WO1992002543A1 - Nouveaux peptides immunosuppresseurs - Google Patents

Nouveaux peptides immunosuppresseurs Download PDF

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Publication number
WO1992002543A1
WO1992002543A1 PCT/EP1991/001420 EP9101420W WO9202543A1 WO 1992002543 A1 WO1992002543 A1 WO 1992002543A1 EP 9101420 W EP9101420 W EP 9101420W WO 9202543 A1 WO9202543 A1 WO 9202543A1
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WIPO (PCT)
Prior art keywords
ala
peptide
thr
amino acid
lys
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PCT/EP1991/001420
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English (en)
Inventor
Federico C. A. Gaeta
Michael F. Powell
Howard M. Grey
Alessandro D. Sette
Thomas S. Arrhenius
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Cytel Corporation
Sandoz Ltd.
Sandoz-Patent-Gmbh
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Application filed by Cytel Corporation, Sandoz Ltd., Sandoz-Patent-Gmbh filed Critical Cytel Corporation
Publication of WO1992002543A1 publication Critical patent/WO1992002543A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to the use of novel peptides which bind to major histocompatibility complex (HHC) molecules and modulate an immune response.
  • HHC major histocompatibility complex
  • T cells An integral part of the mammalian immune response is the ability of T cells to recognize protein antigens.
  • the T cells are presented with processed antigen complexed with the MHC molecules expressed on the surface of certain cells.
  • Antigen presentation appears to be a major, if not the primary, function of MHC molecules.
  • HHC Class I molecules e.g., HLA-A, -B and -C molecules in the human system
  • Class II molecules HLA-DP, -DQ and -DR in humans
  • Class I and Class II MHC molecules play a critical role in regulating an individual's immune response to a protein antigen.
  • autoimmune diseases are characterized by an immune response against "self" antigens. In essence, autoimmune disease results when an individual's immune response is no longer able to distinguish self antigens from non-self (foreign) antigens. It is thought that self-reactive B cells exist in the body for many potential autoantigens, but that the reactivity of the self-reactive B cells and other immune cells is controlled by MHC molecules. Several autoimmune diseases have been shown to be associated with particular MHC alleles. One of the most notable associations has been a rheumatoid syndrome involving the spinal column, ankylosing spondylitis, and the allele HLA-B27. Another, the haplotype HLA-DR2, is common in the autoimmune disease multiple sclerosis. Hashimoto's disease, which affects the thyroid, tends to be associated with
  • HLA-DR5 HLA-DR5.
  • DR4w4 and/or DR4wl4 alleles are genetically predisposed towards rheumatoid arthritis.
  • MHC proteins play a key role in such diseases, perhaps by binding with a self-antigen and presenting it to T cells.
  • autoimmune disease such as rheumatoid arthritis
  • Current treatment for autoimmune disease consists primarily of treating the symptoms, but not intervening in the etiology of the disease.
  • Broad spectrum chemotherapeutic agents are typically employed, which agents are often associated with numerous undesirable side effects.
  • antigens which bind to the DR molecule may serve as a source of binding peptides.
  • Influenza hemagglutinin (“HA”) is a protein identified as specifially reacting with HLA-DR, and portions thereof may be screened for reactivity to provide a sequence which binds the appropriate DR molecule, such as DR1, DR4w4 or DR4wl4, and then screened for the ability to inhibit or otherwise modulate an antigen-specific immune response such as might be associated with the disease.
  • the MHC molecule with which the peptide is being tested may be isolated from cells which express it, or the cells may be used intact.
  • Class II molecules are known and readily available. A variety of assay procedures may be employed in the screening protocol and are also well known in the art. Typically, for instance, the peptide may be labelled, e.g., with a radionuclide, enzyme, fluorescer, etc. and specific binding activity determined.
  • the biological activity of the peptide i.e. the ability to inhibit antigen-specific T cell activation, may also be assayed in a variety of systems.
  • an excess of peptide is incubated with an antigen-presenting cell of known MHC expression, (e.g., DR1) and a T cell clone of known antigen specificity (e.g., tetanus toxin 830-843) and MHC restriction (again, DR1), and the antigenic peptide itself (i.e., tetanus toxin 830-843).
  • the assay culture is incubated for a sufficient time for T cell proliferation, such as four days, and proliferation is then measured using standard procedures, such as pulsing with tritiated thymidine during the last 18 hours of incubation. The percent inhibition, compared to the controls which received no inhibitor, is then calculated.
  • the capacity of peptides to inhibit antigen presentation in an in vitro assay may be correlated to the capacity of the peptide to inhibit an immune response in vivo.
  • In vivo activity may be determined in animal models, for example, by administering an antigen known to be restricted to the particular MHC molecule recognized by the peptide, and the immunomodulatory peptide. T lymphocytes are subsequently removed from the animal and cultured with a dose range of antigen. Inhibition of stimulation is measured by conventional means, e.g., pulsing with [ 3 H]-thymidine, and comparing to appropriate controls.
  • a “core binding region” of the antigen may be determined b sythesizing overlapping peptides or by employing N-terminal or C-terminal deletions (truncations) or additions.
  • core binding region as used herein is meant a sequence of amino acids or amino aci mimetics important in maintaining a binding affinity of the peptide fo an MHC molecule.
  • the core binding region may contain contact residues having characteristics essential for binding.
  • the core binding region comprises those residues of the peptide that define the specificity of the peptide-MHC interaction.
  • a series of peptides with single amino acid substitutions may be employed to determine the effect of electrostatic charge, hydrophobicity, etc. on binding.
  • a series of positively charged (e.g., Lys or Arg) or negatively charge (e.g., Glu) amino acid substitutions may be made along the length of the peptide revealing different patterns of sensitivity towards variou DR molecules.
  • the DR4w4 molecule preferentially binds to peptides that bear little or no electrostatic charge in a core region consisting of residues 309-314 inclusive.
  • substitution of Tyr 309 of peptide A with either Glu or Arg results in substantially decreased binding affinity.
  • critical contact sites i.e., those residues (or their functional equivalents) which must be present in th peptide so as to retain the ability to bind an MHC molecule and inhibit the presentation to the T cell
  • critical contact sites may be identified by single amino acid substitutions, deletions, or insertions.
  • Ala substitutions along the length of HA peptide A reveals that binding to the DR1 molecule is relatively unaffected by such substitutions, except for an Ala substitution of position Tyr-309.
  • Binding to DR4w4 is relatively unaffected by Ala substitutions, except at positions Tyr-309 and Thr-314.
  • Peptides of the invention which are relatively insensitive to single amino acid substitutions with neutral amino acids, except at essential contact sites, have been found to tolerate multiple substitutions. Particularly preferred multiple substitutions are small, S relatively neutral moieties such as Ala, Gly, Pro, or similar residues.
  • substitutions may be homo-oligomers or hetero-oligomers.
