁇ ) (Zymed, South San Francisco, CA) and AP-conjugated goat anti-rabbit IgG were used as primary and secondary detecting Ab, respectively, the p-nitrophenyl phosphate substrate dissolved in diethanolamine buffer (pH 9.8) was added to each well and absorption was read at 405 nm using an automatic microplate reader (Molecular Devices Corp. , Menlo Park, CA) .
  • Lymphokine production Lymphocytes from draining lymph nodes (LNL) were harvested two weeks after f.p. immunization with OVA in CFA.
  • LNL (2 x 10 6 ) were cultured with 2 x 10 6 of irradiated (2,000 rad) syngeneic splenic APC with or without 200 ⁇ g/ml OVA in 1 ml culture medium consisting of RPMI 1640, 1 mM L-glutamine, 1 mM sodium pyruvate, 50 ⁇ M 2-mercaptoethanol plus antibiotics.
  • LNL were incubated with 5 ⁇ g/ml concanavalin A (Con A) in the absence of APC.
  • supematants were harvested, frozen and thawed, then added to the 96-well plates containing 5 x 10 3 per well of IL-2-dependent HT-2 cells (35) .
  • proliferation of HT-2 cells was determined by a calorimetric assay using tetrazolium salt XTT (Diagnostics Chemicals, Ltd., Oxford, CT) and phenazine methosulfate as described elsewhere (36) .
  • Absorbance was read at 450 nm. All assays were performed i triplicate and reported as the mean + standard deviation (SD) .
  • 3 x 10 6 /ml of spleen cells from mice primed with OVA in CFA two weeks earlier were incubated wit medium alone, 200 ⁇ g/ml soluble OVA or 500 ⁇ g/ml soluble OVA(mPEG) conjugate at 37°C for 24 h. Supematants were then tested for lymphokine production by lymphokine ELISA.
  • Lymphokine ELISA Supematants of primed LNL culture with or without OVA were tested for lymphokine pattern by sandwich ELISA (13) . Paired mAb (PharMingen, San Diego, CA) were used as capture and detecting Ab, respectively. Microliter plates were coated with capture Ab in BBS overnight at 4°C. After blocking excess protein-binding sites by PBS plus 1% BSA at 22 °C for 1 h, serially diluted recombinant cytokines (IL-2, IL-4 or IFN ⁇ ) and supematant were added and incubated overnight at 4°C. Biotin- conjugated detecting Ab and avidin-horseradish peroxidase conjugate (Vector Labs, Burlingame, CA) were subsequently added and incubated at 22 °C for 45 min and 30 min, respectively .
  • Mononuclear spleen cells 35 x 10 6 were stimulated in vitro for 6 days with 3 x 10 5 of irradiated (20,000 rad) syngeneic E.G7-OVA cells according to the method described by Moore et al. (6).
  • E.G7-0VA is an la " EL-4 (H-2 b ) thymoma clone transfected with the chicken OVA cDNA gene (6) (provided by Dr. M. J.Bevan, University of Washington, Seattle, WA) .
  • the E.G7-OVA clone provides a model for gene therapy applications of the invention. Cytolytic activity of cultured spleen cells was determined in a standard 4-h 51 Cr-release assay (13). Percent of specific lysis was calculated as: ( 51 Cr release by effector cells - spontaneous 51 Cr release) / (maximal 51 Cr release - spontaneous 51 Cr release) . Maximal 51 Cr release was achieved by adding 1% Triton X-100 to the target cells. Spontaneous 5 Cr release in the absence of effector cells was generally ⁇ 10% of the maximal release in all experiments. All assays were performed in triplicate and reported as the mean + SD.
  • This example shows that mPEG conjugates inhibit specific cytotoxic T cells (CTL) .
  • CTL cytotoxic T cells
  • the above model system for gene therapy utilized tumor cells transfected with the cDNA for the ovalbumin gene (E.G7-0VA) .
  • OVA-specific CTL were engendered by priming mice with OVA in Freund' ⁇ complete adjuvant.
  • the resulting CTL lysed tumor cells that had been transfected with the OVA gene, but not cells transfected with the insulin gene. It was advantageously found that pretreatment of mice with OVA(mPEG) 10 inhibited the priming of CTL by OVA in CFA, as well as OVA-specific Ab responses, whereas unmodified OVA had no effect.
  • the gene therapy model showed suppression of the immune response to OVA protein expressed from the chicken OVA cDNA gene.
  • Transfer of spleen cells from tolerized donor mice donor mice were injected i.p. with PBS, 2.5 mg OVA(mPEG) or 1 mg native OVA and sacrificed 7 days later. Spleens were harvested and 1 x 10 8 of spleen cells were transferred into syngeneic naive mice by i.v. injection (5) .
  • recipients were primed f.p. with 200 ⁇ g OVA in CFA.
  • Spleen cells from recipients were harvested two weeks later and stimulated in vitro with irradiated E.G7-0VA cells as described above.
  • OVA(mPEG) conjugates To determine whether OVA(mPEG) conjugates also inhibited IgG responses in mice immunized with OVA in CFA, sera from pretreated and subsequent immunized mice were tested for OVA-specific IgG production by ELISA. As illustrated in the upper panel of Fig. 1, OVA(mPEG) conjugate profoundly inhibited IgG responses to OVA, whereas native OVA given i.p. did not suppress IgG responses. This was consistent with our earlier observation that Ab responses were inhibited by i.v. but not by i.p. injections of soluble Ag prior immunization with the same Ag in adjuvant (37).
  • OVA(mPEG) conjugates inhibited all IgG isotypes stimulated by OVA in CFA or preferentially, suppressed some, while augmenting other IgG isotypes.
  • all IgG subclasses including predominant IgGl and IgG2b isotypes were inhibited by OVA(mPEG) treatment.
  • OVA(mPEG) conjugate induced tolerance of humoral responses in an antigen-specific and isotype- unspecific manner.
  • OVA (mPEG) conjugate Suppression of lymphokine production by OVA (mPEG) conjugate but not by native OVA .
  • the tolerogenic effect of OVA(mPEG) conjugate on OVA-specific cell-mediated responses was determined next.
  • Mice were pretreated i.p. with OVA(mPEG) or OVA and then primed f.p. with OVA in CFA as described above.
  • OVA(mPEG) When cultured with syngeneic APC and soluble OVA, LNL from untreated control mice primed with OVA in CFA produced IL-2 that supported the growth of IL-2-dependent HT-2 cells (Fig. 3) .
  • Pretreatment with OVA(mPEG) conjugate but not native OVA profoundly suppressed IL-2 production by LNL (Fig. 3).
  • mice were injected i.p. with PBS, 2.5 mg OVA(mPEG) conjugate or 1 mg native OVA.
  • mice were immunized f.p. with 200 ⁇ g OVA in CFA.
  • mice were immunized f.p. with 200 ⁇ g OVA in CFA.
  • Mice were bled two weeks after f.p. immunization and individual sera were tested for OVA-specific IgE by PCA in rats.
  • IgE titer was expressed as the reciprocal of the highest serum dilution giving a blue area greater than 5 mm.
  • c % suppression was calculated as: 100 x (mean IgE titer of control group - mean IgE titer of test group) / (mean IgE titer of control group) .
  • Control mice primed with OVA in CFA developed OVA-specific T cells that lysed E.G7-0VA targets (Fig. 6A) .
  • Pretreatment with OVA(mPEG) conjugate (Fig. 6B) , but not with native OVA (Fig. 6C) , inhibited priming of OVA-specific CTL and prevented development of cytolytic activity in mice that were subsequently challenged with OVA in CFA.
  • Ag(mPEG) conjugates inhibited both arms of cell-mediated immune responses in vivo, that is both humoral and cell mediated responses.
  • Spleen cells from recipients were then stimulated in vitro with irradiated E.G7-OVA cells as described above.
  • transfer of spleen cells from PBS-treated control mice (Fig. 6B) or from OVA-treated mice (Fig. 6D) had no obvious suppressive or enhancing effect on the CTL responses in recipients, in relation to the CTL activity of primed mice that had not received any donor cells (Fig. 6A) .
  • transfer of spleen cells from mice that had been treated with OVA(mPEG) conjugate resulted in a profound suppression of OVA-specific CTL activity (Fig. 6C) .
  • These data suggested that inhibition of cytolytic responses in recipients was mediated by Ts cells rather than a result of diluting CTL precursors by transferred cells, and the magnitude of the suppression effect was correlated to the number of donor cells transferred to the recipients (data not shown) .
  • Ts cells derived from mice treated with OVA(mPEG) or H ⁇ gG(mPEG) conjugates were Thy-1 + , CD3 + , CD4 " , CD5 + , CD8 + and expressed the a ⁇ heterodimer of TCR (26,27).
  • Nonhybridized CD8 + Ts clones were phenotypically distinguishable from the CD8 + CTL by their expression of the carbohydrate epitope detected with the mAb 984D4.6.5 (17), which is expressed by activated Ts cells but not their precursors, CTL precursors, or activated CTL effectors (42) .
  • Ts cells differed from CTL in that they were not cytotoxic and produced Ag-specific TsF, which suppressed in vitro Ab formation (27).
  • CD8 + T cells with cytolytic activity did not account for the biological activity of CD8 + Ts cells induced by Ag(mPEG) conjugates.
  • CD8 + CTL and non-cytolytic, CD8 + Ts cells are currently not clear. They could both be the progeny of a CD8 + T cell precursor with the potential of developing into either subset as demonstrated in the paradigm for development of Thl and Th2 cells from CD4 + Th cell precursors (43) .
  • This idea is supported by the observation that subsets of CD8 + T cells expressing different patterns of lymphokines can be derived from CD8 + T cells obtained from TCR transgenic mice (44) .
  • CD8 + CTL and CD8 + Ts cells could be derived from separate precursors having different functions. Regardless of which model proves to be correct, it is clear that once activated, CD8 + Ts cells inhibit both humoral and cell-mediated immune responses. The mechanisms by which such non cytolytic CD8 + Ts cells downregulate immune responses are not entirely clear. However, the soluble TsF secreted by these CD8 + Ts cells might be a plausible explanation for the observed tolerance (26) .
  • PBS phosphate buffered saline
  • the above example confirms the ability to induce tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses by administering an effective amount of an Ag conjugated with monomethoxypolyethylene glycol (mPEG) conjugate.
  • Ag antigen conjugated with monomethoxypolyethylene glycol
  • the ability to suppress the humoral and cell mediated immune responses has several important applications, particularly in the areas of treating allergies, autoimmune diseases, preventing immune rejection in organ transplants and preventing rejection of genetically engineered cells in gene therapy.
  • Foreign proteins or DNA such as genetic material or vectors for gene therapy, or their derivatives, have therapeutic properties and are administered to patients suffering from certain diseases.
  • the immunogenicity of the said foreign proteins, nucleotides, DNA or vectors, or of their derivatives may vitiate the treatment and hence this invention provides an improved method for the treatment of such diseases.
  • Gene therapy is the insertion of a functioning gene into the cells of a patient (i) to correct an inborn error of metabolism (i.e., genetic abnormality or birth defect resulting in the deficiency of the patient with respect to one or more essential proteins such as enzymes or hormones) , or (ii) to provide a new function in a cell Culver, K.W. , "Gene Therapy", 1994, p. xii, Mary Ann Liebert, Inc., Publishers, New York, NY).
  • the new protein expressed by this gene when the latter is inserted into the appropriate cell of an adult host— would induce in the host an immune response against itself.
  • the host would produce antibodies or cytotoxic cells to the "new" protein, and (ii) this immune response would not only combine and neutralize and thus inactivate the function of the "new" protein, but may also lead to untoward therapeutic complications due to formation of immune complexes.
  • This invention provides a method for overcoming this inherent complication due to the immunogenic capacity of the expressed protein, and is therefore considered to represent a novel and an essential improvement for the treatment of such diseases.
  • Bitoh, S., Lang, G.M. , Kierek-Jaszczuk, D., Fujimoto, S. and Sehon, A.H. disclose "Specific immunosuppression of human anti-murine antibody (MAMA) responses in hu-PBL-SCID mice.”
  • MAMA human anti-murine antibody
  • Bitoh, S., Lang, G.M. and Sehon, A.H. disclose the Suppression of human anti-mouse idiotypic antibody responses in hu-PBL-SCID mice.”
  • allergen refers to a substance capable of eliciting an immune response and ordinarily this is also the substance used for detection of the corresponding antibodies by one of the many in vitro and in vivo immunological procedures available for the demonstration of antigen-antibody interactions.
  • allergen is used to denote an antigen having the capacity to induce and combine with reaginic (i.e., IgE) antibodies which are responsible for common allergies. The latter definition does not exclude the possibility that allergens may also induce reaginic antibodies, which may include immunoglobulins of classes other than IgE.
  • antigenicity is defined as the ability of an antigen (immunogenic material) or allergen to combine in vivo and in vitro with the corresponding antibodies
  • allergenicity or skin activity is defined as the ability of an allergen to combine in vivo with homologous reaginic antibodies hereby triggering systemic anaphylaxis or local skin reactions, the latter reactions being the result of direct skin tests or of passive cutaneous anaphylactic (PCA) reactions
  • immunogenicity in a general sense is the capacity of an antigen or allergen, or of their derivatives produced in vitro or processed in vivo, to induce the corresponding specific antibody response.
  • tolerogens are defined as immunosuppressive covalent conjugates consisting of an antigenic material (immunogenic proteins, such as the expressed protein products of gene therapy vectors, etc.) and a water-soluble polymer (see e.g. Sehon, A.H. , In “Progress in Allergy” (K. Ishizaka, ea.) Vol. 32 (1982) pp. 161-202, Karger, Basel; and U.S. patent No. 4,261,973).
  • an antigenic material immunosuppressive covalent conjugates consisting of an antigenic material (immunogenic proteins, such as the expressed protein products of gene therapy vectors, etc.) and a water-soluble polymer (see e.g. Sehon, A.H. , In “Progress in Allergy” (K. Ishizaka, ea.) Vol. 32 (1982) pp. 161-202, Karger, Basel; and U.S. patent No. 4,261,973).
  • the term "tolerogen” refers to a conjugate consisting of an immunogenic material (protein or polynucleotide) and a nonimmunogenic conjugate, said tolerogen being immunosuppressive in an immunologically specific manner with respect to the antigen which is incorporated into the tolerogenic conjugate irrespective of the immunoglobulin class which is downregulated.
  • the tolerogen may comprise a conjugate of an essentially nonimmunogenic polymer and an immunogenic biologically active product or derivative of the genetic material used for gene therapy.
  • the therapeutic administration of foreign immunogenic material induces an immune response leading to the formation of antibodies of different immunoglobulin classes.
  • the material may form complexes in vivo with such antibodies leading to a poor therapeutic effect by virtue of its being sequestered and neutralized by the antibodies, or to anaphylactic reactions by combination with reaginic antibodies, or to other untoward conditions, i.e. immune complex diseases due to the deposition of antibody-antigen complexes in vital tissues and organs.
  • Wilkinson et al. "Tolerogenic polyethylene glycol derivatives of xenogenic monoclonal immunoglobulins, Immunology Letters, Vol. 15 (1987) pp. 17-22, disclose the administration time of a tolerogenic conjugate to a non-sensitized individual at least one day prior to challenge with an antigen, and optionally about 6 or 7 days.
  • the present invention overcomes deficiencies of the prior art by providing methods of inhibiting humoral and cell mediated immune responses.
  • the invention makes possible the administration of gene therapy, which involves the generation of immunogenic material in a patient deficient of the corresponding gene, possible and effective.
  • Gene therapy procedures as currently practiced involve the administration by itself of a foreign genetic material, or of its biologically active products, and do have certain disadvantages and limitations which are primarily due to their potential immunogenicity in the host deficient of the corresponding gene.
  • This aspect of the present invention aims at overcoming the above mentioned complications by suppressing the production of antibodies to the foreign therapeutic genetic material and of its expression products, and of thus ensuring the efficacy of gene therapy by the prior administration of imunosuppre ⁇ sive doses of tolerogenic conjugates consisting of therapeutically active and potentially immunogenic materials coupled to nonimmunogenic polymers, thus overcoming or minimizing the risk of inducing anaphylactic reactions or immune complex diseases.
  • the invention aims at suppressing substantially an immune response to the protein resulting as a consequence of successful gene therapy, which response would undermine the therapeutic efficacy of a biologically active genetic material and which may also cause untoward physiological reactions (e.g. anaphylaxis and/or immune complex diseases) .
  • the invention provides a method for conducting gene therapy comprising administration to a mammal of an immunosuppressing effective amount of a tolerogenic conjugate comprising the genetic material and/or its expression product (i.e., the protein of which the patient is deficient) and monomethoxypolyethylene glycol having a molecular weight of about 2,500-10,000 daltons, the above administration being at least one day prior to administration of the therapeutic genetic material for gene therapy, wherein said method results in the specific suppression of the immune response and the active development of specific tolerance to said therapeutic genetic material and/or its expression product (s) .
  • a tolerogenic conjugate comprising the genetic material and/or its expression product (i.e., the protein of which the patient is deficient) and monomethoxypolyethylene glycol having a molecular weight of about 2,500-10,000 daltons, the above administration being at least one day prior to administration of the therapeutic genetic material for gene therapy, wherein said method results in the specific suppression of the immune response and the active development of specific tolerance to said therapeutic genetic material
  • the therapeutic genetic material is selected from nucleotides, DNA, RNA, mRNA, which may or may not be attached to or delivered by appropriate vectors for expression of the required therapeutic protein.
  • gene therapy gene delivery vectors may include Moloney murine leukemia virus vectors, adenovirus vectors with tissue specific promotors, herpes simplex vectors, vaccinia vectors, artificial chromosomes, receptor mediated gene delivery vectors, and mixtures of the above vectors.
  • an mPEG conjugate corresponding to a gene therapy protein is administered prior to the administration of the gene therapy vector encoding a gene for a therapeutic protein.
  • an mPEG conjugate corresponding to a vector protein is administered prior to the administration of the gene therapy vector.
  • mPEG conjugates of both the vector protein and gene therapy protein are administered prior to conducting gene therapy with a gene therapy vector encoding a gene for a therapeutic protein.
  • the the vector protein and gene therapy protein mPEG may be conjugated together as a hybrid and administered prior to conducting gene therapy with a gene therapy vector encoding a gene for a therapeutic protein.
  • the objectives of the present discovery are accomplished by a method, wherein an immunosuppressively effective amount of a tolerogen incorporating a foreign genetic material or its active derivative (s) is administered to the mammal prior to the administration of the foreign genetic material or its biologically active derivative (s) .
  • the tolerogenic conjugate is preferably administered to individuals who have not received a prior treatment with the foreign genetic material or its product, i.e. to unsensitized individuals.
  • the invention provides improved methods for gene therapy of different human diseases which can be ameliorated or eliminated by the administration of the appropriate genetic materials, etc. or their therapeutic derivatives, of which the patient is deficient.
  • the tolerogenic conjugates may be synthesized by covalent or noncovalent attachment of nonimmunogenic polymers to natural or synthetic biologically active proteins such as for example (i) murine or rat monoclonal antibodies to human T-cells which have been used to suppress transplant rejection (Colvin, R.B. et al.; Fed. Proc. 41 (1982) p. 363, Abstr. 554) or as "miracle bullets" for the destruction of tumors (Froese, G. et al.; Immunology 45 (1982) p. 303-12, and Immunological Reviews 62 (1982), Ed. G.
  • the tolerogen is a covalent conjugate between monomethoxypolyethylene glycol (mPEG) with molecular weight in the range of 2,500-10,000 daltons and a foreign protein such as ovalbumin (OVA or OA) , which served as a model protein.
  • mPEG monomethoxypolyethylene glycol
  • OVA ovalbumin
  • OA ovalbumin
  • tolerogens of appropriate composition substantially suppress the formation of antibodies of different classes (e.g. IgE and IgG) which are directed specifically against the genetic material per se and/or against its expression product (s).
  • IgE and IgG antibodies of different classes
  • mice The acceptability of the mouse as an experimental model for correlation to human utility in the present experiments is evidenced by Dreborg et al. "Immunotherapy with Monomethoxypolyethylene Glycol Modified Allergens", page 325, which indicates that similar results were achieved in humans and mice and thus confirms mice are an acceptable experimental model for evaluation of mPEG modified allergens. See also Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988, p. 93, which indicates that laboratory mice are an acceptable experimental animal model for examining the immune response, and that mice, in particular, possess appropriate characteristics for studies of the genetics of the immune response.
  • polyethylene glycol ⁇ having molecular weights in the range of 2,000 to 35,000, preferably 4,000 to 20,000, have proved to be effective.
  • Polyethylene glycols in this context also include physiologically acceptable derivatives thereof, such as mono-alky1 ethers, preferably the monomethyl ether, whereby the remaining single terminal hydroxyl groups of the molecules are conveniently used for coupling to the protein.
  • other water-soluble polymers may be used, ⁇ uch a ⁇ polyvinylalcohol ⁇ , polyvinyl pyrrolidones, polyacrylamides and homo- as well as hetero-polymers of amino acids, polysaccharide ⁇ (e.g. pullulan, inulin, dextran and carboxymethyl cellulose) or physiologically acceptable derivatives of these polymers.
  • the coupling reaction i ⁇ made between active groups in the antigen molecules and in the polymer molecule ⁇ . If neces ⁇ ary such groups may have to be introduced into said molecules before the coupling reaction.
  • active groups are for example -NH2 , -NCS, -SH, -OH, -CH 2 I and -COOH and they may be introduced according to well-known method ⁇ , if not already pre ⁇ ent in the molecules u ⁇ ed for the production of tolerogenic conjugate ⁇ .
  • the covalent link between the water-soluble polymer and protein or its active derivative ( ⁇ ) ⁇ hould be as stable as pos ⁇ ible under physiological conditions.
  • the coupling of the polymer onto the antigenic or genetic material mu ⁇ t have been carried out to such an extent that the conjugate i ⁇ rendered tolerogenic, as well as sub ⁇ tantially non allergenic and ⁇ ubstantially non-immunogenic.
  • the tolerogens mu ⁇ t retain a certain number of epitopes of the unmodified antigen, as long as their immunogenicity ha ⁇ been decreased so that they do not induce the formation of antibodies which may cause unacceptable adverse reactions.
  • the degree of substitution (also referred to as the degree of conjugation, which is defined a ⁇ the number of polymer molecules coupled per antigen molecule) , varies from one antigen molecule to another depending on the nature and size of the antigen and on the polymer and it ⁇ molecular eight.
  • One of skill in the relevant art will be able to optimize the degree of sub ⁇ titution u ⁇ ing the di ⁇ clo ⁇ ure example ⁇ .
  • the tolerogenic conjugates be administered in such a manner a ⁇ to lead to the downregulation of the immune response with respect to one or more cla ⁇ ses of immunoglobulins directed against the unconjugated biologically active product of the genetic material.
  • the tolerogenic conjugate ⁇ are to be injected in absence of adjuvants since the adjuvants may counteract their suppre ⁇ ogenic effects.
  • the inclu ⁇ ion of adjuvants along with the unconjugated immunogenic material in the examples given below was justified ⁇ o a ⁇ to stimulate in experimental animals the enhanced production of antibodies in a relatively short time and to thus test under more stringent conditions the capacity of the tolerogenic conjugate ⁇ to suppress the immune respon ⁇ e in the ⁇ e animals even under these extreme conditions which are particularly favorable for enhancing the immune response.
  • proteins and polypeptides are used synonymou ⁇ ly, herein.
  • foreign genetic material refer ⁇ to a nucleotide, DNA, RNA, mRNA, pla ⁇ mid, which are used as carriers of the gene and/or the gene it ⁇ elf re ⁇ pon ⁇ ible for the expre ⁇ ion of the appropriate protein or protein derivative (fragment ⁇ included) , which are ⁇ ubstantially immunogenic in the animal to be treated.
  • the genetic material ⁇ hould be therapeutically effective.
  • Many ⁇ uch proteins, vectors, DNA are known per se (Culver, K.W. , "Gene Therapy", 1994, p. xii, Mary Ann Liebert, Inc., Publishers, New York, NY, incorporated herein by reference in its entirety) .
  • vectors may be selected from the group con ⁇ isting of Moloney murine leukemia virus vectors, adenovirus vectors with tis ⁇ ue ⁇ pecific promotor ⁇ , herpes ⁇ implex vectors, vaccinia vectors, artificial chromosomes, receptor mediated gene delivery, and mixtures of the above vector ⁇ .
  • Gene therapy vectors are commercially available from different laboratories such as Chiron, Inc. , Emeryville, California; Genetic Therapy, Inc., Gaithersburg, Maryland; Genzyme, Cambridge, Massachu ⁇ etts; Somatx, Almeda, California; Targeted Genetics, Seattle, Washington; viagene and Vical, San Diego, California.
  • the effective dose ⁇ (amounts) and formulations commonly used in gene therapy are also known and may be applied to the present invention, although the invention may alternatively employ reduced or increased dose ⁇ .
  • both the biologically active foreign genetic material or its derivatives, a ⁇ well as the corresponding tolerogenic conjugates may be administered parenterally in a soluble form in isotonic solution and after removal of aggregate ⁇ by centrifugation.
  • the protocol followed according to the invention compri ⁇ es the administration initially of an immunosuppre ⁇ ive effective do ⁇ e (amount) of tolerogen, which i ⁇ given prior to the admini ⁇ tration of the gene which encode ⁇ a therapeutically active protein or it ⁇ product. If nece ⁇ ary, thi ⁇ do ⁇ e may be portioned and given on repeated occasions.
  • the immunosuppressive dose which is given may vary from tolerogen to tolerogen, but it has to be administered prior to the entry of the protein into the ho ⁇ t's sy ⁇ tem.
  • the practitioner ⁇ killed in the art can determine the variables such as dose of tolerogen and the minimum interval of time between its administration and the appearance of the immunogenic protein in the ho ⁇ t' ⁇ ⁇ y ⁇ tem. See, for example, references di ⁇ cu ⁇ sed in background of the invention.
  • gene therapy resulting in the production of a "new protein in the protein-deficient patient, should be preceded by administration of the specific tolerogenic conjugate, i.e., the conjugate comprising the same protein and capable of suppre ⁇ sing selectively the immune re ⁇ pon ⁇ e of the ho ⁇ t with re ⁇ pect to the protein in que ⁇ tion.
  • the tolerogenic conjugate is administered prior to the admini ⁇ tration of the gene which expre ⁇ e ⁇ the foreign protein.
  • a time period of at lea ⁇ t one day prior to the admini ⁇ tration of the foreign genetic material is preferred.
  • the tolerogenic conjugate is administered at least about ⁇ ix day ⁇ prior to admini ⁇ tration of the foreign genetic material.
  • the immunosuppressive dose refers to the amount of tolerogen required to ⁇ ubstantially reduce the immune respon ⁇ e of the patient to the protein or to it ⁇ derivative ( ⁇ ) which will be produced a ⁇ a re ⁇ ult of the gene therapy.
  • further doses of the tolerogen may be given in conjunction with the protein or its derivative ( ⁇ ) , i.e. after the primary admini ⁇ tration of the tolerogen.
  • Thi ⁇ mode may repre ⁇ ent one way of ⁇ u ⁇ taining the suppre ⁇ ion of the humoral and cellular immune re ⁇ pon ⁇ e ⁇ and offer ⁇ a more efficient therapeutic regimen for the di ⁇ ea ⁇ e condition for which the treatment ha ⁇ been designed.
  • the invention will now be illu ⁇ trated by some non- limiting, representative examples wherein OVA and its tolerogenic mPEG derivatives have been applied as model sub ⁇ tances to confirm the usefulness of the proposed immunosuppressive treatment of a well-established animal model commonly utilized in the field of immunology.
  • the conjugates are de ⁇ ignated as OVA-(mPEG) where n represents the average degree of conjugation.
  • Figures 8, 9 and 10 show diagrams illustrating the efficiency of the invention. The percentages in brackets of Figs. 1 and 3 represent the degree of ⁇ uppression with re ⁇ pect to the minimal immune response in animals receiving phosphate buffered ⁇ aline (PBS) in lieu of the conjugate ⁇ .
  • PBS phosphate buffered ⁇ aline
  • the mixture was then filtered under dry nitrogen and the filtrate mixed with anhydrous petroleum ether (ca 50 ml, b.pt. 30-60°C) in order to precipitate the polymer.
  • the polymer was separated by filtration under nitrogen, dis ⁇ olved in benzene (20 ml) and reprecipitated with petroleum ether. Thi ⁇ proce ⁇ was repeated seven times to insure that the polymer was free of any re ⁇ idual cyanuric chloride.
