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WO1996010415A1 - Preparations de complexes peptide/systeme majeur d'histocompatibilite - Google Patents

Preparations de complexes peptide/systeme majeur d'histocompatibilite Download PDF

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Publication number
WO1996010415A1
WO1996010415A1 PCT/US1995/012575 US9512575W WO9610415A1 WO 1996010415 A1 WO1996010415 A1 WO 1996010415A1 US 9512575 W US9512575 W US 9512575W WO 9610415 A1 WO9610415 A1 WO 9610415A1
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Prior art keywords
peptide
mbp
mhc
complexes
binding
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PCT/US1995/012575
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English (en)
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WO1996010415A9 (fr
Inventor
Bishwajit Nag
Jeffrey L. Winkelhake
Prabha V. Mukku
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Anergen, Inc.
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Application filed by Anergen, Inc. filed Critical Anergen, Inc.
Priority to AU38874/95A priority Critical patent/AU3887495A/en
Priority to KR1019970702077A priority patent/KR970706014A/ko
Priority to JP8512077A priority patent/JPH10509693A/ja
Priority to EP95938126A priority patent/EP0792157A4/fr
Publication of WO1996010415A1 publication Critical patent/WO1996010415A1/fr
Publication of WO1996010415A9 publication Critical patent/WO1996010415A9/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to methods of preparing MHC-peptide complexes in which essentially all of the complexes comprise the same peptide.
  • the methods comprise incubating an MHC component with a large molar excess of a desired antigenic peptide.
  • the methods comprise incubation of the desired peptide with the MHC component under optimized pH conditions.
  • MHC class II antigens are heterodimeric cell surface (glycoproteins and are crucial in presenting antigenic peptides to CD4 positive T cells (Yewdell and Bennick, Cell 62: 203-206 (1990)).
  • MHC class II antigens occupied with antigenic peptide varied significantly, and in many cases the antigen occupied fraction comprised only a very small portion of the total MHC preparation. This can be explained due to one or a combination of the following reasons: (i) the presence of various prebound endogenous peptides in affinity-purified MHC class II antigens (Chicz et al .
  • the first method described to purify class Il-peptide complexes of defined composition involves a biotin-avidin system where the antigenic peptide contains a long chain thiol cleavable biotin moiety (De otz et al . Proc. Natl . Acad . Sci . USA 88:8730-8734 (1991)). This method has limitations in the sense that it involves several steps and the recovery of defined complexes is significantly low (usually 0.4-4% of the starting samples).
  • the present invention provides methods for the preparation of MHC-peptide complexes useful in ameliorating immunological disorders, such as, for example, autoimmune diseases, allergic responses and transplant responses.
  • These complexes consist essentially of (1) an effective portion of the MHC-encoded antigen-presenting glycoprotein; and (2) a peptide representing a fragment of an autoantigen or other antigenic sequence associated with the disease state to be treated (i.e., an antigenic peptide).
  • the invention provide methods include contacting an MHC component with about a 75 fold to about a 2000 fold molar excess of the peptide, thereby forming an MHC Class Il-peptide complex.
  • the MHC component can be derived from any MHC allele, such as HLA-DR2.
  • the antigenic peptide can be derived from any antigen, for example, myelin basic protein (MBP) .
  • MBP myelin basic protein
  • Preferred peptides include MBP(83-102)Y 83 .
  • the methods may further comprise the step of mixing the MHC Class Il-peptide complex with the pharmaceutically acceptable excipient in a ratio suitable for therapeutic or diagnostic administration of the complex.
  • the invention also provides methods which include contacting the MHC component with the peptide under optimal pH conditions, thereby forming an MHC Class Il-peptide complex. If the MHC component is DR2 and the peptide is MBP(83-102)Y 83 , the optimal pH conditions are about pH 6. If the MHC molecule is DR2 and the peptide is MBP(124-143) , the optimal pH conditions are about pH 8. If the MHC molecule is DR2 and the peptide is MBP(143-168) , the optimal pH conditions are about pH 7.
  • the present invention provides a composition comprising a plurality of MHC-peptide complexes of defined or homogenous composition. These compositions are designed to target T helper cells which recognize a particular antigen in association with a glycoprotein encoded by the MHC. The complexes bind T cell receptors and cause non- responsiveness in target T-lymphocytes and other cells of the immune system.
  • Figures 1A-1C show optimum pH for maximum binding of various MBP peptides to purified HLA- DR2.
  • Affinity purified HLA-DR2 at a concentration of 20 ⁇ g/ml was incubated with 50-fold molar excess of biotinylated MBP(83-102)y 83 peptide
  • Figures 2A-2B show characterization of DR2.MBP(83-102) complexes made at acidic pH. Complex preparations were captured by anti-DR2 dimer specific polyclonal antibody and the presence of heterodimer was detected by L243 coupled peroxidase in an ELISA ( Figure 2B) . Native HLA-DR2 at neutral pH was used as positive control in this assay ( Figure 2A) . Each data point represents an average of triplicate determinants.
  • Figures 3A-3C show a time course of various MBP peptide binding to HLA-DR2.
  • Figures 4A-4C show the effect of increasing peptide concentrations on binding of MBP peptides to DR2.
  • Purified HLA-DR2 at a concentration of 20 ⁇ g/ml was incubated with increasing molar excess of biotinylated-MBP(83- 102)Y83 peptide ( Figure 4A) , biotinylated-MBP (124-143) peptide
  • FIG. 4B and biotinylated-MBP(143-168) peptide (Figure 4C) at pre optimized pH for 72 hours at 37 ⁇ c.