  • the number and types of residues which are substituted or added depend on the spacing necessary between essential contact points and certain functional attributes which are sought (e.g., hydrophobicity versus hydrophilicity). Increased binding affinity for an MHC molecule may also be achieved by such substitutions, compared to the affinity of the parent peptide.
  • such "spacer" substitutions should employ amino acid residues or other molecular fragments chosed to avoid, for example, steric and charge interference which might disrupt binding.
  • D-amino acids may also be probed using D-amino acids. Such substitutions may be made using well known peptide synthesis procedures. Single D-amino acid substitutions in peptide A reveal that those made to the core region produce a peptide having substantially decreased affinity for MHC DR1, whereas those D-form substitutions made outside the core region are relatively well tolerated.
  • a peptide of the present invention will comprise a core binding region comprising a hydrophobic L-amino acid or amino acid mimetic, a spacer sequence of from 2 to 6 residues, and a Thr or Thr mimetic residue, wherein at least one residue is a D-amino acid or an amino acid mimetic.
  • Ri is a spacer sequence of from 0 to 7 amino acid or amino acid mimetic residues
  • R 2 is a hydrophobic L-amino or L-amino acid mimetic residue
  • R 3 is a spacer sequence of from 2 to 6 L-amino acid or
  • R 4 is Thr or a Thr mimetic residue
  • R 5 is a spacer sequence of between 0 and 10 amino acid or amino acid mimetic residues
  • At least one residue is a D-amino acid or an amino acid mimetic.
  • the peptides of the invention display at least one of the following features 1) and 2):
  • R 3 comprises 2-5 residues, two of which constitute an isostere of X 2 -Gln;
  • X_ is a L- or D-amino acid mimetic and X 2 can be an L-amino acid or L-amino acid mimetic.
  • the last residue of R x (Xj) is positively charged.
  • residues are Ala and ala; the isostere of Xi-Phe is typically ala-Phe, Ala-Phe, ala-Cha, Ala-Cha, ala-Ada or Ala-Ada, where Cha is cyclohexyl alanine and Ada is adamantylamine.
  • an isostere of X 2 -Gln is present, it is preferably Arg-Gln.
  • spacer sequence refers to sequences of amino acids or amino acid mimetics, which are substantially uncharged or positively charged under physiological conditions and which may have linear or branched side chains.
  • amino acid mimetic as used herein is meant a moiety, other than a naturally occurring amino acid, that conformationally and functionally serves as a substitute for a particular amino acid in a peptide of the present invention without adversely interfering to a significant extent with, or in some circumstances actually enhancing, the interaction of the peptide with its MHC receptor.
  • Peptides incorporating amino acid mimetics may be synthesized and screened for effect on binding in a variety of assays, e.g., in a competition assay with an unsubstituted parent peptide.
  • Thr mimetic or in general "X mimetic" where X is any specific natural amino acid, includes other natural amino acids which may substitute for the specific amino acid without adversely affecting binding, as described above.
  • X mimetic includes other natural amino acids which may substitute for the specific amino acid without adversely affecting binding, as described above.
  • residue refers to an amino acid or amino acid mimetic incorporated in a polypeptide by an amide bond or amide bond mimetic.
  • isostere refers to a sequence of two or more residues that can be substituted for a second sequence because the steric conformation of the first sequence fits a binding site specific for the second sequence.
  • the term specifically includes peptide back ⁇ bone modifications (i.e., amide bond mimetics) well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the ⁇ -carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks.
  • ⁇ [CH 2 S], ⁇ [CH 2 NHJ, ⁇ [CSNH 2 ], ⁇ .NHCO], ⁇ [C0CH 2 ] and ⁇ [(E) or (Z) CH CH].
  • ⁇ indicates the absence of an amide bond.
  • the structure that replaces the amide group is specified within the brackets.
  • a retro-inverso peptide has a reversed backbone while retaining substantially the original spatial conformation of the side chains, resulting in a retro-inverso isomer with a topology that closely resembles the parent peptide and is able to bind the selected - MHC molecule. See Goodman e_t al. "Perspectives in Peptide Chemistry" pp. 283-294 (1981).
  • amino acid mimetics may be incorporated in the isostere.
  • the side chain of the individual residues may also be modified in a number of ways. For instance, the side chain of a lysine residue isostere may be lengthened or shortened. Other modifications include the alkylation of NH 2 of lysine with a dihydroxypropyl radical.
  • the isosteres described above may comprise either a peptide bond or a peptide bond mimetic, but preferably comprise a peptide bond.
  • each amino acid residue is generally represented by the standard designation, in which the L-form of an amino acid residue is represented by a three-letter symbol having a capital" first letter and the D-form for those amino acids having D-forms is represented by a three-letter symbol having a lower case initial letter.
  • Glycine has no asymmetric carbon atom and is simply referred to as "Gly". It is to be understood that where a sequence is defined as consisting only of L-amino acids, that sequence may contain Gly, or other amino acids lacking an asymmetric carbon atom.
  • the peptides of the invention contain at least one residue selected from D-amino acids and amino acid mimetics, preferably at least one D-amino acid. Such residues are preferably located at one or both of the termini, as this is found to improve the stability of the peptide jLn vivo.
  • the peptides of the present invention preferably have a human serum half life at least about two times that of peptide A, preferably about four to ten time ss greater. Peptides with half lives greater than ten times that of peptide A are particularly preferred. Stability can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, have been used to test stability. A preferred method for determining stability is to measure half life in the 252 human serum assay described in detail below.
  • Certain preferred embodiments of the peptides of the present invention comprise modifications to the N- and C-terminal residues to alter the physical or chemical properties of the peptide, such as, for example, to affect binding, stability, bioavailability, ease of linking, etc.
  • the N-terminal residue can be modified to include alkyl, cycloalkyl, aryl, arylalkyl, and acyl groups.
  • the N-terminal residue may be linked to a variety of moieties other than amino acids such as polyethylene glycols (such as tetraethylene glycol carboxylic acid monomethyl ether), pyroglutamic acid, succinoyl, methoxy succinoyl, benzoyl, phenylacetyl, 2-, 3-, or 4-pyridylalkanoyl, aroyl, alkanoyl (including acetyl and cycloalkanoyl e.g.
  • cyclohexylpropanoyl arylakanoyl, arylaminocarbonyl, alkylaminocarbonyl, cycloalkyl- aminocarbonyl, alkyloxycarbonyl (carbamate caps), and cycloalkoxy- carbonyl, among others.
  • Preferred modifications of the C-terminus include modification of the carbonyl carbon of the C-terminal residue to form a carboxy- terminal amide or alcohol (i.e., as reduced form).
  • the amide nitrogen, covalently bound to the carbonyl carbon on the C-terminal residue will have two substitution groups, each of which can be hydrogen, alkyl or an alkylaryl group (substituted or unsubstituted).