  • the active intermediate wa ⁇ finally dissolved in benzene, the solution frozen and the benzene sublimed away under high vacuum to leave a fine white powder.
  • Elemental analysi ⁇ of the intermediate confirmed that it contained 2 chlorine atoms.
  • the intermediate, corre ⁇ ponding to C 2563 H 3077 0 127 N 3 Cl 2 with an average molecular weight of 5,738 dalton ⁇ would have a theoretical compo ⁇ ition in percentage ⁇ of C, 53.65; H, 8.92; N, 0.73; Cl, 1.24; which agree ⁇ with its determined composition of C, 53.51; H, 8.89; N, 0.77; Cl, 1.08.
  • the chloride content of the intermediate was also determined by hydrolysi ⁇ and titration of the chloride relea ⁇ ed with ⁇ ilver nitrate. Thu ⁇ , the activated intermediate (120 mg) was dis ⁇ olved in water (10 ml) and the pH adju ⁇ ted to 10 with dilute sodium hydroxide. After heating at 90°C for two hours, the solution was cooled and the chloride titrated with silver nitrate (0.001N), using a chloride ion selective electrode to indicate the endpoint. The chloride content of the activated intermediate was found to be 2.1, consistent with the structure shown above.
  • the polymer-protein mixture was stirred for one half hour at 4°C and then one half hour at room temperature.
  • the reaction mixture was desalted by either dialyzing for four day ⁇ against running di ⁇ tilled water or by pa ⁇ ing through a column of Sephadex® G-25 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) .
  • Tolerogen ⁇ of ⁇ aporin, ricin a chain, birch pollen allergen Bet v 1 and recombinant ragweed allergen R8.1 have al ⁇ o been prepared by the above method, with an induction of tolerance to these antigens.
  • n is calculated by the f ormula
  • OVA mPEG 106 and OVA-mPEG. 1 9 inhibited the anti-OVA IgE response, respectively, only to the extent of 94% and 50%.
  • the weights of the conjugate ⁇ given corre ⁇ pond to their protein content are the weights of the conjugate ⁇ given corre ⁇ pond to their protein content.
  • Each OVA-mPEG conjugate was injected into groups of 4 mice each at the four dose ⁇ of 10 ⁇ , 50 ⁇ q , 150 ⁇ q and 600 ⁇ .
  • the control group of mice received PBS as placebo.
  • OVA-mPEG 10 The ⁇ uppressive effect of OVA-mPEG 10 on IgM, IgG, and IgE plaque forming cells (PFC) in spleen and lymph nodes.
  • One mg of OVA-mPEG 10 (containing 10 mPEG groups with an average mol wt of 10,000 dalton ⁇ , which were coupled per OVA molecule by the ⁇ uccinic anhydride method (Wie, S.I. et al., Int. Archs. Allergy appl. Immun. 64, 84 1981)) or PBS was administered intraperitoneally to each group of four (B6D2)F1 mice each one day prior to immunization with 1 ⁇ q of DNP 3 -OVA in 1 mg A1(0H) 3 .
  • Each tissue sampling represents a pool from 4 mice
  • pan-specific suppression of the "human" antibody respon ⁇ es against murine monoclonal antibodies (i.e., HAMA respon ⁇ e ⁇ ) of the IgG cla ⁇ s could be achieved with 8 tolerogenic mPEG preparations, each con ⁇ i ⁇ ting of one of the 4 gamma chain ⁇ and of one of the two type ⁇ of light chain ⁇ of murine IgG (Bitoh, S., Lang, G.M. , Kierek-Ja ⁇ zczuk, D., Fujimoto, S. and Sehon, A.H. Specific immuno ⁇ uppres ⁇ ion of human anti-murine antibody (MAMA) responses in hu-PBL-SCID mice. Hum. Antibod. Hybridomas 4_:144-151, 1993).
  • MAMA human anti-murine antibody
  • the expres ⁇ ed protein material of the cystic fibrosis transmembrane conductance regulatory gene (CFTR) (Genzyme, Cambridge, Mas ⁇ achu ⁇ etts) for the treatment of cystic fibrosis is dissolved in sodium tetraborate buffer (4 ml, 0.1 M, pH 9.2) and the activated mPEG added to the olution at 4°C.
  • CFTR cystic fibrosis transmembrane conductance regulatory gene
  • the amount of activated mPEG is varied to prepare conjugates of differing degrees of polymer sub ⁇ titution. Different mole ratios (mPEG/gene product) are used to prepare specific tolerogenic conjugate ⁇ a ⁇ described earlier.
  • the polymer-gene product mixture is stirred for one half hour at 4°C and then one half hour at room temperature.
  • the reaction mixture is desalted by either dialyzing for four days again ⁇ t running distilled water or by pas ⁇ ing through a column of Sephadex® G-25 (Pharmacia Fine Chemical ⁇ AB, Upp ⁇ ala, Sweden) .
  • phosphate buffer 0.008 M, pH 7.7.
  • the salt free mPEG conjugate ⁇ of the cy ⁇ tic fibro ⁇ i ⁇ gene product are applied in water and the free (unbound) mPEG washed through the column with the pH 7.7 buffer.
  • Free mPEG is detected on thin layer chromatography ⁇ Camag Kieselgel DSF-5, Terochem Lab Ltd, Alberta) eluant 3:1 chloroform/methanol ⁇ using iodine vapor for development.
  • Conjugate ⁇ of the CFTR gene are administered to a patient at least one day prior to transfer of the cystic fibrosis transmembrane conductance regulator gene to lung tissue using recombinant adenoviral vectors or liposomes.
  • the amount of activated mPEG is varied to prepare conjugates of differing degrees of polymer substitution.
  • mPEG/gene product Different mole ratios (mPEG/gene product) is used to prepare specific tolerogenic conjugates as described earlier.
  • the polymer-gene product mixture is stirred for one half hour at 4°C and then one half hour at room temperature.
  • the reaction mixture is desalted by either dialyzing for four day ⁇ against running distilled water or by passing through a column of Sephadex® G-25 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) .
  • the salt free mPEG conjugates of the LDLr-gene products are applied in water and the free (unbound) mPEG washed through the column with the pH 7.7 buffer. Free mPEG is detected on thin layer chromatography ⁇ Camag (Kieselgel DSF-5, Terochem Lab Ltd, Alberta) eluant 3:1 chloroform/methanol ⁇ using iodine vapor for development.
  • Conjugates of the LDLr gene product are administered to a patient.
  • Hepatocytes are grown in the laboratory and genetically altered with a murine retroviral vector containing LDLr gene.
  • the cell ⁇ are reinf sed through the hepatic artery to the liver of the patient at lea ⁇ t one day after admini ⁇ tration of the conjugate.
  • the present invention can readily be adapted to any gene therapy protocol and is generally applicable to the administration of any therapeutic immunogenic material and not just the specific examples listed above.
  • Gene therapy may be applied to ⁇ omatic cell ⁇ or germ line cells by methods known ⁇ uch as gold electroporation, microinjection or jet injection, or other methods a ⁇ ⁇ et forth in Sambrook et al. "Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Pre ⁇ (1989)” incorporated herein by reference in it ⁇ entirety.
  • the invention provide ⁇ for a method for treating by gene therapy a mammal with a therapeutic amount of a biologically active antigenic material or it ⁇ expre ⁇ ion product.
  • This method comprises conducting gene therapy by admini ⁇ tering to a mammal an immuno ⁇ uppre ⁇ ive effective amount of a tolerogenic conjugate consisting of a protein coupled to monomethoxypolyethylene glycol (mPEG) having a molecular weight of about 2,000-10,000 dalton ⁇ , wherein administration of said tolerogenic conjugate is at lea ⁇ t one day prior to admini ⁇ tration of a gene therapy vector encoding a gene for a protein, wherein ⁇ aid protein i ⁇ identical to said protein which is coupled to mPEG, and wherein said method result ⁇ in the ⁇ uppression of an immune response and in the development of tolerance to the protein expressed by said gene encoded by said gene therapy vector.
  • mPEG monomethoxypolyethylene glycol
  • the method of conducting gene therapy includes a) administering to a mammal an immunosuppre ⁇ ive effective amount of a tolerogenic conjugate con ⁇ i ⁇ ting of a protein conjugated to monomethoxypolyethylene glycol (mPEG) having a molecular weight of about 2,000 to 10,000 dalton ⁇ , wherein admini ⁇ tration of ⁇ aid tolerogenic conjugate is at least one day prior to admini ⁇ tration of DNA, RNA or mRNA and encoding a protein administered for gene therapy, wherein the encoded protein i ⁇ identical to ⁇ aid protein which is conjugated to mPEG, and wherein said method re ⁇ ult ⁇ in the suppre ⁇ ion of an immune re ⁇ pon ⁇ e and in the development of tolerance to encoded protein of said DNA, RNA or mRNA admini ⁇ tered for gene therapy.
  • the method ⁇ uppre ⁇ ses the formation of about 98% of antibodies against said antigenic genetic material or its product.
  • the method of ⁇ uppre ⁇ ion of the humoral and cellular immune responses of the invention finds application in ⁇ pecific area of gene and protein replacement therapy.
  • patient ⁇ with Hemophilia A are from birth deficient of the gene which serves as the template for the production of clotting Factor VIII (F.VIII) .
  • the current replacement therapy involves the frequent admini ⁇ tration of F.VIII, which i ⁇ i ⁇ olated from blood of normal volunteers or synthesized by recombinant technology. Either of these therapies is extremely expensive and, most importantly, the delay between the onset of bleeding and administration of the factor may lead to tis ⁇ ue damage.
  • transgenic protein as well as the vector protein ( ⁇ ) , in addition to being excreted as such by the tran ⁇ fected cell ⁇ , may al ⁇ o be degraded with the cell into peptide ⁇ incorporating ⁇ ome of the epitopes of the protein( ⁇ ).
  • These foreign peptide ⁇ when pre ⁇ ented in association with MHC molecules on the cell membrane, are believed to elicit the production of cytotoxic cell ⁇ capable of de ⁇ troying the transfected cells.
  • Immune response can be blunted by a variety of immunosuppressive regimens, which have been reviewed in relation to treatment of hemophilia by Bertrop, et al in an article based on the discu ⁇ ions at a joint WHO and World Federation of Hemophilia meeting (2) .
  • Most of these therapies, as in the case in order transplantation, are nonspecific and involve the continual administration of immunosuppressive drugs which are expensive and may have deleterious side effects.
  • BRMs Biologic Response Modifiers
  • a major challenge for the biotechnology of potentially therapeutic recombinant BRMs is to overcome their immunogenicity, as is the case for natural foreign proteins (e.g., monoclonal antibodie ⁇ , enzyme ⁇ , toxin ⁇ , hormones, heterologous F.VIII) .
  • the therapeutic effectiveness of even the corresponding chimeric or humanized recombinant proteins is undermined by their immunogenicity which is often due to only minor conformational differences with respect to the three-dimensional structures of their corresponding natural progenitors.
  • BRMs some recombinant BRMs elect ⁇ the production of complementary, neutralizing and/or blocking antibodie ⁇ by the hose. These antibodies intercept the BRMs and prevent them from reaching their target cell ⁇ . Moreover, depending on the class of antibodies elicited, the patient may develop serum sickness, renal and hepatic toxicity, and even anaphylactic shock in severe cases.
  • the inventors have developed a method for conversion of a variety of immunogenic proteins or immunogenic fragments of the proteins (P) to tolerogenic derivatives. This conversion involve ⁇ the coupling of an optimal number (n) of molecules of monomethoxypolyethylene glycol (mPEG) onto the protein antigen in question.
  • n an optimal number of molecules of monomethoxypolyethylene glycol
  • Step I Injection of tolerogenic conjugates of the appropriate immunogenic BRM, i.e. P(mPEG) n ;
  • Step II Administration of the unmodified P, about 7 days after injection of the immunosuppre ⁇ ive P(mPEG) n . Thereafter, unmodified biologically active P can be injected repeatedly over extended period ⁇ without further injection of P(mPEG) .
  • unmodified biologically active P can be injected repeatedly over extended period ⁇ without further injection of P(mPEG) .
  • mice ⁇ uppre ⁇ ion of antibody re ⁇ ponses to heat aggregated human monoclonal (myeloma) IgG, referred to as HaHIgG, was shown to persist up to 540 days in spite of multiple injections of HaHIgG at different interval ⁇ over thi ⁇ ⁇ pan of time.
  • the rea ⁇ on for the interval of about 7 day ⁇ betweens Steps I and II is to allow propagation of P-specific suppressor T (Ts) cells which are activated by P(mPEG) n (4). Additional injections of the unmodified P maintain the proliferation of these cell ⁇ , which suppre ⁇ the ⁇ pecific T helper cell ⁇ that al ⁇ o recognize the epitopes of the same P, though not neces ⁇ arily the ⁇ ame epitope ⁇ as those recognized by the Ts cells.
  • Ts P-specific suppressor T
  • mice to a given protein Ag A by treatment with Ag A (mPEG) n result ⁇ in their becoming i munologically unresponsive to an unrelated Ag B , on condition that Ag B is injected into these mice in the form of a covalent adduct with Ag A , i.e., as Ag A -Ag B , but not as a mixture with Ag A .
  • This cognate phenomenon of "linked immunological suppression" is also conferred on naive mice by treating them first with TsF A nd then with the Ag A -Ag B adduct.
  • any clone of T ⁇ cell ⁇ recognize only one epitope (i.e. one antigenic determinant) of the re ⁇ pective high molecular weight multi-determinant protein.
  • hu-PBL-SCID mou ⁇ e denotes a mouse with severe combined immunodeficiencies, which has been engrafted with human peripheral blood leucocytes. This sy ⁇ tem represents the closest in vivo model for a functional human lymphoid system.
  • the inventors demonstrated that (i) mPEG conjugate ⁇ of a foreign P, i.e., murine mAb, induced ⁇ pecific ⁇ uppre ⁇ ion of human anti-P antibodie ⁇ , (ii) this suppression wa ⁇ due to the generation of human P-specific CD8 + T cells, and (iii) the suppression was transferable with these human Ts cells into secondary naive recipient SCID mice that had been engrafted with the leucocytes of the original donor of the cells (9, 10).
  • pan- ⁇ pecific ⁇ uppression of the "human" antibody responses against murine monoclonal antibodies (i.e., HAMA responses, including anti-diotypic responses) of the IgG class could be achieved with only eight tolerogenic mPEG preparations, each consisting of one of the four gamma chains and one of the two types of light chains of murine IgG.
  • the two-step method described above i ⁇ demonstrated to be suitable for ⁇ uppre ⁇ ing the activation of cytotoxic T cells (CTL ⁇ ) , as is the case for gene therapy involving the transfection of a "new" gene into an immunocomponent patient who has been deficient from birth of the particular gene required to express the corre ⁇ ponding protein.
  • CTL ⁇ cytotoxic T cells
  • the gene therapy model ⁇ yste consisted of E.G7-OVA target cells, which had been generated by Dr. M.
  • Recombinant hF.VIII has a molecular weight of 330,000 and consist ⁇ of 2,332 amino acids, 160 of which are lysines (13) .
  • the fir ⁇ t conjugate will be ⁇ ynthe ⁇ ized using one of our proven methods for the preparation of tolerogenic derivatives of diverse protein antigens with molecular weights in the range of 51,000-150,000 Da, vis., recombinant human insulin (rhi) , ⁇ aporin, chain A of ricin, a major birch pollen allergen (Bet v 1) , a recombinant ragweed allergen (r ⁇ .l), ovalbumin (OVA), and human and mouse IgG.
  • mPEG-NPC mPEG p- nitrophenyl carbonate
  • mPEG-NPC is used in an 8-fold higher molar concentration than that of the lysines of a given protein.
  • 47 mg of OVA corresponding to lmM of OVA (which contains 19 lysines per molecule of OVA) is reacted with 486.4 mg (152 mM) of mPEG-NPC for the synthesis of tolerogenic OVA(mPEG) n conjugates containing an average of 10-11 molecules of mPEG per molecule of OVA; the subscript n, refers to the average degree of conjugation.
  • tolerogenic mPEG conjugates of mouse or human IgG 150 mg of IgG corresponding to 1 mM of IgG and containing on the average 90 lysine ⁇ per molecule of IgG, wa ⁇ reacted with 2.3 grams of mPEG-NPC.
  • the resulting tolerogenic IgG(mPEG) n conjugates contained on the average 25 to 35 mPEG molecules per molecule of IgG.
  • hF.VIII (mPEG) n conjugated
  • hF.VIII is dis ⁇ olved at 10 mg/ml in 0.1 M borate buffer, pH 9.7 and then mixed quickly with mPEG-NPC which i ⁇ dissolved in an identical volume of double distilled water (DDW) .
  • DDW double distilled water
  • the reaction mixture i ⁇ transferred to a dialysis bag, which should then be suspended in 4 liters of 0.05M borate buffer (pH 9.7) and the reaction is continued with con ⁇ tant ⁇ tirring for 1 hour at room temperature and overnight in the cold; the large volume of buffer out ⁇ ide the dialysis bag serve ⁇ a ⁇ a pH- stat. Finally, for the isolation of the conjugate, the content of the dialysi ⁇ bag is applied onto a Pharmacia BioPilot gel filtration column which has been equilibrated with DDW. The conjugate is in the void volume, followed by
  • the method of synthesis of the tolerogenic hF.VIII (mPEG) n conjugates in a pure form involves a series of step ⁇ , including isolation of the pure conjugate by gel filtration chromatography, which lead ⁇ to yield ⁇ of the order of 50-70%.
  • hF.VIII In view of the low immunogenicity of hF.VIII, to ensure a consi ⁇ tent immune re ⁇ pon ⁇ e in mice and rat ⁇ , hF.VIII i ⁇ admini ⁇ tered in an adjuvant. Five to ⁇ even week old female mice of both ⁇ train ⁇ receive two ip injection ⁇ of a ⁇ uspension of hF.VIII in Al(OH) 3 at an interval of 21 days. The strain of mice which mount the most consi ⁇ tent anti- hF.VIII antibody titer i ⁇ ⁇ elected for further experiment ⁇ with hF.VIII in Al(OH) 3 .
  • Freund' ⁇ adjuvant i ⁇ u ⁇ ed with the Sprague-Dawley rats because this adjuvant has been proven to induce high titered Ab responses to hF.VIII in this strain of rat ⁇ .
  • the rats are immunized sc with hF.VIII in Freund's adjuvant; the fir ⁇ t injection contain ⁇ complete Freund's adjuvant and the ⁇ econd injection after an interval of 3 weeks contains incomplete Freund's adjuvant.
  • the mice and rats are bled at weekly intervals and the antibody production is determined by ELISA.
  • hF.VIII (mPEG) n conjugates differing in "n" are be synthesized.
  • 4 groups of 4 mice each receive ip, respectively, 100, 200, 400 and 800 mg of hF.VIII (mPEG) n .
  • the mice are be given the first immunizing dose of hF.VIII in adjuvant 7 days later, and are re-immunized after 21 days.
  • control mice (4 mice per group) are injected ip with unmodified hF.VIII, or with diluent in lieu of conjugate. All mice are bled at weekly intervals after primary immunization for assaying their sera by ELISA.
  • rat ⁇ receives ip, respectively, 100, 200, 400 and 800 mg of hF.VIII (mPEG) .
  • Two control groups of 6 rat ⁇ each are injected ip, re ⁇ pectively, with unmodified hF.VIII or diluent in lieu of conjugate.
  • the rat ⁇ are re-immunized 21 days after the primary immunization. All rats are bled at weekly interval ⁇ after primary immunization for the determination of their anti-hF.VIII levels by ELISA. Im unosuppression of an ongoing immune response to hF.VIII by a protocol involving a combination of hF.VIII (mPEG) conjugate and ycophenolic acid or other immunosuppressive drug.
  • Rejection of tran ⁇ planted cells and organs is also an application for the mPEG strategy.
  • Rejection result ⁇ from the recognition of protein ⁇ encoded by the MHC, HLA in human ⁇ , by CD4 + and/or CD8 + T cell ⁇ .
  • Many antigens have been expres ⁇ ed in large amount ⁇ by recombinant technology.
  • Purified HLA antigen ⁇ can be modified with mPEG and the corre ⁇ ponding conjugates injected into patients prior to organ or cell transplantation.
  • the inventor ⁇ can modify peripheral blood cell ⁇ or membranes from these cells with mPEG. This is feasible since it has been ⁇ hown that erythrocyte ⁇ can be modified with mPEG.
  • MHC ⁇ pecific tolerance i ⁇ induced with mPEG modified antigen ⁇ hence ⁇ uppre ⁇ sion of the immune respon ⁇ e and of the rejection of tran ⁇ plant ⁇ by prior pretreatment by the appropriate MHC-mPEG conjugate i ⁇ po ⁇ ible with this treatment or in conjunction with reduced doses of immunosuppressive drugs that have various side effect, including inhibition of all immune responses.
  • the invention provides a method of preventing an immune rejection of organ transplant ⁇ compri ⁇ ing administering an effective amount of Ag(mPEG) conjugate to induce tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses.
  • the present invention targets mPEG to antigen-specific T cells, including CD8 + T cells.
  • the inventors have used published methods for stimulating CD8 + T cells with exogenous antigens by delivery in adjuvant to test this idea.
  • mPEG-OVA complexes were injected before priming mice with OVA in Freund's complete adjuvant. About two weeks later, mice were sacrificed and their spleen cells assayed for development of OVA specific cytolytic T cells (CTL) .
  • CTL OVA specific cytolytic T cells
  • the inventors provide approaches that are effective to induce tolerance and thus could be used as alternatives to treat autoimmunity. mPEG modification of such antigens render them tolerogenic and have the potential to reverse these autoimmune diseases.
  • This therapy can be extended to any autoimmune disease, in which a tissue specific antigen can be identified. It is believed that generations of a particular number of T ⁇ cell ⁇ may reverse the ⁇ e autoimmune di ⁇ ease ⁇ .
  • Thi ⁇ therapy may be extended to any autoimmune di ⁇ ea ⁇ e a ⁇ uming that the culprit ti ⁇ sue specific antigen is identified, isolated or synthe ⁇ ized for preparation of the corresponding mPEG conjugate.
  • T cells can transfer disease in normal syngeneic recipient ⁇ , a ⁇ i ⁇ the case within the same strain of mice or rats.
  • T cells In experimental multiple sclerosis, and the related model of experimental autoimmune encephalomyelitis (58) , T cells have been shown to recognize several antigens. Such T cells can transfer different disea ⁇ e into normal ⁇ yngeneic recipient ⁇ .
  • T cell ⁇ In multiple ⁇ clero ⁇ i ⁇ and in experimental autoimmune encephalomyeliti ⁇ , T cell ⁇ have been ⁇ hown to recognize several antigens that are restricted in expres ⁇ ion to the central nervou ⁇ system. These include: myelin basic protein, proteolipid protein and myelin oligodendrocyte antigen. T cells recognizing insulin, glutamic acid decarboxylase, and other, as yet unidentified antigens expressed by beta cells, have been isolated from non-obese diabetic (NOD) mice, BB rats, and diabetic humans. mPEG treated cells can be u ⁇ ed to induce tolerance to certain cell ⁇ in the body that are known to be the target ⁇ of autoimmunity . For example, beta cell ⁇ from the pancreas are modified with mPEG and injected into nonobese diabetic (NOD) mice before or after the appearance of spontaneous diabetes.
  • NOD non-obese diabetic
  • the tolerogenic properties of the conjugates of the invention have the capacity to inactivate (in vivo in PCA reactions) IgE-sensitized rat skin mast cell ⁇ , and B E cells. (24). Injection of OVA(mPEG) 10 into skin sites inhibited the release of mediators of anaphylaxis on sub ⁇ equent injection of 1 mg DNP 44 -BSA and O/lmg. of Bet v 1. In contrast, injection with unmodified OVA or unrelated mPEG conjugate prior to i.v. challenge with DNP 44 BSA or Bet v 1, did not affect the PCA reactions to either of these two antigens.
  • the conjugates of the invention where the antigen is an allergen, inactivate mast cells. This resulted in specific suppre ⁇ ion of a primary immune re ⁇ ponse after admini ⁇ tration of the conjugate ⁇ of the invention.
  • the allergenicity of the conjugate ⁇ of the invention are 10-500 fold lower than that of the original allergen.
  • the method of the invention may be practiced to treat a condition selected from allergies and autoimmune diseases by inducing tolerance to an antigen (Ag) or ⁇ elf-protein or autoantigen, in both humoral and cell mediated immune re ⁇ pon ⁇ es comprising admini ⁇ tering an effective amount of Ag(mPEG) conjugate to induce tolerance to an antigen (Ag) .
  • a condition selected from allergies and autoimmune diseases by inducing tolerance to an antigen (Ag) or ⁇ elf-protein or autoantigen, in both humoral and cell mediated immune re ⁇ pon ⁇ es comprising admini ⁇ tering an effective amount of Ag(mPEG) conjugate to induce tolerance to an antigen (Ag) .
  • the invention treats organ-specific autoimmune diseases in animal and rejection of DNA transfected cells of their products by administration of mPEG conjugates of autoantigens selected from the group consi ⁇ ting of collagen- induced arthriti ⁇ by type II collagen, experimental autoimmune encephalomyelitis by myelin basic protein, and diabetes in NOD mice by insulin to induce tolerance to an antigen (Ag) in both humoral and cell mediated immune re ⁇ pon ⁇ es.
  • autoantigens selected from the group consi ⁇ ting of collagen- induced arthriti ⁇ by type II collagen, experimental autoimmune encephalomyelitis by myelin basic protein, and diabetes in NOD mice by insulin to induce tolerance to an antigen (Ag) in both humoral and cell mediated immune re ⁇ pon ⁇ es.
  • the principles of the invention provide a method of treating organ-specific autoimmune disea ⁇ e ⁇ in animal and rejection of DNA transfected cells of their products comprising administration of mPEG conjugate ⁇ of autoantigen ⁇ ⁇ elected from the group consi ⁇ ting of collagen-induced arthriti ⁇ by type II collagen, experimental autoimmune encephalomyelitis by myelin basic protein, and diabetes in NOD mice by insulin to induce tolerance to an antigen (Ag) in both humoral and cell mediated immune responses.
  • mPEG conjugate ⁇ of autoantigen ⁇ selected from the group consi ⁇ ting of collagen-induced arthriti ⁇ by type II collagen, experimental autoimmune encephalomyelitis by myelin basic protein, and diabetes in NOD mice by insulin to induce tolerance to an antigen (Ag) in both humoral and cell mediated immune responses.
  • a method of treating established allergic respon ⁇ es or established autoimmune disease states involves a combination therapy as follows: i) wipe out the establi ⁇ hed immune response of the host to all antigens by pharmacological immunosuppres ⁇ ive agent ⁇ for a period of about 2 to about 3 weeks, without destruction of the stem cells, and ii) treatment of the "immunologically revirginized" patient with mPEG conjugates of the auto-antigen ⁇ in anticipation of generating ⁇ pecific Ts cells which would suppre ⁇ the induction of the Th cell ⁇ involved in the auto-immune response.
  • any known immunosuppres ⁇ ive drugs may be used for step i) such as those which specifically target suppres ⁇ ion of T cell ⁇ or B cell ⁇ or both.
  • ⁇ tep i) may be substituted by a bone marrow or stem cell transplant.
  • the invention provides promising result ⁇ in experimental animal models that conjugate ⁇ of immunogenic Ag and mPEG provide for the development of novel therapeutic and clinical application ⁇ .
  • the clinical applications of mPEG conjugates of diverse therapeutic agents is increased.
  • Liposome-encapsulated antigens are proces ⁇ ed in ly ⁇ osomes, recycled, and pre ⁇ ented to T cells. Cell 64:393.
  • Nonionic triblock copolymers facilitate delivery of exogenous proteins into the MHC class I and class II proces ⁇ ing pathways.
  • the ⁇ uppre ⁇ or factor of T ⁇ uppre ⁇ or cells induced by tolerogenic conjugates of ovalbumin and monomethoxypolyethylene glycol is serologically and physicochemically related to the ab heterodimer of the T cell receptor. J. Immunol. 152:3.
  • Antigen-specific suppressor factors of noncytotoxic CD8+ suppre ⁇ sor T cells downregulate antibody re ⁇ ponse ⁇ al ⁇ o to unrelated antigens when the latter are presented as covalently linked adduct ⁇ with the specific antigen.