  • the open circles represent the binding of MBP (1-14) peptide.
  • Figures 5A-5C show competitive binding of biotinylated MBP peptides in presence of non-biotinylated peptides.
  • Figure 5A represents the binding of biotinylated MBP(83-102)Y 83 peptide with increasing concentration of non-biotinylated MBP(83-102)Y(closed circles) and MBP(124-143) peptide (open circles) .
  • Figure 5B represents the binding of biotinylated MBP(124-143) in presence of non-biotinylated MBP(83-102)Y83 (closed circles) and MBP(124-143) (open circle) .
  • Figure 5C represents the binding biotinylated MBP(143-168) with increasing concentrations of non-biotinylated MBP(83-102)Y83 (closed circles) and MBP(143-168) (open circles) at fully optimized conditions.
  • Figures 6A-6C show Sephadex G-75 gel filtration purification of various DR2-peptide complexes. One g of complexes of DR2 and various MBP peptides were prepared and applied on Sephadex G-75 (20ml bed volume) .
  • Figures 7A-7C show stability of DR2.MBP peptide complexes at various temperatures. Purified complexes were incubated at 4°C (open circles) 25°C (closed circles) and 37°C (open square) and at various time, samples aliquotes were removed as described below.
  • Figures 8A-8B show binding of peptide to affinity-purified HLA-DR2.
  • Figure 8A represents the kinetics of biotinylated- MBP(83-102)Y 83 peptide binding to HLA-DR2.
  • Purified HLA-DR2 at a concentration of 2 ⁇ g/ml was incubated with 50-fold molar excess of either biotinylated-MBP (83-102)Y 83 peptide (closed circles) or biotinylated-MBP(1-14) peptide (open circle) at 37°C and at neutral pH. At various times aliquots were removed and frozen at -20°C. At the end of the experiment, samples were analyzed as described below.
  • Figure 8B represents the quantitation of biotinylated-MBP (83-102)Y 83 peptide (closed circles) and biotinylated-MBP (1-14) peptide (open circles) bound to HLA-DR2 at various molar excess peptide concentrations.
  • FIGS 9A-9B show competitive binding of biotinylated-MBP (83-102)Y 83 peptide in the presence of either MBP (83-102)Y 83 or MBP (124-143) peptide.
  • Purified HLA-DR2 at a concentration of 2 ⁇ g/ml was incubated with 300-fold molar excess of biotinylated-MBP (83-102)Y 83 peptide and in the presence of 0-10,000 fold molar excess of either MBP (83-102)Y 83 peptide ( Figure 9A) or MBP (124-143) peptide ( Figure 9B) at 37 ⁇ c for 96 hours.
  • the amount of biotinylated-MBP (83-102)Y 83 peptide associated with HLA-DR2 was then quantitated as described below.
  • Figures 10A-10B show narrowbore HPLC analysis of acid-eluted peptides.
  • One mg of purified DR2 or MBP (83-102)Y 83 -bound DR2 complexes were subjected to acetic acid extraction.
  • the acid-eluted peptides were analyzed on a Waters (Millipore) HPLC system using narrowbore C-18 reverse phase column as described below.
  • Figure 10A shows standard MBP (83-102)Y 83 peptide;
  • Figure 10B endogenous peptides eluted from purified HLA-DR2;
  • Figure 10C blank buffer profile;
  • Figure 10D peptide eluted from fully occupied HLA-DR2.MBP (83-102)Y 83 complexes.
  • Unwanted T cell activation is known to be associated with a number of pathological, immunological disorders such as, for example, autoimmune diseases, allergic responses and transplant rejections.
  • Autoimmune diseases are a particularly important class of the diseases involving deleterious or unwanted immune responses. In autoimmune diseases, self- tolerance is lost and thus, the immune system attacks "self" tissue as if it were a foreign target. More than 30 autoimmune diseases are presently known to exist; myasthenia gravis (MG) rheumatoid arthritis (RA) and multiple sclerosis (MS), for example, are three autoimmune diseases which have received wide-spread public attention.
  • MG myasthenia gravis
  • RA rheumatoid arthritis
  • MS multiple sclerosis
  • the present invention provides methods for preparing a composition comprising a plurality of MHC-peptide complexes of defined composition. Once formed, this composition of homogenous MHC-peptide complexes can be used to modulate T cell function in the treatment of immunological disorders such as, for example, autoimmune diseases, allergic responses and transplant rejections.
  • the purified complexes of the present invention can be used as vaccines to promote immune responses.
  • the MHC component either Class I or Class II
  • the complex may be linked to isolated co- stimulatory ligands such that T cell proliferation is induced.
  • T cells will respond to the antigenic peptide presented by the complexes and an immune response will be initiated.
  • Homogenous complexes are also useful in understanding the kinetics of MHC-peptide interaction, crystallographic analysis, and in generating antibodies specific for a given complex.
  • the complexes mad by the methods of the present invention contain at least two components: (l) a peptide representing a fragment of an autoantigen or other antigenic sequence associated with the disease state to be treated (i.e., an antigenic peptide); and (2) an effective portion of an MHC-encoded glycoprotein involved in antigen presentation.
  • An effective portion of an MHC glycoprotein is one which comprises an antigen binding site and the regions necessary for recognition of the MHC-peptide complex by the appropriate T cell receptor.
