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WO1994018998A1 - Traitements de reactions allergiques au moyen de complexes peptidiques de mhc - Google Patents

Traitements de reactions allergiques au moyen de complexes peptidiques de mhc Download PDF

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Publication number
WO1994018998A1
WO1994018998A1 PCT/US1994/001919 US9401919W WO9418998A1 WO 1994018998 A1 WO1994018998 A1 WO 1994018998A1 US 9401919 W US9401919 W US 9401919W WO 9418998 A1 WO9418998 A1 WO 9418998A1
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Prior art keywords
peptide
mhc
complex
antigen binding
binding site
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PCT/US1994/001919
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English (en)
Inventor
Somesh Sharma
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Anergen, Inc.
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Priority to AU62707/94A priority Critical patent/AU6270794A/en
Publication of WO1994018998A1 publication Critical patent/WO1994018998A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A61K39/36—Allergens from pollen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/605—MHC molecules or ligands thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/622—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier non-covalent binding
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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

Definitions

  • the invention relates to methods and compositions for the modulation of T cell function in the treatment of allergic responses.
  • it concerns isolated complexes which target T cells associated with the disease.
  • MHC major histocompatibility complex
  • MHC molecules are heterodimeric glycoproteins expressed on cells of higher vertebrates and play a role in immune responses. MHC glycoproteins are divided into two groups, class I and class II, which differ structurally and functionally from each other. In general, the major function of MHC molecules is to bind antigenic peptides and display them on the surface of cells. These peptides result from processing of an antigen by a presenting cell (APC) into peptide fragments of between 8 and 20 amino acids.
  • APC presenting cell
  • Class I MHC molecules are expressed on almost all nucleated cells and are recognized by cytotoxic T lymphocytes, which then destroy the antigen-bearing cells.
  • Class II MHC molecules are expressed primarily on cells involved in initiating and sustaining immune responses, such as T lymphocytes, B lymphocytes, macrophages, etc.
  • Class II MHC molecules are recognized by helper T lymphocytes and induce proliferation of helper T lymphocytes and amplification of the immune response to the particular antigenic peptide that is displayed.
  • T cell receptor Engagement of the T cell receptor induces a series of molecular events characteristic of cell activation, such as, increase in tyrosine phosphorylation, Ca ++ influx, PI turnover, synthesis of cytokines and cytokine receptors, and cell division (see, Altman et al., Adv. Immunol . 48:227-360 (1990) .
  • molecular events characteristic of cell activation such as, increase in tyrosine phosphorylation, Ca ++ influx, PI turnover, synthesis of cytokines and cytokine receptors, and cell division
  • PI turnover synthesis of cytokines and cytokine receptors
  • cell division see, Altman et al., Adv. Immunol . 48:227-360 (1990) .
  • T cells recognize antigen see Grey, H.M. , et al., Scientific 2_jnerican pp 56-64, (November, 1989) .
  • Thl clones generally produce IFN ⁇ and IL-2 and tend to stimulate the production of IgG antibodies.
  • Th2 clones in contrast, produce IL-4, IL-5, IL-6, and IL-10 and stimulate production of IgE antibodies, which are primarily involved in allergic responses.
  • the present invention relates to MHC-peptide complexes consisting essentially of an allergenic peptide and an isolated MHC component, typically MHC Class II, having an antigen binding site, wherein the allergenic peptide is associated with the antigen binding site.
  • the peptide can be either covalently or noncovalently associated with the antigen binding site.
  • the MHC molecule may be recombinantly produced or isolated from a spleen cell. If the MHC molecule comprises a hydrophobic region, such as a transmembrane region, the complex may be embedded in a liposome or incorporated in a micelle.
  • the allergenic peptide will comprise an epitope which is recognized by a T cell associated with an allergic response.
  • the T cells are associated with allergic responses to ragweed.
  • the peptide is preferably peptide A5 of Amb a V and the MHC component is preferably HLA-DR2.2.
  • the complexes can be combined with a pharmaceutically acceptable carrier, for example phosphate buffered saline, to produce pharmaceutical compositions.
