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WO1997010711A1 - Procedes et compositions de suppression d'une reponse immune a l'aide de peptides de la classe ii du complexe majeur d'histocompatibilite - Google Patents

Procedes et compositions de suppression d'une reponse immune a l'aide de peptides de la classe ii du complexe majeur d'histocompatibilite Download PDF

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Publication number
WO1997010711A1
WO1997010711A1 PCT/US1996/015662 US9615662W WO9710711A1 WO 1997010711 A1 WO1997010711 A1 WO 1997010711A1 US 9615662 W US9615662 W US 9615662W WO 9710711 A1 WO9710711 A1 WO 9710711A1
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Prior art keywords
peptide
class
cells
sequence
peptides
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Inventor
Mohamed H. Sayegh
Barbara T. Murphy
Charles B. Carpenter
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Autoimmune Inc
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Autoimmune Inc
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Application filed by Autoimmune Inc filed Critical Autoimmune Inc
Priority to IL12375996A priority Critical patent/IL123759A0/xx
Priority to EP96933201A priority patent/EP0914041A4/fr
Priority to CA002230119A priority patent/CA2230119A1/fr
Priority to AU72025/96A priority patent/AU7202596A/en
Priority to KR1019980702086A priority patent/KR19990063637A/ko
Priority to BR9610625A priority patent/BR9610625A/pt
Priority to JP9512976A priority patent/JPH11513377A/ja
Publication of WO1997010711A1 publication Critical patent/WO1997010711A1/fr
Priority to NO981201A priority patent/NO981201L/no
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to methods and compositions for suppressing various immune responses, including but not limited to proliferation of (host) lymphocytes to: (a) encounter of histocompatibility antigen (i.e. an alloimmune response or response to a xenograft) ; and (b) antigens present in specific tissues or organs of the body (which includes but is not limited to autoimmune response) .
  • histocompatibility antigen i.e. an alloimmune response or response to a xenograft
  • antigens present in specific tissues or organs of the body which includes but is not limited to autoimmune response
  • MHC major histocompatibility complex
  • transplant recipients have heretofore been treated with various immunosuppressant and cytotoxic drugs, all of which are nonspecific (i.e. do not selectively suppress alloimmunity) and eventually cause global immunosuppression, which increases the susceptibility of the transplant recipient to opportunistic infections and can lead to malignancies. Additionally, several of these drugs have serious or even dangerous side-effects, such as kidney and liver toxicity, as well as hypertension.
  • the Krensky peptides are believed to work by binding to T- cells. They inhibit cytotoxic cell function (i.e. preformed cytotoxic T-cells) , require the co-presence of cyclosporine to produce an inhibitory effect, and importantly, do not appear to interfere with helper T-cell function. Helper T- cells are responsible for the initiation of all immune responses. The inability to interfere with helper T-cell function is thus a drawback for an agent used to suppress an immune response. The Krensky peptides are likely to be responsible only for a non-specific inhibitory effect.
  • T-cell mediated or T-cell dependent autoimmune diseases in particular, T-cells of the afflicted individuals react with peptides from self antigens presented by self-APC.
  • the conventional approach for suppressing autoimmune responses has also involved administration of nonspecific immunosuppressant and cytotoxic agents with the same types of disadvantages as described above with respect to alloimmunity.
  • 08/472,017 (originally filed as 07/596,936 on October 15, 1990 and corresponding to PCT/US91/07542, published as WO92/06708) , which generally discloses the treatment of autoimmune diseases by oral administration of autoantigens.
  • U.S. Patent Application Ser. No. 08/472,017 (originally filed as Ser. No. 07/595,468 on October 10, 1990, and corresponding to application PCT/US91/07475 published as WO92/06704) discloses oral and aerosol compositions containing insulin for treating mammals suffering from, or at risk for, Type 1 diabetes.
  • U.S. Patent Application Ser. No. 08/419,505 (originally filed as Ser. No.
  • 08/472,017 (originally filed as 07/843,732, on 02/28/92 and corresponding to PCT/US93/01705, published as W093/16724) describes orally administering "bystander" antigens from the locality of a tissue under autoimmune attack to suppress autoimmune response.
  • One object of this invention is to provide novel compounds and compositions that suppress immune responses, and methods for using these materials for this purpose.
  • a more specific object of this invention is to provide compounds and compositions that suppress allorecognition, and therefore abate alloimmunity, and methods for using these materials for this purpose.