  • CF TR Adenosine deaminase T-celis and stem cells
  • Malignant melanoma Tumor cells (liposomes) (HLA-B7 and ⁇ 2 -microglobulin)
  • IL-2 Non-small cell lung Tumor cells (retroviral) cancer
  • MDR-1 Stem cells (retroviral)
  • Emphysema-cirrhosis (1) 14432.1 Glaucoma, congeni l (2) Chr.ll
  • 131800 ( 1 ) 12qll-ql3 GMl-gangitos ⁇ dos ⁇ s (l) 3p21-pl4 2 • Epidermoiysis bullosa, Weber Cockayne type, 131800 (2) 12qll-qI3 GM2-gangl ⁇ os ⁇ dos ⁇ s.
  • F Fabry disease (3) Xq22 Hemochromatosis (2) 6p21.3 Faaoscopuloh merat muscular dystrophy (i) 4fl» Hemodiatysis-related amykHdosis ( 1 ) 16q21-q22 Factor H deficiency (1) i ⁇ 32 Hemotytic anemia due to ADA excess (1) 20ql3.Il Factor V deficiency (1) 1023 Hemolyuc anemia due to adenylau kinase deficiency ( 1 ) »q34.1 Factor VII deficiency (1) 13q34 Hemolyuc anemia due to bisphosphoglycerate mutase Factor X deficiency (1) 13q34 defi ency (l) 7q3l
  • GG6PD deficiency (3) Hereditary persistence of alpha-fetoproteinl (3) 4qll-ql3 Oalactokinase deficiency (1) 17q21-q22 ?Hered ⁇ tary persistence of fetal hemoglobin (3) llpl-i Qalactose epimerase deficiency (1) Ip36-p35 •Hereditary persistence of fetal hemoglobin.
  • Oalactosemia (l) 9pl3 heterocellular, Indian type (2) 7q36 Galactos ⁇ al ⁇ los ⁇ s (l) 20qlll .
  • Gaucher disease (1) lq21 Hers disease, or glycogen storage disease VI (1) Chr.H Gaucher disease, variant form ( 1 ) 10q21-q22 Heterocellular hereditary persistence of fetal hemoglobin (2) llplS ⁇ esdtovruuury dyip a (2) llplS IHex ⁇ pseudodeficiencyl (1) 15q23-q24 Gerstmann-Straussler disease, 137440 (3) 20pter-pl2 ?HHH syndrome (2) I3q34 r ⁇ itt-rt sim-rroiw, 143500 (1) ChrJ IHisttduwmial (1) 12o22-q23 Glanzmann thrombasthenia, type A ( 1 ) 17Q21-32 Hoioprosencephaly, type 3 (2) 7q36 Glanxmann thrombasthenia, type B (1) 17021.32 raeJ ⁇ cOTseaeepbady-l (2) 18p ⁇ r-qll Disorder Location Disorder
  • HPFH. nondeletion type G (1) llp.5.5 ⁇ ..I Keratosis foUiculans spinulosa decalvans (2) Xp22.2-p212
  • Hurler-Scheie syndrome ( 1 ) ⁇ p!6.3 Kr ⁇ oe disease (J) /4of4 o ⁇ 2.7
  • Hyperlipoproteinemia 1 8p22 Leprechaunism (1) !9pl3J yperi ⁇ oprot ⁇ memta, typ lb (l) I9ql3i Lesch-Nyhan syndrome (3) Xq26-q27-2
  • Hyperiipoproteinemia type III (1) 19ql3.2 .Letterer-Siwe disease (2) 13ql4-q31
  • Hypertnglycendemia one form (1) llq23 Leukemia, acute myeloid, US type (1) x ⁇ tx
  • IHypcxeruloplumine u hereditary
  • Hy ophosphuas , mfanUle, 241500 (3) Ip36.1-p34 Leuketr ⁇ r/mphoma, B-cell, 2 (2) 18q2l.3
  • Isolated growth hormone deficiency Iliig type with Macular dystrophy, vltelUfors type (2) IlqIS Disorder Location Disorder Location
  • svndrom ⁇ c-2 with NesraepttkeUoaaa, 1*3460 (1) Ilq23-q24 dysmorphism and eer-bral atrophy (2) Neuroepithelioma (2) 22ql2 Mental reurdauon, X-linked svndrom ⁇ c-3, with Neurofibromatosis. von Recklinghausen (3) 17qlL2 spastic diplegia (2) N v ⁇ ropet y, reearrat, with ⁇ i — u paaetea. Mental reurdauon, X-iinkrd.
  • SAP-l (l) I0q21-q22 Ocular albinism autosomal recessive (2) 6q.8-ql ⁇ Methemoglobtnemia due to cytoehrome b5 deficiency (3) Chr l ⁇ Ocular albinism.
  • Forsius-Erikssoo type (2) Xpll-qll Methemogjobtnemia.
  • enzymopathic ( 1 ) 22ql3-31-qter Ocular albinism, NetUeship-Falls type (2) Xp22J Meihemogtobtnenuas.
  • Miller-Dieker lissencephaty syndrome (2) 17pl3 * 3 155200, 165210, 59420, 155220(3) l7tl2IJI ⁇ 22.05 mtocfcmdrtal coaaplex 1 de£ t-»cy, 262010 (1) llqlS Qst ⁇ ooene xm ⁇ ct ⁇ , 4 dxn ⁇ ljorms, MODV. one form (3) Upl55 165200, 165210, 59420, 156220(3) 7g2tl MOD ⁇ , type 1 (2) 20ql3 Osteopetrosis 259700 (1) Ip2l-pl3 MOD ⁇ , type II.
  • Phenylketonuria due to dihydroptendine reductase deficiency ( I ) 4pl5Jl Reunoschists (2) Xp22.3-p22 1
  • Renal tubular actdosts-osteopetrosis syndrome ( 1 ) 8q22 SpUWu-nd spUWoM deformity, type 2 (2) Xq2 ⁇ 'ReUnal cone dystrophy-1 (2) 6q25-q26 Spondyloepiphvseal dysplasia congeniu (3) 12ql3 1l-ql32 'Re nal cone-rod dystrophy (2) 18q21-q22.2 Spo ⁇ dvloepiphvseal dvsplasia tarda (2) Xp22 RetmUts pigmentosa. autosomal dominant (1) llplS Startle disease (2) 6q3J-q56 Disorder Location Disorder Location
  • Stickler syndrome (3) 12ql3 ll-ql3.2 Uaker syidroaae, type IC (I) HP Sucrose intolerance (1 ) 3q25-q26 Usher svndrome type 2 (2) Se ⁇ ravarrer aortic eteaoats (3) 7qllJ! " " T” van der Woude svndrome (2)
  • T Tay-Sachs disease ( I ) 15q23-q24 ⁇ J Veloca ⁇ dio(ae d ayndroaM (2) 22qll Testicular fetninizauon ( 1 ) Xcen-q22 V Yl-rwretinopa-hy, ex»d*-rve. faj lU ⁇ (2) llqlyq23 Thalassemias.

Abstract

A method of inducing tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses by administering an effective amount of an immunosuppressive Ag(mPEG) conjugate. Methods of treating allergies, autoimmune diseases and preventing an immune rejection of organ transplants and DNA transfected cells or cells transfected with a gene therapy vector encoding a foreign protein for gene therapy are also disclosed.

Description

ANTIGEN-mPEG CONJUGATES SUPPRESS HUMORAL AND CELL MEDIATED IMMUNE RESPONSES
Technical Field
The present invention relates to a method of inducing tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses by administering an effective amount of an Ag conjugated with monomethoxypolyethylene glycol (mPEG) conjugate. Methods of treating allergies, autoimmune diseases and preventing an immune rejection of organ transplants and rejection of genetically engineered cells in gene therapy, are also disclosed.
Background Art
Numerous studies have shown that exogenous protein Ag can activate CD8+ T cells that suppress immune responses in an Ag-specific fashion (1-5) . These observations have been puzzling because exogenous Ag normally are not processed via the endogenous pathway for major histocompatibility complex (MHC) class I presentation in most somatic cells. For example, nonphagocytic EL4 cells do not present soluble ovalbumin (OVA) to MHC class I-restricted CD8+ T cells after pulsing with native OVA (6) . However, exogenous OVA could be presented via MHC class I pathway by these cells if delivered by other means, such as osmotic lysis (6) , electroporation (7) , liposomes (8) , mycobacterial infection (9) , nonionic triblock copolymers (10) or receptor-mediated uptake (11) .
The observations that protein Ag(s) alone generally do not prime CD8+ cytolytic T cells (CTL) in vivo have been taken as evidence that exogenous Ag do not stimulate CD8+ T cells. Whether CD8+ CTL precursors are activated by exogenous Ag administered with traditional adjuvants is more controversial (12) . Nevertheless, the inventors have previously shown that OVA emulsified in adjuvants, such as CFA or nonionic triblock copolymers, primed OVA-specific, MHC class I-restricted CD8+ CTL precursors in vivo (4,13). Priming was not due to direct sensitization of MHC class I-bearing cells by contaminating peptides; instead, phagocytic cells were required for priming CD8+ CTL (4) . These findings indicated that exogenous Ag were taken up and processed via the MHC class I pathway by phagocytic macrophages, as suggested by Rock et al. (14).
Previous data have shown that adoptive transfer of Ag-specific CD8+ CTL suppressed subsequent Ab responses in recipients (4,15). Thus, activated CTL inhibit Ag-specific immune responses in vivo , as had been shown in vitro (2,16). Clearly, such T cells could account for some of the activities attributed to CD8+ Ts cells. More recently, the inventors found that oral administration of soluble protein Ag stimulated CD8+ Ts cells that inhibited humoral responses and priming of both CD4+ and CD8+ T cells (5) . These CD8+ Ts cells were not CTL and phenotypically distinguished from CD8+ CTL by reacting with a mAb specific for Ts cells (17) .
Published PCT application WO 95/12413 mentions T cells, and even cytotoxic T cells, as potential targets for inhibition by mPEG compounds. In the PCT, various proteins, such as OVA (ovalbumin) or IgG, were chemically coupled with mPEG. At the appropriate degree of conjugation of mPEG, some of the epitopes of the proteins are still accessible for interaction with the appropriate specific, antibody and this provides the means to target these molecules to B cells or to granulocytes that have bound IgE to their surface via Fc receptors.
Unlike antibodies, the antigen-specific receptors expressed by T cells do not recognize native, intact proteins. Rather, they bind to protein fragments that are bound by proteins encoded by the Major Histocompatability Complex expressed on the surface of specialized antigen presenting cells. The PCT publication WO 95/12413 does not propose that mPEG antigens would be used to inhibit T cells. This treatment would be non-antigen-specific. Such nonspecific inhibition would have little advantage over the immunosuppressive drugs that are currently in use.
The PCT publication has demonstrated that mPEG modification of proteins renders them non-immunogenic in that they fail to stimulate antibody responses. More importantly, the mPEG modified proteins are tolerogenic rendering exposed individuals unable to respond to a subsequent challenge with the unmodified, protein. Thus, tolerance induced by mPEG modified proteins is due to induction of suppressor T cells that in turn down regulate in an Ag-specific manner the immune response of the recipient if administered at any time prior to challenge with the unmodified antigen. Sehon has obtained several patents on a procedure for preventing IgE mediated allergic responses, and favoring the tilting of the immune response to the production of IgG antibodies in allergic individuals. However, the effects of mPEG modified proteins on cell-mediated immune responses have only recently been studied.
U.S. Patent No. 4,296,097 discloses a process for the suppression of the formation of anti-BPO (benzylpenicilloyl) antibodies by administering a conjugate of penicillin and an amino-derivative of polyvinylalcohol polymers.
U.S. Patent No. 4,261,973 discloses a method of suppressing the induction of reaginic antibodies to an allergan by administering a covalent conjugate of the allergen and non-immunogenic water soluble polymers.
U.S. Patent No. 5,447,722 describes a method of suppression of an IgG immune response to an antigenic protein by administering a tolerogenic conjugate of monomethoxypolyethylene glycol and the antigenic protein one day prior to adminstration of the protein alone.
U.S. Patent No. 5,358,710 describes a method of suppressing an animals antibody-mediated immune response to a second antigenic polypeptide by selecting a mammal which is unsensitized to a first antigenic polypeptide, and administering a tolerogenic conjugate of the first antigenic peptide covalently bound to a water soluble polymer. Then an adduct of the first antigenic polypeptide bound to a second antigenic polypeptide is administered. The administration of the tolerogenic conjugate suppresses the capacity of the mammal to mount a humoral antibody response to the first antigenic peptide so that when the first antigenic peptide is conjugated to the second antigenic peptide the anibody mediated immune response to the second antigenic peptide is also suppressed.
Disclosure of the Invention
The goal of this invention is to provide a method for induction of suppression of both humoral (antibody) , and cellular (cell mediated) immune responses to an Ag, by the administration of the antigen in the form of a conjugate with mPEG, (i.e. Ag(mPEG) containing an optimal number of mPEG molecules coupled covalently onto the antigen) at any time prior to injection of the Ag by itself or in conjunction with an appropriate adjuvant, so as to induce humoral and cellular responses to the Ag in question. Hence, the immunosuppressive Ag(mPEG)n conjugate is also referred to as a tolerogenic conjugate.
Part I of the application generally enables methods of suppressing the humoral and cell mediated immune responses. Part II of the application enables specific applications of the above method to gene therapies, organ transplantation and treatment of autoimmune conditions.
In another embodiment, the invention provides a method of obtaining passive transfer of suppression of an immune response comprising treating an animal, preferably a syngeneic animal with Ag(mPEG) conjugate and transferring lymphocytes from said animal to a recipient animal, wherein said lymphocytes provide suppression of Ag-specific cytotoxic lymphocyte (CTL) activity in said recipient animal.
The invention advantageously provides a method of treating a condition selected from the group consisting of allergies and autoimmune diseases by inducing tolerance to an antigen (Ag) in both humoral and cell mediated immune responses comprising administering an effective amount of Ag(mPEG) conjugate to induce tolerance to an antigen (Ag) .
In still another embodiment, the invention provides a method of preventing an immune rejection of organ transplants comprising administering an effective amount of Ag(mPEG) conjugate to induce tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses.
Finally, the invention provides a method of treating organ-specific autoimmune diseases in animal comprising administration of mPEG conjugates of autoantigens selected from the group consisting of collagen-induced arthritis by type II collagen, experimental autoimmune encephalomyelitis by myelin basic protein, and diabetes in NOD mice by insulin to induce tolerance to an antigen (Ag) in both humoral and cell mediated immune responses. The invention provides a method of conducting gene therapy including administering to a mammal an immunosuppressive effective amount of a tolerogenic conjugate consisting of a protein coupled to monomethoxypolyethylene glycol (mPEG) having a molecular weight of about 2,000-10,000 daltons, wherein administration of said tolerogenic conjugate is at least one day prior to administration of a gene therapy vector encoding a gene for a protein, wherein said protein is identical to said protein which is coupled to mPEG, and wherein said method results in the suppression of an immune response and in the development of tolerance to the protein expressed by said gene encoded by said gene therapy vector. The above and other objects of the invention will become readily apparent to those of skill in the relevant art from the following detailed description and figures, wherein only the preferred embodiments of the invention are shown and described, simply by way of illustration of the best mode of carrying out the invention. As is readily recognized the invention is capable of modifications within the skill of the relevant art without departing from the spirit and scope of the invention.
Abbreviations used herein: Ab, antibody; Ag, antigen, APC, antigen presenting cell; CFA, complete Freund's adjuvant; CTL, cytotoxic T lymphocyte; HIgG, human monoclonal (myeloma) immunoglobulin G; IFN-^ interferon; IL, interleukin, LNL, lymph nodal lymphocyte; MHC, major histocompatibility complex; mPEG, monomethoxypolyethylene glycol; OVA, ovalbumin, PCA, passive cutaneous anaphylaxis; TCR, T cell receptor, Th, helper T cells; Ts suppressor T cell; TsF, Ts factor; f.p., foot pad.
Brief Description of Drawings
FIG. 1 shows that OVA(mPEG) conjugate induces suppression of Ab responses. Groups of C57BL/6 mice were injected i.p. with PBS, 2.5 mg OVA(mPEG) or 1 mg OVA. After one week, mice were primed via the f.p. with 200 μg OVA in CFA and bled two weeks later. Sera from each group were tested individually for OVA-specific IgG (upper panel ) or IgG isotypes (lower panel , at 1:100 dilution) by ELISA and absorbance was read at 405 nm. Normal mouse sera were used as negative control and results are shown as averages of three mice + SD.
FIG. 2 shows specificity of suppression of humoral and cell mediated immune responses induced by conjugates of protein Ag and mPEG. C57BL/6 mice were either untreated, or injected i.p. with 2.5 mg OVA(mPEG) or 2.5 mg HΙgG(mPEG). After one week, half of each group of mice were primed via f.p. with 200 μg OVA in CFA and the other half with 200 μg HIgG in CFA. Mice were bled two weeks later and sera (1:50 dilution) from each group were tested individually for IgG specific for OVA (left panel ) or HIgG (right panel ) by ELISA, and absorbance was read at 405 nm.
Normal mouse serum was used as negative control and the results are shown averages of three mice + SD.
FIG. 3 shows that OVA(mPEG) conjugate induces suppression of lymphokine production by helper T cells. C57BL/6 mice were pretreated and primed as described in Figure 1. Two weeks after immunization, draining lymph nodes were harvested and 2 x 10° of LNL were incubated at 37 °C with 2 x 10° of irradiated APC in the presence or absence of 200 μg/ml OVA. As control, LNL were incubated with 200 μg/ml ConA in the absence of APC. Supematants were harvested 24 h later and tested for lymphokine production using HT-2 cells. Results are shown as averages of triplicates + SD.
FIG. 4 shows that OVA(mPEG) conjugate induces suppression of lymphokine production by T cells. C57BL/6 mice were pretreated and primed as described in Figure 1. Draining lymph nodes were harvested two weeks after immunization and LNL were incubated with APC in the presence or absence of OVA as described in Figure 2. Supematants were tested for IL-2 , IL-4 and IFN- by lymphokine ELISA.
FIG. 5 shows that OVA(mPEG) conjugate inhibits activation of T cells. C57BL/6 mice were primed via f.p. with 200 μg OVA in CFA. Two weeks later, 3 x 106/ml of spleen cells were incubated with medium alone, 200 μg/ml OVA or 500 μg/ml OVA(mPEG) conjugate. After 24-h incubation at 37 °C, the supematants were tested for IFN- lymphokine ELISA.
FIG. 6 shows that OVA(mPEG) conjugate induces suppression of cytolytic responses. C57BL/6 mice were injected i.p. with (A) PBS, (B) 2.5 mg OVA(mPEG) or (C) 1 mg native OVA. After one week, mice were primed via f.p. with 200 μg OVA in CFA. Spleens were harvested two weeks later and spleen cells were stimulated with irradiated E.G7-OVA cells. After six days, cytolytic activity of cultured cells was measured using 51Cr-labeled E.G7-OVA or EL4 targets. Results are shown as % specific lysis at various E:T ratios and represent averages of triplicates + SD.
FIG. 7 shows that immunosuppression could be achieved by transfer of spleen cells of syngeneic mice that had been tolerized by OVA(mPEG) . Donor C57BL/6 mice were injected i.p. with (B) PBS, (C) 2.5 mg OVA(mPEG) or (D) 1 mg OVA. One week later, 1 x 10s of spleen cells were transferred i.v. to syngeneic naive mice. One day after adoptive transfer, control mice (A) and recipients (B to D) were primed via f.p. with 200 μg OVA in CFA. Two weeks after priming, spleen cells from recipients were stimulated with irradiated E.G7-OVA cells for six days and their cytolytic activity was tested in a 4-h standard 51Cr-release assay as described in Figure 6.
FIGS. 8, 9 and 10 show diagrams illustrating the efficiency of the invention. The percentages in brackets of Figs. 8 and 10 represent the degree of suppression with respect to the control in animals receiving phosphate buffered saline (PBS) in lieu of the conjugates.
FIG. 11 shows mice injected with human insulin substituted with 3 mPEG groups and later challenged with human insulin developed significantly lower levels of antibody than the control group (p=0.020) by Student's t- test.
Description of the Invention
Part-I Suppression of Humoral and Cell-Mediated Immune Response
The present invention provides a method for inducing specific suppression to a given antigen (Ag) of both humoral and cell-mediated immune responses comprising administering an effective amount of a tolerogenic Ag(mPEG) conjugate. In one embodiment the tolerance induced by Ag(mPEG) conjugates is Ag specific and the suppression of the humoral response is induced in an isotype-nonspecific manner. The tolerance is mediated by Ag-specific CD8+ suppressor T (Ts) cells.
The conjugate, in another embodiment, suppresses IL-2 production by lymph node lymphocytes (LNL) . In yet another embodiment the conjugate suppresses IL-2 , IFN- and IL-4 lymphokine production. The method does not skew CD4+ T cells toward Thl or Th2 phenotype. The Ag(mPEG) conjugate inhibits in vitro lymphokine production by in vivo primed CD4+ Th cells. The Ag(mPEG) conjugate advantageously inhibits both arms of cell-mediated immune responses in vivo .
The invention also provides a method for obtaining passive transfer of suppression of an immune response comprising treating an animal with Ag(mPEG) conjugate and transferring lymphocytes from said animal to a recipient animal, wherein said lymphocytes provide suppression of Ag-specific cytotoxic lymphocyte (CTL) activity in said recipient animal. In this method the transfer of Ts cells inhibited cytolytic responses in recipients. Also the transfer of splenic Ts cells from an Ag (mPEG) -tolerized animal leads to downregulation of primary IgE and IgG responses in recipient animals.
This ability to suppress the immune response provides a method of treating a condition preferably selected from the group consisting of allergies and autoimmune diseases by inducing tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses comprising administering an effective amount of Ag(mPEG) conjugate to induce tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses.
The ability to suppress the immune response also provides for a method of preventing an immune rejection of organ transplants, or rejection of DNA transfected cells and their expressed protein product, comprising administering an effective amount of Ag(mPEG) conjugate to induce tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses. In this method antibodies of all IgG subclasses are suppressed. IgG isotopes dependent upon Thl and Th2 lymphokines are both inhibited by said Ag(mPEG) conjugates. In addition lymphokines produced by CD4+ Th cells are inhibited by said Ag(mPEG) conjugate. These lymphokines are selected from the group consisting of IL-2 , IL-4 and IFN- .
Finally the invention provides a method of treating organ-specific autoimmune diseases in animal comprising administration of mPEG conjugates of autoantigens selected from the group consisting of collagen-induced arthritis by type II collagen, experimental autoimmune encephalomyelitis by myelin basic protein (58) , and diabetes in NOD mice by insulin to induce tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses.
The inventors demonstrated the induction of CD8+ T cells activated by exogenous protein antigens (Ag) in immune regulation. It has been demonstrated that exogenous ovalbumin (OVA) primed murine CD8+ cytotoxic T lymphocytes (CTL) precursors, if administered with complete Freund's adjuvant (CFA) , these CD8+ CTL suppressed Ag-specific humoral and cell-mediated immune responses in recipients after transfer. Thus, CD8+ CTL are immunosuppressive and can be accounted as regulatory cells in some situations. The inventors have shown that oral administration of protein Ag induced tolerance of both humoral and cell-mediated immune responses. This tolerance was mediated by Ag-specific CD8+ suppressor T (Ts) cells that were phenotypically distinguished from CD8+ CTL by reactivity with a monoclonal antibody (mAb) specific for the murine Ts cells. The present inventors have found that mPEG modified ovalbumin (OVA) , which served as a model antigen, induced suppression of cell-mediated responses stimulated by OVA in CFA, as measured by the inhibition of secretion of the lymphokines, IL-4 and IFN- by CD4+ T cells and inhibition of the development of OVA-specific, CD8+, CTL's. This effect was antigen-specific in that responses to unrelated antigens were not inhibited by mPEG modified OVA (and vice versa) . Furthermore, tolerance could be transferred to normal recipients by spleen cells from syngeneic donor mice which had been treated with mPEG modified OVA, i.e. these spleen cell recipients dramatically reduced the priming of CTL's in the cell. These results demonstrate that to prevent cell-mediated, as well as humoral, immune responses are susceptible to antigen-specific immunosuppression by mPEG conjugates of the appropriate antigen. These observations show that this strategy can be used to prevent, or reverse cell-mediated responses in a variety of clinical conditions.
Correction of genetic defects by the transfer of DNA encoding the corresponding non-defective genes is currently the object of intense investigation. One of the limitations of this technology is that the proteins expressed by foreign genes induce antibody responses in recipients deficient of the gene in question. The antibodies bind to the proteins and inhibit their activity by decreasing their effective half-life in circulation. Pretreatment of such recipients with mPEG modified proteins prior to gene therapy would prevent this response. Moreover, it has become clear that proteins encoded by the transferred genes behave as viral genes (i.e. they are first expressed on the cell membrane) and consequently induce cell-mediated immune responses (i.e. generated CTL's are potentially capable of destroying by cytolytic mechanisms, the cells expressing the transgene). These results demonstrate that both antibody and cell- mediated immune responses to the protein products of the transferred genes is inhibited by pretreatment with mPEG modified proteins. Hence this strategy also extends the usefulness of gene transfer procedures.
In the present invention the inventors investigate the mechanisms of immune regulation by using the tolerogenic conjugates of protein Ag and mPEG. The results show that a single injection of OVA(mPEG) conjugate but not unconjugated OVA suppressed subsequent Ab responses, lymphokine production by CD4+ T cells and inhibited priming of CD8+ CTL precursors to OVA. The tolerance induction by Ag(mPEG) conjugates was Ag specific, and suppression could be transferred by splenic T cells from tolerized mice to syngeneic mice. Thus, the inventors confirmed that Ag(mPEG) conjugates not only suppress Ab (i.e. humoral) responses suppressing different, i.e., Ig classes but also inhibit cell-mediated immune responses in vivo .
Covalent coupling of diverse protein antigens and mPEG resulted in Ag(mPEG) conjugates that have been found to be tolerogenic rather than immunogenic (18-22) . Administration of Ag(mPEG) conjugates is a well-established method to induce long-lasting tolerance (20,23) and has been used to prevent the induction of Ag specific allergic responses which are mediated by IgE Abs (19,24). Ag(mPEG) conjugates stimulated the induction of Ag-specific, non cytotoxic Ts cells (25,26), which expressed Thy-1 and CD8+ markers (26,27), and reacted with a mAb specific for the activated murine Ts cells (17) . Clones of nonhybridized CD8+ Ts cells produced a soluble Ts factor (TsF) (26,27) that was serologically and physicochemically related to the aβ heterodimer of TCR (28) . Functionally, these CD8+ Ts cells and TsF suppressed in vitro and in vivo Ab production in an Ag-specific and MHC class I-restricted manner (27,30,31). However, TsF had no suppressive effect on fully differentiated B cells or plasma cells but exerted its down regulating effect on Ag-specific Th cells through interaction with normal CD8+ T cells in the presence of APC and Ag (31,32). Moreover, CD8+ Ts cells produced a ThO-like pattern of cytokines (IL-2, IL-4 , IFN~ , TGF-?, TNF-α and lymphotoxin) that were found not to be directly responsible for the observed downregulation of Ab responses (30) .
Using this model system, the inventors investigated the tolerance induction of cell-mediated immune responses by Ag(mPEG) conjugates. Indeed, in addition to inducing suppression of humoral responses, OVA(mPEG) conjugate but not the unmodified OVA, inhibited lymphokine production by CD4+ Th cells and priming of CD8+ CTL precursors upon challenge with OVA in CFA. Moreover, tolerance induced by OVA(mPEG) conjugate was mediated by Ts cells that induced also Ag-specific suppression on transfer to naive syngeneic recipients.
Example 1
Animals. Eight- to 12-week-old female C57BL/6, mice (H-2 ) were purchased from Harlan Sprague-Dawley (Indianapolis, IN) and used exclusively in this study. Outbred, 250 to 350 g male Sprague-Dawley or Long Evans hooded rats were obtained from the Central Animal Care Services of the University of Manitoba.