  • the C-terminal is an amido group, such as -C0NH 2 , -C0NHCH 3 , -C0NHCH 2 C_H 5 or -CON(CH 3 ) 2 , but may also be 2-, 3-, or 4-pyridylmethyl, 2-, 3-, or 4-pyridylethyl, carboxylic acid, ethers, carbonyl esters, alkyl, arylalkyl, aryl, cyclohexylamide, piperidine- amide and other mono or distubstituted amides.
  • Other moieties that can be linked to the C-terminal residue include piperidine-4-carboxylic acid or amide and cis- or trans- 4-amino- cyclohexanecarboxylic acid or amide .
  • the compound in forming C-terminal amidated compounds of the present invention, can be synthesized directly, for example, using an amine and an oxime resin, as described by Kaiser e_t al. , Science 243:187 (1989), or using a methyl benzhydryl amine (MBHA) polystyrene resin.
  • the amidated compounds may be chemically amidated subsequent to peptide synthesis using means well known in the art, or enzymatically amidated.
  • C-terminal alcohols and corresponding ethers can be prepared by methods well known to those skilled in the art. Fully protected peptides suitable for C-terminal amidation are conveniently prepared by solid phase synthesis using the SASRIN resin in conjunction with an FMOC synthesis protocol.
  • a typical synthesis procedure is as follows.
  • the carboxy-terminal acid is coupled to a Sasrin resin (Bachem, Switzerland) using dicyclohexylcarbodiimide and 4-dimethylaminopyridine.
  • Subsequent chain elongation is carried out using an Applied Biosystems 430A peptide synthesizer.
  • the final amino acid is incorporated with an alpha-amino-tert-butoxycarbonyl (t-BOC) protecting group.
  • the peptide is then cleaved from the Sasrin resin by treatment with 1 X trifluoroacetic acid in dichloromethane. Upon neutralization with pyridine and evaporation of the solvent, a fully protected peptide with a free carboxy terminus is obtained.
  • This compound is then coupled to an amine of choice using hydroxy ⁇ benzotriazole and dicyclohexylcarbodiimide as the condensing agents. Finally, amino terminal and side chain protecting groups are removed by treatment with trifluoroacetic acid.
  • the group R-. contains 1 or 2 residues, with the first residue being a D-amino acid, preferably Ac-ala, phe or ala, wherein the prefix Ac- before the three-letter symbol for an amino acid signifies an acetylated N-terminus on that amino acid.
  • the second // residue, if present, is preferably Ala or ala.
  • the group may also comprise at least one N-alkyl or ⁇ -alkyl amino acid residue.
  • R_ are ala, ala-Ala, Ac-ala and
  • R 2 is a hydrophobic residue and is thought to be a binding site contact residue. Although its selection is generally dependent on the particular MHC molecule of interest, the R 2 residue is preferably selected from the group of Tyr, Phe, Trp, Met, and their amino acid mimetics, such as ⁇ -2-thienyl-alanine, ⁇ -amino-4-phenylbutyric acid, phenylglycine, 3-(2-naphthyl)alanine, 3-(l-naphthyl)alanine, ⁇ -1-adamantylalanine, ⁇ -9-anthracenylalanine, 3-cyclohexylalanine, 2-amino-3(l,l'-biphenyl-4-yl)-propanoic acid, 2-aminoindane-2- carboxylic acid, tetrahydroisoquinoline carboxylic acid, l-(2-propanoic acid)-3-amino-benzazepine-2-one
  • R 2 is Phe, cyclohexylalanine, or ⁇ -1-adamantylalanine. If R_ is absent, R 2 is preferably cyclohexylpropanoyl.
  • R 3 usually comprises 4 residues, preferably composed of Gln-Ser-Gln-Thr, Gln-Arg-Gln-Thr, Ala-Arg-Ala-Thr, Ala-Ala-Ala-Thr, Ala-Ala-Ala-Ala, Ala-Ala-Ala-Arg, Ala-Ala-Ala-Lys or Ala-Asp-Ala-Ala, of which the last four are particularly preferred.
  • R 4 is also thought to be a binding site contact residue. It is preferably Thr, but may also be a Thr mimetic, such as His, Gly, Val, Pro, Ser, alkyl substituted His, hydroxyproline, hydroxyvaline, 3-acetyl-2,3-diaminopropanoic acid, 4-acetyl-2,4-diaminobutanoic acid, allothreonine, and the like.
  • R 5 is preferably 3-6 residues long, more preferably 4-5 residues long, the last residue being a D-amino acid or an unnatural amino acid not found in proteins.
  • Preferred sequences are Lys-Ala-Ala-Pip-NH 2 , Ala-Ala-Ala-Ala-ala, Lys-Ala-Ala-ala, Leu-Lys-Leu-Ala-thr, Leu-Lys-Ala-Ala-ala, and Ala-Lys-Ala-Ala-ala, wherein Pip-NH 2 is piperidine-4-carboxamide.
  • the peptide has a core binding region composed only of L-amino acids.
  • the core binding region will typically include, in the direction from the N- to the C-terminus, a hydrophobic L-amino acid (preferably Phe) or L-amino acid mimetic, a spacer sequence of from 2 to 6 residues, and a Thr or Thr mimetic residue, corresponding to R 2 , R 3 and R 4 respectively.
  • Preferred sequences of the core binding region are Cha-Ala-Ala-Ala-Ala-Thr, Cha-Ala-Ala-Ala-Lys-Thr, Phe-Ala-Ala-Ala-Thr, Phe-Ala-Ala-Ala-Lys-Thr, or Phe-Gln-Arg-Gln-Thr-Thr, where Cha is cyclohexylalanine.
  • R-. , R 3 , and R 5 are spacer sequences. They comprise neutral and positively charged residues.
  • the neutral residues of the spacer sequences typically include, among others, Ala, ala, Gly, Pro, 3-Ala, substituted ⁇ -ala (e.g.
  • a spacer sequence may also include amino acid mimetics such as ethylene glycol or propylene glycol.
  • Positively charged residues may be incorporated in the spacer sequences to increase aqueous solubility.
  • the presence of these residues generally increases solubility to at least about lyg/ml and in some cases, higher than lmg/ml at pH 7.4.
  • Positively charged spacer residues include, among others, Lys, homo-Lys, Arg, N-alkyl or N,N-dialkyl-Lys, homo-Arg, N-,N-dialkyl-homo-Arg, N-alkyl or N,N-dialkyl-Arg, N,N,N-trialkylammonium salts, ornithine (Orn), 2,3-diaminopropanoic acid (dap), 2,4-diaminobutanoic acid (dab), and may have, for example, molecules such as dimers of ethylene glycol - (polyethylene glycol or propylene glycol (polypropylene glycol) attached thereto, e.g., to Lys, to further enhance solubility.
  • R 3 is a spacer sequence within the core binding region.
  • the core binding region (R 2 to R 4 ) of a peptide of the present invention does not contain D-amino acids.