  • Another specific object of this invention is to provide compounds and compositions that suppress an autoimmune response, and therefore abate autoimmunity, and methods for using these materials for this purpose. Even more specific objects of this invention are to provide
  • a further object of this invention is to provide compounds and compositions derived from alpha chains of Class II MHC that accomplish one or more of the foregoing and that are not allele-specific (and, optionally, not species-specific) and can be used to modulate one or more of the foregoing immune responses in individuals.
  • a still further object of this invention is to provide methods, compounds, and compositions down-regulating immune responses, as mentioned above, without causing the serious adverse effects associated with use of conventional global immunosuppressants or cytotoxic agents.
  • a method for suppressing an immune response that involves using a peptide compound (derived from the alpha chain of Class II major histocompatibility complex) to suppress such response.
  • the peptide is typically a fragment of that alpha chain and includes a highly conserved region or motif of amino acid residues that is maintained across alleles and species. Other than the highly conserved motif, the peptide can contain one or more portions of the alpha chain adjacent to such motif. In a preferred embodiment, the peptide is composed of 13-26 amino acid residues, and most preferably 16-23 amino acid residues.
  • the highly conserved motif preferably consists of four amino acids and the peptide contains at least three and most preferably all four of these amino acids. Most preferred is the motif consisting of all four of amino acid residues 70-73 of the alpha chain of human DQ Class II major histocompatibility complex (KHNL) .
  • Use of the peptides of the present invention encompasses their delivery to the site of reaction of a T- cell with an APC, e.g. oral, mucosal, or parenteral administration to an individual in need of suppression of an immune response.
  • Such individuals include, without limitation, transplant recipients, prospective transplant recipients, xenograft recipients, and persons afflicted with a T-cell mediated or T-cell dependent autoimmune disease, or determined to be at risk for developing such a disease (by genetic screening or by confirmation of an ongoing autoimmune reaction, such as that involved in the pre-onset stages of juvenile diabetes against pancreatic beta cells) .
  • the invention relates to the foregoing peptides and to compositions containing them suitable for oral or parenteral administration according to the above methods.
  • Figure IA depicts the full amino acid sequences of the first 95 amino acid residues of the alpha chain of the class II MHC from rat RTI.Bo. 1 (top), rat RTl.Bo.” (middle) and a human HLA.DQoc 1*0101 (bottom) ;
  • Figure IB depicts the same alpha chain fragments as Figure IA but the homologous residues from the middle and bottom sequences have been omitted (replaced by hyphens) ;
  • Figures IA and IB highlight the sequence of a peptide within the invention consisting of amino acid residues 62-77 in the bottom sequence and 62-78 in the top sequence (underlined in Figure IB) .
  • Figure 2 depicts amino acid sequences for full length HLA DQ ⁇ chains (amino acid residues 1-232) isolated from different human individuals in the same manner as Figure IB.
  • the stars (*) depict unelucidated residues.
  • Figure 3 depicts amino acid sequences for HLA DQ ⁇ chains isolated from different individuals in the same manner as Figure 2.
  • Figure 4 shows the sequences of examples of peptides within the invention: MHC class II ⁇ chain peptides DQ ⁇ l*010l, RTl.D ⁇ , and RTl.B ⁇ , and the MHC class II ⁇ chain peptide DQ ⁇ 0501.
  • Figures 5 and 6 are tables that show the percent inhibition exhibited in one way rat mixed lymphocyte response (MLR) experiments ("LEWxWF”, “LEWxBN” . "BNxLEW”, and “BNxWF”), EAE experiments (“Antigen/MBP”) , mitogen proliferation experiments (“ConA”), and human MLR experiments (“Human”) using the DQ ⁇ l*0l01 (“DQ ⁇ ”) peptide. Negative values denote augmentation of immune response.
  • MLR mixed lymphocyte response
  • Figure 7 is a table showing the percent inhibition of various immune responses exhibited in various experiments using the RTl.D ⁇ peptide.
  • Figures 8 and 9 are tables similar to Fig. 7 showing the percent inhibition exhibited in experiments using the RTl.B ⁇ peptide.
  • Figures 10 and 11 are tables similar to Fig. 7 showing the percent inhibition exhibited in experiments using the DQ ⁇ peptide.
  • Figure 12 is a graph of the percent inhibition of rat (LEWxWF) MLR accomplished by various doses of DQ ⁇ *0101 peptide and DQ)8*0501 peptide.