  • the concentration of the complex in the pharmaceutical compositions is typically between about 0.02% and about 1% by weight.
  • the invention also provides methods for treating allergic responses by, for instance, inducing anergy in T cells associated with the allergic response.
  • the pharmaceutical compositions are typically administered intravenously.
  • the invention further provides methods for preparing the complexes, the methods comprise isolating the MHC component from a cell producing the component. Contacting the MHC component with the peptide such that the peptide is coupled to the antigen binding site, and removing excess peptide not coupled to the antigen binding site, typically by dialysis.
  • the methods may also include the step of dialyzing the complex in the presence of lipids to form liposomes.
  • Fig. 1A shows a diagrammatic representation of a Class I MHC-encoded glycoprotein.
  • Fig. IB shows a digrammatic representation of an antigen binding pocket of an MHC molecule.
  • Fig. 2 shows the ability of various peptides from the ragweed allergen, Amb a V, to activate Amb a V-specific T cells.
  • Fig. 3 shows the ability of various peptides from the ragweed allergen, Amb a V, to induce anergy in Ami? a V- specific T cells.
  • the present invention provides novel treatments for conditions involving unwanted T cell reactivity, in particular, allergic diseases associated with particular histocompatibility haplotypes.
  • allergic diseases associated with particular histocompatibility haplotypes.
  • examples of such conditions include food hypersensitivities such as celiac disease and crohn disease and allergic responses to ragweed, dust mites, cats, honey bee venom, and grass pollen.
  • allergic diseases suitable for treatment using the methods of the present invention see, O'Hehir, et al., supra .
  • the invention provides MHC-peptide complexes which can be used to modulate T cell function associated with these diseases.
  • the invention complexes contain at least two components, 1) an allergenic peptide which comprises a sequence specifically recognized by the target T cell and 2) an effective portion of the MHC-encoded glycoprotein associated with the disease to be treated.
  • the MHC component can be either a Class I or a Class II molecule.
  • the association between the peptide and the antigen binding sites of the MHC protein can be by covalent or by noncovalent bonding.
  • the complexes may also 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.
  • MHC glycoproteins encoded by the MHC have been extensively studied in both the human and murine systems. Many of the histocompatibility proteins have been isolated and characterized. For a general review of MHC glycoprotein structure and function, see Fundamental Immunology, 2d Ed. , W.E. Paul, ed. , Ravens Press N.Y. 1989.
  • isolated or “biologically pure” 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 sequences necessary for recognition by the appropriate T cell receptor) which is in other than its native state, for example, not associated with the cell membrane of a cell that normally expresses MHC.
  • An effective portion of an MHC glycoprotein is one which comprises the antigen binding sites and sequences necessary for recognition of the MHC-peptide complex by the appropriate T cell receptor.
  • the MHC component may be recombinantly produced or solubilized from the appropriate cell source.
  • the MHC components are soluble in vivo (i.e., they retain sufficient solubility to function in vivo) they may be associated with detergents to form micelles or with lipids to form liposomes in certain embodiments.
  • Cells expressing particular MHC molecules are available from a variety of sources well known to those of skill in the art.
  • the cells can be used as a source for the expressed proteins or for the genes to be used in recombinant expression, as described below.
  • Human lymphoblastoid cells are a particularly preferred source for MHC genes and/or proteins.
  • MHC glycoproteins have been isolated from a multiplicity of cells using a variety of techniques including solubilization by treatment with papain, by treatment with 3M KC1.
  • soluble HLA-A2 can be purified after papain digestion of plasma membranes from the homozygous human lymphoblastoid cell line J-Y as described by Turner, M.J. et al., J. Biol . Chem . (1977) 252:7555-7567.
  • Papain cleaves the 44 kD chain close to the transmembrane region yielding a molecule comprised of ⁇ lf ⁇ 2 , ⁇ 3 , and ⁇ 2 microglobulin.
