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WO1996032957A1 - Modulation de sequences cytokiniques de clones de lymphocytes t humains autoreactifs - Google Patents

Modulation de sequences cytokiniques de clones de lymphocytes t humains autoreactifs Download PDF

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Publication number
WO1996032957A1
WO1996032957A1 PCT/US1996/005783 US9605783W WO9632957A1 WO 1996032957 A1 WO1996032957 A1 WO 1996032957A1 US 9605783 W US9605783 W US 9605783W WO 9632957 A1 WO9632957 A1 WO 9632957A1
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Prior art keywords
cells
amino acid
peptide
cell
altered
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David A. Hafler
Howard L. Weiner
Vijay K. Kuchroo
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Brigham and Womens Hospital Inc
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Brigham and Womens Hospital Inc
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Priority to AU58511/96A priority Critical patent/AU5851196A/en
Priority to EP96920109A priority patent/EP0825870A4/fr
Publication of WO1996032957A1 publication Critical patent/WO1996032957A1/fr
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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
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4713—Autoimmune diseases, e.g. Insulin-dependent diabetes mellitus, multiple sclerosis, rheumathoid arthritis, systemic lupus erythematosus; Autoantigens
    • 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 altering the cytokine pattern of T-cells (particularly inflammation-promoting T-cells of patients afflicted with an autoimmune disease) from a first pattern that promotes immune responses (Th0) to an altered version of the first pattern that downregulates immune responses.
  • Autoimmune diseases are characterized by an abnormal immune response directed against normal autologous (self) tissues.
  • autoimmune diseases in mammals can generally be classified in one of two different categories: cell-mediated (i.e., T-cell-mediated) or antibody-mediated disorders.
  • cell-mediated autoimmune diseases include multiple sclerosis (MS), rheumatoid arthritis (RA), autoimmune thyroiditis (AT), the autoimmune stage of diabetes mellitus (juvenile-onset or Type 1 diabetes) and autoimmune uveoretinitis (AUR).
  • Antibody-mediated autoimmune diseases include without limitation myasthenia gravis (MG) and systemic lupus erythematosus (SLE).
  • T-cell-mediated and antibody-mediated autoimmune diseases is not absolute, as many antibody-mediated autoimmune diseases have a T-cell component (i.e. include inflammation of the afflicted organ or tissue).
  • Both categories of autoimmune diseases are currently being treated with drugs that suppress immune responses systemically in a non-specific manner, i.e., drugs incapable of selectively suppressing the abnormal immune response.
  • drugs include methotrexate, cyclophosphamide, Imuran (azathioprine) and cyclosporin A.
  • Steroid compounds such as prednisone and methylprednisolone (also non-specific immunosuppressants) are also employed in many instances. All of these currently employed drugs have limited efficacy against both cell- and antibody-mediated autoimmune diseases. Furthermore, such drugs have significant toxic and other side effects and, more important, eventually induce "global" immunosuppression in the subject being treated.
  • PCT/US93/01705 filed February 25, 1993, PCT/US91/01466 filed March 4, 1991, PCT/US90/07455 filed December 17, 1990, PCT/US90/03989 filed July 16, 1990, PCT/US91/07475 filed October 10, 1991, PCT/US93/07786 filed August 17, 1993, PCT/US93/09113 filed September 24, 1993, PCT/US91/08143 filed October 31, 1991, PCT/US91/02218 filed March 29, 1991, PCT/US93/03708 filed April 20, 1993, PCT/US93/03369 filed April 9, 1993, and PCT/US91/07542 filed October 15, 1991, and PCT US95/04120 filed April 7, 1995 (Attorney Docket No. 1010/27956- WO0) and PCT US95/04512 filed April 7, 1995 (Attorney Docket No. 1010/27956-WO2).
  • autoimmune attack refers to the immune events that lead to destruction by the immune system of self-tissue. Oral administration of these "bystander antigens" causes regulatory
  • GALT gut-associated lymphoid tissue
  • MALT mucosa associated lymphoid tissue
  • T- cells elicited by the bystander antigen are targeted to the locus of autoimmune attack where they mediate the local release of immunomodulatory cytokines, such as transforming growth factor beta (TGF- ⁇ ) interleukin-4 (IL-4) or interleukin-10 (IL-10).
  • TGF- ⁇ transforming growth factor beta
  • IL-4 interleukin-4
  • IL-10 interleukin-10
  • IL-4 and IL-10 are also antigen- nonspecific immunoregulatory cytokines.
  • IL-4 in particular enhances Th2 response, i.e. acts on T-cell precursors and causes them to differentiate preferentially into Th2 cells.
  • IL-4 also indirectly inhibits Th1 exacerbation.
  • IL-10 is a direct inhibitor of Th1 responses.
  • Naive T-cells differentiate into distinct populations defined by their cytokine secretion pattern and regulatory function. Thl cells secrete predominantly Interleukin-2 (IL-2) and interferon ⁇ (IFN ⁇ ) and are involved in classic delayed- type hypersensitivity reactions, while Th2 cells, which secrete predominantly IL-4 and IL-10, induce selected immunoglobulin secretion and can down-regulate Th1-mediated immune responses (Mossmann et al., 1986; Romagnan, S., 1994). Thus, cytokines secreted by T-cells after activation qualitatively influence the nature of the immune response.
  • IL-2 Interleukin-2
  • IFN ⁇ interferon ⁇
  • cytokines particularly IL-2 and IFN ⁇ for Th1 and IL-4 for Th2 (Seder and Paul, 1994).
  • Other factors include the dose and type of antigen, the type of antigen-presenting cell (APC), and co-stimulatory molecules involved in activation.
  • APC antigen-presenting cell
  • T-cell secretion of certain cytokines is also implicated in the induction and regulation of autoimmune inflammatory disease, which is mediated by activated autoreactive T-cells that recognize self-tissue-specific antigen in the context of major histocompatibility complex
  • MHC myelin basic protein
  • TGF- ⁇ 1 secretion appears to be of particular importance in regulating EAE, as anti-TGF- ⁇ 1 monoclonal antibodies inhibit the suppressor effect of regulatory MBP-reactive T-cells (Chen et al., 1994).
  • autoreactive T-cells are not necessarily pathologic and can function to down-regulate immune responses associated with tissue inflammation locally. Similar findings have been made in humans affected with multiple sclerosis. TNF has been isolated from central nervous system plaques of MS patients, IFN- ⁇ exacerbates attacks, and TGF- ⁇ is secreted by T-cells of MS patients tolerized with myelin.