  • Figure 13 is the same type of graph as Figure 12 for human MLR.
  • Figure 14 is a graph of the percent specific lysis of WF blast cells by LEW effector cells generated in the presence of DQ ⁇ peptide.
  • Figures 15 and 16 are graphs of 7-interferon (Fig. 15) or interleukin-2 (Fig. 16) concentrations in culture supernatants (human MLR) on day 3 in the presence of various peptides (DQ ⁇ or DQS) at different concentrations, or in the absence of peptide.
  • MHC major histocompatibility complex
  • alloimmunity histocompatibility (a/k/a transplantation) antigens of a donor react directly with host T-cells.
  • host MHC presents peptide fragments of these antigens to host T-cells only.
  • Class I MHC proteins are present on virtually all tissues. Class II MHC proteins are present on the surface of only certain immune system cells.
  • the human MHC genes i.e. the HLA genetic locus
  • the mouse MHC genes are located in the H-2 genetic locus on mouse chromosome 17.
  • the corresponding rat MHC genes are referred to as "RTI”.
  • Class II MHC molecules are membrane giycoproteins that form part of the MHC and are most important in the initiation of immune responses.
  • T-cells activated T-cells, B- cells, macrophages, brain astrocytes, epidermal Langerhans cells, dendritic cells, and thymic epithelium.
  • Class II MHC molecules play a central role in immune response during tissue graft rejection, graft-versus-host reactions, stimulation of antibody production, and in the recognition of "self" (or autologous) antigens, among other immune events.
  • CD4 + T-cells (helper T-cells) recognize antigen only when it is presented in connection with Class II MHC of antigen-presenting cells (hence the term "Class II - MHC restricted”). In turn, these cells are the initiators of any immune response. For this reason, it is most efficient to target CD4 + T-cells in any attempt to suppress an immune response.
  • one or more peptides of the invention are placed in contact with reacting immune system components (e.g. T-cells and APCs) to suppress an immune response in need of suppression.
  • the peptides are derived from (i.e. are fragments of) the alpha chain of the class II MHC and comprise at least one highly conserved motif of at least 3, and preferably of 4, consecutive amino acids.
  • Oral administration shall mean both oral administration and enteral administration (delivery directly into the stomach) .
  • “Mucosal administration” shall mean administration by delivery to the buccal, nasal, bronchial or pulmonary mucosa.
  • Parenter administration means administration by the intravenous, subcutaneous, or intramuscular route.
  • RTI.Doc The present inventors found that the following peptide from the alpha chain of Class II MHC: RTI.Doc” (51- 75) : FASFEAQGALANIAVDKANLDIMIK inhibited alloimmune response in vitro (MLR) .
  • MLR alloimmune response in vitro
  • Rat peptide 51-75 also inhibited (100% inhibition with 500 ⁇ g/mL of peptide) generation of cytotoxic T-cells (CTL) but not the function of preformed cytotoxic T-cells.
  • CTL cytotoxic T-cells
  • the same peptide did not inhibit lymphocyte proliferation to mitogen (in the absence of stimulator cells) , indicating that the MLR inhibition was directed at the interaction between the T-cell receptor of the responder cells and the MHC of the stimulator cells and not to a nonspecific toxic or inhibitory effect on T-cells.
  • the foregoing results with rat peptide 51-75 have been published by the present inventors in Transplantation Proceedings, 1995 (February issue) 27:409.
  • the present inventors also tested various other peptides from the human HLA DQ ⁇ chain and from other rat alpha chain.
  • NSQKEVLEGARASVDR failed to inhibit all of the MLR and CTL reactions described above, and was used as a negative control.
  • DQoci*oi01 (62-77) was a potent inhibitor, although in the context of autoimmunity in this rat model, the peptide RTI.Boc (62-78) appeared to be significantly more potent.
  • Inhibition of immune response in the autoimmune model demonstrates that the inhibitory activity of the peptides of the invention extends beyond alloimmunity to ("ordinary") immunity against foreign antigen and to autoimmunity.
  • Peptides for use in the present invention can be synthesized using well known solid phase synthesis techniques (such as those of Merrifield, R.B. Fed. Proc. Am. Soc. Ex. Biol.. 21; 412, 1962 and J. Am. Chem. Soc. 85: 2149, 1963; Mitchel, A.R- et al. , J. Am. Chem. Soc. 98: 7357, 1976; Tarn, J. et al. , J. Am. Chem. Soc. 105: 6442, 1983), preferably in commercially available automated peptide synthesizing equipment. They can also be synthesized using recombinant DNA technology that is well known in the art. Aside from the sequences disclosed herein, amino acid sequences of Class II MHC alpha chain antigens are available from the Gene Bank database. They have also been published in various references and are known in the art.