  • the above description of the residues in Ri and R 5 applies to R 3 except that all the R 3 amino acid residues are in the L form. ⁇
  • R 2 may, if R_ is absent, be any of a number of non-amino acid hydrophobic moieties which form a hydrophobic "cap" at the N-terminus. These moieties are preamino acid mimetics which do not interfere with recognition of the peptide by the MHC molecule and increase serum half life by increasing peptidase resistance of the peptide.
  • the N-terminal hydrophobic moiety can be linked to the amino terminus (e.g., an R x residue; R 2 , if Ri is absent; or R 3 , if the hydrophobic moiety is R 2 ) through any of a number of linkages such as peptide backbone modifications known to those skilled in the art (see, Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. VII (Weinstein ed., 1983).
  • Linkages suitable in the present invention include:
  • R is the hydrophobic moiety
  • N-terrainal caps e.g. amino acid mimetics
  • suitable N-terminal moieties include aryl (including phenyl, 1-naphthyl, and 2-naphthyl); arylC__ 7 alkyl; styryl; C 4 _ 8 cycloalkyl; and C 4 _ 8 cycloalkylC 1 _ alkyl groups, which may be mono-, di-, or tri-substituted by substituents selected from hydroxy, methoxy, ethoxy, phenoxy, amino, dimethylamino, chloro, bromo, methyl and methylthio; adamantyl and adamantylC__ 7 alkyl moieties.
  • N-terminal caps include groups of formula II
  • Peptides of the present invention possess various combinations of the above features. For instance, virtually any residue of peptide A may be substituted with one of the D-amino acids or amino acid mimetic discussed above, although, as discussed above, the core binding region should consist exclusively of L-amino acids. Alternatively, one or bot terminal residues may be modified as described above. In certain embodiments both terminal residues are modified D-amino acids, for instance, the C-terminal residue is amidated and the N-terminal residu is acetylated.
  • the C-terminal residue can be an isostere of the dipeptide Ala-Ala (such as piperidine-4-carboxylic acid or amid and cis-4-aminocyclohexanecarboxylic acid or amide) and the N-terminal residue can be a D-amino acid.
  • R-. may be absent and R 2 is a hydrophobic amino acid mimetic (e.g. cyclohexylpropanoyl) which forms a hydrophobic cap. Any combination of the modifications an substitutions discussed can be used so long as MHC binding is not adversely affected.
  • Peptides or analogs thereof having the desired activity may be modified as necessary to provide certain desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to inhibit the preselected immune response.
  • the peptides can be modified by extending, decreasing or substituting in the compound's amino acid sequence, e.g., by the addition or deletion of amino acids on either the amino terminal or carboxy terminal end, or both, of the sequences disclosed above.
  • the peptides or analogs of the invention can be modified by altering the order or composition of certain residues, it being readil appreciated that certain amino acid residues essential for biological activity, e.g., those at critical contact sites, may generally not be altered without an adverse effect on biological activity.
  • the non-critical amino acids need not be limited to those naturally occurring in proteins, such as L- ⁇ -amino acids, or their D-isomers, bu may include non-protein amino acids as well, such as ⁇ -, ⁇ -, or ⁇ -amin ⁇ r acids, &-, ⁇ -, or ⁇ -imino acids (such as piperidine-4-carboxylic acid) as well as many derivatives of L- ⁇ -amino acids.
  • a peptide of the present invention may generally comprise either L-amino acids or D-amino acids, but not D-amino acids within the core binding region.
  • the peptides and analogs thereof of the present invention which bind to MHC molecules and inhibit or block MHC restricted antigen- specific T cell activation comprise at least 4 amino acid residues or the conformational equivalent thereof, more usually at least 6, preferably at least 8 and more preferably at least 11 residues long. They usually will not exceed 25 residues or the equivalent thereof in length, preferably no more than 18, more preferably 14 amino acid residues or the equivalent thereof in length.
  • non-critical amino acids may be substituted with other amino acids or with amino acid mimetics; in instances where spacer molecules are substituted for non-essential residues, the approximate length of the resulting peptide should generally not exceed that which may be accommodated by the binding domain of the selected MHC molecule.
  • the peptides of the present invention and pharmaceutical compositions thereof are particularly useful for administration to mammals, particularly humans, to treat a variety of conditions involving autoimmune diseases.
  • rheumatoid arthritis can be treated with conventional chemotherapeutic agents as mentioned above
  • the peptides of the invention as described herein may be used to treat the symptoms of rheumatoid arthritis or prevent their occurrence in identified patients or those patients at risk of developing the disease.
  • the immunomodulatory peptides which bind to selected MHC molecules are more selective than conventional agents, and generally will be less toxic, they will be more effective and less likely to cause complications than the conventional broad spectrum agents.
  • the peptides may be administered in conjunction with the traditional chemotherapeutic agetns, and may be used to lower the dose of such agents to levels substantially below those typically associated with toxicity.
  • autoimmune associated disorders for which the peptides of i the invention may be employed to relieve the symptoms of, treat or prevent the occurrence or reoccurrence of include, for example, Sjogr syndrome, scleroderma, polymyositis, dermatomyositis, systemic lupus erythematosus, juvenile rheumatoid arthritis, ankylosing spondylitis, myasthenia gravis (antibodies to acetylcholine receptors), bullous pemphigoid (antibodies to basement membrane at dermalepidermal junction), pemphigus (antibodies to mucopolysaccharide protein complex or intracellular cement substance), glomerulonephritis (antibodies to glomerular basement membrane), Goodpasture's syndrome, autoimmune hemolytic anemia (antibodies to erythrocytes), Hashimoto's disease (antibodies to thyroid), pernicious anemia (antibodies to intrinsic factor), idiopathic thrombo
  • the dose of the immu ⁇ omodulatory peptides of the invention for treatment of autoimmune disease will vary according to, e.g., the peptide composition, the manner of administration, the particular disease being treated and its severity, the overall health and condition of the patient, and the judgment of the prescribing physician.
  • the dose is in the range of about 5 ⁇ g to 2,000 mg/day, preferably 5 to 700 mg/day for a 70 kg patient.
  • Administration should begin at the first sign of symptoms or shortly after diagnosis, and continue at least until symptoms are substantiall abated and for a period thereafter. In established cases loading doses followed by maintenance doses may be required.
  • compositions are intended for parenteral, topical, oral or local administration, such as by aerosol or trans- dermally, for prophylactic and/or therapeutic treatment.
  • the compos ⁇ itions are suitable for use in a variety of drug delivery systems.
  • the pharmaceutical compositions are administered parent- erally, e.g. intravenously.
  • the invention provides compositions for parenteral administration which comprise an imraunoraodulatory peptide dissolved or suspended in an acceptable carrier, preferably an aqueous carrier e.g. water, buffered water, 0 ⁇ X saline, 0.3% glycine, hyaluronic acid and the like.