Antigens and tolerogens . Purified chicken egg OVA (grade VI) was purchased from Sigma Chemical Co. (St. Louis, MO) . Human monoclonal IgG (HIgG) was isolated from serum of a myeloma patient by ammonium sulfate precipitation and ion exchange chromatography on DEAE (20) . mPEG (average Mr = 3200 Da) was obtained from Pharmacia AB (Uppsala, Sweden) . The tolerogenic conjugates used in this study, OVA(mPEG)10 and HgG(mPEG)25, were prepared as described earlier (23,25,33 incorporated by reference herein) and dissolved in phosphate-buffered saline (PBS, pH 7.4). Note that any antigen may be used in conjunction with mPEG having a molecular weight in the range of about 3000-25,000, preferably 5000-20,000. The subscript n in the formula Ag(mPEG)n represents the degree of conjugation, i.e. the average number of mPEG molecules coupled per molecule of protein Ag. (Although the example presented herein uses OVA Ag as a model antigen, any antigen to which tolerance is desired can be converted to an immunosuppressive mPEG conjugate and be used according to the method of the present invention. )
In an alternative embodiment the tolerogenic conjugates 0VA(mPEG)u and HIgG (mPEG) 25, were synthesized by a modification of the procedure previously reported (21) . Aggregate free protein is concentrated by ultrafiltration iva an Amicon filter to 5-10 mg/ml and then reacted with a large excess, an 8-molar excess of the electrophilically activated mPEG intermediate e.g., mPEG para-nitrophenol carbonate (which reacts with alpha and epsilon amino groups) , commercially available from Shearwater Polymers, Huntsville, Alabama, with respect to the total lysine content of the protein. This procedure is applicable to any protein.
By way of example, 47 mg of OVA (lmM) , which has 19 lysines per molecule of OVA1 (i.e., 19 mM of lysines) was reacted with 486.4 mg of activated intermediate {152 (8 x 19) illimoles}. For 150 mg of IgG (lmM), with an average lysine content of 90 per molecule of HIgG, the IgG was reacted with 2.3 grams of mPEG. The protein was dissolved at 10 mg/ml in 0.1 M borate buffer (pH 9.7) and the intermediate was dissolved in a similar volume of double distilled water (DDW) . The protein was added quickly with stirring to the intermediate solution. The reaction mixture was then added to a dialysis bag and suspended in 4 liters
Nisbet, A.D., Saundry, R.H. , Moir, J.G., Fothergill, L.A. and Fothergill, J.E. 1981, Eur. J. Biochem. 115:335-345. of 0.05M Borate buffer (pH 9.7) for 1 hour at room temperature and overnight in the cold. The liquid outside the dialysis bag was constantly stirred and served as a pH-stat. The content of the dialysis bag was applied to a Pharmacia BloPilot gel filtration column (Superdex 60/600) equilibrated with DDW for the isolation of the conjugate, which elutes in the void volume, from the hydrolyzed mPEG and from the para-nitrophenol which elutes with the buffer salts, and to place the conjugate in DDW for lyophilization. The conjugate is stored lyophilized at -20°C.
Tolerance induction and immunization protocol . C57BL/6 mice were pretreated by intraperitoneal (i.p.) injection with 2.5 mg OVA(mPEG) or 1 mg native OVA. The control mice received 0.5 ml PBS in lieu of Ag. The OVA(mPEG) conjugates used in this study contained -40% (wt/wt) protein, i.e. 2.5 mg OVA(mPEG) conjugates contained about 1 mg native OVA within the complex. Seven days later, mice were immunized in the hindfoot pads (f.p.) with 200 μg of OVA emulsified in CFA containing M . tuberculosis H37Ra (Difco Labs, Detroit, MI) . Two weeks after immunization, mice were bled and sacrificed. Draining lymph nodes and spleens were harvested for testing lymphokine production and cytolytic activity, respectively (4,13). In some control experiments, mice were pretreated with 2.5 mg HΙgG(mPEG) and then immunized with 200 μg HIgG in CFA.
Passive cutaneous anaphylaxis (PCA) assay . The anti-OVA IgE levels in primed sera mice were determined by the PCA in male Sprague-Dawley or Long Evans hooded rats as described elsewhere (34) . Rats were anesthetized by injecting 1% Nembutal i.p. and the backs were shaved from shoulder to rear haunches. Volumes of 50 μl of two-fold serial dilutions of individual sera were injected intrader ally into the backs of the rats. Rats were challenged 4 h later by an intravenous (i.v.) injection of 1 mg OVA in 1 ml PBS containing 1% Evan's Blue. The PCA titer was expressed as the reciprocal of the highest dilution of the serum giving a blue area of at least 5 mm in diameter. The Student's t-test was used to compare the PCA titer of each group with that of the control. The difference was considered statistically significant only when the p value was smaller than 0.01.
Enzyme-linked immunosorbent assay (ELISA) . A solid-phase ELISA was used to determine Ag-specific IgG production in primed mice (13). Microliter plates were coated with OVA or HIgG at a concentration of 10 μg/ml in borate-buffered saline (BBS, pH 8.2) overnight at 4°C and then blocked with PBS containing 1% bovine serum albumin (BSA) and 0.1% sodium azide. The test sera were added in three-fold serial dilutions and incubated at 37 °C for 1 h. Plates were washed extensively with PBS between each step. Alkaline phosphatase (AP) -conjugated goat anti-mouse IgG (Cappel, Durham, NC) was then added and incubated at 37 °C for 1 h. For determining OVA-specific IgG isotypes, rabbit anti-mouse IgG heavy chains ( γ , γZa , γzb and γ ) (Zymed, South San Francisco, CA) and AP-conjugated goat anti-rabbit IgG were used as primary and secondary detecting Ab, respectively, the p-nitrophenyl phosphate substrate dissolved in diethanolamine buffer (pH 9.8) was added to each well and absorption was read at 405 nm using an automatic microplate reader (Molecular Devices Corp. , Menlo Park, CA) .
Lymphokine production . Lymphocytes from draining lymph nodes (LNL) were harvested two weeks after f.p. immunization with OVA in CFA. LNL (2 x 106) were cultured with 2 x 106 of irradiated (2,000 rad) syngeneic splenic APC with or without 200 μg/ml OVA in 1 ml culture medium consisting of RPMI 1640, 1 mM L-glutamine, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol plus antibiotics. As positive control for T cell stimulation, LNL were incubated with 5 μg/ml concanavalin A (Con A) in the absence of APC. After 24-h incubation at 37°C, supematants were harvested, frozen and thawed, then added to the 96-well plates containing 5 x 103 per well of IL-2-dependent HT-2 cells (35) . After another 40-h incubation at 37°C, proliferation of HT-2 cells was determined by a calorimetric assay using tetrazolium salt XTT (Diagnostics Chemicals, Ltd., Oxford, CT) and phenazine methosulfate as described elsewhere (36) . Absorbance was read at 450 nm. All assays were performed i triplicate and reported as the mean + standard deviation (SD) . In some cases, 3 x 106/ml of spleen cells from mice primed with OVA in CFA two weeks earlier were incubated wit medium alone, 200 μg/ml soluble OVA or 500 μg/ml soluble OVA(mPEG) conjugate at 37°C for 24 h. Supematants were then tested for lymphokine production by lymphokine ELISA.
Lymphokine ELISA. Supematants of primed LNL culture with or without OVA were tested for lymphokine pattern by sandwich ELISA (13) . Paired mAb (PharMingen, San Diego, CA) were used as capture and detecting Ab, respectively. Microliter plates were coated with capture Ab in BBS overnight at 4°C. After blocking excess protein-binding sites by PBS plus 1% BSA at 22 °C for 1 h, serially diluted recombinant cytokines (IL-2, IL-4 or IFN~ ) and supematant were added and incubated overnight at 4°C. Biotin- conjugated detecting Ab and avidin-horseradish peroxidase conjugate (Vector Labs, Burlingame, CA) were subsequently added and incubated at 22 °C for 45 min and 30 min, respectively .
The plates were washed extensively with PBS plus 0.1% Tween 20 between each step. Calorimetric reaction was developed by exposure to the 2 , 2 ' -azino-di [ 3-ethyl- benzathiazoline sulfonate] (ABTS) substrate (Kirkegaard & Perry Labs, Gaithersburg, MD) and absorbance was read at 40 nm. The cytokine concentration In each supernatant was calculated from the standard curve of each recombinant cytokine. Cytotoxicity assay . Spleens from C57BL/6 mice were harvested two weeks after immunization with OVA in CFA (4) . Single cell suspensions were prepared and erythrocytes were lysed. Mononuclear spleen cells (35 x 106) were stimulated in vitro for 6 days with 3 x 105 of irradiated (20,000 rad) syngeneic E.G7-OVA cells according to the method described by Moore et al. (6).
E.G7-0VA is an la" EL-4 (H-2b) thymoma clone transfected with the chicken OVA cDNA gene (6) (provided by Dr. M. J.Bevan, University of Washington, Seattle, WA) . The E.G7-OVA clone provides a model for gene therapy applications of the invention. Cytolytic activity of cultured spleen cells was determined in a standard 4-h 51Cr-release assay (13). Percent of specific lysis was calculated as: (51Cr release by effector cells - spontaneous 51Cr release) / (maximal 51Cr release - spontaneous 51Cr release) . Maximal 51Cr release was achieved by adding 1% Triton X-100 to the target cells. Spontaneous 5Cr release in the absence of effector cells was generally <10% of the maximal release in all experiments. All assays were performed in triplicate and reported as the mean + SD.
This example shows that mPEG conjugates inhibit specific cytotoxic T cells (CTL) . The above model system for gene therapy utilized tumor cells transfected with the cDNA for the ovalbumin gene (E.G7-0VA) . OVA-specific CTL were engendered by priming mice with OVA in Freund'ε complete adjuvant. The resulting CTL lysed tumor cells that had been transfected with the OVA gene, but not cells transfected with the insulin gene. It was advantageously found that pretreatment of mice with OVA(mPEG)10 inhibited the priming of CTL by OVA in CFA, as well as OVA-specific Ab responses, whereas unmodified OVA had no effect. Thus the gene therapy model showed suppression of the immune response to OVA protein expressed from the chicken OVA cDNA gene. Transfer of spleen cells from tolerized donor mice . For cell transfer, donor mice were injected i.p. with PBS, 2.5 mg OVA(mPEG) or 1 mg native OVA and sacrificed 7 days later. Spleens were harvested and 1 x 108 of spleen cells were transferred into syngeneic naive mice by i.v. injection (5) . One day after transfer, recipients were primed f.p. with 200 μg OVA in CFA. Spleen cells from recipients were harvested two weeks later and stimulated in vitro with irradiated E.G7-0VA cells as described above.
Discussion
Suppression of humoral responses by OVA (mPEG) conjugate, but not by native OVA . Although inactivation of sensitized mast cells and prevention of systemic anaphylaxis by Ag(mPEG) conjugates have been reported (19,24), the effect of mPEG-AG conjugates on cell-mediated immune responses had not been studied.
In three separate experiments, the anti-OVA IgE responses of all OVA (mPEG) -pretreated mice were suppressed by more than 80% in comparison with that of control (Table 1) . Although there was a great variation of anti-OVA IgE titers in mice pretreated with native OVA (Table 1) , suppression of IgE responses in these mice was marginal (<28%) as compared to control mice. Th s, these data confirmed that Ag(mPEG) conjugates were more tolerogenic and that the unmodified protein Ag exerted deviation of the immune response to other Ig classes.
To determine whether OVA(mPEG) conjugates also inhibited IgG responses in mice immunized with OVA in CFA, sera from pretreated and subsequent immunized mice were tested for OVA-specific IgG production by ELISA. As illustrated in the upper panel of Fig. 1, OVA(mPEG) conjugate profoundly inhibited IgG responses to OVA, whereas native OVA given i.p. did not suppress IgG responses. This was consistent with our earlier observation that Ab responses were inhibited by i.v. but not by i.p. injections of soluble Ag prior immunization with the same Ag in adjuvant (37). Next, it was questioned whether OVA(mPEG) conjugates inhibited all IgG isotypes stimulated by OVA in CFA or preferentially, suppressed some, while augmenting other IgG isotypes. As shown in the lower panel of Fig. 1, all IgG subclasses including predominant IgGl and IgG2b isotypes were inhibited by OVA(mPEG) treatment. Hence, it was concluded that, consistant with Ts cell-mediated suppression (30) , OVA(mPEG) conjugate induced tolerance of humoral responses in an antigen-specific and isotype- unspecific manner.
The specificity of tolerance induced by Ag(mPEG) conjugates was then verified by pretreating mice with OVA(mPEG) or HΙgG(mPEG) . One half of the mice from each group were then immunized with OVA in CFA and the other half with HIgG in CFA. Sera from tolerized mice showed that OVA(mPEG) inhibited only OVA-specific but not HIgG-specific IgG responses, whereas HΙgG(mPEG) suppressed anti-HIgG but not anti-OVA IgG responses (Fig. 2) . Thus, tolerance induced by Ag(mPEG) conjugates is Ag specific (30).
Suppression of lymphokine production by OVA (mPEG) conjugate but not by native OVA . The tolerogenic effect of OVA(mPEG) conjugate on OVA-specific cell-mediated responses was determined next. Mice were pretreated i.p. with OVA(mPEG) or OVA and then primed f.p. with OVA in CFA as described above. When cultured with syngeneic APC and soluble OVA, LNL from untreated control mice primed with OVA in CFA produced IL-2 that supported the growth of IL-2-dependent HT-2 cells (Fig. 3) . Pretreatment with OVA(mPEG) conjugate but not native OVA profoundly suppressed IL-2 production by LNL (Fig. 3). However, LNL from all three groups responded equally well to the mitogen Con A (Fig. 3), indicating that Ag(mPEG) conjugates are not non- pecific toxic to T cells nor induce a deletion of T cells. To further determine if OVA(mPEG) conjugate might induce an immune deviation by altering pattern of lymphokines produced by CD4+ Th cells, lymphokine production by LNL upon stimulation with OVA was tested. LNL from control mice produced IL-2 and IFN-jv but little IL-4 (Fig. 4) . Pretreating mice with OVA(mPEG) conjugate suppressed production of all three lymphokines rather than altering one or another, suggesting that OVA(mPEG) treatment did not skew up CD4+ T cells toward Thl- or Th2-like phenotype at this stage. It is noteworthy that, in contrast to a recent report from Degermann et al, that administration of soluble protein could divert a clonal population of TCR transgenic T cells from Thl toward Th2-type responses (38) , there was not observed an inhibition of CD4+ T cell responses or a deviation of lymphokine pattern by pretreating normal mice with soluble OVA prior to priming with OVA in CFA, suggesting that soluble OVA may have no obvious effect on priming of naive T cells.
Next, it was questioned whether Ag(mPEG) conjugates had downregulating effect on primed CD4+ Th cells. Spleen cells from mice primed with OVA in CFA were cultured with soluble OVA, OVA(mPEG) conjugate or medium alone as control. Supe atants were harvested after 24-h and tested for lymphokine production. Interestingly, OVA(mPEG) conjugate also inhibited in vitro lymphokine production by in vivo primed CD4+ Th cells (Fig. 5) . These results indicate that Ag(mPEG) conjugates prevent not only in vivo priming of T cells, but also inhibit in vitro activation of primed T cells. TABLE 1 OVA(mPEG) Conjugate Induces Suppression of IgE Responses
Expt . i • P • Treatment6
PBS OVA(mPEG) OVA
TαE Titerb IαE Titer Suppression IgE Titer ^Suppression
320 <40 8 800
160 <40 >81 6 64400
160 <40 160
! >80 <10 >80
>80 <10 >88" >>8800 <25
>80 <10 >20
5 320 <20 40
320 <20 >92" 116600 <28
80 <20 320
a Groups of three C57BL/6 mice were injected i.p. with PBS, 2.5 mg OVA(mPEG) conjugate or 1 mg native OVA. One week later, mice were immunized f.p. with 200 μg OVA in CFA. b Mice were bled two weeks after f.p. immunization and individual sera were tested for OVA-specific IgE by PCA in rats. IgE titer was expressed as the reciprocal of the highest serum dilution giving a blue area greater than 5 mm. c % suppression was calculated as: 100 x (mean IgE titer of control group - mean IgE titer of test group) / (mean IgE titer of control group) .
Statistically significant difference between test group vs. control group (p < 0.01),
Suppression of cytolytic responses by OVA (mPEG) conjugate but not by native OVA . To test if Ag(mPEG) also induced suppression of cytolytic responses, C57BL/10 mice were pretreated with PBS, OVA(mPEG) conjugate, or native OVA and primed with OVA in CFA. Spleen cells from these primed mice were then stimulated in vitro with irradiated E.G7-OVA cells to induce CTL activity (4) .
Control mice primed with OVA in CFA developed OVA-specific T cells that lysed E.G7-0VA targets (Fig. 6A) . Pretreatment with OVA(mPEG) conjugate (Fig. 6B) , but not with native OVA (Fig. 6C) , inhibited priming of OVA-specific CTL and prevented development of cytolytic activity in mice that were subsequently challenged with OVA in CFA. Thus, it was concluded that Ag(mPEG) conjugates inhibited both arms of cell-mediated immune responses in vivo, that is both humoral and cell mediated responses.
Transfer of tolerance by splenic T cells induced by OVA (mPEG) conjugat . It had been shown in previous studies that transfer of splenic T cells from mice tolerized by HΙgG(mPEG) or OVA(mPEG) conjugates into syngeneic recipients led to significant long-lasting suppression of Ab responses, as demonstrated by the specific immunological refractoriness to subsequent immunization of aggregated Ag (25,30). To determine whether spleen cells from mice pretreated with OVA(mPEG) conjugate also transferred tolerance and inhibited CTL responses in recipients, transfer experiments were performed. Donor C57BL/6 mice were injected i.p. with 2.5 mg OVA(mPEG) conjugate, 1 mg native OVA, or PBS as control. Spleen cells were harvested and transferred into naive syngeneic mice. Recipient mice were primed f.p. with OVA in CFA one day later and sacrificed after two weeks.
Spleen cells from recipients were then stimulated in vitro with irradiated E.G7-OVA cells as described above. As illustrated in Fig. 7, transfer of spleen cells from PBS-treated control mice (Fig. 6B) or from OVA-treated mice (Fig. 6D) had no obvious suppressive or enhancing effect on the CTL responses in recipients, in relation to the CTL activity of primed mice that had not received any donor cells (Fig. 6A) . In contrast, transfer of spleen cells from mice that had been treated with OVA(mPEG) conjugate resulted in a profound suppression of OVA-specific CTL activity (Fig. 6C) . These data suggested that inhibition of cytolytic responses in recipients was mediated by Ts cells rather than a result of diluting CTL precursors by transferred cells, and the magnitude of the suppression effect was correlated to the number of donor cells transferred to the recipients (data not shown) .
The possibility of selectively downregulating the host's immune responses to a given Ag represents one of the most formidable challenges of modern immunology in relation to the development of new therapeutics for allergies, autoimmune diseases, and prevention of immune rejection of organ transplants. Similar considerations apply to an increasing number of promising therapeutic modalities for a broad spectrum of diseases, which involve the use of chemical or biologically active agents potentially capable of modulating immune responses, provided they were not allergic or immunogenic. Among these agents, the copolymeric conjugates of protein Ag and mPEG were found not only essentially non-immunogenic but also immunosuppressive and tolerogenic. This observation has been evidenced by the fact that administration of mPEG conjugates of a highly immunogenic protein Ag to rodents resulted in the suppression of their capacity to mount IgE responses to the unmodified Ag and these animals were also systemically anergic to the injection of the same Ag (39,40).
Furthermore, experiments have shown that transfer of spleen cells from mice tolerized by Ag(mPEG) conjugates into naive syngeneic recipients led to significant suppression of IgE or IgG responses in the latter to subsequent injections of the corresponding Ag (25,30,39). The results show that the specific immunosuppression induced by tolerogenic Ag(mPEG) conjugates involves the activation of Ag-specific Ts cells. The tolerogenic Ag(mPEG) conjugates induced a profound suppression of immune responses in animal models (40) . Results from this study have demonstrated that OVA(mPEG) conjugate induced tolerance in both OVA specific humoral and cell-mediated immune responses. Ab of all IgG subclasses were suppressed by pretreating mice with OVA(mPEG) conjugate but not by the native unmodified OVA, suggesting that IgG isotypes dependent upon Thl and Th2 lymphokines were inhibited by Ag(mPEG) conjugates. This finding was confirmed by the observations that all lymphokines produced by CD4+ Th cells, such as IL-2 , IL-4 or IFN~ , were inhibited by OVA(mPEG) treatment. Hence, unlike oral tolerance where a Th2 response was preferentially induced by oral Ag (41) , tolerance induced by Ag( PEG) conjugates may not reflect an immune deviation by promoting one Th cell subset while suppressing another.
Moreover, pretreatment with OVA(mPEG) conjugate also inhibited subsequent priming of CD8+ CTL precursors and suppressed their cytolytic activity. This tolerance of cell-mediated responses was mainly attributed to the activation of Ag-specific Ts cells that transferred the tolerance to naive syngeneic recipients. Thus, these data evidence that transfer of splenic Ts cells from Ag(mPEG) -tolerized mice led to downregulation of primary IgE and IgG responses in recipients (26,27).
Earlier studies have revealed that Ts cells derived from mice treated with OVA(mPEG) or HΙgG(mPEG) conjugates were Thy-1+, CD3+, CD4", CD5+, CD8+ and expressed the aβ heterodimer of TCR (26,27). Nonhybridized CD8+ Ts clones were phenotypically distinguishable from the CD8+ CTL by their expression of the carbohydrate epitope detected with the mAb 984D4.6.5 (17), which is expressed by activated Ts cells but not their precursors, CTL precursors, or activated CTL effectors (42) . Moreover, these Ts cells differed from CTL in that they were not cytotoxic and produced Ag-specific TsF, which suppressed in vitro Ab formation (27). Thus, in this tolerance model, CD8+ T cells with cytolytic activity did not account for the biological activity of CD8+ Ts cells induced by Ag(mPEG) conjugates.
The relationship between CD8+ CTL and non-cytolytic, CD8+ Ts cells is currently not clear. They could both be the progeny of a CD8+ T cell precursor with the potential of developing into either subset as demonstrated in the paradigm for development of Thl and Th2 cells from CD4+ Th cell precursors (43) . This idea is supported by the observation that subsets of CD8+ T cells expressing different patterns of lymphokines can be derived from CD8+ T cells obtained from TCR transgenic mice (44) . Alternatively, CD8+ CTL and CD8+ Ts cells could be derived from separate precursors having different functions. Regardless of which model proves to be correct, it is clear that once activated, CD8+ Ts cells inhibit both humoral and cell-mediated immune responses. The mechanisms by which such non cytolytic CD8+ Ts cells downregulate immune responses are not entirely clear. However, the soluble TsF secreted by these CD8+ Ts cells might be a plausible explanation for the observed tolerance (26) .
Numerous studies have shown that treatment of organ specific autoimmune diseases in animal models could be achieved by oral administration of respective autoantigens {reviewed in (41)}, such as collagen-induced arthritis by type II collagen (45,46), experimental autoimmune encephalomyelitis by myelin basic protein (47,48), experimental autoimmune uveoretinitis by S-antigen (49) or diabetes in NOD mice by insulin (50). However, Heath's group has recently reported that under certain circumstances, feeding mice with autoantigens can cause rather than prevent autoimmune diseases (51,52). Thus, the present inventors' data indicated that conjugates of autoantigens and mPEG could bypass this effect of Ag administered orally and provide an alternative means to prevent cell-mediated autoimmunity .
Example 2
Human insulin is a protein that has been considered for gene therapy as a treatment for patients with type I diabetes. Human insulin was conjugated with one, two or three mPEG groups/molecule. Three BALB/c mice per group were treated with phosphate buffered saline (PBS) or 20 micromoles human insulin conjugates in PBS intraperitoneally on days -14 and -7. All mice were challenged with 20 microM unmodified human insulin in complete Freund's adjuvant subcutaneously on day 0. Mice were bled and serum tested for insulin-specific antibody by ELISA. Despite the number of mice in the experiment, human insulin substituted with 3 mPEG groups developed significantly lower levels of antibody than the control group (p=0.020) by Student's t-test.
The above example confirms the ability to induce tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses by administering an effective amount of an Ag conjugated with monomethoxypolyethylene glycol (mPEG) conjugate.
Part II - Gene Therapy
The ability to suppress the humoral and cell mediated immune responses has several important applications, particularly in the areas of treating allergies, autoimmune diseases, preventing immune rejection in organ transplants and preventing rejection of genetically engineered cells in gene therapy.
Foreign proteins or DNA, such as genetic material or vectors for gene therapy, or their derivatives, have therapeutic properties and are administered to patients suffering from certain diseases. However, the immunogenicity of the said foreign proteins, nucleotides, DNA or vectors, or of their derivatives, may vitiate the treatment and hence this invention provides an improved method for the treatment of such diseases.
Gene therapy is the insertion of a functioning gene into the cells of a patient (i) to correct an inborn error of metabolism (i.e., genetic abnormality or birth defect resulting in the deficiency of the patient with respect to one or more essential proteins such as enzymes or hormones) , or (ii) to provide a new function in a cell Culver, K.W. , "Gene Therapy", 1994, p. xii, Mary Ann Liebert, Inc., Publishers, New York, NY).
When the host is totally deficient of the inserted gene from birth, the new protein expressed by this gene —when the latter is inserted into the appropriate cell of an adult host— would induce in the host an immune response against itself. Hence, (i) the host would produce antibodies or cytotoxic cells to the "new" protein, and (ii) this immune response would not only combine and neutralize and thus inactivate the function of the "new" protein, but may also lead to untoward therapeutic complications due to formation of immune complexes. It is, therefore, not surprising that gene therapy has proven successful in adenosine deaminase (ADA) deficiency, i.e., in children deficient of ADA from birth, which is manifested by the absence of functional T lymphocytes and consequently to the severe combined immunodeficiency (SCID) syndrome.
The reported success of gene therapy in young children deficient of ADA from birth is related to the immunodefi- cient status of the child, as no immune response can be generated against the foreign therapeutic genetic material. As a corollary, gene therapy would be successful if it is instituted from birth, when it is relatively easy to induce immunological tolerance to a foreign immunogenic material.
This invention provides a method for overcoming this inherent complication due to the immunogenic capacity of the expressed protein, and is therefore considered to represent a novel and an essential improvement for the treatment of such diseases.
As background to the invention, Bitoh, S., Takata, M. , Maiti, P.K. , Holford-Stevens, V., Kierek-Jaszczuk, D. and Sehon, A.H. , disclose that "Antigen-specific suppressor factors of noncytotoxic CD8+ suppressor T cells downregulate antibody responses also to unrelated antigens when the latter are presented as covalently linked adducts with the specific antigen." Cell. Immunol. 150:168-193, 1993.
Bitoh, S., Lang, G.M. , Kierek-Jaszczuk, D., Fujimoto, S. and Sehon, A.H., disclose "Specific immunosuppression of human anti-murine antibody (MAMA) responses in hu-PBL-SCID mice." Hum. Antibod. Hybridomas 4:144-151, 1993. Bitoh, S., Lang, G.M. and Sehon, A.H. , disclose the Suppression of human anti-mouse idiotypic antibody responses in hu-PBL-SCID mice." Hum. Antibod. Hybridomas 4:144-151, 1993. Dreborg, S. and Akerblom, E. , disclose the safety in humans of "Immunotherapy with monomethoxypolyethylene glycol modified allergens." In: S.D. Bruck (Ed.), CRC Crit. Rev. Ther. Drug Carrier Syst. 6:315-363, (1990).
Generally the term "antigen" refers to a substance capable of eliciting an immune response and ordinarily this is also the substance used for detection of the corresponding antibodies by one of the many in vitro and in vivo immunological procedures available for the demonstration of antigen-antibody interactions. Similarly, the term allergen is used to denote an antigen having the capacity to induce and combine with reaginic (i.e., IgE) antibodies which are responsible for common allergies. The latter definition does not exclude the possibility that allergens may also induce reaginic antibodies, which may include immunoglobulins of classes other than IgE.
As used herein, the term "antigenicity" is defined as the ability of an antigen (immunogenic material) or allergen to combine in vivo and in vitro with the corresponding antibodies; the term "allergenicity" or skin activity is defined as the ability of an allergen to combine in vivo with homologous reaginic antibodies hereby triggering systemic anaphylaxis or local skin reactions, the latter reactions being the result of direct skin tests or of passive cutaneous anaphylactic (PCA) reactions; and the term immunogenicity in a general sense is the capacity of an antigen or allergen, or of their derivatives produced in vitro or processed in vivo, to induce the corresponding specific antibody response.
In relation to this invention, tolerogens are defined as immunosuppressive covalent conjugates consisting of an antigenic material (immunogenic proteins, such as the expressed protein products of gene therapy vectors, etc.) and a water-soluble polymer (see e.g. Sehon, A.H. , In "Progress in Allergy" (K. Ishizaka, ea.) Vol. 32 (1982) pp. 161-202, Karger, Basel; and U.S. patent No. 4,261,973).