  • the peptide selected for immunosuppression has a highly conserved region (or "motif"), not necessarily the K- NL motif.
  • “highly conserved” means that the motif is conserved among several alleles and preferably across species as well.
  • the peptide is derived from the alpha chain of the class II MHC. Although the peptide is preferably 13-26 amino acids in length, it is understood that longer peptides that are to be exposed to degradation, such as through the gastrointestinal tract, can present a smaller peptide of this length. Thus, an orally administered peptide could be longer, and could be naturally processed in the body to reach the preferred length.
  • the active peptide can also be in conjugated form, i.e. conjugated to other peptide sequences or carriers.
  • a peptide selected according to the above criteria can be tested for operability by experimentally determining whether it is suppressive in a T-cell allo-proliferation assay (e.g., an MLR), is suppressive in an antigen specific T-cell proliferation assay, suppresses the generation of cytotoxic T-cells that recognize an alloantigen, or suppresses (e.g., in MLR) the production of stimulatory cytokines, particularly IFN-gamma (gamma interferon) or IL-2 (interleukin 2) .
  • T-cell allo-proliferation assay e.g., an MLR
  • an antigen specific T-cell proliferation assay suppresses the generation of cytotoxic T-cells that recognize an alloantigen
  • suppresses e.g., in MLR
  • stimulatory cytokines particularly IFN-gamma (gamma interferon) or IL-2 (interleukin 2) .
  • Peptides for screening in the invention include, for example, those that are 13-26 amino acids long, and contain a sequence shown in Figure 2 that contains the tetrapeptide KHNL.
  • Examples of such peptides include FDPQGTLR MAVAKHNLNIM, TNIAVLKHNLNILIKRS and KHNLNSLIKRSNSTAATN.
  • the peptides can be administered to individuals who are to receive an allograft, such as an organ transplant, or who have already received an allograft. It is believed that to be most effective, the peptides of the present invention should be administered no later than 7 days after transplant, and preferably less than one day. A peptide is preferably first administered between about 7 and about 14 days before the transplantation procedure. The treatment is preferably continued longer than about 6 months after e.g., an organ or tissue has been transplanted into the individual.
  • the peptides When it is desired to use the peptides to suppress antigen-specific response (e.g. in autoimmunity) , they may be administered either prior to the onset of symptoms of autoimmune disease, or after such symptoms have appeared. In the case of Type I diabetes, for example, the peptides might be administered to an individual found to exhibit an autoimmune reaction against pancreatic ⁇ cells (or at high risk for developing such a reaction) but not yet exhibiting any hyperglycemia or hypoinsuline ia. Suppression of autoimmune response is preferably carried out before the substantial destruction of tissues that the response is directed against.
  • autoimmune diseases in which treatment with the present peptides is useful include without limitation multiple sclerosis (for which EAE is a model) , rheumatoid arthritis (for which collagen-induced arthritis is a model) , uveoretinitis (for which EAU, i.e.
  • autoimmune thyroiditis is a model) , autoimmune thyroiditis, systemic lupus erythematosus, myasthenia gravis, glomerulonephritis, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, pemphigus vulgaris, Grave's disease, mixed connective tissue disease, ulcerative colitis, primary biliary cirrhosis, myositis, Wegener's granulomatosis, male infertility (sperm antigen immunity) , Type II-diabetes and more generally insulin resistance and pernicious anemia.
  • a peptide according to the invention When a peptide according to the invention is to be ingested by the individual, via the oral or enteral route, it is preferably administered in an amount of between about 100 ⁇ g/kg per kg of body weight and about 200 mg per kg of body weight per day. It may be administered as a single dose or in multiple doses daily. Preferably, it is administered in an amount between about l mg and about 50 mg, and most preferably between about lOmg and about 50mg, per kg body weight of the individual per day.
  • the exact amount to be administered can vary depending on the specific activity of a particular peptide, as well as the severity and stage of a patient's disease and/or the physical condition of the patient, and is subject to optimization as is well-known in the art.