  • the MHC component can be either a Class I or a Class II molecule.
  • the association between the peptide antigen and the antigen binding site of the MHC protein can be by covalent or noncovalent bonding.
  • the MHC- peptide complex may contain an effector component which is generally a toxin or a label.
  • the effector portion may be conjugated to either the MHC-encoded glycoprotein or to the autoantigenic peptide.
  • Complexes containing an effector component are disclosed and claimed in U.S. Patent No. 5,194,425, supra .
  • the present invention provides a method for preparing a composition comprising a plurality of MHC-peptide complexes of defined composition.
  • the term "of defined composition” refers to a plurality of MHC-peptide complexes wherein at least 60 percent, usually above 70 percent, preferably about 75 percent, and more preferably about 95 percent or more of the complexes are identical and free from endogenous MHC-peptide complexes.
  • An endogenous MHC-peptide complex is one comprising a peptide which is associated with the MHC molecule when the molecule is isolated from a cell that expresses the MHC molecule.
  • an MHC component having an antigen binding site or sites, is isolated from a cell which produces such components.
  • the MHC component can be readily isolated using the methods and procedures set forth herein.
  • the MHC component is isolated from a natural antigen presenting cell (e.g., a B cell, a dendritic cell, or a macrophage) or an immortalized cell line derived from such a cell.
  • a natural antigen presenting cell e.g., a B cell, a dendritic cell, or a macrophage
  • an immortalized cell line derived from such a cell.
  • glycoproteins encoded by the major hi ⁇ tocompatibility complex have been extensively studied in both the human and murine systems. In general, they have been classified as Class I glycoproteins, which are found on the surfaces of all cells and primarily recognized by cytotoxic T cells; and Class II glycoproteins, which are found on the surface of several cells, including accessory cells such as acrophages, and which are involved in the presentation of antigens to T helper cells. Some of the histocompatibility proteins have been isolated and characterized. For a general review of MHC glycoprotein structure and function, see, e.g., Fundamental Immunology (3d Ed., W.E. Paul, (ed.), Ravens Press, N.Y. (1993)).
  • isolated MHC component refers to an MHC glycoprotein or an effective portion of an MHC glycoprotein (i.e., one comprising an antigen binding site or sites and the sequences necessary for recognition by the appropriate T cell receptor) which is in other than its native state (i.e., not associated with the cell membrane of the cell that normally expresses MHC) .
  • the MHC component is preferably solubilized from an appropriate cell source.
  • human ly phoblastoid cells are particularly preferred as sources for the MHC component.
  • the MHC glycoprotein portions of the complexes of the invention can be obtained by isolation from lymphocytes and screened for their ability to bind the desired peptide antigen.
  • the lymphocytes are from the species of individual which will be treated with the complexes once formed. They may be isolated, for example, from the human B ceils of an individual suffering from the targeted autoimmune disease, which have been immortalized by transformation with a replication deficient Epstein-Barr virus, utilizing techniques known to those in the art.
  • MHC glycoproteins have been isolated from a multiplicity of cells using a variety of techniques including, for example, solubilization by treatment with papain, by treatment with 3M KC1 and by treatment with detergent.
  • detergent extraction of Class II protein from lymphocytes followed by affinity purification is used.
  • the detergent can subsequently be removed by dialysis or through the use of selective binding beads, e .g. , Bio Beads.
  • Methods for purifying the murine I-A (Class II) histocompatibility proteins have been disclosed by Turkewitz, et al . , Molecular Immunology (1983) 20:1139-1147.
  • MHC molecules are then purified by affinity chromatography, using a column containing antibodies raised against the desired MHC molecule.
  • the isolated antigens encoded by the I-A and I-E subregions have been shown to consist of two noncovalently bonded peptide chains: an alpha chain of 32-38 kD and a beta chain of 26-29 kD.
  • a third, invariant, 31 kD peptide is noncovalently associated with these two peptides, but it is not polymorphic and does not appear to be a component of the antigens on the cell surface (Sekaly, J. Exp. Med . (1986) 164:1490-1504).
  • the alpha and beta chains of seven allelic variants of the I-A region have been cloned and sequenced.
  • HLA human Class I histocompatibility proteins
  • the MHC of humans (HLA) on chromosome 6 has three loci, HLA-A, HLA-B, and HLA-C, the first two of which have a large number of alleles encoding alloantigens. These are found to consist of a 44 kD subunit and a 12 kD beta -microglobulin subunit which is common to all antigenic specificities. Isolation of these detergent-soluble HLA antigens was described by Springer, et al . , Proc. Natl . Acad. Sci . USA (1976) 73:2481-2485; Clementson, et al . , in "Membrane Proteins" (Azzi, A., ed.); Bjorkman, P., Ph.D. Thesis Harvard (1984) .
  • the Anti ⁇ enic Peptide Antigenic proteins or tissues for a number of autoimmune diseases are known.
  • experimentally induced autoimmune diseases for example, the following antigens involved in pathogenesis have been characterized: native type-II collagen has been identified in collagen-induced arthritis in rat and mouse, and mycobacterial heat shock protein in adjuvant arthritis (Stuart, et al . , (1984), Ann. Rev. Immunol . 2:199-218; van Eden, et al . , (1988), Nature 331:171-173.); thyroglobulin has been identified in experimental allergic thyroiditis (EAT) in mouse (Maron, et al . , (1988), J. Exp. Med .