In the present context, the term "tolerogen" refers to a conjugate consisting of an immunogenic material (protein or polynucleotide) and a nonimmunogenic conjugate, said tolerogen being immunosuppressive in an immunologically specific manner with respect to the antigen which is incorporated into the tolerogenic conjugate irrespective of the immunoglobulin class which is downregulated. Furthermore, the tolerogen may comprise a conjugate of an essentially nonimmunogenic polymer and an immunogenic biologically active product or derivative of the genetic material used for gene therapy.
The therapeutic administration of foreign immunogenic material induces an immune response leading to the formation of antibodies of different immunoglobulin classes. Hence, on repeated administration, the material may form complexes in vivo with such antibodies leading to a poor therapeutic effect by virtue of its being sequestered and neutralized by the antibodies, or to anaphylactic reactions by combination with reaginic antibodies, or to other untoward conditions, i.e. immune complex diseases due to the deposition of antibody-antigen complexes in vital tissues and organs. Wilkinson et al. "Tolerogenic polyethylene glycol derivatives of xenogenic monoclonal immunoglobulins, Immunology Letters, Vol. 15 (1987) pp. 17-22, disclose the administration time of a tolerogenic conjugate to a non-sensitized individual at least one day prior to challenge with an antigen, and optionally about 6 or 7 days.
The present invention overcomes deficiencies of the prior art by providing methods of inhibiting humoral and cell mediated immune responses. The invention makes possible the administration of gene therapy, which involves the generation of immunogenic material in a patient deficient of the corresponding gene, possible and effective.
Gene therapy procedures as currently practiced involve the administration by itself of a foreign genetic material, or of its biologically active products, and do have certain disadvantages and limitations which are primarily due to their potential immunogenicity in the host deficient of the corresponding gene.
This aspect of the present invention aims at overcoming the above mentioned complications by suppressing the production of antibodies to the foreign therapeutic genetic material and of its expression products, and of thus ensuring the efficacy of gene therapy by the prior administration of imunosuppreεsive doses of tolerogenic conjugates consisting of therapeutically active and potentially immunogenic materials coupled to nonimmunogenic polymers, thus overcoming or minimizing the risk of inducing anaphylactic reactions or immune complex diseases.
Thus, the invention aims at suppressing substantially an immune response to the protein resulting as a consequence of successful gene therapy, which response would undermine the therapeutic efficacy of a biologically active genetic material and which may also cause untoward physiological reactions (e.g. anaphylaxis and/or immune complex diseases) .
The invention provides a method for conducting gene therapy comprising administration to a mammal of an immunosuppressing effective amount of a tolerogenic conjugate comprising the genetic material and/or its expression product (i.e., the protein of which the patient is deficient) and monomethoxypolyethylene glycol having a molecular weight of about 2,500-10,000 daltons, the above administration being at least one day prior to administration of the therapeutic genetic material for gene therapy, wherein said method results in the specific suppression of the immune response and the active development of specific tolerance to said therapeutic genetic material and/or its expression product (s) . Alternatively, multiple copies of a single gene may be insertedinto a gene therapy vector.
In a preferred embodiment the therapeutic genetic material is selected from nucleotides, DNA, RNA, mRNA, which may or may not be attached to or delivered by appropriate vectors for expression of the required therapeutic protein.
In a more preferred embodiment of gene therapy gene delivery vectors may include Moloney murine leukemia virus vectors, adenovirus vectors with tissue specific promotors, herpes simplex vectors, vaccinia vectors, artificial chromosomes, receptor mediated gene delivery vectors, and mixtures of the above vectors.
In an alternative embodiment of the invention which overcomes the immunogenicity of the gene therapy protein, an mPEG conjugate corresponding to a gene therapy protein, is administered prior to the administration of the gene therapy vector encoding a gene for a therapeutic protein.
In still another embodiment of the invention which overcomes the immunogenicity of the gene therapy vector, an mPEG conjugate corresponding to a vector protein, is administered prior to the administration of the gene therapy vector. In a preferred embodiment, mPEG conjugates of both the vector protein and gene therapy protein are administered prior to conducting gene therapy with a gene therapy vector encoding a gene for a therapeutic protein. The the vector protein and gene therapy protein mPEG may be conjugated together as a hybrid and administered prior to conducting gene therapy with a gene therapy vector encoding a gene for a therapeutic protein.
The objectives of the present discovery are accomplished by a method, wherein an immunosuppressively effective amount of a tolerogen incorporating a foreign genetic material or its active derivative (s) is administered to the mammal prior to the administration of the foreign genetic material or its biologically active derivative (s) . The tolerogenic conjugate is preferably administered to individuals who have not received a prior treatment with the foreign genetic material or its product, i.e. to unsensitized individuals.
The invention provides improved methods for gene therapy of different human diseases which can be ameliorated or eliminated by the administration of the appropriate genetic materials, etc. or their therapeutic derivatives, of which the patient is deficient.
The tolerogenic conjugates may be synthesized by covalent or noncovalent attachment of nonimmunogenic polymers to natural or synthetic biologically active proteins such as for example (i) murine or rat monoclonal antibodies to human T-cells which have been used to suppress transplant rejection (Colvin, R.B. et al.; Fed. Proc. 41 (1982) p. 363, Abstr. 554) or as "miracle bullets" for the destruction of tumors (Froese, G. et al.; Immunology 45 (1982) p. 303-12, and Immunological Reviews 62 (1982), Ed. G. Moller, Munksgaard, Copenhagen) , (ii) enzymes, such as superoxide dismutase (Kelly, K. et al.; Cdn. J. of Physiol. Pharmacol., 609 (1982) p. 1374-81) or L-asparaginase (Uren, J.r. et al. : Cane. Research 39 1979) p. 1927-33) , or (iii) natural or synthetic hormones.
In the preferred mode of the invention, the tolerogen is a covalent conjugate between monomethoxypolyethylene glycol (mPEG) with molecular weight in the range of 2,500-10,000 daltons and a foreign protein such as ovalbumin (OVA or OA) , which served as a model protein.
According to this modality, tolerogens of appropriate composition (i.e. consisting of the genetic material or its expression product and an optimal number of mPEG chains attached to it covalently) substantially suppress the formation of antibodies of different classes (e.g. IgE and IgG) which are directed specifically against the genetic material per se and/or against its expression product (s). The latter case is exemplified by OVA.
Animal model
The acceptability of the mouse as an experimental model for correlation to human utility in the present experiments is evidenced by Dreborg et al. "Immunotherapy with Monomethoxypolyethylene Glycol Modified Allergens", page 325, which indicates that similar results were achieved in humans and mice and thus confirms mice are an acceptable experimental model for evaluation of mPEG modified allergens. See also Antibodies: A Laboratory Manual, Cold Spring Harbor Press, 1988, p. 93, which indicates that laboratory mice are an acceptable experimental animal model for examining the immune response, and that mice, in particular, possess appropriate characteristics for studies of the genetics of the immune response.
The Tolerogen Employed
As water-soluble polymers to be used for the preparation of a tolerogen, polyethylene glycolε, having molecular weights in the range of 2,000 to 35,000, preferably 4,000 to 20,000, have proved to be effective. Polyethylene glycols in this context also include physiologically acceptable derivatives thereof, such as mono-alky1 ethers, preferably the monomethyl ether, whereby the remaining single terminal hydroxyl groups of the molecules are conveniently used for coupling to the protein. Also other water-soluble polymers (macromolecules) may be used, εuch aε polyvinylalcoholε, polyvinyl pyrrolidones, polyacrylamides and homo- as well as hetero-polymers of amino acids, polysaccharideε (e.g. pullulan, inulin, dextran and carboxymethyl cellulose) or physiologically acceptable derivatives of these polymers.
For the covalent coupling of εuch polymerε to the genetic material or its antigenic expresεion molecules, chemical methods normally uεed for coupling of biologically active materialε to polymers may be used. Such methods include coupling by means of mixed anhydride, cyanuric chloride, isothiocyanate, reaction between SH derivatives and CH2I derivativeε of the reacting moleculeε. However, it iε obvious to the workerε skilled in the art that other appropriate chemical methods may be used to lead to the production of conjugateε of desired compoεitionε.
The coupling reaction iε made between active groups in the antigen molecules and in the polymer moleculeε. If necesεary such groups may have to be introduced into said molecules before the coupling reaction. Such active groups are for example -NH2 , -NCS, -SH, -OH, -CH2I and -COOH and they may be introduced according to well-known methodε, if not already preεent in the molecules uεed for the production of tolerogenic conjugateε.
In order to minimize the liberation in vivo of the immunogenic and/or allergenic conεtituent (ε) of the tolerogenic conjugateε and to maximize their effectiveneεε at a low dose, it is desirable that the covalent link between the water-soluble polymer and protein or its active derivative (ε) εhould be as stable as posεible under physiological conditions.
The coupling of the polymer onto the antigenic or genetic material muεt, as mentioned above, have been carried out to such an extent that the conjugate iε rendered tolerogenic, as well as subεtantially non allergenic and εubstantially non-immunogenic. In other words the tolerogens muεt retain a certain number of epitopes of the unmodified antigen, as long as their immunogenicity haε been decreased so that they do not induce the formation of antibodies which may cause unacceptable adverse reactions.
To achieve tolerogenicity , the degree of substitution, (also referred to as the degree of conjugation, which is defined aε the number of polymer molecules coupled per antigen molecule) , varies from one antigen molecule to another depending on the nature and size of the antigen and on the polymer and itε molecular eight.
Therefore, for the εynthesis of a tolerogenic conjugate of a given antigen it iε esεential to εyntheεize a series of conjugates with different degreeε of εubεtitution and then eεtabliεh the εpecial range wherein the above mentioned requirementε are fulfilled.
Too low a degree of subεtitution may reεult in conjugates still endowed with allergenic and immunogenic properties, and too high a degree of substitution may result in conjugates which are not tolerogenic. One of skill in the relevant art will be able to optimize the degree of subεtitution uεing the diεcloεure exampleε. The optional εubεtitution range iε one in which tolerogenicity iε achieved.
One of skill in the art can perform the steps outlined herein and arrive at the appropriate degree of coupling of the nonimmunogenic polymer onto any antigenic protein so as to achieve immunosuppressive properties.
In view of the finely tuned homeostatic balance of the immune response, which may be easily perturbed either upwards or downwards by the administration of a given antigen depending on its dose, state of aggregation and route of administration, as well as the presence or abεence of adjuvants, it is critical when practicing the invention for treatment of appropriate diseaεe conditions, that the tolerogenic conjugates be administered in such a manner aε to lead to the downregulation of the immune response with respect to one or more claεses of immunoglobulins directed against the unconjugated biologically active product of the genetic material.
Hence, in practicing this invention for treatment of appropriate diseaseε, the tolerogenic conjugateε are to be injected in absence of adjuvants since the adjuvants may counteract their suppreεεogenic effects. However, the incluεion of adjuvants along with the unconjugated immunogenic material in the examples given below was justified εo aε to stimulate in experimental animals the enhanced production of antibodies in a relatively short time and to thus test under more stringent conditions the capacity of the tolerogenic conjugateε to suppress the immune responεe in theεe animals even under these extreme conditions which are particularly favorable for enhancing the immune response.
The terms proteins and polypeptides are used synonymouεly, herein. In the preεent context the term "foreign genetic material" referε to a nucleotide, DNA, RNA, mRNA, plaεmid, which are used as carriers of the gene and/or the gene itεelf reεponεible for the expreεεion of the appropriate protein or protein derivative (fragmentε included) , which are εubstantially immunogenic in the animal to be treated.
According to one aspect of the invention the genetic material εhould be therapeutically effective. Many εuch proteins, vectors, DNA are known per se (Culver, K.W. , "Gene Therapy", 1994, p. xii, Mary Ann Liebert, Inc., Publishers, New York, NY, incorporated herein by reference in its entirety) .
For the purposeε of example only, vectors may be selected from the group conεisting of Moloney murine leukemia virus vectors, adenovirus vectors with tisεue εpecific promotorε, herpes εimplex vectors, vaccinia vectors, artificial chromosomes, receptor mediated gene delivery, and mixtures of the above vectorε. Gene therapy vectors are commercially available from different laboratories such as Chiron, Inc. , Emeryville, California; Genetic Therapy, Inc., Gaithersburg, Maryland; Genzyme, Cambridge, Massachuεetts; Somatx, Almeda, California; Targeted Genetics, Seattle, Washington; viagene and Vical, San Diego, California.
The effective doseε (amounts) and formulations commonly used in gene therapy are also known and may be applied to the present invention, although the invention may alternatively employ reduced or increased doseε. In principle, both the biologically active foreign genetic material or its derivatives, aε well as the corresponding tolerogenic conjugates, may be administered parenterally in a soluble form in isotonic solution and after removal of aggregateε by centrifugation.
Time Intervalε for Adminiεtration
For the induction of immunological tolerance to a given protein the protocol followed according to the invention compriεes the administration initially of an immunosuppreεεive effective doεe (amount) of tolerogen, which iε given prior to the adminiεtration of the gene which encodeε a therapeutically active protein or itε product. If neceεεary, thiε doεe may be portioned and given on repeated occasions. The immunosuppressive dose which is given may vary from tolerogen to tolerogen, but it has to be administered prior to the entry of the protein into the hoεt's syεtem.
In accordance with principleε outlined in the exampleε, the practitioner εkilled in the art can determine the variables such as dose of tolerogen and the minimum interval of time between its administration and the appearance of the immunogenic protein in the hoεt'ε εyεtem. See, for example, references diεcuεsed in background of the invention. However, gene therapy, resulting in the production of a "new protein in the protein-deficient patient, should be preceded by administration of the specific tolerogenic conjugate, i.e., the conjugate comprising the same protein and capable of suppreεsing selectively the immune reεponεe of the hoεt with reεpect to the protein in queεtion.
The tolerogenic conjugate is administered prior to the adminiεtration of the gene which expreεεeε the foreign protein. A time period of at leaεt one day prior to the adminiεtration of the foreign genetic material is preferred. In a more preferred embodiment, the tolerogenic conjugate is administered at least about εix dayε prior to adminiεtration of the foreign genetic material.
The immunosuppressive dose refers to the amount of tolerogen required to εubstantially reduce the immune responεe of the patient to the protein or to itε derivative (ε) which will be produced aε a reεult of the gene therapy. According to one mode of the invention, further doses of the tolerogen may be given in conjunction with the protein or its derivative (ε) , i.e. after the primary adminiεtration of the tolerogen.
Thiε mode may repreεent one way of εuεtaining the suppreεεion of the humoral and cellular immune reεponεeε and offerε a more efficient therapeutic regimen for the diεeaεe condition for which the treatment haε been designed. The invention will now be illuεtrated by some non- limiting, representative examples wherein OVA and its tolerogenic mPEG derivatives have been applied as model subεtances to confirm the usefulness of the proposed immunosuppressive treatment of a well-established animal model commonly utilized in the field of immunology.
The conjugates are deεignated as OVA-(mPEG) where n represents the average degree of conjugation. Figures 8, 9 and 10 show diagrams illustrating the efficiency of the invention. The percentages in brackets of Figs. 1 and 3 represent the degree of εuppression with reεpect to the minimal immune response in animals receiving phosphate buffered εaline (PBS) in lieu of the conjugateε.
Example 3
Preparation of OVA-mPEG conjugates having different degrees of substitution2
The conjugates used in the experiments given below have been prepared by coupling mPEG molecules to OVA essentially according to the procedure described by Abuchowεki et al., J . Biol . Chem . , Vol. 252, p. 3518 (1977) utilizing cyanuric chloride as one of the possible coupling agents. To begin with, in the experiment described the "active intermediate" consiεting of an mPEG molecule attached to cyanuric chloride waε prepared. It waε found that the moεt important condition of thiε reaction was that all reagentε be completely anhydrouε and that the reaction mixture be protected from atmoεpheric moiεture because of its high εuεceptibility to hydrolysis.
Among various methods used for the syntheεis of the "active intermediate", the example given below illustrates the general procedure. (See also Jackson, C. J.C.,
2 Note this is an older method of preparing mPEG conjugateε, newer method are set for in the specification aatt ppaaggee 14. Charlton, J.L., Kuzminski, K. , Lang, G.M. and Sehon, A.H. "Synthesiε, isolation and characterization of conjugates of ovalbumin with monomethoxypolyethylene glycol using cyanuric chloride as the coupling agent.", Anal. Biochem . , 165: 114, 1987, incorporated herein by reference in its entirety.) Monomethoxypolyethylene glycol (2.5 g. mol wt 5590, Union Carbide) was diεεolved with warming in anhydrous benzene (40 ml) and a portion of the benzene (20 ml) was removed by diεtillation to azeotrope off any water in the polymer. Cyanuric chloride {(CNC1)3, 0.83 g, Aldrich, recryεtallized from benzene} was added under nitrogen followed by potassium carbonate (0.5 g. anhydrous powdered) and the mixture stirred at room temperature for 15 hours.
The mixture was then filtered under dry nitrogen and the filtrate mixed with anhydrous petroleum ether (ca 50 ml, b.pt. 30-60°C) in order to precipitate the polymer. The polymer was separated by filtration under nitrogen, disεolved in benzene (20 ml) and reprecipitated with petroleum ether. Thiε proceεε was repeated seven times to insure that the polymer was free of any reεidual cyanuric chloride. The active intermediate waε finally dissolved in benzene, the solution frozen and the benzene sublimed away under high vacuum to leave a fine white powder.
Elemental analysiε of the intermediate confirmed that it contained 2 chlorine atoms. The intermediate, correεponding to C2563H30770127 N3Cl2 with an average molecular weight of 5,738 daltonε would have a theoretical compoεition in percentageε of C, 53.65; H, 8.92; N, 0.73; Cl, 1.24; which agreeε with its determined composition of C, 53.51; H, 8.89; N, 0.77; Cl, 1.08.
The chloride content of the intermediate was also determined by hydrolysiε and titration of the chloride releaεed with εilver nitrate. Thuε, the activated intermediate (120 mg) was disεolved in water (10 ml) and the pH adjuεted to 10 with dilute sodium hydroxide. After heating at 90°C for two hours, the solution was cooled and the chloride titrated with silver nitrate (0.001N), using a chloride ion selective electrode to indicate the endpoint. The chloride content of the activated intermediate was found to be 2.1, consistent with the structure shown above.
The OVA {40 mg, purified by chromatography on Ultrogel® AcA-54 (LKB, Bromma , Sweden)} was disεolved in sodium tetraborate buffer (4 ml, 0.1 M, pH 9.2) and the activated mPEG added to the solution at 4°C. The amount of activated mPEG was varied to prepare conjugates of differing degrees of polymer subεtitution. Mole ratios ( PEG/OVA) uεed to prepare εpecific conjugates are given in Table 2.
The polymer-protein mixture was stirred for one half hour at 4°C and then one half hour at room temperature. The reaction mixture was desalted by either dialyzing for four dayε against running diεtilled water or by paεεing through a column of Sephadex® G-25 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) .
A DEAE-cellulose or DEAE-Sephacryl® (Pharmacia Fine Chemicalε AB, Uppsala, Sweden) column (5 cm by 30 cm) was equilibrated with phosphate buffer (0.008 M, pH 7.7). The salt free OVA conjugates were applied in water and the free (unbound) mPEG washed through the column with the pH 7.7 buffer. Free mPEG was detected on thin layer chromatography {Camag (Kieselgel DSF-5, Terochem Lab Ltd, Alberta) eluant 3:1 chloroform/methanol} using iodine vapor for development. After removal of the free mPEG from the ion-exchange column, sodium acetate buffer (0.05 M pH 4.0) was uεed to elute the conjugate. The conjugate fractionε were dialyzed and lyophilized to give the dry conjugateε.
Tolerogenε of εaporin, ricin a chain, birch pollen allergen Bet v 1 and recombinant ragweed allergen R8.1 have alεo been prepared by the above method, with an induction of tolerance to these antigens. TABLE 2
Preparation of OA-mPEGn Con jugates
Conjugates'1 Preparation ratiob % mPEGc ,e %0Ad'β
OA-mPEG3.2 10 : 1 26 " 70
OA-mPEG6.6 25 : 1 36 47
OA-mPEG7.6 25 : 1 42 47
OA-ιτ.PEGio.6 50 : 1 51 4 1
OA- PEGu .9 50 : 1 52 . 4 38
a The degree of substitution , n , is calculated by the f ormula
% mPEG mol wt OA
% OA X mol wt mPEG Mole ratio mPEG:OA based on a molecular weight of
5.740 for mPEG-dichlorocyanurate and 44.460 daltons for OA. c The percentages of mPEG by weight were determined by nuclear magnetic resonance (NMR) . d The percentages of protein by weight were determined by the biuret method.
Determination of the immunosuppressive effect of the IgE response of different OVA-mPEGr conjugateε
The reεultε of experiments illustrated in Fig. 8 clearly demonstrate the stringent dependency of the suppreεsogenicity of mPEG conjugates on their molecular composition. Thus, whereas treatment of groupε of four (B6D2)F1 mice each with 50 μq of OVA-mPEG32, or OVA-mPEG66, or OVA-mPEG76 one day prior to intraperitoneal immunization with the εenεitizing dose, consisting of iμq of OVA and 1 mg Al(OH)3, led to esεentially complete (99-100%) abrogation of the primary anti-OVA IgE reεponεe, as measured —on day 14 after immunization— by PCA in hooded rats, the more substituted conjugates, i.e. OVA mPEG106 and OVA-mPEG.1 9, inhibited the anti-OVA IgE response, respectively, only to the extent of 94% and 50%. In this and the following examples, the weights of the conjugateε given correεpond to their protein content.
Long lasting εuppression of the IgE reεponse by protein mPEG conjugates in contraεt to a tranεient εuppreεεive effect of unconjugated protein
Even unmodified OVA was capable of downregulating the primary IgE response in relation to the response of control mice which had received PBS instead of OVA or conjugates. In this experiment three groups of four (B6D2)F1 mice each received phosphate buffered εaline, or 50 μq of OVA-mPEG, 5 or 50 μq of OVA. All animalε were bled on day 10, 14, 21, 27, 35, 42 and 49 and their IgE titerε were determined by PCA in hooded ratε . As illustrated in Table 3, it is important to point out that whereas the suppressogenic effect of OVA-mPEG conjugates was long-lasting, the down regulating effect of free OVA was of short duration and, in actual fact, its administration predispoεed the animalε to an anamnestic response which reached, after booster immunization (administered on day 28) , IgE antibody levelε equivalent to those of control animalε which had received PBS and the two εensitizing doεeε of one antigen. The reεultε given in Table 3 clearly demonεtrate that a tolerogenic conjugate injected prior to repeated adminiεtration of the corresponding free protein essentially abrogated the immune response.
TABLE 3
Effect of administering 50 μg of 0A-mPEG<,.5 or of free OA one day prior to immunization
Day of bleeding after PCA titers for groups of primary iπvmurlization mice treated with
PBS OA OA-mPEGi ■,
10 5,120 40 < 4
14 1,940 40 < 4
21 1,280 40 < 4
27 640 40 < 4
35 1,920 1,920 160
42 2,560 1,280 160
49 5,120 N.D.* 160 On day 28 all three groups received a booster dose of the sensitizing OA preparation. * N.D. = not determined.
The effect of different doseε of the tolerogen on the IgE response
Each OVA-mPEG conjugate was injected into groups of 4 mice each at the four doseε of 10 μ , 50 μq , 150 μq and 600 μ . The control group of mice received PBS as placebo.
As is evident from Fig. 9, treatment with different conjugates at doses of 10 μq and 50 μq per mouse revealed marked differences in their suppressogenic capacity. It is alεo to be noted that at a doεe of 150 μ , all conjugateε were highly suppressive and at 600 μq , all the compounds tested suppreεsed completely the IgE response.
The effect of different doseε of the tolerogen on the IaE response
The sera used in Fig. 10 to illustrate the effect of tolerogenic conjugates on the IgG responεe were the same as thoεe uεed in Fig. 8. Aε illuεtrated in Fig. 10, adminiεtration of 50 μq of 0VA-mPEG76 resulted in the maximal εuppreεεion, i.e. of the order of 98% of the primary anti-OVA IgG response, which was determined 14 days after the first injection of the εenεitizing doεe of OVA by a radio-immunoaεsay employing the paper radio immunosorbent procedure (Kelly, K.A. et al.; J. Immunol. Meth. 39 (1980) p. 317-33) utilizing OVA bound to the paper and with 125I-labelled affinity purified sheep antiεerum to ouεe IgG.
Example 4
The εuppressive effect of OVA-mPEG10 on IgM, IgG, and IgE plaque forming cells (PFC) in spleen and lymph nodes. One mg of OVA-mPEG10 (containing 10 mPEG groups with an average mol wt of 10,000 daltonε, which were coupled per OVA molecule by the εuccinic anhydride method (Wie, S.I. et al., Int. Archs. Allergy appl. Immun. 64, 84 1981)) or PBS was administered intraperitoneally to each group of four (B6D2)F1 mice each one day prior to immunization with 1 μq of DNP3-OVA in 1 mg A1(0H)3.
On several days thereafter the spleen, as well as the mesenteric, parathymic and inguinal lymph nodeε were removed and aεεayed for IgM, IgG, and IgE anti-DNP PFC (Rector, E.S. et al., Eur. J. Immunol. 10, p. 944-49 (1980). In Table 4 are given the numbers of PFC in the above tisεueε 10 days after immunization; from theεe data it is evident that treatment with this tolerogen markedly reduced the number of IgM, IgE, and IgG PFC in all tissues examined. Therefore, these results support the claim that the tolerogens shut off the immune response rather than neutralize the circulating antibodies.
TABLE 4
The effect of OA-mPEG10 on the suppression of IgM, IgG, and IgE plaque forming cells (PFC) in spleen and lymph nodes
Anti-DNP PFC per 10B cells from different tissues* Antibody Parathymic Mesenteric Inguinal
Class Treatment Spleen Nodes Nodes Nodes
IgM PBS 2,150 2,950 Nd Nd **
OA-mPEG 900 200 Nd Nd
IgG PBS 15,350 78,550 5,000 Nd
OA-mPEG Nd 1,300 Nd Nd
IgE PBS 10,410 16,530 11,140 300
OA-mPEG 500 950 400 Nd
Each tissue sampling represents a pool from 4 mice
The above experiments establish the immunosuppressive effects discussed above and the effectε at variouε doεages.
In addition, utilizing the hu-PBL-SCID mice, it was demonstrated that in accordance with the phenomenon of "linked immunological suppresεion", cross-specific suppresεion of the human antibody reεponse could be induced to murine mAbs which differ in their antigen binding specificitieε from those of the murine mAbs which had been incorporated into the tolerogenic conjugateε, on condition that both mAbs shared the same heavy and light chains. Thus, that pan-specific suppression of the "human" antibody responεes against murine monoclonal antibodies (i.e., HAMA responεeε) of the IgG claεs could be achieved with 8 tolerogenic mPEG preparations, each conεiεting of one of the 4 gamma chainε and of one of the two typeε of light chainε of murine IgG (Bitoh, S., Lang, G.M. , Kierek-Jaεzczuk, D., Fujimoto, S. and Sehon, A.H. Specific immunoεuppresεion of human anti-murine antibody (MAMA) responses in hu-PBL-SCID mice. Hum. Antibod. Hybridomas 4_:144-151, 1993).
Thus, in accordance with the preεent invention, prior to beginning of gene therapy, i.e., prior to insertion of a new gene into a host which is required for expresεion of a protein beneficial to the hoεt, e.g., one of the deficient clotting factorε or enzymeε, it iε essential to render the host tolerant to the protein in question by the use of the invention described.
Example 5
The expresεed protein material of the cystic fibrosis transmembrane conductance regulatory gene (CFTR) (Genzyme, Cambridge, Masεachuεetts) for the treatment of cystic fibrosis is dissolved in sodium tetraborate buffer (4 ml, 0.1 M, pH 9.2) and the activated mPEG added to the olution at 4°C.
The amount of activated mPEG is varied to prepare conjugates of differing degrees of polymer subεtitution. Different mole ratios (mPEG/gene product) are used to prepare specific tolerogenic conjugateε aε described earlier. The polymer-gene product mixture is stirred for one half hour at 4°C and then one half hour at room temperature. The reaction mixture is desalted by either dialyzing for four days againεt running distilled water or by pasεing through a column of Sephadex® G-25 (Pharmacia Fine Chemicalε AB, Uppεala, Sweden) .