  • the oral pharmaceutical formulations of the present invention may contain inert constituents including pharmaceutically acceptable carriers, diluents, fillers, solubilizing or emulsifying agents and salts of the type that are well-known in the art.
  • tablets and caplets may be formulated in accordance with conventional procedures employing solid carriers, such as starch and bentonite, that are well-known in the art.
  • solid carriers include bentonite, silica, dextrose and other commonly used carriers.
  • carriers and diluents which may be used in the formulations of the present invention include saline and any physiologically buffered saline solution such as phosphate buffered saline, pH 7-8 and water.
  • Capsules containing the peptide may be made from any pharmaceutically acceptable material, e.g, gelatin or a cellulose derivative.
  • the peptide may be administered in the form of sustained release oral delivery systems and/or enteric coated oral dosage forms, such as is described in U.S. Patent No. 4,704,292 issued November 3, 1987, U.S. Patent No. 4,309,404 issued January 5, 1982, or U.S. Patent No. 4,309,406 issued January 5, 1982.
  • the amount of peptide contained in an individual dose need not in itself constitute an effective amount for suppressing immune response, since the necessary effective amount can be reached by administration of more than one dose.
  • the active compounds of the invention can be incorporated into a physiologically acceptable solution or suspension. These preparations preferably contain from about 100 ⁇ g of active compound per kg to about 200 mg per kg of body weight thereof. Preferred compositions and preparations according to the present inventions are prepared so that a parenteral dosage unit contains from about 1 mg to about 50 mg of active compound per kg body weight. Most preferred is from about 10 mg to about 50 mg active compound per kg of body weight.
  • the solutions or suspensions can also include the following components: a sterile diluent such as, for example, water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol, other synthetic solvents, and the like; antibacterial agents such as, for example, benzyl alcohol, methyl parabens, and the like; antioxidants such as, for example, ascorbic acid, sodium bisulfite, and the like; chelating agents such as, for example, ethylenediamine tetraacetic acid and the like; buffers such as, for example, acetates; citrates, phosphates, and the like, and agents for the adjustment of toxicity such as, for example, sodium chloride, dextrose, and the like.
  • a sterile diluent such as, for example, water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol, other synthetic solvents, and the like
  • the parenteral multiple dose vials can be of glass or plastic materials.
  • the active protein is placed in contact with the buccal, nasal, bronchial or pulmonary mucosa.
  • Formulations useful for mucosal administration include those suitable for administration of polypeptides across the mucosal membrane.
  • U.S. Patent Nos. 4,226,848 and 4,690,683 describe polymeric matrices useful in administering pharmaceuticals into the nasal cavity.
  • U.S. Patent No. 4.952,560 discloses an ointment formulation comprising a water-soluble protein and a monohydric alcohol which may be suitable for use in administering the present invention because it increases absorption of drugs across epithelial barriers. Methods of improving transcutaneous absorption of materials is described in U.S. Patent No. 4,272,516. Each of these formulations and others well known in the art may be used for mucosal delivery of bystander antigen as described in the present invention.
  • Additional suitable formulations include commercially available vehicles and formulations which may but need not include surface active agents and other skin penetrants as absorption promoters.
  • U.S. Patent No. 5,407, 911 describes the use of axacycloalkane derivatives as absorption promoters for high molecular weight polypeptides.
  • U.S. Patent No. 5,397,771 describes the use of n-glycofurols in methods of administering pharmaceutical compositions across the mucosal membrane.
  • U.S. Patent No. 4,548,922 discloses the use of water-soluble amphophilic steroids to increase absorption.
  • Gel-based compositions such as those described in Morimoto et al. ( Chem. Pharm. Bull . 35(7) .3041-3044) are also suitable for the present invention.
  • the quantity of peptide administered in, e.g., an aerosol dosage form by inhalation is preferably between about 100 ⁇ g and 200 mg per kg body weight per day, preferably between 1-50 m 9/ k 9 of body weight, and most preferably between 10-50 mg/kg of body weight.
  • the by-inhalation forms of the present invention may be administered to a patient in a single dosage form or multiple dosage forms. The exact amount to be administered may vary depending on the state and severity of any disease to be treated, the activity of the patient's immune system and the physical condition of the patient.
  • Inhalable aerosol or spray pharmaceutical formulations may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are well- known in the art.
  • pharmaceutically acceptable carriers include water, normal saline and physiologically-acceptable buffered saline solutions such as phosphate buffered saline solutions, pH 7.0-8.0.