  • EAT experimental allergic thyroiditis
  • acetyl choline receptor has been identified in experimental allergic myasthenia gravis (EAMG) (Lindstrom, et al. (1988), Adv. Immunol . 42:233-284); and myelin basic protein (MBP) and proteolipid protein (PLP) have been identified in experimental allergic encephalo yelitis (EAE) in mouse and rat (See Acha- Orbea, et al . , supra) .
  • target antigens have been identified in humans: type-II collagen has been identified in human rheumatoid arthritis (Holoshitz, et al . , (1986), Lancet ii:305-309); and acetyl choline receptor in myasthenia gravis (Lindstrom, et al . , (1988), supra) .
  • antigen-presenting cells APCs
  • the location of these smaller segments within the antigenic protein can be determined empirically. These segments are thought to be about 8 to about 18 residues in length and to contain both the agretope (recognized by the MHC molecule) and the epitope
  • peptides capable of binding an MHC molecule can vary. Thus, peptides of greater length, e.g., up to 100 residues can also be used in the complexes. Usually, the peptides will be less than about 50 residues in length, preferably less than about 30.
  • MBP myelin basic protein
  • EAG experimental allergic encephalitis
  • systemic lupus erythematosus has a complex systemology, it is known to result from an autoimmune response to red blood cells. Peptides which are the antigenic effectors of this disease are found in the proteins on the surface of red blood cells.
  • Rheumatoid arthritis RA
  • IDM Insulin-dependent diabetes mellitus
  • Critical peptides in eliciting the immune response in IDDM are believed to be portions of the insulin sequence and the beta cell membrane surface proteins.
  • the relevant antigenic peptide subunits can be readily synthesized using standard automated methods for peptide synthesis being that they are relatively short in length. Alternatively, they can be made recombinan ly using isolated or synthetic DNA sequences, but this is not the most efficient approach for peptides of this length.
  • the complexes of the invention can be designed to destroy the immune response to the peptide in question.
  • the MHC-peptide complex will contain an effector component.
  • the effector portion of the MHC-peptide molecule can be, for example, a toxin, a chemotherapeutic agent, an antibody to a cytotoxic T-cell surface molecule, a lipase, or a radioisotope emitting "hard” radiation (e.g., beta radiation).
  • a number of protein toxins are well known in the art and include, for example, ricin, diphtheria, gelonin, Pseudomonas toxin, and abrin.
  • Chemotherapeutic agents include, but are not limited to, doxorubicin, daunorubicin, methotrexate, cytotoxin, and anti- sense RNA. Moreover, antibiotics can also be used as the effector component. Antibodies have been isolated to cytotoxic T-cell surface molecules and these may thus operate as toxins. In addition, radioisotopes such as yttrium-90, phosphorus-32, lead-212, iodine-131, or palladium-109 can be used. The emitted radiation effects the destruction of the target T-cells. O 96/10415 PC_7US95/12575
  • the active portion of the effector component is entrapped in a delivery system such as a liposome or dextran carrier; in these cases, either the active component or the carrier may be bound in the complex.
  • a delivery system such as a liposome or dextran carrier; in these cases, either the active component or the carrier may be bound in the complex.
  • a gamma-emitting radioisotope such as technetium-99 or indium- Ill can be used.
  • other types of labeling such as fluorescence labeling by, for example, fluorescein can be used.
  • the effector component can be attached to the MHC glycoprotein or, if its nature is suitable, to the peptide portion. Iodine 131 or other radioactive labels, for example, can often be included in the peptide determinant sequence.
  • Complexes containing an effector component are disclosed and claimed in U.S. Patent No. 5,194,425, supra.
  • the MHC component has been isolated and the antigenic peptide has been synthesized, these two elements can be associated with one another to form an MHC-peptide complex using the methods of the invention.
  • the antigenic peptides are preferably associated noncovalently with the pocket portion of the MHC protein by, for example, mixing the two components together. Excess peptide can be removed using a number of standard procedures, such as, for example, by ultrafiltration or by dialysis.
  • the present invention is based in part on the discovery that large molar excess of peptide can be used to produce 100% loaded, homogenous MHC-peptide complexes, that is complexes of defined composition.
  • large molar excess of peptide can be used to produce 100% loaded, homogenous MHC-peptide complexes, that is complexes of defined composition.
  • about a 50 to about a 100-fold molar excess of peptide is used.
  • About 75- fold molar excess is usual.
  • higher levels of between about a 200 and about a 300-fold excess can be used.
  • No more than about a 2000-fold excess of peptide is used, usually less than about 1000-fold excess and preferably less than about 500-fold excess.
  • homogenous compositions of MHC- peptide complexes can be prepared by optimizing the pH conditions in which the MHC component and peptide are incubated. As shown below, such an approach has provided for increased peptide loading using three different MHC-peptide complexes.
  • compositions made the methods of the invention are usually systemic and is effected by injection, preferably intravenous.
  • Formulations compatible with the injection route of administration may, therefore, be used. Suitable formulations are found in Remington ' s Pharmaceutical Sciences , (Mack Publishing Company, Philadelphia, PA, 17th ed. (1985)).
  • a variety of pharmaceutical compositions comprising complexes of the present invention and pharmaceutically effective carriers can be prepared.
  • the pharmaceutical compositions are suitable in a variety of drug delivery systems. For a brief review of present methods of drug delivery, see , e .g. , Langer, Science 249:1527-1533 (1990).