  • T-cell activation in general is not a simple on/off response.
  • Allen and coworkers (Evavold and Allen, 1991; Evavold et al., 1993a) demonstrated that stimulation of a T-cell clone with modified peptide-antigens that trigger a suboptimal T-cell receptor (TCR) signal can induce cytokine secretion or IL-2 receptor expression and cytolysis without thymidine incorporation.
  • Altered peptides have also been shown to act as TCR antagonists or to induce anergy in Th1 and Th2 clones (Alexander et al., 1993; De Magistris et al . , 1992 ; Jameson et al . , 1993 ; Ostrov et al . , 1993 ; Rupper et al., 1993; Sloan-Lancaster et al., 1993, 1994).
  • none of these publications involved work with an autoimmune disease or an animal model thereof.
  • One object of the invention is to provide additional methods and compositions for combatting autoimmune disease. Another object is to provide additional methods and compositions for combatting immune responses associated with autoimmune disease.
  • a third object is to provide methods and compositions for converting T-cells mediating immune response associated with autoimmune disease to a benign or regulatory type, thereby reducing immune response and/or suppressing it.
  • FIG. 1 shows the T-cell receptor (TCR) and major histocompatibility complex (MHC) contact points for native (cognate) peptide ligand MBP p85-99; (arrows up indicate TCR contact points; arrows down indicate MHC contact points).
  • TCR T-cell receptor
  • MHC major histocompatibility complex
  • Figure 2 is a graph of the ability of cognate and altered peptide ligands to bind to T-cell receptor (counts per minute of tritiated thymidine incorporation) at various peptide concentrations ( ⁇ g/ml); dark triangles: p(91A); dark squares: p(90A); dark diamonds: p(90D); dark circles: p(90K); open circles: p(92A); open triangles: cognate peptide ligand MBP p(85-99); and dark squares: no antigen (negative control).
  • Figure 3A is an autoradiograph showing phosphorylation of CD4-associated tyrosine kinase p56 lck stimulated by: no antigen (control), MBP p(85-99), and altered peptides p(90A), p(91A) and p(93A).
  • Figure 3B is a fluorograph depicting Ca ++ flux induction (410nm/480nm fluorescence ratio vs. time in seconds) in T-cells presented with APC pulsed with MBP p(85-99) and p(90A); no antigen was used as a negative control and ionomycin and EGTA were used as positive controls.
  • Figure 3C is a bar diagram showing IL-4 secretion by T-cells presented with: no antigen; MBP p(85-99); no antigen in the presence of PMA and ionomycin; p(90A); p(90K); no antigen + PMA; p(90A) + PMA; p(90K) + PMA; no antigen + ionomycin; p(90A) + ionomycin; p(90K) + ionomycin.
  • Figure 4 is a Southern Blot showing modulation of m- RNA encoding TGF- ⁇ 1 and IL-4 after T-cell stimulation with: no antigen; MBP p(85-99); p(90A); and p(93A).
  • FIG. 5 is a series of bar graphs showing the secretion (in pg/ml) by T-cells of various cytokines (TGF- ⁇ 1,
  • IL-2, IL-4, IL-10 and ⁇ -IFN upon stimulation with: no antigen; cognate peptide ligand p (85-99); and altered peptide ligands
  • Figure 6 contains three graphs depicting the secretion in picograms/ml of IL-4, IFN- ⁇ and TGF- ⁇ 1 by T-cells stimulated with APCs pulsed with various concentrations of MBP p(85-99) and p(90A). Levels generated by p(90a) are shown as dark squares; those generated by native Mbp p(85-99) are shown as open circles.
  • Figure 7 is a graph showing mean disease scores for animals immunized with a native peptide W144 alone (open boxes) or coimmunized with W144 and Q144 at a ratio of 1:5 (open squares) and assessed daily for signs of disease.
  • Figure 8 consists of three graphs showing the proliferative response of LNC from mice immunized with W144 (a), Q144(b) or W144 and Q144(c).
  • Figure 9 consists of four graphs showing the proliferative response and cytokine production of T-cell lines specific for native W144(WLI) or Q144(QL1) peptides.
  • Graphs a and b show proliferative response while graphs c and d show cytokine production.
  • W is W144 (the native peptide)
  • Q is Q144
  • LR is L144/R147
  • A is A144.
  • Figure 10 shows cytokine production for T-cells specific for Q144.
  • the amount of cytokine produced for clones of QL1 is displayed in terms of IFN ⁇ versus IL-10 and IL-4 versus IL-10 to determine whether the Q144-specific clones were of the THO (IFN ⁇ , IL-10) or TH2 (IL-4, IL-10) phenotype.
  • Figure 11 is a graph of mean disease scores for SJL mice immunized with W144, Q144 or PLP 190-209. Results shown are the mean disease scores for each group from three independent experiments.
  • Cytokine pattern of a T-cell means the cytokines secreted by that T-cell.
  • Immuno response enhancing cytokine pattern or "Th-0 cytokine pattern” means cytokines secreted by an activated (immune attack) T-cell and including one or more of IL-2, TNF and IFN- ⁇ , but not substantial amounts of TGF- ⁇ , IL-4 or IL-10.
  • Immuno response regulating cytokine pattern means cytokines secreted by a regulatory T-cell including one or more of TGF- ⁇ , IL-4, or IL-10 but not substantial amounts of IL-2, TNF or IFN- ⁇ .
  • Cognate peptide ligand means a peptide consisting essentially of an antigenic determinant against which a T-cell has been elicited, and which a T-cell recognizes and proliferates to.
  • altered peptide ligand means a peptide differing in amino acid sequence from a cognate peptide ligand in at least one amino acid residue.
  • TCR contact point or "TCR contact residue” means an amino acid residue within a peptide ligand that participates in binding of the ligand to a T-cell receptor that recognizes it.
  • MHC contact point or "MHC contact residue” means an amino acid residue within a peptide ligand that participates in presentation of the ligand in connection with the major histocompatibility complex (MHC) of an antigen-presenting all
  • Cytokine-affecting TCR contact point is a TCR contact residue which upon conservative or non-conservative substitution with another amino acid residue causes the cytokine pattern of the T-cell to switch towards a regulatory cytokine pattern.
  • MBP p(85-99) or p(85-99) means the cognate peptide ligand MBP peptide having the sequence ENPWHFFKNIVTPR, i.e. amino acid residues 85-99 of myelin basic protein (MBP).
  • MBP p. (82-104) which has the sequence: DENPWHFFKNIVTPRTPP.