  • the isolated antigens encoded by the murine I-A and I-E subregions have been shown to consist of two noncovalently bonded peptide chains: an ⁇ chain of 32-38 kD and a ⁇ 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, R.P., J. Exp. Med.
  • HLA human Class I proteins
  • the MHC of humans (HLA) on chromosome 6 has three loci, HLA-, 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 /3 2 -microglobulin subunit which is common to all antigenic specificities. Isolation of these detergent-soluble HLA antigens was described by Springer, T.A., et al., Proc . Natl . Acad . Sci . USA (1976) 73:2481-2485.
  • MHC antigens is not known in such detail, it is thought that Class II glycoproteins have a domain structure, including an antigen binding site, similar to that of Class I. It is formed from the N-terminal domain portions of two class II chains which extend from the membrane bilayer. The N-terminal portion of one chain has two domains of homology with the ⁇ 2 and ⁇ regions of the MHC Class I antigen sequence. Cloning of the Class II genes permits manipulation of the Class II MHC binding domains for example, as described below.
  • the MHC glycoprotein portions of the complexes of the invention then, can be obtained by isolation from lymphocytes or other cells capable of producing them and then screened for the ability to bind the desired peptide.
  • the lymphocytes are typically from the species of individual which will be treated with the complexes.
  • they may be isolated from human B cells from an individual suffering from the targeted allergic response, which have been immortalized by transformation with a replication deficient Epstein-Barr virus, utilizing techniques known in the art.
  • Class II protein from lymphocytes followed by affinity purification as described in U.S. Patent No. 5,130,297 is used. Detergent can then be removed, preferably by dialysis. Alternatively, the amino acid sequence of each of a number of Class II proteins are known, and the genes have been cloned. The proteins can thus be made using recombinant methods. Generally, the nomenclature used hereafter and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for DNA and RNA isolation, amplification, and cloning. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer's specifications.
  • the DNA constructs will typically include an expression control DNA sequence (including naturally- associated or heterologous promoter regions) operably linked to protein coding sequences.
  • the term "operably linked” as used herein refers to linkage of a promoter upstream from one or more DNA sequences such that the promoter mediates transcription of the DNA sequences.
  • the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the MHC molecules.
  • Human MHC DNA sequences can be isolated in accordance with well known procedures from a variety of human cells. MHC cDNA and genomic clones from a variety of cells have been extensively characterized (see, e.g., Paul, Chapter 17, and Kabat et al., Sequences of Proteins of Immunological Interest , (U.S. Dept. of Health and Human Services, NIH, 1987) , which is incorporated herein by reference) . Typically, traditional screening of a cDNA library prepared from RNA isolated from appropriate cells is used. PCR amplification of the desired sequences can also be used (See, PCR Protocols , Innis et al., eds. Academic Press, 1990).
  • Suitable source cells for the DNA sequences and host cells for expression and secretion can be obtained from a number of sources, such as the American Type Culture Collection ("Catalogue of Cell Lines and Hybrido as,” 6th edition (1988) Rockville, Maryland, U.S.A. and NIGMS Human Genetic Mutant Cell Repository 1990/1991 Catalogue of Cell Lines , 15th edition, NIH Publication No. 91-2011, which are incorporated herein by reference) .
  • the nucleotide sequences used to transfect the host cells can be modified according to standard techniques to yield MHC molecules with a variety of desired properties.
  • the molecules of the present invention can be readily designed and manufactured utilizing various recombinant DNA techniques well known to those skilled in the art and described in detail, below.
  • the chains can vary from the naturally- occurring sequence at the primary structure level by amino acid, insertions, substitutions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain.
  • the amino acid sequence variants can be prepared with various objectives in mind, including increasing the affinity of the molecule for target T cells, or for facilitating purification and preparation of the MHC molecule.
  • the modified molecules are also useful for modifying plasma half life, improving therapeutic efficacy, and lessening the severity or occurrence of side effects during therapeutic use.
  • the amino acid sequence variants are usually predetermined variants not found in nature.
  • the variants typically exhibit the same biological activity as naturally occurring MHC molecule.