  • MHC-peptide complexes it is frequently desirable to modify the complexes to alter their pharmacokinetics and biodistribution.
  • pharmacokinetics see , Remington 's Pharmaceutical Sciences, supra , Chapters 37-39.
  • methods for altering pharmacokinetics and biodistribution are known to one of ordinary skill in the art (see, e .g. , Langer, supra) .
  • the pharmaceutical compositions are intended for parenteral, topical, oral or local administration, such as by aerosol or transder ally, for prophylactic and/or therapeutic treatment.
  • the pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration.
  • unit dosage forms suitable for oral administration include powder, tablets, pills, and capsules.
  • compositions for intravenous administration comprise a solution of the complex dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, and the like.
  • PBS phosphate buffered saline
  • a preferred formulation is PBS containing 0.02% TWEEN-80.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous solution prior to administration.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • concentration of the complex can vary widely, i.e., from less than about 0.05%, usually at or at least about 1% to as much as 10 to 30% by weight and will be selected primarily by fluid volumes, viscosities, etc. , in accordance with the particular mode of administration selected.
  • Preferred concentrations for intravenous administration are about 0.02% to about 0.1% or more in PBS.
  • nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient.
  • the complexes are preferably supplied in finely divided form along with a surfactant and propellant.
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as, for example, caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride such as, for example, ethylene glycol, glycerol, erythritol, arabitol, mannitol, sorbitol, the hexitol anhydrides derived from sorbitol, and the polyoxyethylene and polyoxypropylene derivatives of these esters.
  • the surfactant may constitute 0.1%-20% by weight of the composition, preferably 0.25-5%.
  • the balance of the composition is ordinarily propellant.
  • Liquefied propellants are typically gases at ambient conditions, and are condensed under pressure.
  • suitable liquefied propellants are the lower alkanes containing up to 5 carbons, such as butane and propane; and preferably fluorinated or fluorochlorinated alkanes. Mixtures of the above may also be employed.
  • a container equipped with a suitable valve is filled with the appropriate propellant, containing the finely divided compounds and surfactant. The ingredients are thus maintained at an elevated pressure until released by action of the valve.
  • compositions containing the complexes can be administered for therapeutic, prophylactic, or diagnostic applications.
  • compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications.
  • An amount adequate to accomplish this is defined as "therapeutically effective dose.” Amounts effective for this use will depend on the severity of the disease and the weight and general state of the patient. As discussed above, this will typically be between about 0.5 mg/kg and about 25 mg/kg, preferably about 3 to about 15 mg/kg.
  • compositions containing the complexes of the invention are administered to a patient susceptible to or otherwise at risk of a particular disease.
  • a patient susceptible to or otherwise at risk of a particular disease is defined to be a "prophylactically effective dose.”
  • prophylactically effective dose the precise amounts again depend on the patient's state of health and weight.
  • the doses will generally be in the ranges set forth above.
  • compositions containing the appropriately complexes or a cocktail thereof are administered to a patient suspected of having an autoimmune disease state to determine the presence of autoreactive T cells associated with the disease.
  • efficacy of a particular treatment can be monitored.
  • An amount sufficient to accomplish this is defined to be a "diagnostically effective dose.” In this use, the precise amounts will depend upon the patient's state of health and the like, but generally range from 0.01 to 1000 mg per dose, espe ⁇ cially about 10 to about 100 mg per patient.
  • Example 1 high and low affinity immunodominant peptide epitopes from MBP were selected and shown to have pH dependent binding characteristics to puri ied HLA-DR2.
  • the hybridoma cell line L243 producing monoclonal antibodies against monomorphic human HLA-DR molecules was obtained from American Type Culture Collection, Bethesda, MD.
  • Homozygous lymphoblastoid cell line GM 03107 expressing HLA-DR2 (DRB1*1501 and DRB5*0101) was obtained from the National Institute of General Medical Sciences (NIGMS) human genetic mutant cell repository (Coriell Institute of Medical Research, NJ) .
  • Rabbit polyclonal antibody against HLA-DR2 heterodimer was obtained from Zymogenetics, WA. Para-nitrophenyl phosphate disodium hexahydrate was purchased from Sigma Chemicals, MO.
  • Immunopure biotinylated bovine serum albumin contai ing known amount of biotin molecules was purchased from Pierce Chemicals. Streptavidin conjugated purified alkaline phosphatase was obtained from Tropix, inc. MA. Purification of human HLA-DR2 from lymphoblastoid cells Purification of HLA-DR2 from EBV-transformed lymphoblastoid cells was carried out as described earlier (Nag B. et al, Proc . natn . Acad. Sci . U.S.A. 90:1604-1608 (1993) with some minor modifications.
  • Triton X-100 cell lysate was applied on to L243 coupled sepharose-4B column and the bound DR2 was eluted in phosphate buffer containing 0.05% n-dodecyl /3-D-maltoside (DM) detergent at pH 11.3. Fractions were immediately neutralized with 1M acetic acid and the DR2 pool was collected through a DEAE ion exchange column in a phosphate buffer containing 0.5M NaCl and 0.05% DM, pH 6.0. Purified protein was then filtered through a 180 kD membrane and characterized by 13.5% SDS polyacrylamide gel electrophoresis followed by silver staining (LabLogix silver stain kit, Belmont, CA) . Synthesis of various MBP peptides
  • the peptide resin (0.25 mmoles) was suspended in 15 ml N-methylpyrrolidinone (NMP) containing 122 mg of D-biotin, 79.6 mg of 1-hydroxybenzotriazole hydrate (HOBT) and 81 ⁇ l of 6.4 M diisopropylcarbodiimide (DIPCDI) solution. The suspension was gently mixed overnight at room temperature. A small sample of resin was washed with NMP and subjected to ninhydrin test to confirm the completion of the reaction. The resin was then filtered, washed with 50 ml NMP and methanol alternately twice and then with methanol and dichloromethane (DCM) alternately twice. The resin was dried under vacuum.