  • useful solubilizing and emulsifying agents are physiologically balanced salt solutions, phosphate buffered saline and isotonic saline.
  • the salts that may be employed in preparing mucosal dosage forms of the invention include the pharmaceutically acceptable salts of sodium and potassium.
  • Aerosol compositions can be administered, e.g., as a dry powder or preferably in an aqueous solution.
  • Preferred aerosol pharmaceutical formulations may comprise, for example, a physiologically-acceptable buffered saline solution containing between about 7 mg and about 700 mg of the peptide of the present invention.
  • Dry aerosol in the form of finely divided solid particles that are not dissolved or suspended in a liquid are also useful in the practice of the present invention.
  • the compositions used in the present invention may be in the form of dusting powders and comprise finely divided particles having an average particle size of between about 1 and 5 microns, preferably between 2 and 3 microns.
  • Finely divided particles may be prepared by pulverization and screen filtration using conventional techniques that are well known to those skilled in the art.
  • the particles may be administered by inhaling a predetermined quantity of the finely divided material, which can be in the form of a dry atomized powder.
  • the invention can also be used to suppress immune response in veterinary medicine, animals used in laboratory research, and in suitable human patients.
  • the present invention is illustrated by the following examples, which are intended to illustrate the invention without limiting its scope.
  • Example 1 Inhibition of Rat Mixed Lymphocyte Response (MLR)
  • Lymph nodes were taken from LEW (typically used as responders) and WF or BN rats (typically used as stimulators) . Rats of the Lewis (LEW) , Wistar Furth (WF) and Brown Norway (BN) variety were used. The excised nodes were then pressed through stainless steel mesh and suspended in medium containing RPMI with L-glutamine, 10% fetal calf serum, IM HEPES, penicillin and streptomycin, and 5 xlO "5 M of 2-ME (mercaptoethanol) . The lymph node cells were then washed twice and resuspended in the same medium at 3 x IO 6 cells/mL. Stimulator cells were irradiated (3000 Rads) .
  • Results of the rat MLR experiments are shown in Figures 5, 7, 8 and 10 for different combinations of rat responder and stimulator cells.
  • Figure 5 for example, “LEWxWF” results are shown for the human DQ ⁇ peptide (i.e. the "DQ ⁇ fl 0101" peptide shown in Figure 4) .
  • “LEW” is the responder cell, while “WF” is the stimulator cell.
  • Results of three experiments for this (responder)x(stimulator) combination are shown. Results are also shown for the combinations "LEWxBN", “BNxLEW”, “BNxWF” and "LEWxBN” .
  • the rat peptides derived from the alpha chain of the class II MHC, RTl.D ⁇ and RTlBo. also inhibited rat MLRs.
  • the RTl.B ⁇ peptide achieved 100% inhibition in the LEWxWF and BNxWF MLRs at 10 ⁇ g/mL.
  • the RTI.Do. peptide was somewhat less inhibitory. It achieved 100% inhibition of the BNxWF MLR at 100 ⁇ g/mL but was less inhibitory in the other rat MLRs.
  • Figure 10 shows the results obtained in rat MLRs using the DQ ⁇ (i.e. the "DQ ⁇ l 0501" peptide) . No inhibition occurred.
  • PBMC peripheral blood mononuclear cells
  • 20 L of blood was placed in a 50 mL NuncTM tube and diluted to 32 mL with medium containing RPMI, penicillin/streptomycin and HEPES. This was underlayed with 12 mL of FicollTM and spun in a centrifuge at 2000 RPM for 30 minutes. An interface layer formed of a "Buffy coat" of cells was removed, and those cells placed in another 50 mL NuncTM tube. These tubes were filled with 50 mL of the medium described above and spun at 1500 RPM for 10 minutes.
  • Liquid was then removed from the tubes and the pellet resuspended in 5 mL of the same medium, combining cells from 2 separate tubes. Medium was added to arrive at 50 mL. The suspension was then spun at 1200 RPM for 10 minutes. Liquid was removed and the pellet, resuspended in 4 mL of 10% medium (the above described serum in medium at a ratio of 1:10) and the cells counted. Further 10% medium was added to arrive at a concentration of 4 x 10° cells/ml.