A DEAE-celluloεe or DEAE-Sephacryl® (Pharmacia Fine Chemicalε AB, Uppsala, Sweden) column (5 cm by 30 cm) is equilibrated with phosphate buffer (0.008 M, pH 7.7). The salt free mPEG conjugateε of the cyεtic fibroεiε gene product are applied in water and the free (unbound) mPEG washed through the column with the pH 7.7 buffer.
Free mPEG is detected on thin layer chromatography {Camag Kieselgel DSF-5, Terochem Lab Ltd, Alberta) eluant 3:1 chloroform/methanol} using iodine vapor for development.
After removal of the free mPEG from the ion-exchange column, sodium acetate buffer (0.05 M, pH 4.0) is used to elute the conjugate. The conjugate fractions are dialyzed and lyophilized to give the dry conjugateε. Conjugateε of the CFTR gene are administered to a patient at least one day prior to transfer of the cystic fibrosis transmembrane conductance regulator gene to lung tissue using recombinant adenoviral vectors or liposomes.
Example 6
The expressed protein material of the low density lipoprotein receptor (LDLr) gene used in the treatment of familial hypercholesterole ia iε dissolved in sodium tetraborate buffer (4 ml, 0.1 M, pH 9.2) and the activated mPEG added to the solution at 4°C. The amount of activated mPEG is varied to prepare conjugates of differing degrees of polymer substitution.
Different mole ratios (mPEG/gene product) is used to prepare specific tolerogenic conjugates as described earlier. The polymer-gene product mixture is stirred for one half hour at 4°C and then one half hour at room temperature. The reaction mixture is desalted by either dialyzing for four dayε against running distilled water or by passing through a column of Sephadex® G-25 (Pharmacia Fine Chemicals AB, Uppsala, Sweden) .
A DEAE-cellulose or DEAE-Sephacryl® (Pharmacia Fine Chemicals AB, Uppsala, Sweden) column (5 cm by 30 cm) is equilibrated with phosphate buffer (0.008 M, pH 7.7).
The salt free mPEG conjugates of the LDLr-gene products are applied in water and the free (unbound) mPEG washed through the column with the pH 7.7 buffer. Free mPEG is detected on thin layer chromatography {Camag (Kieselgel DSF-5, Terochem Lab Ltd, Alberta) eluant 3:1 chloroform/methanol} using iodine vapor for development.
After removal of the free mPEG from the ion-exchange column, sodium acetate buffer (0.05 M, pH 4.0) is used to elute the conjugate. The conjugate fractions are dialyzed and lyophilized to give the dry conjugates.
Conjugates of the LDLr gene product are administered to a patient. Hepatocytes are grown in the laboratory and genetically altered with a murine retroviral vector containing LDLr gene. The cellε are reinf sed through the hepatic artery to the liver of the patient at leaεt one day after adminiεtration of the conjugate.
Table 5 εhowε a liεt of gene therapy εyste ε which have been approved by the Recombinant DNA Activities Committee of the National Instituteε of Health. However, no consideration appears to have been given to overcoming the potential complications due to the host mounting an immune response against the reεpective gene products.
Thus the present invention can readily be adapted to any gene therapy protocol and is generally applicable to the administration of any therapeutic immunogenic material and not just the specific examples listed above.
Gene therapy according to the existing art may be applied to εomatic cellε or germ line cells by methods known εuch as gold electroporation, microinjection or jet injection, or other methods aε εet forth in Sambrook et al. "Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Preεε (1989)" incorporated herein by reference in itε entirety. Thus the invention provideε for a method for treating by gene therapy a mammal with a therapeutic amount of a biologically active antigenic material or itε expreεεion product. To retain the effectiveneεε of εaid antigenic material (ε) from counteraction by an antibody (ies) produced against it (them); it is eεεential to εuppreεs the capacity of the recipient of the gene to mount an antibody responεe(s) to εaid biologically active antigenic material (ε) .
This method comprises conducting gene therapy by adminiεtering to a mammal an immunoεuppreεεive effective amount of a tolerogenic conjugate consisting of a protein coupled to monomethoxypolyethylene glycol (mPEG) having a molecular weight of about 2,000-10,000 daltonε, wherein administration of said tolerogenic conjugate is at leaεt one day prior to adminiεtration of a gene therapy vector encoding a gene for a protein, wherein εaid protein iε identical to said protein which is coupled to mPEG, and wherein said method resultε in the εuppression of an immune response and in the development of tolerance to the protein expressed by said gene encoded by said gene therapy vector.
Alternatively the method of conducting gene therapy includes a) administering to a mammal an immunosuppreεεive effective amount of a tolerogenic conjugate conεiεting of a protein conjugated to monomethoxypolyethylene glycol (mPEG) having a molecular weight of about 2,000 to 10,000 daltonε, wherein adminiεtration of εaid tolerogenic conjugate is at least one day prior to adminiεtration of DNA, RNA or mRNA and encoding a protein administered for gene therapy, wherein the encoded protein iε identical to εaid protein which is conjugated to mPEG, and wherein said method reεultε in the suppreεεion of an immune reεponεe and in the development of tolerance to encoded protein of said DNA, RNA or mRNA adminiεtered for gene therapy. The method εuppreεses the formation of about 98% of antibodies against said antigenic genetic material or its product.
Example 7
The method of εuppreεεion of the humoral and cellular immune responses of the invention finds application in εpecific area of gene and protein replacement therapy. In particular, patientε with Hemophilia A are from birth deficient of the gene which serves as the template for the production of clotting Factor VIII (F.VIII) . The current replacement therapy involves the frequent adminiεtration of F.VIII, which iε iεolated from blood of normal volunteers or synthesized by recombinant technology. Either of these therapies is extremely expensive and, most importantly, the delay between the onset of bleeding and administration of the factor may lead to tisεue damage.
J. Walter and K.A. High report encouraging results obtained for the in vivo and ex vivo syntheεiε of F.VIII (and of F.IX) by the lateεt methodε of gene therapy with the aid of naturally occurring animal (mouεe and dog) modelε of the diεeaεe. In vivo gene therapy is the treatment of choice, since it would provide sustained synthesis of the missing clotting factor and obviates the need for frequent treatments. Therapies involving the adminiεtration of natural or recombinant clotting factor, the transgenic clotting factor, as well as the proteins expresεed by the companion adenoviral vector, elicit the formation of the respective anti-factor antibodies ("inhibitors") and anti- vector antibodies which may undermine the effectiveness of gene therapy.
The transgenic protein as well as the vector protein (ε) , in addition to being excreted as such by the tranεfected cellε, may alεo be degraded with the cell into peptideε incorporating εome of the epitopes of the protein(ε). These foreign peptideε, when preεented in association with MHC molecules on the cell membrane, are believed to elicit the production of cytotoxic cellε capable of deεtroying the transfected cells.
Immune response can be blunted by a variety of immunosuppressive regimens, which have been reviewed in relation to treatment of hemophilia by Bertrop, et al in an article based on the discuεεions at a joint WHO and World Federation of Hemophilia meeting (2) . Most of these therapies, as in the case in order transplantation, are nonspecific and involve the continual administration of immunosuppressive drugs which are expensive and may have deleterious side effects.
The same gene therapy protein complicating featureε apply also to adenoviral vector proteins. Although it is possible that lesε immunogenic or totally non-immunogenic adeno-associated virus (AAV) vectors will be developed and that vectorless inεertion of tranεgenes into the hoεt's DNA are achieved, the immunogenicity of the transgenic protein still represents a serious problem. Indeed, this phenomenon is evidence in canine anti-Factor VIII antibody generated in hemophilic animals treated with a homologous factor VIII transgene.
Evidence for the utility of his discovery for the induction of long term antigen (Ag) -specific suppression of both antibody and cytotoxic cell responses by pretreatment of the host with the tolerogenic conjugate of the particular Ag and an optimal number (n) of molecules of monomethoxypolyethylene glycol (mPEG) , i.e., Ag(mPEG)n is presented below. The administration of only a single dose of 100-200 μq of tolerogenic mPEG conjugates of a variety of antigens sufficed to induce specific long term suppression of the immune response in mice and rats, in spite of multiple injections of the respective antigens over extended periods. Most importantly, mPEG has been shown to be a biocompatible polymer and has been used for the synthesis of diverse pharmaceutical products.
The method described below iε readily adapted for induction of (F.VIII) -εpecific suppresεion of the immune response in (F.VIII) -deficient recipients irrespective of the origin of F.VIII.
Prevention of Therapeutic Complications due to the Immunogenicity of Recombinant Biologic Response Modifiers (BRMs)
A major challenge for the biotechnology of potentially therapeutic recombinant BRMs is to overcome their immunogenicity, as is the case for natural foreign proteins (e.g., monoclonal antibodieε, enzymeε, toxinε, hormones, heterologous F.VIII) . The therapeutic effectiveness of even the corresponding chimeric or humanized recombinant proteins is undermined by their immunogenicity which is often due to only minor conformational differences with respect to the three-dimensional structures of their corresponding natural progenitors.
Consequently, administration of some recombinant BRMs electε the production of complementary, neutralizing and/or blocking antibodieε by the hose. These antibodies intercept the BRMs and prevent them from reaching their target cellε. Moreover, depending on the class of antibodies elicited, the patient may develop serum sickness, renal and hepatic toxicity, and even anaphylactic shock in severe cases.
The inventors have developed a method for conversion of a variety of immunogenic proteins or immunogenic fragments of the proteins (P) to tolerogenic derivatives. This conversion involveε the coupling of an optimal number (n) of molecules of monomethoxypolyethylene glycol (mPEG) onto the protein antigen in question. The protocol for the effective induction of εpecific immunoεuppression in mice and ratε conεists of two εtepε:
Step I: Injection of tolerogenic conjugates of the appropriate immunogenic BRM, i.e. P(mPEG)n; Step II: Administration of the unmodified P, about 7 days after injection of the immunosuppreεεive P(mPEG)n. Thereafter, unmodified biologically active P can be injected repeatedly over extended periodε without further injection of P(mPEG) . For example, in mice εuppreεεion of antibody reεponses to heat aggregated human monoclonal (myeloma) IgG, referred to as HaHIgG, was shown to persist up to 540 days in spite of multiple injections of HaHIgG at different intervalε over thiε εpan of time.
The reaεon for the interval of about 7 dayε betweens Steps I and II is to allow propagation of P-specific suppressor T (Ts) cells which are activated by P(mPEG)n (4). Additional injections of the unmodified P maintain the proliferation of these cellε, which suppreεε the εpecific T helper cellε that alεo recognize the epitopes of the same P, though not necesεarily the εame epitopeε as those recognized by the Ts cells.
It is also noted that pretolerization of mice to a given protein AgA by treatment with AgA(mPEG)n resultε in their becoming i munologically unresponsive to an unrelated AgB, on condition that AgB is injected into these mice in the form of a covalent adduct with AgA, i.e., as AgA-AgB, but not as a mixture with AgA. This cognate phenomenon of "linked immunological suppression" is also conferred on naive mice by treating them first with TsFA nd then with the AgA-AgB adduct. Obviously, any clone of Tε cellε recognize only one epitope (i.e. one antigenic determinant) of the reεpective high molecular weight multi-determinant protein. Application to suppresεion of the antibody response of the human lymphoid syεtem and gene therapy
Experimentε were performed in hu-PBL-SCID mice. The abbreviation "hu-PBL-SCID mouεe" denotes a mouse with severe combined immunodeficiencies, which has been engrafted with human peripheral blood leucocytes. This syεtem represents the closest in vivo model for a functional human lymphoid system. The inventors demonstrated that (i) mPEG conjugateε of a foreign P, i.e., murine mAb, induced εpecific εuppreεεion of human anti-P antibodieε, (ii) this suppression waε due to the generation of human P-specific CD8+ T cells, and (iii) the suppression was transferable with these human Ts cells into secondary naive recipient SCID mice that had been engrafted with the leucocytes of the original donor of the cells (9, 10).
In addition, utilizing the hu-PBL-SCID mice, it was demonstrated that, in accordance with the phenomenon of "linked immunological suppression', cross-specific suppresεion of the human antibody reεponse could be induced to murine mAbs which differ in their antigen binding specificities from those of the murine mAbs which had been incorporated into the tolerogenic conjugated, on condition that both mAbs shared the same heavy and light chains.
Thuε, it waε concluded that pan-εpecific εuppression of the "human" antibody responses against murine monoclonal antibodies (i.e., HAMA responses, including anti-diotypic responses) of the IgG class could be achieved with only eight tolerogenic mPEG preparations, each consisting of one of the four gamma chains and one of the two types of light chains of murine IgG.
The two-step method described above iε demonstrated to be suitable for εuppreεεing the activation of cytotoxic T cells (CTLε) , as is the case for gene therapy involving the transfection of a "new" gene into an immunocomponent patient who has been deficient from birth of the particular gene required to express the correεponding protein. It iε eεtabliεhed that the εame mPEG conjugate, i.e., OVA(mPEG) 10, had the dual capacity of εuppressing the antibody response and the activation of Ag-specific CTLs (5) . For these experimentε, the gene therapy model εyste consisted of E.G7-OVA target cells, which had been generated by Dr. M. Bevan by transfecting syngeneic (C57BL/6) Ia-EL-(H-2b) thymoma cells with the OVA cDNA gene. Thus, whereas control mice which had been primed with OVA in CFA developed OVA- specific CTLs that lysed the target cells, induction of both Abs and CTLs to OVA was abrogated in mice which had been immuno-suppreεεed by pretreatment with tolerogenic OVA(mPEG)10 conjugated, but not with unmodified OVA.
Experiments with Human Factor VIII (hF.VIII) Preparation of hF.VIII-mPEG conjugates
Recombinant hF.VIII has a molecular weight of 330,000 and consistε of 2,332 amino acids, 160 of which are lysines (13) . The firεt conjugate will be εyntheεized using one of our proven methods for the preparation of tolerogenic derivatives of diverse protein antigens with molecular weights in the range of 51,000-150,000 Da, vis., recombinant human insulin (rhi) , εaporin, chain A of ricin, a major birch pollen allergen (Bet v 1) , a recombinant ragweed allergen (rδ.l), ovalbumin (OVA), and human and mouse IgG. With the exception of rhi , the common feature shared by the other tolerogenε iε that their compositionε conεiεted of 2 to 3 mPEG moleculeε (coupled via e-amino groups of lysines) per 100 amino acids. Human F.VIII has 6-7 lysineε per 100 amino acids and it is anticipated, on the basis of our experience with other tolerogens, that at least 50% of them would be available for coupling.
Each of the aggregate-free proteins is reacted with a large excess of the activated mPEG derivative, viz., mPEG p- nitrophenyl carbonate (mPEG-NPC) , which reacts preferen- tially with e-amino groups and which was custom-synthesized by Shearwater Polymers Inc. (Huntεville, AL) utilizing a highly purified preparation of mPEG (average Mr-3200 Da) containing less than 2% of diols. The mPEG-NPC iε used in a large molar excess in relation to the total lysine content of each protein because of its substantial hydrolysis at the relatively high pH which is required for coupling with e-NH2 groups . mPEG-NPC is used in an 8-fold higher molar concentration than that of the lysines of a given protein. For example, 47 mg of OVA corresponding to lmM of OVA (which contains 19 lysines per molecule of OVA) is reacted with 486.4 mg (152 mM) of mPEG-NPC for the synthesis of tolerogenic OVA(mPEG)n conjugates containing an average of 10-11 molecules of mPEG per molecule of OVA; the subscript n, refers to the average degree of conjugation. For the synthesis of tolerogenic mPEG conjugates of mouse or human IgG, 150 mg of IgG corresponding to 1 mM of IgG and containing on the average 90 lysineε per molecule of IgG, waε reacted with 2.3 grams of mPEG-NPC. The resulting tolerogenic IgG(mPEG)n conjugates contained on the average 25 to 35 mPEG molecules per molecule of IgG.
For the synthesiε of hF.VIII (mPEG) n conjugated, hF.VIII is disεolved at 10 mg/ml in 0.1 M borate buffer, pH 9.7 and then mixed quickly with mPEG-NPC which iε dissolved in an identical volume of double distilled water (DDW) . Prior to initiating this procedure it is establiεhed that hF.VIII iε stable at pH 9.7 in borate buffer. The reaction mixture iε transferred to a dialysis bag, which should then be suspended in 4 liters of 0.05M borate buffer (pH 9.7) and the reaction is continued with conεtant εtirring for 1 hour at room temperature and overnight in the cold; the large volume of buffer outεide the dialysis bag serveε aε a pH- stat. Finally, for the isolation of the conjugate, the content of the dialysiε bag is applied onto a Pharmacia BioPilot gel filtration column which has been equilibrated with DDW. The conjugate is in the void volume, followed by
(i) hydrolyzed mPEG-NPC, and (ii) the buffer saltε and the p-nitrophenyl released in the reaction. The isolated conjugate is lyophilized and stored at -20°C.
The method of synthesis of the tolerogenic hF.VIII (mPEG)n conjugates in a pure form involves a series of stepε, including isolation of the pure conjugate by gel filtration chromatography, which leadε to yieldε of the order of 50-70%.
Immunization of normal inbred BALB/C mice, normal out-bred CD-I mice and normal outbred Sprague-Dawley rats with hF.VIII in the presence of adjuvant
In view of the low immunogenicity of hF.VIII, to ensure a consiεtent immune reεponεe in mice and ratε, hF.VIII iε adminiεtered in an adjuvant. Five to εeven week old female mice of both εtrainε receive two ip injectionε of a εuspension of hF.VIII in Al(OH)3 at an interval of 21 days. The strain of mice which mount the most consiεtent anti- hF.VIII antibody titer iε εelected for further experimentε with hF.VIII in Al(OH)3. Freund'ε adjuvant iε uεed with the Sprague-Dawley rats because this adjuvant has been proven to induce high titered Ab responses to hF.VIII in this strain of ratε. The rats are immunized sc with hF.VIII in Freund's adjuvant; the firεt injection containε complete Freund's adjuvant and the εecond injection after an interval of 3 weeks contains incomplete Freund's adjuvant. The mice and rats are bled at weekly intervals and the antibody production is determined by ELISA.
Repeated treatment of normal mice and rats with hF.VIII in the absence of adjuvant
This immunization regimen resembles more closely that administered to hemophilic patientε. Both BALB/C and CD-I ice and the Sprague-Dawley ratε are injected with hF.VIII without adjuvant and their sera is asεayed for the preεence of antibody by ELISA. The doεe of hF.VIII and the interval of time between repeated injections of hF.VIII is refined in this series of experiments. The presence of antibody with a significant titer in at least 50% of the animals justifies continuation with this immunization strategy. Induction of tolerance
Several hF.VIII (mPEG) n conjugates differing in "n" are be synthesized. Each of the hF.VIII (mPEG) conjugateε iε tested for its degree of tolerogenicity in mice as described In step I, 4 groups of 4 mice each receive ip, respectively, 100, 200, 400 and 800 mg of hF.VIII (mPEG) n. The mice are be given the first immunizing dose of hF.VIII in adjuvant 7 days later, and are re-immunized after 21 days. control mice (4 mice per group) are injected ip with unmodified hF.VIII, or with diluent in lieu of conjugate. All mice are bled at weekly intervals after primary immunization for assaying their sera by ELISA.
Next, the effective tolerogenic doεe of each conjugate required for suppreεsing the immune response when hF.VIII is injected with or without adjuvant is established by the above protocol. Control animals are given unmodified hF.VIII or diluent in lieu of conjugate. The animalε are be bled at weekly intervalε and the εera aεεayed by ELISA. The moεt immunoεuppreεεive conjugate in mice are teεted in Sprague-Dawley ratε. In accordance with the procedures used for mice, the conjugate is injected 7 days prior to primary immunization. Four groups of 6 ratε each receive ip, respectively, 100, 200, 400 and 800 mg of hF.VIII (mPEG) . Two control groups of 6 ratε each are injected ip, reεpectively, with unmodified hF.VIII or diluent in lieu of conjugate. The ratε are re-immunized 21 days after the primary immunization. All rats are bled at weekly intervalε after primary immunization for the determination of their anti-hF.VIII levels by ELISA. Im unosuppression of an ongoing immune response to hF.VIII by a protocol involving a combination of hF.VIII (mPEG) conjugate and ycophenolic acid or other immunosuppressive drug.
It was establiεhed in experiments, utilizing OVA aε the antigen and mycophenolic acid (MPA) , as the immunosuppressive drug, that (i) whereas the ongoing antibody response was abrogated while MPA was administered, the responεe reappeared after the administration of MPA had been diεcontinued, and (ii) adminiεtration of MPA did not affect the induction of εuppreεεion by mPEG conjugateε.
Therefore, the doεe of MPA required for abrogation of an ongoing immune response to hF.VIII in mice and in Sprague-Dawley rats is establiεhed.
Conεidering that some of the patients may have been given hF.VIII prior to receiving the tolerogenic mPEG- conjugate of hF.VIII it is important to establish if co- administration of an immunosuppreεεive doεe of MPA or other immunoεuppreεεive drug and of a tolerogenic doεe of hF.VIII (mPEG)n may lead to long-term εuppreεsion of the ongoing immune response to hF.VIII.
Tolerance in hemophilic mice
Recent studieε in hemophilic mice indicate that they too develop neutralizing antibodies to human factor VIII upon exposure to this protein (17) . With this as background, as the tolerogenic capacity of one or more hF.VIII (mPEG)n conjugates is established their immunosuppressive capacity in hemophilic mice when administered prior to injection of hF.VIII iε tested. The degree of immunoεuppreεεion both by ELISA and in terms of the effect on clotting time as measured by the Bethesda test is established. If co-administration of MPA or other immunosuppressive drug with the tolerogenic hF.VIII (mPEG) conjugate leads to effective suppression of an ongoing anti- hF.VIII antibody response in mice and rats, the usefulness of this regimen in hemophilic mice producing "inhibitors" is establiεhed. Hemophilic dogε are alεo uεed in the inveεtigationε .
Example 8 Tranεplantation Example
Rejection of tranεplanted cells and organs is also an application for the mPEG strategy. Rejection resultε from the recognition of proteinε encoded by the MHC, HLA in humanε, by CD4+ and/or CD8+ T cellε. Many antigens have been expresεed in large amountε by recombinant technology. Purified HLA antigenε can be modified with mPEG and the correεponding conjugates injected into patients prior to organ or cell transplantation. For antigens that have not yet been identified or for which recombinant proteins are not yet available, the inventorε can modify peripheral blood cellε or membranes from these cells with mPEG. This is feasible since it has been εhown that erythrocyteε can be modified with mPEG. MHC εpecific tolerance iε induced with mPEG modified antigenε; hence εuppreεsion of the immune responεe and of the rejection of tranεplantε by prior pretreatment by the appropriate MHC-mPEG conjugate iε poεεible with this treatment or in conjunction with reduced doses of immunosuppressive drugs that have various side effect, including inhibition of all immune responses.
The invention provides a method of preventing an immune rejection of organ transplantε compriεing administering an effective amount of Ag(mPEG) conjugate to induce tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses.
The present invention targets mPEG to antigen-specific T cells, including CD8+ T cells. The inventors have used published methods for stimulating CD8+ T cells with exogenous antigens by delivery in adjuvant to test this idea. Hence, mPEG-OVA complexes were injected before priming mice with OVA in Freund's complete adjuvant. About two weeks later, mice were sacrificed and their spleen cells assayed for development of OVA specific cytolytic T cells (CTL) .
The results showed that mPEG inhibited priming for OVA-specific CTL and thus caused tolerance that prevented normal εtimulation. In addition, thiε technique is extended to coupling of various cells with mPEG. Cells conjugated with mPEG are uεed to induce tolerance to specific MHC antigens of a given organ graft donor prior to organ transplantation.
Example 9
Autoimmunity Example
The inventors provide approaches that are effective to induce tolerance and thus could be used as alternatives to treat autoimmunity. mPEG modification of such antigens render them tolerogenic and have the potential to reverse these autoimmune diseases. This therapy can be extended to any autoimmune disease, in which a tissue specific antigen can be identified. It is believed that generations of a particular number of Tε cellε may reverse theεe autoimmune diεeaseε. Thiε therapy may be extended to any autoimmune diεeaεe aεεuming that the culprit tiεsue specific antigen is identified, isolated or syntheεized for preparation of the corresponding mPEG conjugate.
Several autoimmune diεeases are known to involve cytokine producing CD4+ T cells and/or CD8+ T cellε that recognize auto-antigenε. Such T cells can transfer disease in normal syngeneic recipientε, aε iε the case within the same strain of mice or rats. In experimental multiple sclerosis, and the related model of experimental autoimmune encephalomyelitis (58) , T cells have been shown to recognize several antigens. Such T cells can transfer different diseaεe into normal εyngeneic recipientε.
In multiple εcleroεiε and in experimental autoimmune encephalomyelitiε, T cellε have been εhown to recognize several antigens that are restricted in expresεion to the central nervouε system. These include: myelin basic protein, proteolipid protein and myelin oligodendrocyte antigen. T cells recognizing insulin, glutamic acid decarboxylase, and other, as yet unidentified antigens expressed by beta cells, have been isolated from non-obese diabetic (NOD) mice, BB rats, and diabetic humans. mPEG treated cells can be uεed to induce tolerance to certain cellε in the body that are known to be the targetε of autoimmunity . For example, beta cellε from the pancreas are modified with mPEG and injected into nonobese diabetic (NOD) mice before or after the appearance of spontaneous diabetes.
The tolerogenic properties of the conjugates of the invention have the capacity to inactivate (in vivo in PCA reactions) IgE-sensitized rat skin mast cellε, and BE cells. (24). Injection of OVA(mPEG)10 into skin sites inhibited the release of mediators of anaphylaxis on subεequent injection of 1 mg DNP44-BSA and O/lmg. of Bet v 1. In contrast, injection with unmodified OVA or unrelated mPEG conjugate prior to i.v. challenge with DNP44BSA or Bet v 1, did not affect the PCA reactions to either of these two antigens.
The conjugates of the invention, where the antigen is an allergen, inactivate mast cells. This resulted in specific suppreεεion of a primary immune reεponse after adminiεtration of the conjugateε of the invention. The allergenicity of the conjugateε of the invention are 10-500 fold lower than that of the original allergen.
The method of the invention may be practiced to treat a condition selected from allergies and autoimmune diseases by inducing tolerance to an antigen (Ag) or εelf-protein or autoantigen, in both humoral and cell mediated immune reεponεes comprising adminiεtering an effective amount of Ag(mPEG) conjugate to induce tolerance to an antigen (Ag) .
The invention treats organ-specific autoimmune diseases in animal and rejection of DNA transfected cells of their products by administration of mPEG conjugates of autoantigens selected from the group consiεting of collagen- induced arthritiε by type II collagen, experimental autoimmune encephalomyelitis by myelin basic protein, and diabetes in NOD mice by insulin to induce tolerance to an antigen (Ag) in both humoral and cell mediated immune reεponεes.
The principles of the invention provide a method of treating organ-specific autoimmune diseaεeε in animal and rejection of DNA transfected cells of their products comprising administration of mPEG conjugateε of autoantigenε εelected from the group consiεting of collagen-induced arthritiε by type II collagen, experimental autoimmune encephalomyelitis by myelin basic protein, and diabetes in NOD mice by insulin to induce tolerance to an antigen (Ag) in both humoral and cell mediated immune responses.
Example 10
A method of treating established allergic responεes or established autoimmune disease states involves a combination therapy as follows: i) wipe out the establiεhed immune response of the host to all antigens by pharmacological immunosuppresεive agentε for a period of about 2 to about 3 weeks, without destruction of the stem cells, and ii) treatment of the "immunologically revirginized" patient with mPEG conjugates of the auto-antigenε in anticipation of generating εpecific Ts cells which would suppreεε the induction of the Th cellε involved in the auto-immune response.
Any known immunosuppresεive drugs may be used for step i) such as those which specifically target suppresεion of T cellε or B cellε or both. Alternatively, εtep i) may be substituted by a bone marrow or stem cell transplant.