  • MBP p85-99(93A) or p(93A) means an altered peptide ligand wherein the 93d residue of MBP (K in the cognate peptide) has been replaced by A (alanine).
  • MBP immunodominant region peptide p(143 -162) has the amino acid sequence FKGVDAQGTLSKIFKLGGRD.
  • MBP minimum immunodominant peptide p(148-162) has the sequence AQGTLSKIFKLGGRD.
  • PLP immunodominant region peptide p (31-50) has the sequence LFCGCGHEALTGTEKLIETY.
  • PLP immunodominant region peptide p (181-200) has the sequence WTTCQSIAFPSKTSASIGSL.
  • An altered PLP peptide consisting of p(139-151) (which is immunodominant in mice) with a substitution at residue 144 has suppressed immune responses associated wtih EAE (experimental autoimmune encephalomyelitis).
  • amino acids or residues
  • the present inventors have found that stimulation of human autoreactive cell clones previously determined to recognize immunodominant epitopes (in humans) of myelin basic protein and to be of the Th0 type (i.e. secreting immune response promoting cytokines) with certain altered peptide ligands presented by antigen-presenting cells (i.e. altered- ligand/MHC complexes) induce a switch in the cytokine pattern of these activated (immune attack) T-cells, and cause them to secrete suppressive cytokines.
  • the altered peptide ligands that could effect the cytokine pattern switch differ in amino acid sequence from the cognate peptide ligand of the T-cell clones by one amino acid at a TCR contact point.
  • Phenylalanine at residue 91 of MBP p(85-99) represents a primary TCR contact residue for MBP p(85-99) .
  • Altered ligands substituted at this residue do not elicit any response from T-cells recognizing MBP p(85-99). It is accordingly preferred to use as altered peptide ligands analogs of MBP p (85-99) that are not substituted at position 91.
  • Histidine at residue 90 and lysine at residue 93 are secondary TCR contact residues. Any amino acid substitution at those residues (both conservative and nonconservative) will cause a change in the cytokine pattern of the inflammation- promoting T-cells towards the suppressive cytokine pattern.
  • the preferred altered peptide ligands will be the ones that will cause patients' T-cells to secrete the highest levels of suppressive cytokines, especially TGF- ⁇ .
  • the T-cell clone not proliferate in response to the altered peptide ligand but merely switch its cytokine pattern.
  • the altered peptide ligands efficiently inhibit antigen-dependent T-cell proliferation and can induce IL-4 or
  • the altered peptide ligands appear to engage the TCR with comparable affinity to the cognate peptide but trigger a different set of signals, leading to an altered cytokine pattern, a regulatory cytokine pattern.
  • Th0 type T-cells to a TGF- ⁇ or IL-4 secreting T-cell has important physiological consequences because suppressive cytokines inhibit immune response including immune responses to self-antigens.
  • Altered peptide ligands parenterally administered can thus confer attenuation of autoimmune responses by causing autoreactive T-cells to switch to a suppressive cytokine pattern, and thereby suppress inflammatory immune responses.
  • Effective amounts for administration will range generally from about 1 to about 50 mg/patient/treatment for any autoimmune disease and any altered peptide ligand.
  • Preferred amounts for altered peptide ligands derived from MBP p (85-99) will be from 5-15 mg/patient/treatment.
  • Treatment frequency can be once a month. Treatment duration can be as long as benefits persist for a period of months or even several years.
  • the therapy dose, frequency of administration and regimen will be subject to optimization according to the disease, the altered peptide ligand employed, the physical condition of the patient, and the levels of regulatory cytokines elicited in the T-cells with the altered cytokine patterns, as is well-appreciated by those skilled in the art.
  • More than one altered peptide variant can be administered to a patient simultaneously with or separately from another altered peptide ligand.
  • the altered peptide ligands are preferably administered in a physiologically acceptable medium or diluent, suitable for injectable preparations. Any parenteral route of administration may be employed, e.g. subcutaneous, intraperitoneal or intramuscular or intravenous injection.
  • the altered peptide ligands of the present invention can be identified by using the following procedure.
  • T-cells that recognize a known antigen are isolated. If not already known, the immunodominant region(s) of the antigen or bystander antigen are thereafter determined by synthesizing overlapping peptides that span the amino acid sequence of the entire antigen. It is contemplated that such peptides will be of at least about 15 residues and preferably about 20 residues (and more preferably between 18 and 20 residues) in length. Each of the overlapping peptides is then tested by exposing such peptide to a panel of T-cell clones that are isolated from patients afflicted with the autoimmune disease to be treated and which recognize the antigens .
  • T-cell isolation is (as well as for identification of the immune dominant epitopes) disclosed in more detail for MS in PCT US93/03369, filed April 9, 1993 may be employed to create T-cell panels for other autoimmune diseases by appropriate substitution of patients and antigens.
  • the procedure can also be used for individual patients, if desired.
  • the T-cell panels are used to identify the immunodominant epitope of the antigen, as follows:
  • Each of the overlapping peptides is exposed to an antigen presenting cell (APC), i.e. a cell which presents that antigen to the T-cells that recognize such antigen.
  • APC antigen presenting cell
  • the T-cell lines from the panel are tested for binding to each overlapping peptide.
  • the overlapping peptide (or peptides) that are recognized by a substantial number of the T-cell panel members are then identified using this procedure. That is, the overlapping peptide or peptides selected are those recognized by the largest number of T-cell panel members.
  • the smallest immunodominant fragment of the overlapping peptide i.e. the smallest fragment that binds to T-cells which recognize the antigen with nearly the same affinity as the entire overlapping peptide containing the immunodominant epitope
  • MIEP minimum immunodominant epitope peptide
  • MIEP amino acid residues of the MIEP that are (a) MHC contact points or (b) TCR contact points must be identified.
  • a panel of MIEP analogs each differing from the MIEP by a single amino acid substitution at a different position is then constructed.
  • Each MIEP analog is thereafter tested for binding affinity to the MHC of an APC (for the antigen or bystander antigen) and for binding (as an analog/MHC complex) to a T-cell clone that recognizes the immunodominant epitope of the antigen.
  • APC for the antigen or bystander antigen
  • binding as an analog/MHC complex
  • T-cell clone that recognizes the immunodominant epitope of the antigen.
  • the measured binding affinity of each MIEP analog to the APC and to the TCR is compared to the measured binding affinity of the MIEP to the APC (MHC) and the TCR. If substitution of one amino acid residue of the MIEP results in reducing the binding affinity for the MHC, that residue is an MHC contact point. In creating the altered peptide ligand residues that have been identified as MHC contact points should not be substituted.