  • 10% medium the above described serum in medium at a ratio of 1:10
  • a human MLR was performed as follows. Stimulator PBMC were irradiated at 3000 rads. 100 ⁇ L of the 10% medium described above was added to each well of a 96 well U- bottomed plate. Experimental groups were set up in quadruplicate. In the first row of each plate, to which the highest concentration of peptide was to be added, a further 60 ⁇ L of medium was added. To four of these wells, 40 ⁇ l of DQo; peptide was added at a concentration of 1 mg.ml. In the adjacent four wells, 40 ⁇ l of DQ ⁇ peptide was added at a concentration of 1 mg/ml. The contents of each well was mixed with a multichannel pipette.
  • 100 ⁇ l was removed from each well, and mixed with 100 ⁇ l of media in each well of the next row of the plate. This was repeated for each row in the plate. 50 ⁇ l of irradiated stimulator cells (2 x 10 6 cells) was then added to each well, except for the control wells (which contained only media) . 50 ⁇ l of responder cells (2 x 10° cells) were then added to all wells, including the control wells. The plates were incubated at 37°C. On the evening of the 5th day following the start of incubation, each plate was pulsed with 3 H thymidine. 18 hours later the plates were harvested.
  • Figure 13 is a graph of the combined results showing percent inhibition of human MLR using the DQo. and DQ ⁇ peptides.
  • Preformed LEW effector cytotoxic T-cells were generated from LEWxWF bulk cultures as in Example 3, except that no peptides were present in the bulk cultures.
  • Harvested LEW effector cells were then pre-incubated with 10 ⁇ g/mL of peptides for 1 hour.
  • the effector cells were then tested for their ability to lyse 51 Cr labelled WF target blasts in a 4-hour cytotoxic assay as in Example 3. Neither peptide was found to inhibit the preformed effector cytotoxic T-cells at 10 ⁇ g/ml.
  • the effects of the DQ ⁇ and DQ ⁇ peptides on upregulatory cytokine production in the human MLR was measured by ELISA on each day for seven days of incubation using supernatants from cell cultures and ENDOGEN kits for human cytokines. Both dose-response and time course experiments were performed. The dose-response results, illustrated in Figures 15 and 16 for day 3, demonstrate complete suppression of each of IFN-7 and IL-2 by the DQ ⁇ peptide but not by the DQ ⁇ peptide. (The presence of these cytokines is associated with upregulation of immune responses.) In the time-course experiment (data not shown) 100 ⁇ g/mL of peptide was used in the cultures. Supernatants were collected each day from quadruplicate cultures for seven days and assayed at the end of that period. The human DQ ⁇ peptide at this concentration effectively inhibited both IL-2 and IFN-7 over the seven-day period.
  • EAE allergic encephalomyelitis
  • CFA Freund's Adjuvant
  • Example 8 Additional Peptides from the class II MHC a chain
  • Two additional peptides derived from the non- polymorphic RT1.D" (DR or IE like) alpha chain of the rat class II MHC were also tested as described above in Examples 1-4.
  • Peptide 1 consisted of residues 26 to 50 and peptide 2 consisted of residues 51 to 75.
  • Peptide 2 had the following sequence: FASFEAQGALANIAVDKANLDIMIK Peptide 2 was found to inhibit the LEWxWF MLR at about 75% inhibition with 500 ⁇ g/mL. It also inhibited LEWxBN MLR at about 60% inhibition with 500 ⁇ g/mL, and human MLR at about 72% inhibition with 500 ⁇ g/mL.
  • MLR inhibition by peptide 2 was neither strain nor species specific. Although Peptide 2 achieved substantial inhibition, it did so at concentrations 5- to 50-fold higher than the human DQ ⁇ , rat RTl.D ⁇ and rat RTl.B ⁇ peptides tested in Examples l and 2. Peptide 1 was not inhibitory in these systems.
  • Peptide 2 inhibited generation of cytotoxic lymphocytes in a dose-response fashion (100% inhibition with 500 ⁇ g/mL of peptide) . Incubation of preformed effector cytotoxic T-cells with peptide 2 prior to addition of targets failed to inhibit lysis.

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Abstract

L'invention se rapporte à des procédés et à des compositions permettant de supprimer une réponse immune mettant en oeuvre ou comprenant des fragments peptidiques de la chaîne alpha du complexe majeur d'histocompatibilité de la classe II, ces fragments peptidiques contenant un motif extrêmement bien conservé et efficace pour supprimer un et de préférence tous les phénomènes suivants: une réaction lymphocitaire mixte ou d'autres réactions d'alloreconnaissance des lymphocytes T; la génération des lymphocytes T cytotoxiques reconnaissant un alloantigène; la prolifération lymphocitaire contre l'antigène tissulaire; et la production de cytokine stimulatrice par des lymphocytes. Les réponses immunes qui peuvent être atténuées ou déprimées sont l'alloimmunité et l'autoimmunité.