  • compositions may be sterilized by If- conventional sterilization techniques, or may be sterile filtered.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier 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, detergents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • the concentration of immunomodulatory peptides in the pharmaceutical formulations can vary widely, i.e., from less than about 0.01%, usually at or at least about 5% to as much as 50 to 75% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 10 mg of peptide.
  • Two or more peptides of the invention may be combined to form a peptide "cocktail" under certain circumstances for increased efficacy.
  • the peptides of the invention may also be used in conjunction with other pharmaceutically active agents.
  • 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, that is, one or more peptides of the invention, preferably 25-75%.
  • the immunomodulatory peptides are preferably supplied in finely divided form along with a conventional non-toxic surfactant and a suitable propellant. Typical percentages of peptides are 0.01%-20% by weight, preferably 1%-10%; and of surfactant from 0.1%-20% by weight, preferably 0.25%-5%.
  • the compositions containing the immunomodulatory peptides can be administered for prophylactic and/or therapeutic treatments. In therapeutic 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.
  • Amounts effective for this use will depend on the severity of the disease and the weight and general state of the patient, but generally range from about 0.1 mg to about 2,000 mg of peptide per day for a 70 kg patient, with dosages of from about 0.5 mg to about 700 mg of peptide per day being more commonly used. It must be kept in mind that the materials of the present invention may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations.
  • compositions containing the peptides of the invention are administered to a patient susceptible to or otherwise at risk of a particular autoimmune disease to enhance the patient's own immunoregulatory capabilities.
  • a patient susceptible to or otherwise at risk of a particular autoimmune disease to enhance the patient's own immunoregulatory capabilities.
  • Such an amount is defined to be a "prophylactically effective dose.”
  • the precise amounts again depend on the patient's state of health and weight, but generally range from about 0.1 mg to about 500 mg per 70 kg patient, more commonly from about 0.5 mg to about 200 mg per 70 kg of body weight.
  • the peptides may also find use as diagnostic reagents.
  • a peptide may be used to determine the susceptibility of a particular individual to a treatment regimen which employs the peptide or related peptides, and thus may be helpful in modifying an existing treatment protocol or in determining a prognosis for an affected individual.
  • cells bearing the selected MHC antigen e.g., white blood cells
  • the peptide(s), an immunostimulatory peptide, and a collection of T cells from the same individual will be obtained from the individual, incubated in vivo with " the peptide(s), an immunostimulatory peptide, and a collection of T cells from the same individual.
  • the proliferation of the T cells can then be assayed in the presence or absence of said immunomodulatory peptide or peptide mixture. 13
  • This Example illustrates the method used to identify peptides which bind human MHC HLA-DRl and -DR4w4.
  • a library of about one hundred eighty synthetic peptides is tested.
  • the library screened is non- redundant in the sense that care is taken to eliminate amino acid sequences too closely related; overlapping peptides are allowed but only if they overlap less than 50% of total residues.
  • EBV-transformed homozygous cell lines are used as a source of DR molecules.
  • Cell lines are routinely monitored for DR expression by FACS analysis. Their DR types are confirmed by serological typing (Terasaki et al., Amer. J. Clin. Path. 69:103-120 (1978)) and RFLP analysis (Schreuder, J. Expt. Med. 164:938 (1986)).
  • Cell lines used are maintained jln vitro by culture in RPMI 1640 medium (Flow Labs, McLeon, VA), supplemented with 2 mM L-glutamine (Gibco, Grand Island, NY), 50 mM 2-mercaptoethanol (Sigma Chemical Co., St.
  • DR molecules are purified essentially as described by Gorga et al. (Gorga et al., J. Biol. Chem. 262:16087-16094 (1987)) using the monoclonal antibody LB3.1 (Gorga et al., Cell. Immunol. 103:160-173 (1986)), covalently coupled to protein A-Sepharose CL-4B. Epstein-Barr virus-transformed human B cell lines, homozygous at the DR locus, are used as a source of DR molecules. The LG-2 (DR1) and Preiss (DR4, Dw4) lines were obtained from Dr. J.C.
  • Sepharose CL-4B (10 ml), protein-A-Sepharose (5 ml), V6/32-protein-A-
  • the columns are washed with 10 column volumes of 10 mM
  • Amicon YM-30 membrane Protein content is evaluated by a BCA protein assay (Pierce) and confirmed by SDS PAGE electrophoresis.
  • Peptide synthesis Peptides are synthesized on an Applied Biosystems (Foster City, CA) 430A peptide synthesizer according to well known protocols. The synthesis of the peptide ala-Cha-Ala-Ala-Ala-Lys-Thr-Ala-Ala-Ala-alaNH 2 (Example 10 below) is typical.
  • the hydroxybenzotriazole ester of tert-butyloxycarbonyl-d- alanine (Boc-d-alanine) is prepared by treatment of Boc-d-alanine with equimolar amounts of hydroxybenzotriazole and dicyclohexylcarbodiimide in dimethylformamide for thirty minutes at room temperature.
  • the mixture is then filtered to remove precipitated dicyclohexylurea.
  • the resulting solution is added to a methylbenzhydrylamine resin and allowed to react for 60 minutes with gentle shaking or rocking.
  • the resin is thoroughly washed with dimethylformamide followed by dichloromethane.
  • the t-butyloxycarbonyl protecting group of the resin-bound alanine is then removed by mixing the resin together with a 50 % solution of trifluoroacetic acid in dichloromethane for 30 min. , followed by thorough washing with dichloromethane.
  • the resulting resin- bound amine trifluoroacetate salt is neutralized by treatment with a 20 % solution of diisopropylethylamine in dichloromethane.
  • Chain elongation is accomplished by repeating the above sequence of 1) amino acid activation, 2) coupling and 3) deprotection for each amino acid residue.
  • the resin is dried and the peptide is cleaved from the resin and completely deprotected by exposure to hydrogen fluoride in the presence of anisole for 90 minutes at 0°C.
  • the hydrogen fluoride is evaporated with a nitrogen stream and the peptide is washed with cold diethylether. Finally the peptide is redissolved in 25 % aqueous acetic acid and the 2) solvent is removed by lyophilization.
  • the synthesis of peptides containing amino acid mimetics is carried out in the same way.
  • amino acid mimetics are incorporated according to the above procedures.
  • the peptides are then purified by reverse-phase high- performance liquid ehromatography (HPLC).
  • HPLC reverse-phase high- performance liquid ehromatography
  • the purity of the peptides is substantiated by amino acid sequence and/or composition analysis. They are routinely >95% pure after HPLC.
  • Peptides are radiolabeled as described below.
  • DR-peptide binding assay Purified DR molecules (10-1,000 nM) are incubated with 10 nM 125 I-radiolabeled peptide for 48 hours in the presence of added protease inhibitors. HPLC-purified peptides are radioiodinated ( 125 I) using the Chloramine T method, (Buus et al., Science 235:1353-1358 (1987)).