  • each TCR contact point can be a substitution site.
  • Preferred substitution sites are those at which substitution of a non-native amino acid residue does not permit the MIEP recognizing T-cell to proliferate upon encountering the MIEP analog, but merely cause the T-cell to switch to a regulatory cytokine pattern.
  • Altered peptide ligands for use in the present invention are created by substitution of a single MIEP amino acid residue with a different amino acid residue at a TCR contact point.
  • substitutions at positions 90, 91 and 93 yield ligands that bind to the T-cells recognizing the MIEP MBP p (85-99) with affinity comparable to that of the native MBP p(85-99) differing by less than one order of magnitude. Substitutions at position 90 and 93 are preferred because the T-cells do not proliferate upon encounter of the altered peptide ligand.
  • a panel of altered peptide ligands is assembled. Each panel member is then assayed for binding to a T-cell that recognizes the MIEP, ability to cause such a T-cell to proliferate, and for the cytokine pattern that it elicits in such T-cell.
  • Altered peptide ligands that bind to the T-cell receptor with comparable affinity to that of the nature MIEP and that cause the T-cell to switch to a regulatory cytokine pattern are selected.
  • the assay for determination of the T-cell cytokine pattern before and after exposure to an altered peptide ligand is described below with specific reference to T-cell from DR2+ patients that recognize MBP P (85-99) and altered peptide ligands derived from MBP p (85-99). This procedure can be readily adapted to T-cells of different specificities, different antigens involved in different autoimmune diseases.
  • each altered peptide ligand is contacted with peripheral T-cells isolated from a patient afflicted with the target autoimmune disease in the presence of irradiated mononuclear cells (used as antigen presenting cells).
  • Preferred candidate peptides for therapy are those which induce the highest levels of release of TGF- ⁇ 1 (or IL-4) without inducing cell division (monitored by thymidine incorporation) or the release of any other immune response-enhancing cytokines.
  • the techniques described herein can be used for identification of immunods mirant epitopes of other antigens involved in other autoimmune diseases.
  • Antigens specific to the afflicted tissue or organ recognized by activated (immune altered) cells from patients suffering from an autoimmune disease can be subjected to the foregoing procedure for identification of immunodominant epitopes, MIEP's and altered peptide ligands that cause activated autoreactive T-cells to switch to a regulatory cytokine pattern.
  • Non- limiting examples of antigens to which there is autoimmu ⁇ ity in other autoimmune diseases include Type II and Type I collagen for rheumatoid arthritis and glutamic acid decarboxylase for autoimmune (e.g.
  • T cell clones Ob1A12 and Ob3D1 that recognize MBP p were generated from peripheral blood mononuclear cells of a MS patient by limiting dilution cloning as described previously (Ota, et al., 1990). The clones were maintained in fetal calf serum, 10% dimethyl sulfoxide (DMSO) in liquid nitrogen.
  • DMSO dimethyl sulfoxide
  • MBP p85-99 EBPVVHFFKNIVTPR
  • altered peptides were synthesized in the Biopolymer Laboratory, Harvard Medical School, by automated solid phase methods using FMOC-protected amino acid precursors and purified by reverse-phase HPLC. Peptides were greater then 98% pure on the basis of HPLC analysis. Binding of MBP peptides to purified DRB1*1501 (human TCR) molecules was determined as previously described (Wucherpfennig, et al., 1994).
  • the homozygous B cell line 9010 (DR2/DQ6) was used as APCs.
  • APCs were pulsed with peptide at 1 - 100 ⁇ g/ml for 2 hours, washed and irradiated with 5000 rads.
  • T cells (5x10 4 ) and APCs (2x10 4 ) were then co- cultured in triplicate for 3 days in 200 ⁇ l complete medium at 37°C.
  • 1 ⁇ Ci of 3 H-thymidine was added to each well and T-cell proliferation measured as 3 H- thymidine incorporation.
  • +++ represents proliferation (stimulation index >3) at 1 ⁇ g/ml
  • ++ represents proliferation at 10 ⁇ g/ml
  • APCs (B cell line 9010) were prepulsed with a suboptimal dose of 2 ⁇ g/ml MBP p85-99 peptide for 2 hours, irradiated and washed, followed by a second pulse with the altered peptide ligands at 0.1, 1, 10 and 100 ⁇ g/ml for 2 hours. After another wash the APCs were added to the T-cell clone Ob1A12 (5x10 4 APCs and 2x10 5 T cells per well in triplicates) and co-cultured for 3 days. Proliferation was measured as thymidine incorporation (in cpm, see Fig. 1) after an 18 hour pulse. Native MBP p85-99 and MBP p85-99(93A), which are agonist peptides, were used as positive controls.
  • the immunoprecipitate was washed twice in lysis buffer and incubated in a [ ⁇ 32 P] -ATP kinase buffer for 10 minutes. The reaction was stopped by the addition of 2 volumes of sample buffer. Samples were boiled for 5 minutes and separated on a 10% SDS gel. Autoradiography was done for 30 minutes on Kodak XAR 5 film, and the results are shown in Fig. 3A.
  • T cells (10 7 /ml) were loaded with indo-1 acetoxymethyl ester (Molecular Probes, Junction City, OR) at 2 ⁇ g/ml in culture medium for 45 min. at 37°C and then diluted 1:10 in culture medium.
  • APCs were pulsed with peptide at 100 ⁇ g/ml for 2 hours, washed in RPMI and resuspended in culture medium.
  • T cells (10 6 ) and APCs (5x10 5 ) were run on an Epics V flow cytometer to establish a baseline. Then cells were spun for 1 min. at 1200 rpms to establish cell to cell contact and rerun on the flow cytometer for up to 600 seconds.
  • the indo-1 dye After Ca 2+ binding the indo-1 dye exhibits changes in fluorescence emission wavelength from 480 to 410 nM. The ratio of 410:480 nM indo-1 fluorescence was recorded vs time and expressed in arbitrary units, one arbitrary unit representing approximately 200 nM increase of Ca 2+ . Addition of ionomycin (4 ⁇ g/ml) was used as a positive control and EGTA (5 ⁇ g/ml) as a negative control. The results are shown in Fig. 3B. Measurement of cytokine mRNA
  • RNAzol B cells (9010) were pulsed with the peptides as described above. 10 5 T cells were then co-cultured with 4x10 4 APCs in 200 ⁇ l medium in U-bottomed wells for 4 hours followed by RNA extraction and cDNA synthesis. Total cellular RNA was extracted using the RNAzol B method (Teltest, Inc., Friendswood, TX). RNA was co-precipitated with 10 ⁇ g of transfer RNA in isopropanol overnight. For cDNA synthesis the pellet was resuspended in 6 ⁇ l of sterile double distilled water, 5 ⁇ l of oligo-dT (Sigma) and random hexamers (Promega) were added and the samples heated to 70°C for 10 minutes.