  • the variants and derivatives that are not capable of binding are useful, for instance, as im unogens for raising antibodies to MHC alleles so long as at least one MHC epitope remains active.
  • Polypeptide fragments comprising only a portion (usually at least about 60-80%, typically 90-95%) of the primary structure may be produced.
  • MHC genes contain separate functional regions, each having one or more distinct biological activities.
  • the MHC proteins may be modified so as to retain certain functions (e.g., T cell receptor binding) while exhibiting lower immunogenicity.
  • modifications of the genes encoding the MHC molecule may be readily accomplished by a variety of well- known techniques, such as site-directed mutagenesis (see,
  • Insertional variants of the present invention are those in which one or more, amino acid residues are introduced into a predetermined site in the protein and which displace the preexisting residues.
  • cleavable sequences may be fused to the protein (e.g., sequences form viral proteins) which allow ready affinity chromatographic purification of the fusion protein. Once isolated, the cleavable sequences are removed by treatment with an appropriate protease and the desired MHC molecule is recovered.
  • Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place.
  • Non-natural amino acid i.e., amino acids not normally found in native proteins
  • isosteric analogs as well as isosteric analogs (amino acid or otherwise) are also suitable for use in this invention.
  • Substantial changes in function or immunological identity are made by selecting substituting residues that differ in their effect on the structure of the polypeptide backbone (e.g., as a sheet or helical conformation), the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chain.
  • substitutions which in general are expected to produce the greatest changes in function will be those in which (a) a hydrophilic residue, e.g., serine or threonine, is substituted for (or by) a hydrophobic residue, e.g.
  • electropositive side chain e.g., lysine, arginine, or histidine
  • an electronegative residue e.g., glutamine or aspartine
  • a residue having a bulky side chain e.g., phenylalanine
  • Substitutional variants of the subunits also include variants in which functionally homologous (having at least about 70% homology) domains of other proteins are substituted by routine methods for one or more of the MHC domains.
  • Particularly preferred proteins for this purpose are other members of the immunoglobulin superfamily.
  • Deletions are characterized by the removal of one or more amino acid residues from the MHC sequence. Typically, the transmembrane and cytoplasmic domains are deleted. Deletions of cysteine or other labile residues also may be desirable, for example in increasing the oxidative stability of the protein. Deletion or substitutions of potential proteolysis sites, e.g., Arg Arg, is accomplished by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.
  • the transmembrane domain is inactivated by deletion of all the transmembrane domain residues. Inactivation of the membrane binding function is also accomplished by deletion of sufficient residues (not necessarily all the residues) to produce a substantially hydrophilic hydropathy profile at this site or by substituting with heterologous residues which accomplish the same result.
  • Glycosylation variants are included within the scope of this invention. They include variants completely lacking in glycosylation (unglycosylated) and variants having at least one less glycosylated site than the native form (deglycosylated) as well as variants in which the glycosylation has been changed.
  • deglycosylated and unglycosylated amino acid sequence variants include substitutional or deletional mutagenesis, e.g., the asparagine residue is deleted or substituted for by another basic residue such as lysine or histidine.
  • flanking residues making up the glycosylation site are substituted or deleted, even though the asparagine residues remain unchanged, in order to prevent glycosylation by eliminating the glycosylation recognition site.
  • unglycosylated subunits which have the amino acid sequence of the native subunits are produced in recombinant prokaryotic cell culture because prokaryotes are incapable of introducing glycosylation into polypeptides.
  • Glycosylation variants are conveniently produced by selecting appropriate host cells or by in vitro methods.
  • Yeast for example, introduce glycosylation which varies significantly from that of mammalian systems.
  • bacterial or other procaryotic organisms can be used to produce deglycolysated molecules.
  • Mammalian cells from a different species (e.g., hamster, murine, insect, porcine, bovine or ovine) or tissue than the MHC source are routinely screened for the ability to introduce variant glycosylation as characterized for example by elevated levels of mannose or variant ratios of mannose, fucose, sialic acid, and other sugars typically found in mammalian glycoproteins.