  • NMP N-methylpyrrolidinone
  • HOBT 1-hydroxybenzotriazole hydrate
  • DIPCDI diisopropylcarbodiimide
  • the peptide was cleaved from the resin using trifluoroacetic acid (TFA) containing scavengers.
  • TFA trifluoroacetic acid
  • the crude biotinylated peptides were isolated by precipitation with ether and dried under vacuum.
  • the biotinylated peptides were then purified by reverse-phase HPLC and the identity of the purified peptides were confirmed by mass spectrometry.
  • affinity- purified HLA-DR2 at a concentration of 2 ⁇ g/ml was incubated with increased molar excess of biotinylated-MBP peptides at 37°C for 96 hours at pH 7.0.
  • the resulting complex preparations were analyzed by antibody capture plate assay using an enzyme-conjugated avidin system as described earlier (Jensen, J. Exp. Med 171:1779-1784(1991); Reay et al, Eur. Molec. Biol . Org. J. 11:2829-2839 (1992)) with some modifications.
  • the standard curve was generated using the BSA-biotin conjugate ranging between 0.014-1.80 pmoles.
  • association parameters such as pH, peptide concentration and the duration of peptide incubation were tested using affinity purified HLA-DR2 ( containing DRB1* 1501/DRB5* 0101) and four different MBP peptides. These peptides were selected based on their immunodominant characteristics along with affinities toward HLA-DR2 (Valli et al, J. clin . Invest . 91:616-628 (1993)). As shown in example 2, the MBP (1-14) peptide had almost no affinity to purified HLA-DR2 and was used as a control peptide in all binding assay.
  • HLA-DR2 was incubated with 50 fold molar excess of various MBP peptides in binding buffer with pH ranging from 5 to 10. As shown in Figure 1A, only the MBP(83-102) peptide showed increased binding at acidic pH. In contrast, the MBP(124-143) peptide showed maximum binding at basic pH ( Figure IB) . The maximum peptide binding of the third MBP peptide MBP(143-168) was observed at neutral pH ( Figure 1C) . Since acidic conditions (pH 4 or below) are known to dissociate MHC class II heterodimers into monomeric ⁇ and ⁇ chains (Passmore et al, J. Immunol . Meth . 155:193-200 (1992)), we sought to evaluate the molecular characteristics of
  • DR2.MBP(83-102) complexes prepared at pH 5 and 6 have by non- reduced SDS-PAGE analysis.
  • the gel electrophoresis result shows that complexes prepared at pH 5 and 6 have significant dissociation of hetrodimers into monomers.
  • One possibility of such dissociation could be due to the effect of electrophoresis conditions.
  • complex preparations made at various pH values were analyzed by heterodimeric specific ELISA. Ninety six well plates were coated with anti-DR2 polyclonal antibody that has been characterized to recognize only the heterodimeric DR2.
  • the time course for maximum peptide binding was examined by incubating various biotinylated MBP peptides to HLA-DR2 at respective optimum pH of the binding buffers.
  • HLA-DR2 was incubated with increasing amount of three MBP peptides at their optimum pH for 72 hours. An increase in peptide concentration showed increase in binding in case of all three MBP peptides tested. Results presented in Figure 4 show that approximately 50-100 fold molar excess of each MBP peptide over DR2 concentration was sufficient enough for complete saturation of DR2. In case of both high affinity MBP peptides [MBP(83-102) and MBP(124-143) ] , 50 fold molar excess peptide concentration lead to almost 100% occupancy of DR2 at their optimum pH ( Figure 4A and 4B) .
  • results presented here clearly demonstrate that the optimization of the in vitro binding conditions can maximize the loading of antigenic peptides to purified MHC class II molecules.
  • pH of the binding buffer appears to be the most critical in peptide loading.
  • the optimum pH for maximum peptide binding differs for each peptide and MHC class II molecule based on the net charge of the peptide and the binding groove of MHC class II molecule.
  • changing pH of the binding buffer can result in 100% occupancy of MHC class II molecules.
  • Such binding of peptides at altered pH appears to be specific as demonstrated by both competitive assay in this report.
  • Example 2 This example describes an alternative method of loading purified MHC class II antigens with synthetic peptide with 100% recovery by co-incubating MHC class II and antigenic peptide at higher peptide concentrations at neutral pH.
  • the hybridoma cell line L243 producing monoclonal antibodies against monomorphic human HLA-DR molecules was obtained from American Type Culture Collection, Bethesda, MD.
  • Homozygous lymphoblastoid cell line GM 03107 expressing HLA-DR2 was obtained from the National Institute of General Medical Sciences (NIGMS) human genetic mutant cell repository (Coriell Institute of Medical Research, NJ) .