  • the PCR for TGF- ⁇ and IL-4 was semiquantitative with 28 amplification cycles at 94°C denaturation (1 min.), 60°C annealing (1 min.), 72°C extension (90 sec), which was determined to be within the linear range of the PCRs. Concentrations in the PCRs were as previously reported (Wucherpfennig, et al., 1990), except for the addition of 0.6 ⁇ Ci of 32 P labeled dCTP to each reaction.
  • Forward primer for TGF- ⁇ 1 was 5'-GCC CTG GAC ACC AAC TAT TGC-3', reverse primer 5'-GCT GCA CTT GCA GGA GCG CAC-3'; PCR product length is 336 base pairs.
  • Primers for IL-4 were as follows; forward primer 5'-CTG CTA GCA TGT GCC GGC AAC TTT GTC CAC-3' , reverse primer 5'-GAA GTT TTC CAA CGT ACT CTG GTT GGC TTC-3', length of PCR product is 365 bp.
  • Radioactive PCR products were separated on 5% polyacrylamide gel and visualized by autoradiography. Cytokine ELISA
  • T cells from Ob1A12 were estimated in 5 ml polypropylene tubes with 4x10 5 peptide pulsed APCs (9010 B cells) pulsed with peptide for 2 hours.
  • Culture supernatants for IL-2, IL-4, IL-10 and IFN- ⁇ were taken after 40 hours.
  • To measure secretion of active TGF- ⁇ 1 the cells were then washed carefully 3 times in RPMI in order to remove all serum and resuspended in serum-free medium X-Vivo 20 supplemented with 2U rIL-2/ml and 2U rIL-4/ml (Boehringer Mannheim).
  • Supernatant for TGF- ⁇ 1 ELISA was harvested after 72 hours from the start of experiment.
  • the ELISA was done for active TGF- ⁇ 1 and was performed as described previously (Miller, et al., 1993).
  • primary and secondary antibodies were purchased from Pharmingen and used following the manufacturer's directions.
  • IL-2 was assayed using the ELISA kit from Endogen.
  • IFN- ⁇ the primary antibody was obtained from Genzyme, the secondary from Biosource International. Assays were done in duplicate.
  • TCR antagonist properties of altered peptide ligands were assessed to aid in screening peptide ligands that bound to the TCR.
  • the biologic activity of peptides with substitutions at position 90 was examined in a prepulse TCR- antagonist experiment in which T-cells are stimulated by APCs that were pulsed with a limited concentration of native peptide, followed 2 hours later by stimulation with increasing concentrations of peptide antagonist. This assay was developed to avoid competition between antigen and antagonist at the MHC level and thus allow measurement of events at the TCR level (De Magistris et al., 1992). TCR antagonism has been attributed to active signaling events resulting in the inhibition of early biochemical events required for T-cell activation rather than just competition for TCR binding with the native peptide
  • MBP p85-99 peptides altered at position 90 could inhibit proliferation induced by the native peptide, suggesting that they could act as a TCR ligand and signal in the context of class II MHC.
  • These altered peptide ligands did not induce clonal unresponsiveness as the T-cells but showed a full proliferative response to MBP p85-99 7 days after the initial peptide stimulation (data not shown).
  • MBP p85-99[91A] inhibited proliferation of Ob1A12 to a substantially lower degree, even at concentrations of 100 ⁇ g/ml, suggesting that this peptide- MHC complex had a low TCR affinity as compared with the peptides with substitutions at position 90 ( Figure 1).
  • peptide MBP p85-99(93A) acted as an agonist similar to the native peptide, indicating that position 93 is not a critical TCR contact point.
  • Antigen-specific activation of T-cells results in a cascade of events involving Ca 2+ influx and activation of the CD4-associated protein tyrosine kinase p56 lck leading to a mobility shift of p56 lck to p60 lck on gel electrophoresis (Barridge, 1993; Rudd et al., 1994).
  • CD4-associated p58 lck was immunoprecipitated and tested in an in vitro autophosphorylation assay.
  • MBP p85-99 induced a strong Ca ++ influx in contrast with p(90A) ( Figure 3B).
  • T-cells can be activated by a combination of ionomycin and phorbol myristate acatate (PMA), which induce a Ca ++ flux and direct protein kinase C activation (resulting in p58lck activation), respectively.
  • PMA phorbol myristate acatate
  • TGF- ⁇ 1 mRNA was induced after stimulation of Ib1A12 with MBP p85-99(90A) ( Figure 4A).
  • a second human MBP p85-99 -reactive T-cell clone, Ob3D1 was examined. This clone also has a Th0 cytokine profile and proliferates to MBP p85-99 and (90A), while substitutions at position 93 result in loss of reactivity, indicating that the lysine at this position is a critical TCR contact residue.
  • Stimulation with MBP p85-99(93A) but not MBP p85-99 or (90A) induced significant MRNA expression of TGF- ⁇ 1 ( Figure 4B). Switch in Cytokine Secretion after T-Cell
  • MBP p85-99(90A) and (90D) induced TGF- ⁇ 1 secretion, corresponding to the induction of mRNA synthesis.
  • these MBP analog peptides switched cytokine secretion patterns by selectively inducing mRNA synthesis and protein secretion of TGF- ⁇ 1 in the absence of thymidine incorporation or secretion of IL-2, IL-4, IL-10, or IFN ⁇ .
  • TGF- ⁇ 1 secretion could not be detected by ELISA in clone Ob3D1 in spite of specific induction of TGF- ⁇ 1 mRNA synthesis, which might be due to either postranscriptional regulation or autocrine usage of the secreted TGF- ⁇ 1 by this cell clone.
  • TGF- ⁇ 1 secretion could not be detected by ELISA in clone Ob3D1 in spite of specific induction of TGF- ⁇ 1 mRNA synthesis, which might be due to either postranscriptional regulation or autocrine usage of the secreted TGF- ⁇ 1 by this cell clone.