  • In vitro processing of the subunit typically is accomplished by enzymatic hydrolysis, e.g., neuraminidase digestion.
  • the heavy ( ⁇ ) ana light ( ⁇ ) chains are synthesized using a carboxy terminal truncation which deletes the hydrophobic domain, and the carboxy termini can be arbitrarily chosen to facilitate the conjugation of toxins or label.
  • cysteine residues near the carboxy termini are included to provide a means to form disulfide linkage of the chains; the synthetic gene can also include restriction sites to aid in insertion into expression vectors and in manipulating the gene sequence to encode analogs.
  • the ⁇ and ⁇ chains are then inserted into expression vectors, expressed separately in an appropriate host, such as E. coli , yeast, or other suitable cells, and the recombinant proteins obtained are recombined in the presence of the peptide antigen.
  • the nucleotide sequences encoding the MHC component will be expressed in hosts after the sequences have been operably linked to an expression control sequence (i.e., positioned to ensure the translation of the structural gene) along with other sequences (e.g., enhancers, polyadenylation sites, etc.) necessary for efficient transcription and translation of the desired sequences.
  • an expression control sequence i.e., positioned to ensure the translation of the structural gene
  • other sequences e.g., enhancers, polyadenylation sites, etc.
  • This collection of sequences is referred to here as an "expression cassette" which is typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
  • expression vectors comprising the expression cassette will contain selection markers, e.g., tetracycline or neomycin, to permit detection of those cells transformed with the desired sequences (see, e.g., U.S. Patent 4,704,362).
  • selection markers e.g., tetracycline or neomycin
  • Expression vectors and recombinant production from the appropriate DNA sequences are performed by methods known in the art.
  • Expression can be in procaryotic or eucaryotic systems. Procaryotes most frequently are represented by various strains of E. coli . However, other icrobial strains may also be used, such as bacilli, for example Bacillus subtilis , various species of Pseudomonas , or other bacterial strains.
  • plasmid vectors which contain replication sites and control sequences derived from a species compatible with the host are used. For example, E. coli is typically transformed using derivatives of PBR322, a plasmid derived from an E .
  • procaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, including such commonly used promoters as the ⁇ -lactamase (penicillinase) and lactose (lac) promoter systems (Change et al., Nature 198:1056 (1977)) and the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res .
  • promoters for transcription initiation optionally with an operator
  • ribosome binding site sequences including such commonly used promoters as the ⁇ -lactamase (penicillinase) and lactose (lac) promoter systems (Change et al., Nature 198:1056 (1977)) and the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res .
  • the expression systems useful in the eucaryotic hosts comprise promoters derived from appropriate eucaryotic genes.
  • a class of promoters useful in yeast include promoters for synthesis of glycolytic enzymes, including those for 3-phosphoglycerate kinase (Hitzeman, et al., J. Biol . Chem . 255:2073 (1980)).
  • Other promoters include those from the enolase gene (Holland, M.J., et al. J. Biol . Chem . 256:1385 (1981)) or the Leu2 gene obtained from YEpl3 (Broach, J. , et al., Gene 8:121 (1978)).
  • Suitable mammalian promoters include the early and late promoters from SV40 (Fiers, et al., Nature 273:113 (1978)) or other viral promoters such as those derived from polyoma, adenovirus II, bovine papilloma virus or avian sarcoma viruses.
  • Eukaryotic transcription can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting sequences of between 10 to 300bp that increase transcription by a promoter. Enhancers can effectively increase transcription when either 5' or 3 ' to the transcription unit. They are also effective if located within an intron or within the coding sequence itself.
  • viral enhancers are used, including SV40 enhancers, cytomegalovirus enhancers, polyoma enhancers, and adenovirus enhancers.
  • Enhancer sequences from mammalian systems such as the mouse immunoglobulin heavy chain enhancer are also commonly used.
  • Mammalian expression vector systems will also typically include a selectable marker gene.