Thus the invention provides promising resultε in experimental animal models that conjugateε of immunogenic Ag and mPEG provide for the development of novel therapeutic and clinical applicationε. mPEG derivativeε of naturally derived protein Ag alεo εuppreεε the autoimmunity and prevent the onεet of autoimmune diεeaεeε. The administration of mPEG conjugateε of immunogenic proteins, εuch as recombinant lymphokines (53) or xenogeneic enzymes (54,55), haε proven to be a εafe procedure in man. Hence, the clinical applications of mPEG conjugates of diverse therapeutic agents is increased. REFERENCES
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TABLE 5
Disorder (gene used) Cells altered (vector)
Cystic fibrosis3 Respiratory epithelium
(CF TR) (adenoviral)
Cystic fibrosis3 Respiratory epithelium
(CF TR) (adenoviral)
Disorder (gene used) Cells altered (vector) Cystic fibrosis3 Respiratory epithelium
Adenosine deaminase T-celis and stem cells (CF TR) (adenoviral) deficiency3 (ADA) (retroviral)
Brain tumors Stem cells (retroviral) Familial Liver cells (retroviral)
(MDR-1) hypercholesterolemia3
Brain tumors (primary and Tumor cells (retroviral) (LDLr) metastatic) (HS-tk) Gaucher disease* Stem cells (retroviral)
Brain tumors (primary)3 Tumor cells (retroviral) (glucocerebrosidase)
(HS-tk)
Brain tumors (primary and Tumor cells (retroviral) Gaucher disease* Stem cells (retroviral) metastatic) (HS-tk) (glucocerebrosidase)
Brain tumors (primary and Tumor cells (retroviral) metastatic) (HS-tk) Gaucher disease* Stem cells (retroviral)
Brain tumors (primary) Tumor cells (glucocerebrosidase)
(anti-sense 1GF-1) (DNA transfection) Gaucher disease Stem cells (retroviral)
Brain tumors (primary)3 Tumor cells (retroviral) (glucocerebrosidase)
(HS-tk) HIV infection T cells (retroviral)
Brain tumors (primary and Tumor cells (retroviral) (Mutant Rev) metastatic) (HS-tk) HIV infection Muscle (retroviral)
Breast cancer Fibroblasts (retroviral) (HIV-1 III env)
(IL-4) HIV infection Muscle (retroviral)
Breast cancer Stem cells (retroviral) (HIV-1 IIIB Env and Rev)
(MDR-1) HIV infection T cells (retroviral)
Breast cancer Stem cells (retroviral) (HIV-1 ribozyrne)
(MDR-1) Leptomeningeal Tumor cells (retroviral)
Coiorectal cancer Fibroblasts (retroviral) carαnomatosis
(IL-4) (HS-tk)
Coiorectal cancer Tumor cells (retroviral) Malignant melanoma Tumor cells (retroviral)
(IL-2 or TNF-α gene) (IL-4)
Malignant melanoma Tumor cells (retroviral)
Coiorectal cancer Tumors cells (liposomes) (IL-2)
(HLA-B7 and β2-mιcroglobulin) Malignant melanoma Tumor cells (retroviral)
Coiorectal cancer Fibroblasts (retroviral) (IL-2)
(IL-2)
Cystic fibrosis* Respiratory epithelium Malignant melanoma Tumor cells (retroviral)
(CF TR) (adenoviral) (IL-2)
Cystic fibrosis* Respiratory epithelium Malignant melanoma Fibroblasts (retroviral)
(CF TR) (adenoviral) (IL-4)
Cystic fibrosis Respiratory epithelium Malignant melanoma Tumor cells (liposomes)
(CF TR) (liposomes) (HLA-B7) Disorder (gene used) Cells altered (vector)
Malignant melanoma Tumor cells (liposomes) (HLA-B7 and β2-microglobulin)
Malignant melanoma T cells or tumor cells (TNF-α or IL-2) (retroviral)
Malignant melanoma Tumor cells (retroviral)
(interferon-7) Malignant melanoma Tumor cells (retroviral)
(B7) . Neuroblastoma* Tumor cells (retroviral)
(IL-2) Non-small cell lung Tumor cells (retroviral) cancer
(p53 or antisense K-ras) Ovarian cancer Tumor cells (retroviral)
(HS-tk) Ovarian cancer (MDR-1) Stem cells (retroviral)
Ovarian cancer (MDR-1) Stem cells (retroviral)
Renal cell carcinoma Tumor cells (retroviral)
(IL-2) Renal cell carcinoma Fibroblasts (retroviral)
(IL-4) Renal cell carcinoma Fibroblasts (retroviral)
(TNF-α or IL-2)
Renal cell carcinoma Tumor cells (retroviral)
(GM-CSF) Small cell lung cancer Tumor cells
(IL-2) (DNA transfection) Solid tumors Tumor cells (liposomes)
(HLA-B7 and β2-microglobulin) Disorder Location Disorder Location
Chr.16 Colorblindness, tntan (2) 7q22-qter
8q24.12-q24 13 Coiorectal adenoma (1) 12pl2.1 lp36.3-p3 Coiorectal cancer (1) 12pl2.1
12pl3 Coiorectal cancer ( 1 ) I8q23.3
Figure imgf000076_0001
βp2U Coiorectal cancer (1) 5q21
CJ deficiency (1) 19pl3.3-pl3i Coiorectal cancer. 114500 (3) I7pl3 1
C3b iiu tivitor deficiency ( 1 ) Coiorectal cancer (3) iα-V-ββ
Ot deficiency (3) βpZ1.3 Combined C6 C7 deficiency ( 1 ) δpl3
C5 deficiency (1) 9q34.1 TCombined variable rrrøijammaglobulinemia ( 1 ) 14q32 3
C6 deficiency ( 1) .pl3 Coaujeaύtal bilateral ftbeeace of vu d« ereaβ ( 1 ) IcilΛ
C7 deficiency ( I) 6pl3 TCoewαuea. cardiac aanaaaliea (2) 22qll
C8 deficiency, type 1 (2) lp32 Contractural arachnodactyty, congenital (3) Chr.5
C8 deficiency, type II (2) Ip32 Coproporphyna (l) Chr.S
C9 deficiency ( 1) 5pl3 TCornelia de ange syndrome (2) 3q26.3
CaatposeeUc θj.pl.iii 1 (2) 17qMJ-αl5.1 (Coronary artery disease, susceptibility to} (1) 6q27
Carbamoyiphosphate synlheuse 1 deficiency ( 1 ) if Corusol resistance (1) iq3I
ICar omc anhydrase 1 defictencyl (1) 8q22 C 1 deficiency (1) lq32
Carborypeoddaje B defle sKy (1) Otis TCπuϋofrwtoais l ctrapuuU (2) SfUrtttΛ ardιomyopalhy (l) 2o35 Craualotrawcoaat, type n (2) IqS qter
Cardiomyopathy. dilated. X-linked (I) Xp21.2 Craniosynosiosi*, type 1 (2) 7p21J-p2U
Cirf astyopatky, fusilial kypennpUb 1, l«MO0 (S) 14qlZ ICreatine kinase. brain type, ectopic expression of | (2) 14q32
Caritαfayopatfey, t sslUaJ kypeftnpkic, 1 (2) lqS Creuαfeldt-Jakob disease. 123400 (3) 20pter-pl2
Cardtomyopa-hy. familial hypertrophic, 3 (2) 16ς2 CnglerNajjar tynάrorru, tape 1, 118800(1) CM
Carttlaa»*»ir hypop aU (I) »plS-qIl TCryptorcrudism (2) Xp21
Cat-eye syndrome (2) 22qll ?Cutιs laxa. marfanoid neoutal type (1 ) 7q31.1^U
TCataxact, uteri r polar, 1 (2) 14α.24-<)t-r ICystathioninuna) (1) Chr.16
Ouaratt, congenital total (2) Xp Cystic fibπm (3) t≠U
Cataract, cesujesdial, wren s kroptatuύstU (2) lβplSJ TCrstismriai (2) 14(122
Cataract, Coppock-like (3) 2o33-o35 TCyaOsπrla, 220100 (1) 2pte*α32J
Cataract. Marnertype (2) 160-2.1 "¥— ^ Deafness, conductive, with stapes fixation (2) XqI3-q21.1
Cataract, zonular pulverulent- 1 (2) lo2 1 1 Deafness, low-tone (2) 6q31-q33
CDS. tea c imrt. dtfinncy (I) .o»β«.... A J Dtbntoςume wttvuy (1) OqlS.l
Ceatral core disease (S) 14412 Dentinogenesis unperfecu-1 (2) 4ql3-q21
Central core dura* q/muxle (I) I9ql3.l Denys-Drash syndrome ( 1) Ilpl3
Cenuocytic lymphoma (2) llςl3 Diabetes lnsintdus. nephrogenic (3) Xq28
Cerebral amyloid angiopathy ( 1 ) ZOpll Diabetes mtφiήus, ntwrohypaphyseal, 115700(1) OpIS
Cerebral utertoptttgr wttk a* oort al be-araa aad .Diabetes mellitus, lnsuluvdependent-l (2) 6p2 leuuαeetiu opUhT (2) UqU Diabetes meUuus, tuuJm-nsutαnt, nth ααmlλosu
Cer-brotendinous xanthomatotu (2) 2q3- qter ntffneatu(l) 1 pl3J
CeroKl upofuscinosis. neuronal-1. infantile (2) lp32 Diabetes meUitus, rare form (1) UplLδ
Cervical carcinoma (2) llqlS Diastrophic dysplana (2) oq31^)34
ICETP defi-ιen-yl (l) 16q21 DiOeorge syndrome (2) 22qll
Charcot-Mane-Tooth neuropathy, flow nerve conduction Diphenylhydantoin toxicity ( 1 ) lpll-ςter type la (2) 17pi (Diphtheria, susceptibility to | (1) (q23
Charcot.Mane- Tooth neuropathy, alow nerve conduction D A lU« I deOdeιιcy (l) ltqlt-2-qlU type lb (2) Iq21.2-q23 TDubin-Johnson syndrome (2) 13q3
Charcot-Mane-Tooth neuropathy. X-bnked-l, dominant (2) Xq.3 Duchenne muscular dystrophy (3) Xp2U
Chareot-Mane-Tooth neuropathy, X-linked-2, receasrre (2] 1 Xp22-2 I DvsalbuminemK hyperthrroxinemi l (1) 4qll-ql3
Cholesteryl ester storage disease (1) 10q24-q25 I Dysalbuminemic hypenineemial (1) 4qll-ql3
TCtoadrodyiplajii tacttta, rklaoMUc (2) 4t>lCf>14 Dyauruanaaia, tualltu (2) •<ιβι-«»
Chondrodyspiasia punetau- X-ltnked dominant (2) Xq28 Dysfibπnogenemia. alpha types (1) 4q28
Chondrodyiplasia punctata. X-hnted recessive (2) Xp22J Dysfibπnogenemia. beta types (1) 4q28
ChorOHleremla (2) X021.2 Dysfibnnogenemia, gamma types ( 1 ) 4q28
Chraruc granulomstous diseaae. autosorαal due to Dyskeratosi congenita (2) Xq28 deficiency o YBΛ (3) Mq24 .Dyslexιa-1 (2) lδqll
Chronic granulomatous disease due to deficiency of Dyspiasminogenemic thrombophilia (1) 6q26-fl27
NCF-1 (I) Tql SS Dysprothrombine ia ( 1 ) UplH12
Chronic granulomatous disease due to deficiency of I Dystransthyretinemic hyperthyroxineπu* 1(1) 18qll.2-ql2.1
NCF-2 (1) lq25 ■ ^ ?EECsyrκirome (2) qllΛ^U
Chroruc granulomatous disease, X-unked (3) Xp21.1 H Ehlers-Danlos syndrome, type IV, 1300M (3) 2qSI
IChronic infections, due to opsonin defect) (1) 10qll-2-q21 Λ-J Ehlers-Danlos syndrome, type VI.225400 (1) Ip3d3-p36j
CitruUuiemla (1) »q34 Ehlers-Danlos syndrome, type VIIΛ1, 130060 (3) 17q21Jl-q22.05
Ocft palate, XJmkt β) X l3^ I εiitrrs-Dantost ndrσme, type VlUi. 130060(3) f≠ l
TO Wo-ru- l d asUa (2) βq» TEhimϋanlos syndrome, ivpeX(l) 1≠
CMO II deficiency (1) 8q21 lEIliptocytosis Malaysian-Melanesian typel (1) I7q21-α22
Csduryie syButraste-t, Use owt, 21(410 (2) lOqll Ellιptocytosιs-1 (3) Ip360-p34
Coffin- owry syndrome (2) Xp22ip22.1 Ellιpto-ytosu-2 (2) lq21
Colon αuHet, faai- l, ittsiKtyposts type 1 (2) 2plβ-pl. Ellιptocytosιs-3 (2) 14q22-q23.2
Colorblindness, blue monochromatie (3) Xq28 Emery-Dreifuss muscular dystrophy (2) Xq28
Colorblindness deutan (3) Xq28 Emphysema (1) 14032.1
Colorblindness, proun (3) Xq28 Emphysema due to alpha-2-macroglobulin deficiency (1) 12pl3-3-pl2.3 Disorder Location Disorder Location
Emphysema-cirrhosis (1) 14432.1 Glaucoma, congeni l (2) Chr.ll
Endocardial fibroelastosιs-2 (2) Xq28 Glmacosaa, prinuuy open angle (2) lqil-qβl
Enolase deficiency (I) lptβr-pSβ.13 Glioblastoma muluforme (2) 10pl2-q23 2
TEosinophdic myeloprollferative disorder (2) 12pl3 Giucose galactose malabsorption (1) Z2qll-ai-qtcr
Epidermoiysis oullosα dystroplucα, domnααt, 131750 (3) 3ptU Gtαtaateaddeaau. type DC (!) 4q3&tjttr
Epidermotysis bulhβa dystrophies, recessive, 226600 (3) 3p2 Glutancaciduru, type I1A (1) Iδq23-q25
Epidermorysis bullosa, Ogna type (2) 8q24 -H«tιri-»-War-a, type HB (2) Or.M
Epidermoiysis bullosa simplex. 131900 (3) 17ql2-q21 Glutathιonιnuna (l) 22qll.l-qll2
Ep4detw)iyris bolloMa-atr «x. Dcwtta»Me«ratιp-, Glycerol kinase deficiency (2) Xp21.3-p21.2
181760 (S) 12qll-qlS Gtr-oe-a storage dlaeaje ID (8) lp21 Epidermoiysis bullosa simplex, Dowtmg- eara type, Glycogen storage disease VII ( 1 ) lcen-q32
131760 (3) 17ql2-q21 Glycogen storage disease, X-linked hepatic (2) Xp22-2-p22.1 Eptdermoiyps bullosa smp ∑, penerαi ed, 131900 (I) ltqI IS IGlyoxalase II deficiency] (1 ) 16pl3 TEpidermotysis bullosa simplex, localised. 131800 ( 1 ) 12qll-ql3 GMl-gangitosιdosιs (l) 3p21-pl4 2 • Epidermoiysis bullosa, Weber Cockayne type, 131800 (2) 12qll-qI3 GM2-ganglιosιdosιs. AB variant ( 1 ) Chr5 Epid-r»»Wtelrypeetec te^», ll«800 (l) 17q21-q22 GM2-ganglιosιdosιs, juvenile, adult ( 1 ) 16q23-q24 t deewtytte kyperitentoaai, 111800 (I) llqll-qlj βoeminne TKCR syndrome (2) Xq28 Epxierraotyuc palmoplantar keratoderma (2) 17qll-q23 Goiter, adolescent, mulunodular (1) 8q24^-q24J EpUepay. ees-qp. s∞aatal (2) tOqlJ-x-qlSJ Goiter, aou-adeaalc, abapie (1) 8qM-Z-OΪ4J Epilepsy, juvenile myoclomc (2) 6p2 TOoldenhar syndrome (2) 7p Epilepsy, progressive myockmrc (2) 21q22J Gonadal dysgenesis. XY female type (2) Xp22-p21 Epitheϋoma, self-healing, aquamous 1, Goudal dyageaeaia, XT type (1) ϊplLS
Ferguson-Smith type (2) 9q31 Gonadoblsstoma (2) Upl3 .Erythremιa (l) 7q21 Oonadotropin deficiency (2) Xp21 Erythremias, alpha- (1) 16pteτ-pl3.3 Greig -raniopolysyndactyty syndrome (3) 7pl3 Erythremias. beu- (1) llpl.5 Oynecomastta, familial, due to increased Er throblastosis (etaiis ( 1 ) Ip36.2-p34 aromatase activity ( 1 ) 16021.1 |Es7tb»cτtaeis, (aaaJUal), 133100 (2) MplUplU Gyrate atrophv of choroid and retina with ornithinemia. Erythrokeratodermia vaπabius (2) lp38J-p34 B6 responsive or unresponsive ( 1 ) I0ς26 /EuUiyroid l hyper- and hypothyrcxmema/ (1) XifH "y TT Harderoporphynnuria (1) ChrJ Ewing sarcoma (3) 22ql2 I — 1 THar-nup disease, 234600 (1) 2)X-tvqS2-t
Exeruonal myoglobinuna due to deficiency of LDH-A ( 1 ) llplδ.4 JL JL Heinz body anemias, alpha- (1) I6pter-pll3 Exadstrn vtcreor-t-oopat y, X-Unked (2) Xq21JI Hemi body anemias, beu- ( 1 ) llplS 5
F Fabry disease (3) Xq22 Hemochromatosis (2) 6p21.3 Faaoscopuloh merat muscular dystrophy (i) 4fl» Hemodiatysis-related amykHdosis ( 1 ) 16q21-q22 Factor H deficiency (1) iθ32 Hemotytic anemia due to ADA excess (1) 20ql3.Il Factor V deficiency (1) 1023 Hemolyuc anemia due to adenylau kinase deficiency ( 1 ) »q34.1 Factor VII deficiency (1) 13q34 Hemolyuc anemia due to bisphosphoglycerate mutase Factor X deficiency (1) 13q34 defi ency (l) 7q3l |34 Factor XI deficiency (I) 4q3S Hemolyuc anemu due to G6PD deficiency ( 1 ) Xq28 Factor XII deficiency (1) δq33-qter emotytic anemta due to ftucosephosphate isomerase Factor XII1A deficiency (3) 6p2S-p24 deficiency (1) 19013.1 Factor XII1B deficiency (1) Iq31-q32.) Hemolyuc anemia due to glutathione peroxidase Fam tat Medtinπuuanjmer (I) lOpl! deficiency (1) 3qll-ql2 TFancom anβma (I) l<rU Hemotytic anemia due to glutathione reductase Fancont cmemtα-. (2) t0qlSΛ*13J deficiency (1) 8p21.1 Farum (l) Xq28 Heaaotytlc ajwasia die to KxeUsuuw deOcieacjr (1) lOqM
|?Fetal alcohol syndrome) (1) 12q24.2 Hemotytic anemia due to PGK deficiency (1) Xql3 ΪTetal hydantoin syndrome ( 1 ) lpll-qter Hemotytic anemia due to phosphofructolunase deficiency (1) 21q22-3 ffVjrodytplana oss c ns μtυ muυa (I) tOpll Hemolyuc anemia due to tπosephosphate isomerase Fish-eye disease (3) 16q22.1 deficiency (1) 12pl3 I Fu -odor syndrome I (1) iq Hemophilia A (3) Xq28 Fletcher factor deficiency ( 1 ) 4q35 Hemophilia B (3) Xq27.1-q27-Σ Focal dermal hypoplasia (2) Xp22Jl Hemorrhagic diathesis due to 'anuthrombin* Pittsburgh (1) 14q32-l Fnedreich ataxia (2) «qlS-«2U Hemorrhagic diathesis due to PAI 1 deficiency ( 1 ) 7q21^«22 Fructose intolerance (1) q22 .Hepatic lipase deficiency (I) 18q21-q23 Fucoudosts (l) lp34 ?Hepatocarcιnoma ( 1 ) 2qI4-q21 Fumarase deficiency (1) lcHZ-1 Hepatocellular carcinoma (3) 4q32.1
GG6PD deficiency (3) Xq28 ( Hereditary persistence of alpha-fetoproteinl (3) 4qll-ql3 Oalactokinase deficiency (1) 17q21-q22 ?Heredιtary persistence of fetal hemoglobin (3) llpl-i Qalactose epimerase deficiency (1) Ip36-p35 •Hereditary persistence of fetal hemoglobin. Oalactosemia (l) 9pl3 heterocellular, Indian type (2) 7q36 Galactosιalκlosιs (l) 20qlll .Hereditary persistence of fetsl hemoglobin, Swiss type (2) XpllΛ Gardner syndrome (3) 4o*f-β« » Herauuaky-ridlak sradrotae, Ϊ0S800 ( 1) ltejl. Gaucher disease (1) lq21 Hers disease, or glycogen storage disease VI (1) Chr.H Gaucher disease, variant form ( 1 ) 10q21-q22 Heterocellular hereditary persistence of fetal hemoglobin (2) llplS βesdtovruuury dyip a (2) llplS IHexΛ pseudodeficiencyl (1) 15q23-q24 Gerstmann-Straussler disease, 137440 (3) 20pter-pl2 ?HHH syndrome (2) I3q34 røitt-rt sim-rroiw, 143500 (1) ChrJ IHisttduwmial (1) 12o22-q23 Glanzmann thrombasthenia, type A ( 1 ) 17Q21-32 Hoioprosencephaly, type 3 (2) 7q36 Glanxmann thrombasthenia, type B (1) 17021.32 raeJαcOTseaeepbady-l (2) 18pβr-qll Disorder Location Disorder Location
?Holoprosenceρhιly-2 (2) 2p21 absent GH and owarski type with biouiactive GH (3) 17q22-q24
14qlLl-qU Isovalenca demia ( 1 ) 15ql4-ql5
?Holt-Oram syndrome (2) 14q23-α24.2 T ?Jacobsen syndrome (2) Ho
?Holt-Oram syndrome (2) 20pl3
Homocystinuna. B6*responsιve and nonresponsive types ( 1 ) 21q22-3 1
HPFH. deletion type (1) llplS.6 u T y Kallmann syndrome (2) Xp22.3
HPFH. nondeletion type A ( 1) llplδ.6 1^ (Kippa light chain deficiency) (1) 2pl2
HPFH. nondeletion type G (1) llp.5.5 Λ..I Keratosis foUiculans spinulosa decalvans (2) Xp22.2-p212
HPRT-related gout (l) Xq2β-q27.2 I Kuunogen deficiency! (I) 3q26-qter
?Humoral hypercalcemia of malignancy (1 ) 12pl2.1-plU tKltpeeM'eU aywrraaae (2) tqll .
HunUngton disease (2) 4pl63 Knιest dysplasιa (l) 12ql3.ll-ql3.2
Hurter syndrome (1) 4plβ.3 ?Kostmann agranulocytosis (2) βp21.3
Hurler-Scheie syndrome ( 1 ) <p!6.3 Krαύoe disease (J) /4of4 oΛ2.7
HydT∞epka u dae to aswedact of Syrrbsa, 207000 (8) Xq28 -r ruαase defiαency, adult, 223100 (1) Chri
Hydrops/etatts, one form (I) 19013.1 1 tLactase deficiency, congenital (1) Chr.2
Hyperammonema du to CTPase deficiency (1) IplS-pU 1 J 7Lactoferrιn-dencιent neutrophUs, 245480 ( 1 ) 3q21-q23
Hyperbetalipoproteinemia (1) 2p24 Langer-Gied on syndrome (2) 8q24.ll-q24.13
Hypertalcesaia, ypoeaietiri-, faa-Ual (2) *«21-q24 Langer-Saldino achondrogenesis-rrypoctnrKlrogenesis (1) .2ql3.11-ql3.2
Hyperchoiesteroieraia, faπulial (3) I«pl3\2-pl3.1 Laron dwarfism (l) 8pl3-pl2 fHypergtyαnemia, isolated nonketouc, type I (2) »p22 TLaryngeal adductor paralysis (2) 6p21Λ-p21.2
?Hypenmmunoglobulιn 01 syndrome (2) 14q32-33 1 Lead poisoning, susceptibility to ) (1) Sq34
Hyperkalemic periodic paralysis (3) 17q23,l-q25.3 TLeramyomata, mui ple hereditary cutaneous (2) lβpll.32
?Hyperteuαneπua.ιsoteucιneπua or hypervalinemia ( 1 ) 12pter-qI2 LeiorrQomatosis-nephropathy syndrome, 308940 ( 1 ) Xq22
Hyperlipoproteinemia 1 (1) 8p22 Leprechaunism (1) !9pl3J yperiφoprotβmemta, typ lb (l) I9ql3i Lesch-Nyhan syndrome (3) Xq26-q27-2
Hyperiipoproteinemia, type III (1) 19ql3.2 .Letterer-Siwe disease (2) 13ql4-q31
IHyperphenytalaninemia, mιld| (3) 12q24.1 Leukemia, acute /mphobtashc (1) 19pl3J
|?Hyperproglucagonemiaj (1) 2q36-q37 Leukemia, acute tymphoblasUc (2) 9p22-p21
Hyperproinsulineπua. familial (1) llpl5 .Leukemia, acute rymphocyUc, with 4/11
7Hyperteaaioα, MS wi till, 146400 (1) 17q21-qϊ2 translocauon (3) 4q21
(Hrpetteaatoa. eaiinrlal, piiLLptfbUity to) (8) lq42-q4S Leukemia, acute myetout (3) tlqtSJ
Hypertnglycendemia. one form (1) llq23 Leukemia, acute myeloid, US type (1) x≠tx
?Hypervalιnemιa or hyperleucineHsoleucinemia ( 1 ) Ctu-,19 Leukemia, acute pre-6-cell (2) lq23
Hypoalphalipoproteinemia (1) Uq23 Leukemia, acute promyelocytic (1) 17q21.1
Hypobetaiipoproteinemia (1) 2p24 Leukemia, acute promyeiocyuc (2) lbq22
Hypoc ler-rtc aypero-l-earia, type D (2) MplU Leukemia, acute. T-cell (2) llplS
IHypcxeruloplumine u, hereditary | (1) Jq21-q24 Leukemia, chronic myeloid (3) 22qll-.l
Hypofibnnogenenua, gamma types (1) 4q28 Leukemia, chronic myeloid (3) tq34.1
THypotfr-eaaiatrse w P-Xl oefl-ieβc O) tOqlS-31 LMkcaaia, asmrtluaeage (2) Ckr.4
Hypogonadisra, roφergonadotropic (1) 19q 13-32 Leukemia, mye ud lymphoid or raued-lmeage (2) Uq23
7Hypogonadιsm, hypogonadotropic due to GNRH Leukemia. T-cell acute r/mphoblastic (2) llplS deficiency, 227200 (1) 8p21-plU Leukemia, T-cell acute rsmphoblastK (2) »q34.3
Hypomagnesemia, X-bnked pπmary (2) Xp22 Leukemia, T tli acute t mpKoblasloid (2) 19pl3J-pl31
* Hypomelanosis of lu> (2) 16ςll-ql3 Leukemia. T-cell acute rymphocyUc (2) 10q24
?Hypomelanosιs of ltd (2) Sq33-qter .Leukemia, transient (2) 2lqll2
Hypoparat yroidism, familial ( 1 ) Hpl5.S-pl5.1 Leukenua-l. T-cell acute rymphoblastic (3) lp32
Hypoparathyrotdum, X-unked (2) Xqϊβ-q27 Leukemιs-2, T-cell acute r/mphobUstic (3) <q31
•rlypophosphatasta, adult, 148300 (1) Ip36.1-p34 Leukernia rymphorαa, B-eelL I (2) llqlSJ
Hy ophosphuas , mfanUle, 241500 (3) Ip36.1-p34 Leuketrω r/mphoma, B-cell, 2 (2) 18q2l.3
Hypophosphatemia, hereditary (2) Xp22J2-p22.1 Leukeraia/rymphoma, B-oell, 3 (2) 19ql3
?Hypophosphatemιa with deafness (2) Xp22 Leukernja rympfwma, T-cell (2) 144)32.1
Hypoprothrombinenua ( 1 ) llpll-ql2 leukemia ympnoma, T-cell (2) 2q34
?Hypospadιas-dy3phagιa syndrome (2) 6plS-pI2 Leukemia rymphoma, T-cell (3) 14qlU2
Hypot-tyrotdim, bβr-ditary -βsujeeital (1) •q24_J-q24J Leukocyte adhesion deficiency ( 1 ) 21q223
Hypothyroidisrα, nongoitrous (1) lplS U-Fraumeni syndrome (1) 17pl3.1
Hypothyroidism, nongoitrous, due to TSH resistance (1) 14q31 Upotmide dehydrogenase deficiency (1) 7q3l- 32
-rJI-hthyosis rulgaris, 146700 (I) lq21 Upoma (2) 12ql3-qU
1 lchthyosιs. χ.tinked (3) Xp22-32 Dw cell carcinoma (1) Ilpl4-pl3
X Tbsatotlle dlla, syadroaae (2) •p Long QT syndrome (2) llplSi
Immunodeficiency, X-linked, with hyper-lgM (3) Xq24-q27 Lowe syndrome (3) Xq26.1
Inconunentia ptgmentl, fmπϋtial (2) Xq27-q28 Lupus erytheπatosus, systemic 152700 (1) lq23
InconUnenUa pigmen , sporadic type (2) XplUl iΛTnphoproliferative syndrome. X-bnked (2) Xq25 infertile male syndrome (I) Xcen-q22 .Lynch cancer family syndrome II (2) 18qll-ql2 f lnosine tnphosphalase deficiency] (1) 20p . Lysosomat acid phosphatase deficiency ( 1 ) llpl2-pll
Insomnia, fatal familial (3) 20pter-pl2 "It rMacrocytK ajwmu ofbq-syndrome. refractory (2) 6q 12-032 llt a*<i<QeaAt*t tiM)xu* meWCm-l (t) llq |\/l Maerocytlc aaeaaia, refractory, of toeydiut, lnterferon, alpha, deficiency (1) Sp21 lYl 163660 (3) lqJt.1
Interferon, immune, deficiency (1) 12q24.1 Maalar dysorop«τ (l) ♦pll.l-ee. Isolated growth hormone deficiency due to defect in acular dvstrophy, atypical vitelllform (2) 8q24
0HRF (1) 20pll.23-qter Macs ar dystrophy, North Cvolbu type (2) tqH-qltU
Isolated growth hormone deficiency, Iliig type with Macular dystrophy, vltelUfors type (2) IlqIS Disorder Location Disorder Location
22qlS-qter Multiple endocrme neoplasia II (I) lOqllS
'Male infertility, familial (I) Multiple endocrme ruoplasia III (2) lOαllJ
'Male pseudohermaphrodiusm due to defective LH ( 1 ) 19ql3J2 'Muluple exostoses (2) 8q23-q24 1
Malignant hypertnermιasusceptibιlιty-1. 145600 (3) 'MulUple lipomatosis (2) 12ql3-ql4 taJlgsuu-t irpertberaa aaaeeptnnilty-2, 145400 (2) 17qlL2-qX4 f Martlpie acteraeis, —.ctptibUUy to) (2) 18q22-qter
Malignant melanoma, cutaneous (2) 'Muscle glycogenosιs (l) λq 12-c 13
'Manic-depressive illness. X*lιnked (2) Mtacmlar dystrophy, DMkeauwϋke, arto.oo.al (2) Uqlϊ-qlS
Mannosιdosιs ( l) I9pl3.2-ql2 Muscular dystrophy, ttml gtrdle auiosomal
Maple syrup urine disease, type 1 (3) dominant (2) q22 q31J
Maple syrup urine disease, type 2 (3) lp31 Muscular dystrophy, limb-girdle, autosomal recessive (2) 15ql5-q22
Maple syrup urine disease, type 3 ( 1 ) 6p22 p2l Mτel«ryιc4aιtte sym roa»e, r*-le»k-mie (3) 6q31.1
Marian syndrome 154700 (3) I5q21 1 Myelogenous leukemia, acute (3) 5q31 1
Maroteaux Lamy syndrome several forms (1) 5qll-ql3 Myeloperoxidase deficiency ( 1 ) 17q21.3-q22
Marun Bell syndrome (2) Xq27J Myoadenylate deaminase deficiency ( 1 ) Ip21-pl3
MASA syndrome (2) Xq28 (M ocaπUal Imtarβioe- oio-eptibUity to) (3) l.qH ll l3 Myoglobinuπa tiemotysu due to PGK deficiency ( 1 ) Xql3
McCune-Albnght potyostouc fibrous dysplasia. 174800 ( 1 ) 20qO2 Myopathy due to CT se deficiency (1) Ipl3.pl!