  • TGF- ⁇ 1 secretion could not be detected by ELISA in clone Ob3D1 in spite of specific induction of TGF- ⁇ 1 mRNA synthesis, which might be due to either postranscriptional regulation or autocrine usage of the secreted TGF- ⁇ 1 by this cell clone.
  • Ob1A12 was stimulated with either the native MBP peptide or (90A) peptide at lower concentrations and the secretion of IL-4, IFN- ⁇ , and TGF- ⁇ 1 measured. With decreasing peptide concentrations, T-cell activation was attenuated as measured by IL-4 of IFN- ⁇ secretion ( Figure 6) or [ 3 H] thymidine incorporation (data not shown) without the induction of TGF- ⁇ 1 secretion. This suggests that the induction of TGF- ⁇ 1 secretion by altered peptide ligands was not due to attenuated T-cell activation. In addition, costimulation with ionomycin and PMA with the altered ligands did not induce TGF- ⁇ 1 secretion.
  • TGF- ⁇ 1 The production of active TGF- ⁇ 1 is not exclusively transcriptionally regulated, since a latent form of the cytokine is secreted from the cell and postsecretional activation must occur (Wahl, 1991). Thus, in these experiments, we measured the production of the active TGF- ⁇ 1 by ELISA. Since TGF- ⁇ 1 can be released from dying cells, cell viability after 3 days in culture was examined by trypan blue uptake and was found to be similar in all conditions, indicating that the altered peptide ligands did not induce cell death.
  • a patient (DR2 + ) is suffering from relapsing remitting multiple sclerosis and has activated autoreactive T- cells that recognize MBP p(85-99). It is determined that in vitro exposure of such T-cells from the patient to APCs presenting altered peptide ligand p(90A) induce these cells to secrete substantially increased amounts of TGF- ⁇ , without causing them to proliferate. It is also determined that a p93A/MHC complex binds to the TCR of such T-cells with comparable affinity (different by less than an order of magnitude) as the native MBP p (85-99). This patient is treated parenterally with 10 mg of p(90A) suspended in saline once a month for three months.
  • mice Female (4 to 6 week-old) SJL mice were purchased from the Jackson Laboratory (Bar Harbor, Maine) and housed under virus-free conditions.
  • Peptide antigens for in vi tro studies were synthesized on a MilligenTM model 9050 synthesizer using Fmoc chemistry. MilligenTM PAL amide resins were used to produce peptides with C terminal amides . Most peptides were >90% pure , as determined by high pressure liquid chromatography (HPLC), and were not purified further. For disease induction, HPLC- purified peptide PLP 139-151, obtained from Alkermeres, Incorporated (Cambridge, Massachusetts) was used in some experiments.
  • the peptides used in these experiments were PLP 139-151 designated W144 (HSLGKWLGHPDKF), Q144 (HSLGKQLGHPDKF), A144 (HSLGKALGHPDKF), T144 (HSLGKTLGHPDKF), L144/R147 (HSLGKLLGRPDKF), and PLP 190-209 (SKTSASIGSLCADARMYGVL).
  • W144 HLGKWLGHPDKF
  • Q144 HLGKQLGHPDKF
  • A144 HLGKALGHPDKF
  • T144 HLGKTLGHPDKF
  • L144/R147 HLGKLLGRPDKF
  • PLP 190-209 Single letter abbreviations for the amino acids are the same as those indicated above.
  • mice were injected subcutaneously at two sites with W144 emulsified in CFA and supplemented with Mycobacterium tuberculosis H37 RA (500 ⁇ g/mouse: Difco, Detroit, Michigan). On day 0 and 3, each mouse was also injected intravenously with 10 9 heated-killed Bordetella pertussis bacilli (pertussis vaccine lot number 264, Massachusetts Public Health Biological Laboratories, Boston, Massachusetts). The concentration of W144 was titrated in each set of experiments to give optimal disease and was between 50-100 ⁇ g/mouse. In the preimmunization experiments, SJL mice were initially immunized at two sites with peptide emulsified in CFA supplemented with M.
  • mice were immunized with the peptide or a mixture of peptides shown, keeping the concentration of disease-inducing peptide constant. Mice were examined daily beginning on day 9 for disease, which was assessed clinically according to the following criteria: 0, no disease; 1, limp tail; 2, hindlimb weakness; 3, hindlimb paralysis; 4, hindlimb plus forelimb paralysis; 5, moribund or dead. When animals were moribund or at the end of the experiment (day 30-40), mice were sacrificed and brains and spinal cords were fixed, processed for histologic analysis, and evaluated (Kuchroo et al., 1994). Culture Media
  • DMEM fetal bovine serum
  • sodium pyruvate 1 mM
  • L-glutamine 2 mM
  • MEM essential vitamin mixture 1 x
  • penicillin 100 U/ml
  • streptomycin 100 U/ml
  • gentamicin 0.1 mg/ml
  • 10% heat- inactivated fetal bovine serum Bio Whittaker, Incorporated, Walkersville, Maryland
  • asparagine 0.1 mM
  • folic acid 0.1 mg/ml
  • 2-mercaptoethanol 5 x
  • T-cell growth factor 0.6% T-cell growth factor (T-StimTM, Collaborative Biomedical Research, Bedford, Massachusetts) and 0.06% recombinant IL-2.
  • mice were injected subcutaneously at five sites with antigen emulsified in CFA (Difco Laboratories, Inc.) containing a total of 250 ⁇ g M. tuberculosis H37 RA. Mice immunized with a single peptide received a total of 100 ⁇ g of antigen; mice immunized with a mixture of W144 and Q144 received 100 ⁇ g of W144 and either 100 ⁇ g or 300 ⁇ g of Q144 (i.e. a total of 200 or 400 ⁇ g of antigen per mouse). On day 10, lymph nodes were removed and LNCs prepared from them. LNCs (4 x 10 5 per well) were cultured in triplicate in 96-well round-bottomed plates
  • Supernatants were collected from LNCs (4 x 10 5 per well). T-cell lines (5 x 10 4 T-cells plus 5 x 10 5 syngeneic irradiated spleen cells per well) or T-cell clones 24 or 40 hr after activation in vitro.
  • the concentration of IL-4 in the supernatants of LNC was measured by ELISA or by using CT4S cells, which were maintained in culture in medium supplemented with recombinant IL-4. Prior to assay, CT4S cells were kept overnight in IL-4-free medium, harvested, and washed three times, then resuspended at 1 x 10 5 cells/ml.
  • the concentrations of IFN ⁇ , IL-2, IL-10 (and IL-4 in the supernatants collected from T-cell lines or T-cell clones) were measured by quantitative capture ELISA according to the guidelines of the manufacturers.