  • protease inhibitors are: 1 mM PMSF, 1.3 mM 1,10-phenanthroline, 73 uM pepstatin A, 8 mM EDTA, 6mM N-ethyl maleimide, and 200 ⁇ M N ⁇ -p-tosyl-L-lysine chloromethyl ketone (TLCK).
  • the final detergent concentration in the incubation mixture is 0.05% NP-40.
  • Several of the peptides screened are hydrophobic and require DMS0 to maintain solubility and reduce peptide adsorption to surfaces. In these instances peptide stock solutions are prepared in neat DMS0, and the final DMS0 concentration in the incubation mixture is adjusted to 5%.
  • the ⁇ is corrected by subtracting the negative control ( 125 I-peptide incubated for two days at room temperature in the presence of protease inhibitors and 120 ⁇ g/ml of unlabeled peptide, but in the absence of any DR).
  • Competitive inhibition by unlabeled peptide is used to determine ID 50% values.
  • Peptide HA 307-319 having good binding activity for both DR1 and DR4w4 molecules, is chosen from the screening assays for further study.
  • the DR1, DR2, Dr4w4 and DR4wl4 binding capacities of a series of peptide A analogs with single or multiple amino acid substitutions are determined as described above.
  • the peptides are tested for their ability to block an antigen-specific,
  • HLA-DR restricted T cell proliferative response The protocol is as follows:
  • An EBV positive, DR1+ homozygous typing cell line, LK.-2 is washed 3X with Hank's Balanced Salt Solution (HBSS), suspended to 5 x 10 6 /ml in HBSS, and fixed with 0.5% p-formaldehyde (w/v) at room temperature for 20 minutes.
  • HBSS Hank's Balanced Salt Solution
  • the fixed LG-2 cells are washed IX with HBSS, washed 2X with RPMI media supplemented with L-glutamine, non-essential amino acids, sodium pyruvate, antibiotics, and 10% human sera type AB (Complete Media, CM), resuspended at 10 6 /ml in CM, and then plated as 100 ⁇ l into individual wells of a 96 well microtiter dish.
  • the fixed LG-2 cells are then pulsed simultaneously for 2 hours at 37°C with 50 ⁇ l of test peptide and 50 ⁇ l of stimulatory peptide which have been dissolved in CM.
  • the amount of stimulatory peptide added to each test well is constant and is calculated so as to yield 60-80% of the maximal T cell proliferative response.
  • the potential inhibitor peptide is evaluated in duplicate at several concentrations corresponding to multiples of the stimulatory peptide concentration, usually 3X, 10X, 50X, and 250X.
  • concentrations corresponding to multiples of the stimulatory peptide concentration usually 3X, 10X, 50X, and 250X.
  • the plates are centrifuged, and the media carefully aspirated and replaced with fresh medium.
  • the plates are washed in this manner three times in order to ensure complete removal of unbound peptide.
  • 50 ⁇ l of CM remains in the well.
  • Antigen-specific T cells are washed 2X in CM, suspended to 2 x 10 5 /ml in CM, and plated as 150 ⁇ l into the wells containing the peptide-pulsed LG-2 cells. The cultures are incubated for three days at 37°C, and pulsed with 0.1 ⁇ Ci/well of 3 H-thymidine during the last 16 hours of culture. At the termination of the cultures the cells are harvested onto glass fiber and the amount of 3 H-thymidine, which has been incorporated by the responder T cell, determined using liquid scintillation counting.
  • each peptide is determined relative to the peptide of Example 29 below. For each test peptide the amount of inhibition is plotted as a function of the inhibitor concentration, and this graph is used to determine a 50% inhibitory dose. The inhibitory activity is then expressed as the ratio of the 50% inhibitory dose of the standard peptide (determined in a parallel experiment) to that of the test peptide. These data are presented in the last th 2 columns of Table 1. This inhibitory value constitutes an additional means by which the MHC binding peptides can be compared.
  • the half life of the peptides is determined using a 25% human serum (v/v) assay. Using the procedure described below, the 25% human serum half life of peptide A is determined to be about 100 minutes.
  • the protocol is as follows. Pooled human serum (Type AB, non-heat inactivated) is obtained from Irvine Scientific and is delipidated by centrifugation before use. Human serum is diluted to 25% with RPMI tissue culture media. All chemicals (reagent or HPLC grade) are purchased commercially from Sigma or Aldrich and used without further purification.
  • reaction solution in a 1.5 ml Eppendorf tube is temperature-equilibrated at 37 + 1°C for 15 minutes before adding 5 ⁇ l of peptide stock solution (10 mg/ml in DMS0) to make the final peptide concentration 40 ⁇ g/ml.
  • the initial time is recorded.
  • 100 ⁇ l of reaction solution is removed and added to 200 ⁇ l of either 6% aqueous trichloracetic acid or ethanol.
  • the cloudy reaction sample is cooled (4°C) for 15 minutes and then spun at 14,000 rpm (Eppendorf centrifuge) for 2 minutes to pellet the precipitated serum proteins.
  • TCA shows the highest peptide recovery, but where less than 90% recovery in TCA is observed, EtOH is used; all peptides show 85% or greater recovery in their optimal precipitation media and iv) peptide degradation is carried out under conditions where the degradation rate constant is independent of peptide concentration (demonstrated, for example, by lowering the peptide concentration or using radiolabeled peptide only), and proportionately dependent on biological media concentration.
  • a different brand of C-18 column for example, Waters Bondapak
  • HPLC linearity is determined for a representative group of peptides up to 50 ⁇ g/mL (three-fold higher than the concentration actualy assayed for in the biological media Examples) in DMS0/H 0 (1:1). This mixed solvent system is chosen to minimize peptide loss due to adhesion to the reaction vessel.
  • HPLC stability-specificity is carried out by traditional methods including: separate analysis of non-precipitated media peaks, analysis of peptide peak shape and peak absorbance ratioing at 50 % peptide remaining, and kinetic analysis where first order loss of parent peptide should be observed (degradation of the parent peptide cannot display a first order decay curve [when run under pseudo first order conditions] if B or C [of same retention time and similar molar absorptivity] build up to any appreciable extent).
  • Kinetic analysis is carried out by least squares analysis of log (integration peak area) versus time. When necessary, correction is made for small, interfering media peaks that coelute with the parent peptide (subtraction of background). Most reactions are followed for at least two halflives (except for the most stable peptides) and all pseudo first order plots are linear showing correlation coefficients > .98 (8 points).
  • NH 2 at end signifies a carboxyamide modified C-terminus
  • Cha L-cyclohexylalanine
  • Hpr trans-4-hydroxyproline
  • Ada L-adamantylalanine
  • Dpi N-(dihydroxypropyl)-L-lysine
  • Binding affinity relative to peptide A 1, where affinity greater than 1.0 is “++", 0.51 to 1.0 is “+”, 0.1 to 0.5 is “ ⁇ ", and less than 0.1 is "-”.