I McLeod phenotype | (2) Xp21.2-p21 1 Myopathv due to phosphogtycerate muuse deficiency (1) 7pl3-pllS
Medullary thyroid carcinoma (2) lO llJ Myopιa-I (2) Xq28
Megacolon (2) lOqlU Myotonia congenita, atypical acetazolamide-responsrve (2; 1 17q23 1-q253
Megalocornea, X-linked (2) )a>otoaiιeoe«eauι<- dcβlEant, lβOβOO (2) 7q36
Melaaosaa, αttaaeoM -*"f ■■' (2) tp21 Myotonia conρemla, recesswe, 255700 (3) 7oW
Menιngιoma (2) 22ql23-qler Myotonic dystrophy (2) 19ql3-2-ql3J3
Meningioma (3) 22qHL3-ql31 Myotubular myopathy, X-linked (2) Xq28
Myxoid liposarcoma (2) 12ql3-ql4
Mental retardation of WAGR (2) *X T ' syndrome 310465 (1) Xp22J-p21 1
Meets! reurdauJos, S«yder-Bobtι»o« type (2) Xp21 |\ j Nail-patella syndrome (2) 9q34
J-, Nance-Horan svndrome (2) Xp22-2-p21 1 witk aφkaa (2) Xpll Nemaiine myopathy-1 (2) Iq21-q23 Mental retardation, X'linked, svndromιc-1. with epkroaopUl-uds, Jweenlie (2) 2p2J-ce, dystonic movements, ataxia. and seizures (2) Neuroblasto a (2) lp36J2-p361 Mental reurda on X-linked. svndromιc-2, with NesraepttkeUoaaa, 1*3460 (1) Ilq23-q24 dysmorphism and eer-bral atrophy (2) Neuroepithelioma (2) 22ql2 Mental reurdauon, X-linked svndromιc-3, with Neurofibromatosis. von Recklinghausen (3) 17qlL2 spastic diplegia (2) Nvβropet y, reearrat, with μi — u paaetea. Mental reurdauon, X-iinkrd. svndromu , with 162600 (3) 17plU congenital contraetures and low fingertip arches (2) Neutropenia, immune (2) lq23 Mental retardauon. X-linked. syndromιc-5. with Niemann-Pick disease, type A ( 1 ) llplS.4 15.1
Dandy-Walker malformation, basal ganglia disease Niemann-Pick disease, type B ( 1) llplδ 4-15 1 and seizures (2) Xq25-q27 Niem»π»-.V* disease, type C (2) 18p Mental retardation X-linked, syndronuc-C. with Nightbhndness, congenital suuonary, type 1 (2) XpllJ gynecomastia and obesity (2) Xp21.1-q22 {Ha*ύ lin *eptmΛaa.άuύ>aιeM »tmuM, Mental reurdauon X-ltnked-1 non-dysmorphlc (2) aeanρtib0iq>to| (2) lβqlS-S 'Menul reurdauon, X-lιnked-2. non-dysmorphic (2) Xqll-ql2 Nome disease (2) Xpll 4 Menul reurdauon λ-lιnked-3 (2) Norum disease (3) 16q22.1 Menul reurdation-skeletal dysplasia (2) Xq28 Nucleoside phosphorylase deficiency, immunodeficiency Meuchromatic leukodystrophy ( 1 ) due to (1) 14ql3 1 Meuchromatic kukodystrophy due to deficiency of Obesity (2) 7q3l
SAP-l (l) I0q21-q22 Ocular albinism autosomal recessive (2) 6q.8-qlδ Methemoglobtnemia due to cytoehrome b5 deficiency (3) Chr lβ
Figure imgf000079_0001
Ocular albinism. Forsius-Erikssoo type (2) Xpll-qll Methemogjobtnemia. enzymopathic ( 1 ) 22ql3-31-qter Ocular albinism, NetUeship-Falls type (2) Xp22J Meihemogtobtnenuas. alpha- ( 1 ) 16pter-pl3J Ornithine trinscarba ytase deficiency (3) Xp21 1 Methemoglobinemias. beu- ( 1 ) Orofacul deft (2) opter-023 Methytmalonicaciduru, muuse deficiency type ( 1 ) Oroucacιdurιa (l) 3ql3 MevaOoedesciduia (1) Ckr.12 Osteoarthrosis, precocious (3) 12ql3.ll-q.3J! 'Microphthaimia with linear skin delects (2) Ontogenesis mperfectα, 4 dsnuαljorms. Miller-Dieker lissencephaty syndrome (2) 17pl3*3 155200, 165210, 59420, 155220(3) l7tl2IJI^22.05 mtocfcmdrtal coaaplex 1 de£ t-»cy, 262010 (1) llqlS Qstβooene xmφβ ctα, 4 dxn αljorms, MODV. one form (3) Upl55 165200, 165210, 59420, 156220(3) 7g2tl MODΪ, type 1 (2) 20ql3 Osteopetrosis 259700 (1) Ip2l-pl3 MODΪ, type II. 125851 (3) 7pl6-pl3 Osteoporosis, kttop«tnle,16e710 (3) 17q2Ml-q22-06 'Moebius syndrome (2) 13ql2-2-qIJ Oseeαtaraaaa, 159600 (2) I3ql4U-ql4U 'Monocyie carboxyesterase deficiency ( 1 ) 16ql3-q22.1 Otopalatodigiul syndrome, type 1 (2) Xq28 Mu olιpιdosιs ll (l) Orariu ceuτ4awtu,l«7000 (2) lβqlM-q.3-2 Mucolipidosis 111 (1) Ovarian carcinoma (2) 9p24 Mucoporysacchaπdosis 1 (1) 4pl6J Ovarian failure, premature (2) Xq26-q27 Mucoporysacchaπdosis II (2) Xq28 Oxalosu l (l) 2o36-q37 ■4»copotywrtιrkto«tι IVλ (3) 16q24-l "|-"V 'Paget disease of bone (2) 6p2 Mucopolvsacrhaπdosis IVB ( I ) Y-f TPalllΛerβall eyndroaK (2) tol&Λ Mucopolv -charidosisMI (1) 7q21 l l JL Pancreatic lipase deficiency ( 1 ) 10q26 < Multiple endocrine neoplasia 1 (1) 'Panhypopiluitaram (1) 3q Disorder Location Disorder Location
'Panhypopituiunsm, X-linked (2) Xq21.3-q22 Eetjadtie pigaoeoto or a-toanetal icmealrt (3) *q21-oJU
Pαrαgαnglαtnα (2) llq22J <fai Eetiiiia plfsaeatoea- pertpfce-ta-r- ted (3) βpZLl-cea
Paramyotonia congeniu. 168300 (3) 17q23.l-q253 ReUnitis pιgmentosa-1 (2) 8pll-q21
Parathyroid adenomatosis 1 (2) llq!3 Reunius pιgmentosa-2 (2) Xpll.3
?Partetal .oraaafau (2) Ilpl2-pll.l2 Kettmtιspιgmenιosα3(2) Xp21.1
If srk nsonism, susceptibility to/ (1) 22013.1 leϋ-ltti p-faκιtou-4. aatnanial doaaisuutt (8) »q21-qM
Paroxysmal nocuirnal hemoglobinuru (1) Xq22-1 Ke-taltts ttfsteaitαakt (2) *q
Pelizaeus-Meπbacher disease (3) Xq22 TReunius putmentosa-6 (2) Xp21Λ-p2U reMareter juacttoai cootrtctiot, (2) •p RetlaΛb p-s»e u>«a-9 (2) 7pl..l-plS
'Pendred syndrome (2) 8q24 leUnl s pigsee toaarlO (2) 'q
Peπodonuus, juvenile (2) 4qll-ql3 leHnltia paaecttta albeoceaui (1) (p2Ll<α
Persistent Mulleπan duct syndrome (1) 19pI3.3-pl3.2 Reunoblastoma (3) 13ql4 l-qH.2
Phenylkeununa (3) I2q24.l ?Reunol binding protein, deficiency of ( 1) 10q23-q24
Phenylketonuria due to dihydroptendine reductase deficiency ( I ) 4pl5Jl Reunoschists (2) Xp22.3-p22 1
Flwoei_ntaoeytosu (2) ip ?Rett syndrome (2) Xp
Phosphoπbosyl pyrophosphate synthetase-relaled gout ( 1 ) Xq22-q24 Rhabdomyosarcoma (2) llpl5.5
TPhosphorylase kinase deficiency of liver and muscle, Rhabdomyosarcoπ . alveolar (2) 2q37
261750 (2) 16ql2-ql3.1 Rhabdomyosarcoma. αttmtαr (3) 2t)35 Piebαldism (3) 4 l2 Rh-null disease (1) 3cen-q22
Pituitary tumor, growth-hor one-fecreung ( 1 ) 20ql32 TRh-null hemolyuc anemia ( 1 ) lp36 2-p34 PK deficiency hemolyuc anemia ( 1 ) lq21 Rickets, vitamin D-resisum ( 1 ) 12ql2-ql4 ( Placental lactogen deficiency | (1) 17q22-q24 gieger syndrome (2) 4 25^ Placental steroid sulfatase deficiency (3) Xp2232 Rod monochromacy (2) Chr.14 Plasmin inhibitor deficiency ( 1 ) 17pter-pl2 TRothmund-Thomson syndrome (2) Chrβ Plasminogen activator deficiency ( 1 ) 8pl2 Rubinstetn-Tavbt syndrome (2) I6pl3.3 Plasminogen deficiency, types 1 and II (1) 6q2β-q27 TtUtseellSUver syndroate (2) 17q26 Plasminogen Tochigi disease ( I ) 6q26-q27 " < -teetkre-Ckotzeα eyndroaae (2) iv te t oiphαMβllα storage pool deficiency (1) l≠3≠5 ^■oa, Salivary gland pleomorphic adenoma (2) 8ql2 (Polio, susceptibility to/ (!) 19ql3tαl3S _ Sandhoff disease d) 5ql3 Polycysuc kidney disease (2) 16pl3.3l.pl3.12 TSaiffllppo disease, type H.C (2) Or.14 Potycystic ovarian d isesse ( I ) 17qll-ql2 Sanfilippo syndrome D ( 1 ) I2ql4 Poiyposis coli, familial (3) 5q2Hι22 Sarcoma, synovul (2) xpiπ Pompe disease (I) 17q23 Scheie syndrome ( I ) 4pl6J Porphyna, acute hepatic (1) qJ4 TSchrzophrenia (2) 5qll.2-ql3.3 Porphyna. acute intermittent (1) llq24.)- 242 8-tuιophr-sia. ehroiie (3) 21q2lΛq22.05 Porphyna, Chester type (2) llq (TScr-iιop«r-ι-U. sMcepabUlt t-.) (2) Jql3.S Porphyna. congenital erythropoieUc (1) 10q25-2-q263 Sclerotylosis (2) 4q28-q31 Porphyna cutanea tarda ( I ) Ip34 Severe coeeblswd tssM«βodeflciency, 202SO0 ( 1) 10pl6-pl4 Porphyna, hepatoerythropoieUc (1) Ip34 Severe combined immunodeficiency due to Porphyna vanegau (2) 14q32 ADA deficiency (1) 20ql3.ll Posianestheuc apnea ( 1 ) 3q26.1-q262 Severe combined immunodeficiency due to Prader-Willi syndrome (2) lδqll 1L2 deficiency (1) 4q26-q27 (rYe-eela-apsta, auiceptlbUlty to) (3) Iq42-q43 &r*re combtneoC fntmunodςfi-i-Ti-p, Progressive cone dystrophy (2) Xp21.1-pll.3 HLA doss ll-ne∞twe type (1) 19pl3.1 F oudase deficiency ( I ) I9cen-ql3.ll Severe combined immunodeficiency. X-linked.300400 (3) Xql3 Properdin deficiency. X-ltnked (3) Xpll.4-pll.23 Sbortstan-re (2) Xpter-α22 S2 Propunicacidemia, type I or pccλ type ( 1 ) 13q32 rSialidosis (2) 6p21 3 Propwnicacidemia, type II or pccB type ( 1 ) 3q21-q22 Sickle cell anemia (1) llplS.S Protein C deficiency ( 1 ) 2ql3-ql4 7-UMpeoe-G.laM-Beamel sysKtroaae (2) XoeaHl2 3 rrotebi C bUMtor deflcteaKj (2) 14qS2.1 TS us nuvrsus ns eπim (2) I4q32 Protein s deficiency (1) Spll.l-qiπ ?SLE (1) lq32 Frotoporphyna, erythropoietK ( J) IBpterpll ll Small-cell cancer of lung (2) 3p23-p21 Pseudohermaphrodiusm. male, with s^mecomastia ( 1 ) 17qll-ql2 TSmith-Lemii-Opitz syndrome (2) 7q34-qter Pseudoroφoaldosteronisro (1) 4031.1 Smith-Magenis syndrome (2) tfplU Pseudohypbparathyroidism, type la (1) 20qI3 2 Spastic paraplegia, X*lιnked, uncomplicated (2) Xq21-q22 Pieodotatlnal peslaooaerotal hrp opa in (1) Chr-2 -tøfeerocytosie, awrediury (S) 17q21-q22 Pseudo-vitamin 0 dependency rickets I (2) 12ql4 Spherocytosis, hereditary, Japanese type ( 1 ) 15ql5 Pseudo-Zelhveger syndrome ( 1 ) 3p23-p22 Spherocvtosis. recessive ( 1 ) Iq21 'Pyridoxine dependency with seizures (I) 2q31 Spherocytosιs-1 (3) 14q22-q23_2 Pyropctktlocytosis (1) lq21 Spherocytosιs-2 (3) 8pllJ2 Pymvate carboxyiase deficiency ( 1 ) llq Spinal and bulbar muscular atrophy of Kennedy, Pyruvate dehydrogenase deficiency (1) Xp22-2-p22.1 313200 (3) Xcen-q22
R ?Rabson-Mendenhail syndrome ( I ) 19p Spinal muscular atrophy II (2) &ql2-2-ql3.3 !Ragweed sensitivity (2) 6p21.3 Spinal muscular atrophy HI (2) 5ql2JJ-ql3.3 Reifenstem syndrome (1) Xcen-q22 Spιnocerebellarataxιa-1 (2) 6p21.3-p21.2 Renal cell carcinoma (2) 3pl4.2 laocerebeUar atrophy D. (2) 12q24 IRenal lucosunal (2) βp21.3 Split-hand/foot deformity, type 1 (2) 7q21.2-q2l 3
Renal tubular actdosts-osteopetrosis syndrome ( 1 ) 8q22 SpUWu-nd spUWoM deformity, type 2 (2) Xq2β 'ReUnal cone dystrophy-1 (2) 6q25-q26 Spondyloepiphvseal dysplasia congeniu (3) 12ql3 1l-ql32 'Re nal cone-rod dystrophy (2) 18q21-q22.2 Spoπdvloepiphvseal dvsplasia tarda (2) Xp22 RetmUts pigmentosa. autosomal dominant (1) llplS Startle disease (2) 6q3J-q56 Disorder Location Disorder Location
Stickler syndrome (3) 12ql3 ll-ql3.2 Uaker syidroaae, type IC (I) HP Sucrose intolerance (1 ) 3q25-q26 Usher svndrome type 2 (2) Seφravarrer aortic eteaoats (3) 7qllJ! " " T" van der Woude svndrome (2)
T Tay-Sachs disease ( I ) 15q23-q24 \J Velocaιdio(ae d ayndroaM (2) 22qll Testicular fetninizauon ( 1 ) Xcen-q22 V Yl-rwretinopa-hy, ex»d*-rve. faj lUϋ (2) llqlyq23 Thalassemias. alpha- (1) 16pter-pl33 Vltzooretiaopatay, aweva-Kαlar tnflaaraiatory (2) llqlj Thalassemias beu (1) Upl55 (Vrvax malaria susceptibility to) (1) Iq21-q22 Thrombocytoperua. λ linked (2) Xp21.pl 1 von Hippel ϋndau syndrome (2) 3p26-p25 Thrombophϋia due to elevated HRO ( I ) 3pl4-qter von Willebrand disease ( 1 ) 12pter-pl2 Thrombophtlta due to excessive plasminogen activator TT "T aardenburg syndrome, type 1 (3) 2q3 inhibitor (1) 7q21.3-q22 \l\l VVajrdealK-rg aymdruaae, type m, Thrombophilia due to hepann cofactor II T 48820 (3) ZqSS deficiency (1) 22qll waisman parkinsomtm-menul retardauon syndrome (2) Xq28 Thyroid hormone resistance.274300, 188570 (3) 3p24.3 Watson svndrome. 193520 (3) 17qll_> Thyroid wdme peπmdαse deficiency (1) 2pl3 Werdπig-Hoflmann disease (2) 5012-2-0,13-3 Thyroid papillary carcinoma ( 1 ) 10qll-ql2 Werner svndrome (2) 8pl2-pll Thyrotropin-releasing hormone deficiency ( 1 ) ChrJ ( eπtteke-KorsaJiotf eyndroeee, awoctptlbUlty to) (1) *pl4J Torsion dystonia (2) 9q32-q34 Wieacker-Wolff syndrome (2) Xql3-q21
•brawn dystomαiiαrktnsonism, F φmo type (2) Xql2-≠l 1 TWIIHaaas Rπimi syidrotae (2) 4qJ»-qJ5.I TTourette syndrome (2) 18q22.1 Wilms tumor (2) llpl3 T anscobalamin II deficiency (1) 22qlL2-qter Wilms tumor, type 2 (2) llplS S Prαnscortm deficiency I (1) 14q321 Wilson disease (2) 13ql4-q21 Trencher Collαs mαndibulofαααl dysostosα (2) 5o32<#31 Wiskott-Aldnch syndrome (2) XplU-plU Tnchorhtnophalangeal syndrome, type 1 (2) 8q24.12 'Wolf-Hirschhorn syndrome, 194190 (3) 4pl6 1 Ttypsinogen deficiency ( 1 ) 7q32-qter Wolf-Htrschhom syndrome (2) 4pl6J (TflsbenoJoate. aoaeetit-btirt to) (2) to Wol an disease (1) 10q24-q25 Tuberous sclerosιs-1 (2) 9q33-q34 Wrinkly akin ayadroaae (2) 2qS2 Tuberous sclerosιs-2 (2) 11023 "T 'Xeroderma pigmentosum ( I ) 1042 .Tuberous sclerosιs-3 (2) 12q23.3 Xeroderma pigmentosuir group B (3) 2q21 Tiberoaa aderoaia-i (2) lopl- Turner syndrome (1) Xql3 1 groin C (2) Ckct Tyrosuiemii, type 1 (1) 16q23-q25 Xeroderma pigmentosum, group D.278730 ( 1 ) 19ql3-2-ql3.3 Tyrosinemia, type II (1) 164)22.1-422.3 Xeroderma pigmentosum. type A ( 1) 8q34 1
U Urate oxidase deficiency ( I ) lp22 'Xeroderma pigmentosum, type F (2) Chr.15 Urotuhiasis, 2J)-dιhydroxysderune (1) 16q24 PTf Zeltweger syndrome, type II (1) Ip22-p21 Uakβr STStttroaae, type IΛ (2) 14q32 t Zellweger syndrome- 1 (2) 7qll23 Uoher syndrosae, type IB (2) llqlS-5 LA
The purpose of the above description and examples is to illustrate some embodiments of the present invention without implying any limitation. It will be apparent to those of skill in the art that various modifications and variations may be made to the composition and method of the present invention without departing from the spirit or scope of the invention. All patents and publications cited herein are incorporated by reference in entirety.

Claims

WE CLAIM : 1. A method for inducing tolerance to an antigen (Ag) which is manifested by suppressing both humoral and cell-mediated immune responses, comprising administering an effective amount of an Ag(mPEG) conjugate for the induction of tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses.
2. The method of claim 1, wherein said Ag(mPEG) conjugate inhibits activation of cytolytic activity by primed CD8+ CTL.
3. The method of claim 1, wherein said tolerance of the humoral immune response is induced in an isotype-pan- specific manner.
4. The method of claim 1, wherein the tolerance is mediated by Ag-specific CD8+ suppressor T (Ts) cells.
5. The method of claim 1, wherein said conjugate suppresses IL-2 production by lymph node lymphocytes (LNL) .
6. The method of claim 1, wherein said conjugate suppresses IL-2, IFN-^ and IL-4 lymphokine production.
7. The method of claim 6 wherein said method does not influence CD4+ T cells to express the characteristics of their Thl or Th2 phenotype.
8. The method of claim 1, wherein said Ag(mPEG) conjugate inhibits lymphokine production by primed CD4+ Th cells.
9. The method of claim 1, wherein said Ag(mPEG) conjugate inhibits both arms of humoral and cell-mediated immune responses in vivo and said tolerance induced by Ag(mPEG) conjugates is Ag specific.
10. A method of obtaining passive transfer of suppression of an immune response comprising treating an animal with Ag( PEG) conjugate and transferring lymphocytes from said animal to a syngeneic recipient animal, wherein said lymphocytes provide suppression of Ag-specific cytotoxic lymphocyte (CTL) activity in said recipient animal.
11. The method of claim 10, wherein said transfer inhibited humoral and cytolytic responses in recipients are mediated by Ts cells.
12. A method of treating a condition selected from the group consisting of allergies and autoimmune diseases by inducing tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses comprising administering an effective amount of Ag(mPEG) conjugate to induce tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses.
13. A method of preventing an immune rejection of organ transplants or transplants of DNA transfected cells comprising administering an effective amount of Ag(mPEG) conjugate which is xenogeneic or allogeneic to a patient in need of said organ transplant, to induce tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses prior to the administration of the (Ag) .
14. A method of claim 13, wherein antibodies of all IgG subclasses are suppressed.
15. The method of claim 14, wherein IgG isotypes dependent upon Thl and Th2 lymphokines are both inhibited by said Ag(mPEG) conjugates.
16. The method of claim 14, wherein lymphokines produced by CD4+ Th cells are inhibited by said Ag(mPEG) conjugate.
17. The method of claim 16 wherein said lymphokines are selected from the group consisting of IL-2, IL-4 and IFN- -
18. A method of treating organ-specific autoimmune diseases in animal comprising administration of mPEG conjugates of autoantigens selected from the group consisting of collagen-induced arthritis by type II collagen and diabetes in NOD mice by insulin to induce tolerance to an antigen (Ag) in both humoral and cell-mediated immune responses prior to adminstration of said (Ag) .
19. A method of conducting gene therapy comprising the step of administering to a mammal an immunosuppressive effective amount of a tolerogenic conjugate consisting of a protein coupled to monomethoxypolyethylene glycol (mPEG) having a molecular weight of about 2,000-10,000 daltons, wherein administration of said tolerogenic conjugate is at least one day prior to administration of a gene therapy vector encoding a gene for a protein, wherein said protein is identical to said protein which is coupled to mPEG, and wherein said method results in the suppression of an immune response and in the development of tolerance to the protein expressed by said gene encoded by said gene therapy vector.
20. A method of conducting gene therapy comprising the steps of a) administering to a mammal an immunosuppressive effective amount of a tolerogenic conjugate consisting of a protein conjugated to monomethoxypolyethylene glycol (mPEG) having a molecular weight of about 2,000 to 10,000 daltons, wherein administration of said tolerogenic conjugate is at least one day prior to administration of DNA, RNA or mRNA and encoding a protein administered for gene therapy, wherein the encoded protein is identical to said protein which is conjugated to mPEG, and wherein said method results in the suppression of an immune response and in the development of tolerance to encoded protein of said DNA, RNA or mRNA administered for gene therapy.
21. Method of conducting gene therapy according to claim 20, wherein mPEG conjugates of both a vector protein and protein administered for gene therapy are administered prior to conducting gene therapy with a gene therapy vector encoding a gene for a therapeutic protein.
22. Method of conducting gene therapy according to claim 21, wherein the vector protein and protein administered for gene therapy are conjugated together as a hybrid mPEG conjugate and administered prior to conducting gene therapy with a gene therapy vector encoding a gene for a therapeutic protein.
23. Method of conducting gene therapy according to claim 20, wherein said protein administered for gene therapy is blood factor VIII protein.
24. A method of treating hemophaelia comprising the steps of administering an effective amount of a human blood factor protein (mPEG) conjugate for the induction of tolerance to said human blood clotting factor protein in both humoral and cell-mediated immune responses prior to administering a gene for said clotting factor encoded by a gene therapy vector.
25. A method according to claim 20 wherein said human blood factor is selected from the group consisting of human clotting blood factor VIII and human blood factor IV.
26. A method of overcoming the immunogenicity of a gene therapy vector, comprising administering an mPEG conjugate corresponding to a vector protein administered prior to the administration of the gene therapy vector.
27. A method according to claim 24 wherein the vector protein is a vector coat protein.
PCT/US1998/009786 1997-05-14 1998-05-14 ANTIGEN-mPEG CONJUGATES SUPPRESS HUMORAL AND CELL MEDIATED IMMUNE RESPONSES WO1998051341A1 (en)

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US7858749B2 (en) 2003-02-26 2010-12-28 Nektar Therapeutics Polymer-factor VIII moiety conjugates
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