  • purified rat MAb to mouse cytokine IL-2 (clone JES5-1A12), IL-4 (clone BVD4- 1D11), IL-10 (clone JES5-2A5), and IFN ⁇ (clone R4-6A2) were obtained from Pharmingen (San Diego, California) and used to coat ELISA plates (ImmulonTM 4, Dynatech Laboratories Inc., Chantilly, Virginia) .
  • IL-2, IL-4, IL-10 and IFN ⁇ : Pharmingen were used to construct standard curves and biotinylated rat MAb to mouse cytokine IL-2 (clone JES6-5H4), IL-4 (clone BVD6-24G2), IL-10 (clone SXC-1), and IFN ⁇ (clone XMG1.2) (Pharmingen) were used as the second antibody.
  • Assays were developed with TMB Microwell Peroxidase Substrate (Kirkegaard and Perry Laboratories Inc., Maryland) and read after the addition of stop solution at 450 nm using a model 2550 Microplate Reader (Bio-Rad Laboratories, California). Derivation of T-Cell Lines and Clones
  • T-cell lines were generated from LNCs from mice immunized with W144 (WL1) or Q144 (QL1). LNCs were prepared and cultured in syngeneic serum with the appropriate antigen (20 ⁇ g/ml) for 5 days. T-cell blasts were purified over a Ficoll-HypaqueTM gradient and fed with culture medium containing 0.6% T-cell growth factor (T-StimTM, Collaborative Biomedical Research) and 0.06% recombinant IL-2. Cells were fed every 2-3 days and restimulated every 10-18 days by the immunizing antigen (20 ⁇ g/ml) plus irradiated syngeneic spleen cells (5 x 10 6 cells/ml) as a source of APCs.
  • T-StimTM T-cell growth factor
  • Clones were obtained by culturing cells from QL1 at limiting dilution (Kuchroo et al. 1994). Cells were fed with culture medium plus T-cell growth factors every 2-3 days and restimulated with a mixture of antigen (20 ⁇ g/ml) plus irradiated syngeneic spleen cells (5 x 10 6 cells/ml) after 10 days. Wells that contained growing cells were identified 4 days later and the cells transferred to 48-well plates (SumilonTM, Sumitomo Bakelite Company, Tokyo, Japan), fed with medium containing T-cell growth factors every 2-3 days, and expanded by activation with antigen and APCs every 2-4 weeks.
  • mice were immunized using the same protocol as the in vitro proliferation assays. Lymph nodes were removed on day 10 and LNCs were resuspended at a concentration of 6-10 x 10 6 cells per ml in culture medium containing 0.5% syngeneic serum in place of fetal bovine serum. Cells were cultured in the presence of various antigens 920 ⁇ g/ml) for 4 days, then harvested and purified over a Ficoll-HypaqueTM gradient.
  • the APL 0144 Protects Mice from EAE
  • the Peptide Q144 is Not an MHC Blocker but Induces T-cells that are Cross-Reactive with the Native Peptide.
  • LNCs from animals immunized with Q144 showed a much more degenerate response in that they proliferated to the same extent when stimulated in vitro with the native peptide, W144 or Q144, and responded even more vigorously to a mixture of active peptide and Q144.
  • the LNCs responded well both to W144 and to a mixture of native peptide and Q144, but the response to Q144 alone was reduced.
  • Q144 When these LNCs were activated by Q144, significant levels of IL-2, IFN ⁇ , and IL-10 were detected.
  • IL-4 was measured by enzyme-linked immunosorbent assay (ELISA) or using CT4S cells and was less than 2x background in all assays. The data suggest that immunization with Q144 alters the phenotype of the T-cells induced, leading to cells producing IL-10 in addition to cells producing IL-2 and IFN ⁇ .
  • MAbs monoclonal antibodies specific for anti- CD4, anti-CD8, and anti-TCR V ⁇ 8.1/V ⁇ 8.2 (as an isotope-matched control antibody) were added to the LNC at the time of in vi tro activation.
  • T-cell lines were generated from SJL mice immunized with W144 or Q144-
  • a T-cell line derived from animals immunized with native peptide W144 (WL1) has properties that recapitulate those found in primary LNC cultures, with a dominant proliferative response to W144 and production of the Thl cytokine IFN ⁇ ( Figure 9).
  • the T-cell line WL1 also produced IFN ⁇ upon activation with the altered peptides Q144 and A144 and, in some experiments, low levels of IL-10 were detected when the line was stimulated with Q144.
  • the T-cell line derived from animals immunized with Q144 (QL1) demonstrated greater cross-reactivity in that it showed a significant proliferative response to the Q144, native, and
  • the line produced high levels of both IFN ⁇ and IL-10 in response to these antigens.
  • QL1 Q144-specific T-cell line
  • Th0 cells have a particularly desirable cytokine profile because they produce both IL-10 (which is suppressive of Thl responses) and IFN ⁇ (which is suppressive of Th2 responses).
  • mice were immunized with either W144, Q144, or a nonencaphalitogenic peptide PLP 190-209.
  • Short-term T-cell lines were generated from LNCs prepared from mice 10 days after immunization and activated in vitro with immunizing or control peptides.
  • LNCs from mice immunized with W144 and Q144 were also activated with Q144 and W144, respectively, to generate cross-reactive T-cell lines.
  • T-cell blasts were harvested and transferred into naive mice, which were then actively immunized with W144/CFA to induce EAE. The course of the disease was followed for 26 days ( Figure 11).
  • mice preimmunized with Q144 were further immunized with PLP 139-151 and followed for disease progression in the same experiment.
  • T-cell lines generated from the LNCs of mice immunized with native W144 and activated with W144 (data not shown) or Q144 into mice that were then immunized with W144 accelerated the onset of clinical disease by 1-2 days and enhanced the maximum disease severity when compared with the control mice that were injected with a T-cell line specific for an unrelated PLP peptide 190-209.
  • the T-cell lines generated from mice immunized with Q144 and activated in vitro with Q144 conferred some protection from clinical disease but the greatest protection was seen after transfer of the T- cell line generated from mice immunized with Q144 and activated in vi tro with W144 (Fig. 11). This demonstrates that immunization with Q144 induces cells that protect mice from EAE, but that these T-cells are cross-reactive and should be activated by the native peptide to best mediate protection.