  • K D of peptide A is approx. 5.6 nM vs DRl and 42 nM vs DR4w4.
  • Binding affinity relative to peptide B 1; notation as above.
  • K D of peptide B is approx.67 nM.
  • Peptide B is the peptide having the sequence set out in Seq. Id. No.2.
  • the 50% inhibitory dose for the peptide of Example 29 is approx. 10 ⁇ M vs DRl, 30 ⁇ M vs DR4w4, and 30 ⁇ M vs DR4wl4.
  • R 2 may be replaced by a hydrophobic "end cap” comprising an amino acid mimetic without affecting the binding (Table 4).
  • Chp 3-cyclohexylpropionic acid
  • Ada L-adamantyl alanine
  • Cha L-cyclohexylalanine
  • Aac adamantylacetic acid
  • Ppa 3-phenylpropionic acid
  • ada D-adamantylalanine
  • 2Na 2-naphthoic acid
  • INa 1-naphthoic acid TABLE 4

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Abstract

Compositions peptidiques aptes à lier les molécules intéressantes du complexe majeur d'histocompatibilité (MHC) et à inhiber l'activation des lymphocytes T. Les peptides ont une longueur de 4 à 25 restes d'acides aminés, et possèdant une région de liaison centrale comportant, de l'extrémité N vers l'extrémité C, un L-amino-acide hydrophobe ou un mimétique hydrophobe d'amino-acide, une séquence espaceur de 2 à 6 restes, et un Thr ou un mimétique de Thr. L'un au moins des restes est un D-amino-acide ou un mimétique d'amino-acide. Les compositions peuvent servir au traitement des maladies associées à certains allèles de DR, par exemple les maladies auto-immunes telles que la polyarthrite rhumatoïde.
PCT/EP1991/001420 1990-08-01 1991-07-29 Nouveaux peptides immunosuppresseurs WO1992002543A1 (fr)

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WO1993005011A1 (fr) * 1991-08-29 1993-03-18 Sandoz Ltd. Nouveaux immunosuppresseurs
EP0610743A1 (fr) * 1993-02-09 1994-08-17 Bayer Corporation Nouveaux aminométhylène-peptides immunosuppressifs
EP0670727A1 (fr) * 1992-11-13 1995-09-13 Thomas Jefferson University Composes inhibant la proliferation des lymphocytes t et leurs procedes d'utilisation
EP0735893A1 (fr) * 1993-09-14 1996-10-09 Cytel Corporation Alteration de la reponse immunitaire a l'aide de peptides se liant a des alleles pan dr
WO1996040753A1 (fr) * 1995-06-07 1996-12-19 G.D. Searle & Co. Derives d'acide pipecolique obtenus a partir d'amides de threoline et de proline utiles dans le traitement de l'arthrite rhumatoide
WO1997026538A1 (fr) * 1996-01-19 1997-07-24 Virginia Mason Research Center Strategie de determinant antigenique de peptide specifique d'un allele aux fins de la mise au point de vaccin
US5695994A (en) * 1992-08-31 1997-12-09 Ludwig Institute For Cancer Research Isolated cytolytic T cells specific for complexes of MAGE related peptides and HLA molecules
WO1998017680A1 (fr) * 1996-10-19 1998-04-30 Zeneca Limited Analogues peptidiques contenant un noyau lactame a 7 chainons
WO1998050423A2 (fr) * 1997-05-07 1998-11-12 Centre National De La Recherche Scientifique Analogues peptidiques et leurs utilisations notamment dans des compositions pharmaceutiques et pour le diagnostic
WO2000005249A2 (fr) * 1998-07-23 2000-02-03 The President And Fellows Of Harvard College Peptides synthetiques et procedes d'utilisation de ceux-ci dans des therapies de maladies auto-immunes
US6034214A (en) * 1992-08-31 2000-03-07 Ludwig Institute For Cancer Research Isolated nonapeptides which bind to HLA molecules and provoke lysis by cytolytic T cells
US6087336A (en) * 1996-02-23 2000-07-11 Zeneca Limited Peptide derivatives useful in treating autoimmune diseases
US6222012B1 (en) 1992-08-31 2001-04-24 Ludwig Institute For Cancer Research Isolated nonapeptides presented by HLA molecules, and uses thereof
EP1098902A1 (fr) * 1998-07-23 2001-05-16 Yeda Research And Development Co. Ltd. Traitement de maladies auto-immunes a l'aide du copolymere 1 et des copolymeres et peptides apparentes
EP1128839A1 (fr) * 1998-11-12 2001-09-05 Yeda Research And Development Company, Ltd. Compositions pharmaceutiques comprenant des copolymeres de peptides synthetiques et methodes de prevention et de traitement de la reaction du greffon contre l'hote (gvhd) et de la reaction de l'hote contre le greffon (hvgd)
US6355617B1 (en) 1996-11-27 2002-03-12 Syngenta Limited Peptide derivatives
US6413517B1 (en) 1997-01-23 2002-07-02 Epimmune, Inc. Identification of broadly reactive DR restricted epitopes
US6413935B1 (en) 1993-09-14 2002-07-02 Epimmune Inc. Induction of immune response against desired determinants
US6509165B1 (en) 1994-07-08 2003-01-21 Trustees Of Dartmouth College Detection methods for type I diabetes
US6541453B2 (en) 1996-06-07 2003-04-01 Syngenta Limited Peptide derivatives
EP1438061A2 (fr) * 2001-10-03 2004-07-21 President And Fellows of Harvard College Copolymeres destines a la suppression de maladies auto-immunes, et methodes d'utilisation
US6800287B2 (en) 1998-09-25 2004-10-05 Yeda Research And Development Co., Ltd. Copolymer 1 related polypeptides for use as molecular weight markers and for therapeutic use
US6800285B2 (en) 2000-06-20 2004-10-05 Moses Rodriguez Treatment of central nervous system diseases by antibodies against glatiramer acetate
US7022663B2 (en) 2000-02-18 2006-04-04 Yeda Research And Development Co., Ltd. Oral, nasal and pulmonary dosage formulations of copolymer 1
US7026443B1 (en) 1999-12-10 2006-04-11 Epimmune Inc. Inducing cellular immune responses to human Papillomavirus using peptide and nucleic acid compositions
US7033582B2 (en) 2000-06-05 2006-04-25 Teva Pharmaceutical Industries, Ltd. Use of glatiramer acetate (copolymer 1) in the treatment of central nervous system disorders
US7053043B1 (en) 1999-07-23 2006-05-30 Yeda Research And Development Co.Ltd. Pharmaceutical compositions comprising synthetic peptide copolymers and methods for preventing and treating GVHD and HVGD
US7252829B1 (en) 1998-06-17 2007-08-07 Idm Pharma, Inc. HLA binding peptides and their uses
US7425332B2 (en) 1998-07-23 2008-09-16 Yeda Research And Development Co., Ltd. Treatment of autoimmune conditions with Copolymer 1 and related Copolymers