  • Ampholines and various isoelectric point markers for two-dimensional electrophoresis were purchased from Bio-Rad Laboratories, Inc. Purification of human HLA-DR2 from lymphoblastoid cells
  • EBV-transformed lymphoblastoid cells were cultured in RPMI 1640 medium containing 2 mM L-glutamine and 10% heat inactivated FBS, and were harvested at a density of lxlO 6 cells/ml. Purification of monoclonal antibody and coupling to CNBr-activated Sepharose 4B was carried out as described earlier (Nag et al, J. Immun . 148:3483-3491 (1992)). HLA-DR2 molecules were purified from Triton X-100 membrane extracts of cultured GM 03107 lymphoblastoid cells on L243 monoclonal antibody-coupled Sepharose 4B column as described earlier (Nag et al, J. Immunol .
  • the peptide resin (0.25 mmoles) was suspended in 15 ml N-methylpyrrolidinone (NMP) containing 122 mg of D-biotin, 79.6 mg of 1-hydroxybenzotriazole hydrate (HOBT) and 81 ⁇ l of 6.4 M diisopropylcarbodiimide (DIPCDI) solution. The suspension was gently mixed overnight at room temperature. A small sample of resin was washed with NMP and subjected to ninhydrin test to confirm the completion of the reaction. The resin was then filtered, washed with 50 ml NMP and methanol alternately twice and then with methanol and dichloromethane (DCM) alternately twice. The resin was dried under vacuum.
  • NMP N-methylpyrrolidinone
  • HOBT 1-hydroxybenzotriazole hydrate
  • DIPCDI diisopropylcarbodiimide
  • the peptide was cleaved from the resin using trifluoroacetic acid (TFA) containing scavengers.
  • TFA trifluoroacetic acid
  • the crude biotinylated peptides were isolated by precipitation with ether and dried under vacuum.
  • the biotinylated peptides were then purified by reverse-phase HPLC and the identity of the purified peptides were confirmed by mass spectrometry.
  • affinity-purified HLA-DR2 For the quantitation of bound peptide, affinity- purified HLA-DR2 at a concentration of 2 ⁇ g/ml was incubated with increased molar excess of biotinylated-MBP peptides at 37°C for 96 hours at pH 7.0. The resulting complex preparations were analyzed a plate assay using an enzyme-conjugated avidin system as described earlier (Reay et al, EMBO J. 11:2829-2839 (1992)) with some modifications. One ⁇ g per 50 ⁇ g affinity-purified L243 monoclonal antibody was coated per well of a 96-well plate in PBS.
  • the plate was incubated at for 18 hours at 4°C and wells were blocked with 1% fish gelatin at 25°C for 30 minutes. Preformed complexes (0.78-100 ng) at a concentration of 15.6 ng/ml-2.0 ⁇ c/ml (0.013 - 1.66 pmoles) were applied to each well in a PBS buffer containing 0.1% fish gelatin, 0.01% Tween-80 and 0.02% azide. The plate was incubated at 25°C for 2 hours and washed with PBS containing 0.1% Tween-20.
  • the bound biotinylated peptide was detected by incubating the plate at 25 ⁇ C for 30 minutes in the presence of streptavidin-alkaline phosphatase conjugate (Tropix, MA) diluted to 5000 fold in PBS containing 0.1% fish gelatin. Wells were washed with 50 mM Tris HC1, pH 7.0 containing 0.1% Tween-20 and developed with 200 ⁇ l/well of 1 mg/ml of p-Nitrophenyl phosphate disodium (Sigma Chemicals) dissolved in 0.1M Diethanolamine pH 10. The percent of DR2 antigens with bound peptide was then calculated from the standard curve.
  • the standard curve was generated using the BSA-biotin conjugate ranging between 0.014-1.80 pmoles. Equivalent amounts of biotinylated peptides in the absence of MHC class II antigens were used as controls which showed less than 1% non-specific binding and was subtracted for calculating the percent peptide occupancy.
  • the first dimensional gels were poured to a height of 6.5 cm in a glass tube (l mm inner diameter x 7.5 cm length) .
  • the gel solution contained 9.2 M urea, 5.5% aery1amide/bis, 2% Triton X-100 and a mixture of 1.5% ampholines pH 5-7 and 0.5% pH 3-10 which was degassed and polymerized by adding 50 ⁇ l of 10% ammonium persulfate and 18 ⁇ l of
  • TEMED N,N,N' ,N'-tetramethylethy-lenediamine
  • sample overlay solution (9 M urea, 1% ampholine pH 5-7, 0.5% ampholine pH 3-10 and 0.05% bromophenol blue) was used to overlay the sample solution.
  • the IEF electrophoresis was carried out at 900 V for 3.5 hours.
  • the lower chamber buffer contained 10 mM phosphoric acid and the upper chamber contained 20 mM sodium hydroxide.
  • the IEF tube gels were incubated for 5 minutes with reducing sample buffer (62.5 mM Tris HC1, pH 6.8, 10% glycerol, 2% SDS and 25 mM DTT and 0.05% bromophenol blue), preheated to 95°c for 5 minutes and then electrophoresis was performed at a constant current of 50 mA for 2 minutes followed by 25 mA for 45 minutes. Gels were stained for analysis using LabLogix silver stain kit. Results
  • HLA-DR2 containing both DRB1*1501 and DRB5*0101 class II molecules were purified from lymphoblastoid cells on an antibody-coupled affinity column followed by ion-exchange chromatography. Silver staining of the purified proteins showed purity greater than 98%. Peptide binding was measured by plate assay using biotinylated-MBP peptides and quantitated by colorimetric method using alkaline-phosphatase coupled streptavidin. An equivalent amount of biotinylated peptide incubated under identical conditions but in the absence of HLA-DR2 was used as a control.