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Abstract

L'invention porte sur des compositions et procédés modifiant la séquence cytokinique de lymphocytes T autoréactifs activés, et notamment celle de lymphocytes T favorisant les inflammations chez des patients atteints de maladies auto-immunes, et atténuant la réponse immunitaire liée à des maladies auto-immunes. L'invention porte également sur une méthode de modification de séquences cytokiniques à partir d'une séquence initiale, Th0 induisant des réponses immunitaires, pour aboutir à une version modifiée abaissant la réponse immunitaire.
PCT/US1996/005783 1995-04-20 1996-04-19 Modulation de sequences cytokiniques de clones de lymphocytes t humains autoreactifs Ceased WO1996032957A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0896999A4 (fr) * 1996-03-13 2001-03-14 Shionogi & Co Clone de cellules t humaines specifique a la polyarthrite rhumatoide
WO2002016410A3 (fr) * 2000-08-21 2002-09-12 Univ Bristol Procede de selection de peptide
WO2005080985A3 (fr) * 2004-02-18 2005-12-15 Enteron Ltd Partnership Methodes et compositions permettant de detecter et de traiter des maladies auto-immunes
EP2157101A1 (fr) 2002-01-31 2010-02-24 Andromeda Bio Tech Ltd. Peptides HSP et analogues pour la modulation de réponses immunes via des cellules présentant l'antigène
KR20150107766A (ko) * 2013-01-15 2015-09-23 아피토프 인터내셔날 엔브이 펩타이드

Family Cites Families (1)

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WO1993025661A1 (fr) * 1992-06-10 1993-12-23 President And Fellows Of Harvard College Clones heterogenes de cellules t specifiques contre le peptide proteolipidique 139-151

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CELL, 28 January 1994, Vol. 76, PAUL et al., "Lymphocyte Responses and Cytokines", pages 241-251. *
IMMUNOLOGY TODAY, December 1993, Vol. 14, No. 12, EVAVOLD et al., "Tickling the TCR: Selective T-Cell Functions Stimulated by Altered Peptide Ligands", pages 602-609. *
IMMUNOLOGY TODAY, June 1993, Vol. 14, ADORINI et al., "Selective Immunosuppression", pages 285-289. *
J. EXP. MED., November 1992, Vol. 176, KHOURY et al., "Oral Tolerance to Myelin Basic Protein and Natural Recovery from Experimental Autoimmune Encephalomyelitis are Associated with Downregulation of Inflammatory Cytokines and Differential Upregulation of Transforming Growth Factor Beta, Interleukin 4 and Prostaglandin E *
JOURNAL OF CLINICAL INVESTIGATION, February 1993, Vol. 91, VALLI et al., "Binding of Myelin Basic Protein Peptides to Human Histocompatibility Leukocyte Antigen Class II Molecules and Their Recognition by T Cells from Multiple Sclerosis Patients", pages 616-628. *
JOURNAL OF IMMUNOLOGY, 01 January 1993, Vol. 150, No. 1, ALEXANDER et al., "Functional Consequences of Engagement of the T Cell Receptor by Low Affinity Ligands", pages 1-7. *
JOURNAL OF IMMUNOLOGY, 15 April 1993, Vol. 150, No. 8, EVAVOLD et al., "Separation of T Helper 1 Clone Cytolysis from Proliferation and Lymphokine Production Using Analog Peptides", pages 3131-3140. *
JOURNAL OF IMMUNOLOGY, 15 July 1990, Vol. 145, No. 2, MARTIN et al., "Fine Specificity and HLA Restriction of Myelin Basic Protein-Specific Cytotoxic T Cell Lines from Multiple Sclerosis Patients and Healthy Individuals", pages 540-548. *
JOURNAL OF IMMUNOLOGY, 15 May 1993, Vol. 150, No. 10, OSTROV et al., "T Cell Receptor Antagonism Mediated by Interaction Between T Cell Receptor Junctional Residues and Peptide Antigen Analogues", pages 4277-4283. *
NATURE, 12 July 1990, Vol. 346, OTA et al., "T-Cell Recognition of an Immunodominant Myelin Basic Protein Epitope in Multiple Sclerosis", pages 183-187. *
PROC. NATL. ACAD. SCI. U.S.A., January 1992, Vol. 89, MILLER et al., "Suppressor T Cells Generated by Oral Tolerization to Myelin Basic Protein Suppress Both in Vitro and in Vivo Immune Responses by the Release of Transforming Growth Factor Beta After Antigen-Specific Triggering", pages 421-425. *
See also references of EP0825870A4 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0896999A4 (fr) * 1996-03-13 2001-03-14 Shionogi & Co Clone de cellules t humaines specifique a la polyarthrite rhumatoide
US8343500B2 (en) 2000-08-21 2013-01-01 Apitope Technology (Bristol) Limited Peptide composition
AU2001278637B2 (en) * 2000-08-21 2006-05-25 Apitope Technology (Bristol) Limited Peptide selection method
EP1731912A3 (fr) * 2000-08-21 2006-12-27 Apitope Technology (Bristol) Limited Procédé de sélection de peptide
WO2002016410A3 (fr) * 2000-08-21 2002-09-12 Univ Bristol Procede de selection de peptide
US8961986B2 (en) 2000-08-21 2015-02-24 Apitope Technology (Bristol) Limited Peptide composition
NO337988B1 (no) * 2000-08-21 2016-07-18 Apitope Tech Bristol Ltd Tolerogent peptid, farmasøytisk preparat som omfatter det samme, samt dets anvendelse.
CZ307202B6 (cs) * 2000-08-21 2018-03-21 Apitope Technology (Bristol) Limited Způsob in vitro pro identifikaci peptidů
EP2157101A1 (fr) 2002-01-31 2010-02-24 Andromeda Bio Tech Ltd. Peptides HSP et analogues pour la modulation de réponses immunes via des cellules présentant l'antigène
WO2005080985A3 (fr) * 2004-02-18 2005-12-15 Enteron Ltd Partnership Methodes et compositions permettant de detecter et de traiter des maladies auto-immunes
KR20150107766A (ko) * 2013-01-15 2015-09-23 아피토프 인터내셔날 엔브이 펩타이드
EP2945965B1 (fr) * 2013-01-15 2018-07-25 Apitope Technology (Bristol) Limited Peptide
RU2667428C2 (ru) * 2013-01-15 2018-09-19 Эпитоп Текнолоджи (Бристоль) Лимитед Пептид
KR102129763B1 (ko) * 2013-01-15 2020-07-03 아피토프 테크놀러지 (브리스톨) 리미티드 펩타이드

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