US7429374B2 (en) 2001-12-04 2008-09-30 Teva Pharmaceutical Industries, Ltd. Process for the measurement of the potency of glatiramer acetate
US7462354B2 (en) 1999-12-28 2008-12-09 Pharmexa Inc. Method and system for optimizing minigenes and peptides encoded thereby
US7611713B2 (en) 1993-03-05 2009-11-03 Pharmexa Inc. Inducing cellular immune responses to hepatitis B virus using peptide compositions
US7655221B2 (en) 2004-05-07 2010-02-02 Peptimmune, Inc. Methods of treating disease with random copolymers
US8741576B2 (en) 1999-11-18 2014-06-03 Epimunne Inc. Heteroclitic analogs and related methods
US9340577B2 (en) 1992-08-07 2016-05-17 Epimmune Inc. HLA binding motifs and peptides and their uses
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WO1993005011A1 (fr) * 1991-08-29 1993-03-18 Sandoz Ltd. Nouveaux immunosuppresseurs
US9340577B2 (en) 1992-08-07 2016-05-17 Epimmune Inc. HLA binding motifs and peptides and their uses
US6222012B1 (en) 1992-08-31 2001-04-24 Ludwig Institute For Cancer Research Isolated nonapeptides presented by HLA molecules, and uses thereof
US6488932B1 (en) 1992-08-31 2002-12-03 Ludwig Institute For Cancer Research Method for treating cancer by administering MAGE-derived peptides
US6034214A (en) * 1992-08-31 2000-03-07 Ludwig Institute For Cancer Research Isolated nonapeptides which bind to HLA molecules and provoke lysis by cytolytic T cells
US5695994A (en) * 1992-08-31 1997-12-09 Ludwig Institute For Cancer Research Isolated cytolytic T cells specific for complexes of MAGE related peptides and HLA molecules
US6379901B1 (en) 1992-08-31 2002-04-30 Ludwig Institute For Cancer Research Method for screening for cancer using binders for MAGE related peptides and complexes of the peptide and HLA molecules
EP0670727A1 (fr) * 1992-11-13 1995-09-13 Thomas Jefferson University Composes inhibant la proliferation des lymphocytes t et leurs procedes d'utilisation
EP0670727A4 (fr) * 1992-11-13 1997-07-30 Univ Jefferson Composes inhibant la proliferation des lymphocytes t et leurs procedes d'utilisation.
US5958882A (en) * 1992-11-13 1999-09-28 Thomas Jefferson University Compounds that inhibit T cell proliferation and methods using the same
EP0610743A1 (fr) * 1993-02-09 1994-08-17 Bayer Corporation Nouveaux aminométhylène-peptides immunosuppressifs
US7611713B2 (en) 1993-03-05 2009-11-03 Pharmexa Inc. Inducing cellular immune responses to hepatitis B virus using peptide compositions
US7202351B1 (en) 1993-09-14 2007-04-10 Pharmexa Inc. Alteration of immune response using pan DR-binding peptides
EP0735893A4 (fr) * 1993-09-14 1998-02-04 Cytel Corp Alteration de la reponse immunitaire a l'aide de peptides se liant a des alleles pan dr
US6413935B1 (en) 1993-09-14 2002-07-02 Epimmune Inc. Induction of immune response against desired determinants
EP0735893A1 (fr) * 1993-09-14 1996-10-09 Cytel Corporation Alteration de la reponse immunitaire a l'aide de peptides se liant a des alleles pan dr
US6509165B1 (en) 1994-07-08 2003-01-21 Trustees Of Dartmouth College Detection methods for type I diabetes
WO1996040753A1 (fr) * 1995-06-07 1996-12-19 G.D. Searle & Co. Derives d'acide pipecolique obtenus a partir d'amides de threoline et de proline utiles dans le traitement de l'arthrite rhumatoide
WO1997026538A1 (fr) * 1996-01-19 1997-07-24 Virginia Mason Research Center Strategie de determinant antigenique de peptide specifique d'un allele aux fins de la mise au point de vaccin
US6087336A (en) * 1996-02-23 2000-07-11 Zeneca Limited Peptide derivatives useful in treating autoimmune diseases
US6541453B2 (en) 1996-06-07 2003-04-01 Syngenta Limited Peptide derivatives
US6207644B1 (en) 1996-10-19 2001-03-27 Zeneca Limited Peptide analogues containing a 7-membered lactam ring
WO1998017680A1 (fr) * 1996-10-19 1998-04-30 Zeneca Limited Analogues peptidiques contenant un noyau lactame a 7 chainons
US6355617B1 (en) 1996-11-27 2002-03-12 Syngenta Limited Peptide derivatives
US6413517B1 (en) 1997-01-23 2002-07-02 Epimmune, Inc. Identification of broadly reactive DR restricted epitopes
FR2763071A1 (fr) * 1997-05-07 1998-11-13 Centre Nat Rech Scient Analogues peptidiques, et leurs utilisations notamment dans des compositions pharmaceutiques et pour le diagnostic
WO1998050423A2 (fr) * 1997-05-07 1998-11-12 Centre National De La Recherche Scientifique Analogues peptidiques et leurs utilisations notamment dans des compositions pharmaceutiques et pour le diagnostic
WO1998050423A3 (fr) * 1997-05-07 1999-08-19 Centre Nat Rech Scient Analogues peptidiques et leurs utilisations notamment dans des compositions pharmaceutiques et pour le diagnostic
US7252829B1 (en) 1998-06-17 2007-08-07 Idm Pharma, Inc. HLA binding peptides and their uses
WO2000005249A2 (fr) * 1998-07-23 2000-02-03 The President And Fellows Of Harvard College Peptides synthetiques et procedes d'utilisation de ceux-ci dans des therapies de maladies auto-immunes
WO2000005249A3 (fr) * 1998-07-23 2000-10-05 Harvard College Peptides synthetiques et procedes d'utilisation de ceux-ci dans des therapies de maladies auto-immunes
EP2848627A3 (fr) * 1998-07-23 2015-04-01 Yeda Research And Development Co., Ltd. Traitement des pathologies auto-immunes avec le copolymère 1 et copolymères et peptides associés
EP1098902A4 (fr) * 1998-07-23 2002-07-24 Yeda Res & Dev Traitement de maladies auto-immunes a l'aide du copolymere 1 et des copolymeres et peptides apparentes
US7566767B2 (en) * 1998-07-23 2009-07-28 President And Fellows Of Harvard College Synthetic peptides and methods of use for autoimmune disease therapies
US7425332B2 (en) 1998-07-23 2008-09-16 Yeda Research And Development Co., Ltd. Treatment of autoimmune conditions with Copolymer 1 and related Copolymers
US7279172B2 (en) 1998-07-23 2007-10-09 Yeda Research And Development Co., Ltd. Treatment of autoimmune conditions with copolymer 1 and related copolymers
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