  • bitinylated-MBP (83-102)Y 83 peptide binding with DR2 at higher peptide concentration was demonstrated in a competition assay.
  • purified HLA-DR2 was co-incubated with 300 fold molar excess of biotinylated-MBP (83-102)Y 83 peptide in the presence of increasing concentrations of non- biotinylated-MBP (83-102)Y 83 peptide.
  • biotinylated-MBP (83-102)Y 83 binding was competed out with increasing concentrations of nonbiotinylated MBP (83-102)Y 83 peptide and was completely inhibited at a concentration of 33-fold over biotinylated-MBP (83-102)Y 83 peptide.
  • another epitope from the same human myelin basic protein MBP (124-143) which has higher binding affinity to HLA-DR2 (Valli et al, (1993), supra) was able to compete for the binding of biotinylated-MBP (83-102) 83 peptide (Figure 8B) .
  • MHC class II molecules Beside endogenously bound peptides, a significant portion of purified MHC class II molecules are often known to be associated with invariant chain polypeptides.
  • the association of the invariant chain in the endoplasmic reticulum serves two important functions. First it prevents class II molecules from binding peptides in the early stage of transport (Roche and Cresswell, Proc. Natl . Acad . Sci . USA 88:8730-8734 (1991); Lotteau et al, Nature 348:600-605 (1990); Roche and Cresswell, Nature 345:615-618 (1990)).
  • IEF pH 5-7
  • polyacrylamide-SDS 13.5% polyacrylamide-SDS

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Abstract

La présente invention concerne des procédés pour préparer des complexes peptide/système majeur d'histocompatibilité ayant une composition définie. Le peptides sont utilisés en fort excès molaire. On peut également optimiser le pH de préparation.
PCT/US1995/012575 1994-09-30 1995-09-29 Preparations de complexes peptide/systeme majeur d'histocompatibilite WO1996010415A1 (fr)

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AU38874/95A AU3887495A (en) 1994-09-30 1995-09-29 Preparation of mhc-peptide complexes
KR1019970702077A KR970706014A (ko) 1994-09-30 1995-09-29 엠에이치씨-펩티드 복합체의 제조 방법(preparation of mhc-peptide complexes)
JP8512077A JPH10509693A (ja) 1994-09-30 1995-09-29 Ｍｈｃ−ペプチド複合体の調製法
EP95938126A EP0792157A4 (fr) 1994-09-30 1995-09-29 Preparations de complexes peptide/systeme majeur d'histocompatibilite

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EP0792157A1 (fr) * 1994-09-30 1997-09-03 Anergen, Inc. Preparations de complexes peptide/systeme majeur d'histocompatibilite
EP0973547A1 (fr) * 1996-04-30 2000-01-26 Anergen, Inc. Amelioration de l'affinite de liaison des peptides antigeniques vis-a-vis des molecules de cmh

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WO2002081683A2 (fr) * 2001-04-05 2002-10-17 Nextgen Sciences Ltd. Analyse de proteines

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5130297A (en) * 1988-06-23 1992-07-14 Anergen, Inc. Conjugates useful in ameliorating autoimmunity MHC-II-peptide
US5260422A (en) * 1988-06-23 1993-11-09 Anergen, Inc. MHC conjugates useful in ameliorating autoimmunity
US5284935A (en) * 1988-06-23 1994-02-08 Anergen, Inc. MHC-mediated toxic conjugates useful in ameliorating autoimmunity
US5468481A (en) * 1988-06-23 1995-11-21 Amergen, Inc. MHC class II-peptide conjugates useful in ameliorating autoimmunity

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US5736142A (en) * 1993-09-14 1998-04-07 Cytel Corporation Alteration of immune response using pan DR-binding peptides
WO1996010415A1 (fr) * 1994-09-30 1996-04-11 Anergen, Inc. Preparations de complexes peptide/systeme majeur d'histocompatibilite

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5130297A (en) * 1988-06-23 1992-07-14 Anergen, Inc. Conjugates useful in ameliorating autoimmunity MHC-II-peptide
US5260422A (en) * 1988-06-23 1993-11-09 Anergen, Inc. MHC conjugates useful in ameliorating autoimmunity
US5284935A (en) * 1988-06-23 1994-02-08 Anergen, Inc. MHC-mediated toxic conjugates useful in ameliorating autoimmunity
US5468481A (en) * 1988-06-23 1995-11-21 Amergen, Inc. MHC class II-peptide conjugates useful in ameliorating autoimmunity

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP0792157A4 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0792157A1 (fr) * 1994-09-30 1997-09-03 Anergen, Inc. Preparations de complexes peptide/systeme majeur d'histocompatibilite
EP0792157A4 (fr) * 1994-09-30 1999-12-01 Anergen Inc Preparations de complexes peptide/systeme majeur d'histocompatibilite
EP0973547A1 (fr) * 1996-04-30 2000-01-26 Anergen, Inc. Amelioration de l'affinite de liaison des peptides antigeniques vis-a-vis des molecules de cmh
EP0973547A4 (fr) * 1996-04-30 2004-12-29 Anergen Inc Amelioration de l'affinite de liaison des peptides antigeniques vis-a-vis des molecules de cmh

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KR970706014A (ko) 1997-11-03
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