PCT/US1996/015662 1995-09-21 1996-09-23 Procedes et compositions de suppression d'une reponse immune a l'aide de peptides de la classe ii du complexe majeur d'histocompatibilite Ceased WO1997010711A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
IL12375996A IL123759A0 (en) 1995-09-21 1996-09-23 Methods and compositions for suppressing an immune response using major histocompatibility complex (MHC) class II peptides
EP96933201A EP0914041A4 (fr) 1995-09-21 1996-09-23 Procedes et compositions de suppression d'une reponse immune a l'aide de peptides de la classe ii du complexe majeur d'histocompatibilite
CA002230119A CA2230119A1 (fr) 1995-09-21 1996-09-23 Procedes et compositions de suppression d'une reponse immune a l'aide de peptides de la classe ii du complexe majeur d'histocompatibilite
AU72025/96A AU7202596A (en) 1995-09-21 1996-09-23 Methods and compositions for suppressing an immune response using major histocompatibility complex (mhc) class ii peptides
KR1019980702086A KR19990063637A (ko) 1995-09-21 1996-09-23 주요 조직적합 복합체(mhc) 클래스 ⅱ 펩티드를 이용한 면역응답 억제방법 및 조성물
BR9610625A BR9610625A (pt) 1995-09-21 1996-09-23 Composição farmacêutica para suprimir uma resposta imunitária peptídeo e método para preparar uma composição farmacêutica
JP9512976A JPH11513377A (ja) 1995-09-21 1996-09-23 主要組織適合遺伝子複合体（ｍｈｃ）クラスｉｉペプチドを使用する免疫反応を抑制するための組成物及び方法
NO981201A NO981201L (no) 1995-09-21 1998-03-17 Fremgangsmåter og sammensetninger for å undertrykke en immunrespons ved anvendelse av hovedhistokompatibilitetskompleks (MHC) klasse II peptider

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US411795P 1995-09-21 1995-09-21
US60/004,117 1995-09-21

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CA (1) CA2230119A1 (fr)
HU (1) HUP9901795A2 (fr)
IL (1) IL123759A0 (fr)
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7247438B1 (en) 1997-02-18 2007-07-24 Dana-Farber Cancer Institute Methods of identifying agents which enhance caspase activity

Families Citing this family (1)

* Cited by examiner, † Cited by third party
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JP2014133703A (ja) * 2011-03-30 2014-07-24 Clio Inc 生体組織から単離できるｓｓｅａ−３陽性の多能性幹細胞を含む他家移植用細胞治療用組成物

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH08502244A (ja) * 1992-08-11 1996-03-12 プレジデント アンド フェローズ オブ ハーバード カレッジ 免疫調節ペプチド

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF EXPERIMENTAL MEDICINE, May 1993, Vol. 177, HURTENBACH et al., "Prevention of Autoimmune Diabetes in Non-Obese Diabetic Mice by Treatment with a Class II Major Histocompatibility Complex-Blocking Peptide", pages 1499-1504. *
JOURNAL OF EXPERIMENTAL MEDICINE, November 1990, Vol. 172, BENICHOU et al., "Immunogenicity and Tolerogenicity of Self-Major Histocompatibility Complex Peptides", pages 1341-1346. *
See also references of EP0914041A4 *
THE JOURNAL OF IMMUNOLOGY, 15 July 1991, Vol. 147, No. 2, AGRAWAL et al., "T Cells Recognize Peptide Sequences of Self MHC Class II Molecules Exist in Syngeneic Mice", pages 383-390. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7247438B1 (en) 1997-02-18 2007-07-24 Dana-Farber Cancer Institute Methods of identifying agents which enhance caspase activity

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KR19990063637A (ko) 1999-07-26
HUP9901795A2 (hu) 1999-09-28
IL123759A0 (en) 1998-10-30
NO981201D0 (no) 1998-03-17
NO981201L (no) 1998-03-17
EP0914041A4 (fr) 2000-06-28
JPH11513377A (ja) 1999-11-16
AU7202596A (en) 1997-04-09
CA2230119A1 (fr) 1997-03-27
BR9610625A (pt) 1999-03-16

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