  • suitable markers include, the hypoxanthine-guanine phosphoribosyl transferase gen (HGPT) , the thymidine kinase gene (TK) , or various prokaryotic genes conferring drug resistance.
  • Amplifiable marker genes such as dihydrofolate reductase gene (DHFR) , may also be used (see, generally,
  • prokaryotic drug resistance genes useful as markers include genes conferring resistance to neomycin, G418, and hygromycin.
  • the vectors containing the nucleotide segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, cationic liposomes, or electroporation may be used for other cellular hosts. Other methods used to transform mammalian cells include the use of Polybrene, protoplast fusion, microprojectiles and microinjection. See, generally, Sambrook et al., supra .
  • the whole MHC proteins, or individual chains of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity and fraction column chromatography, gel electrophoresis and the like, (See, generally, Scopes, R. , Protein Purification , Springer-Verlag, N.Y. (1982) .
  • the polypeptides may then be used therapeutically or in developing and performing assay procedures, immunofluorescent stainings, and the like. (See, generally, Immunological Methods , Vols. I and II, Eds. Lefkovits and Pernis, Academic Press, New York, N.Y. (1979 and 1981) .
  • Allergenic Peptides The proteins or tissues associated with a number of allergic responses are known. Methods for mapping particular T cell epitopes within the allergens are also known, as described below. For instance, T cell epitopes have been identified in honey bee venom allergens, dust mite allergens, and ragweed allergens have been identified.
  • antigen-presenting cells APCs
  • the location of these smaller segments within the protein can be determined empirically. These segments are thought to be 8-18 residues in length, and contain both the agretope (recognized by the MHC molecule) and the epitope (recognized by T cell receptor on the T-helper cell) .
  • the epitope itself is a contiguous or non-contiguous sequence of 5-6 amino acids which recognizes the antigen-specific receptor of T-helper cells.
  • the agretope is a continuous or non-contiguous sequence which is responsible for the association of the peptide with the MHC glycoproteins.
  • the empirical process of determining the relevant 8- 18 amino acid subunits typically involves using purified MHC Class II proteins incorporated into phospholipid vesicles by detergent dialysis. The resultant vesicles are then allowed to fuse to clean glass cover slips to produce on each a planar lipid bilayer containing MHC molecules (Brian and McConnell, Proc . Natl . Acad . Sci . USA (1984) 81:6159, which is incorporated herein by reference) .
  • One cover slip containing MHC Class II molecules embedded in the adherent planar lipid membrane is placed in each well of several 24-well culture plates.
  • Synthetic peptides corresponding to overlapping sequences from the allergenic protein and containing one or more radiolabeled amino acid residues are placed in the wells with a cover slip and PBS and allowed to incubate several days.
  • the extent of binding of peptide in the MHC Class II glycoprotein antigen binding site is measured by the amount of radioactivity incorporated into the MHC Class II-planar lipid membrane on the cover slip versus planar lipid membrane alone.
  • each of the synthetic peptide segments that contain an agretope is again incorporated into the antigen binding site of isolated MHC Class II proteins embedded in planar lipid membranes on cover slips.
  • One cover slip is added to each well of a 24-well culture plate, and spleen cells from an animal immunized against the allergen (and from which strain the adherent MHC Class II proteins were isolated) are added to each well.
  • T cell hybridoma proliferation as measured by tritiated thymidine uptake into DNA, indicates that the MHC Class II protein-bound peptide contains both an agretope and an epitope for binding to the T cell.
  • Activation of T cell clones is determined by measuring IL-3 production (see, Quill et al., supra) .
  • the Dupont apparatus and technique for rapid multiple E e Pti de .synthesis (RAMPS) is used to synthesize the members of a set of overlapping (10 residue overlap) , 20- residue peptides from the allergen.
  • One or more radioactive amino acids is incorporated into each synthetic peptide.
  • the pentafluorphenyl active esters of side chain-protected, FMOC amino acids are used to synthesize the peptides, applying standard stepwise solid phase peptide synthetic methods, followed by standard side chain deprotection and simultaneous release of the peptide amide from the solid support.
  • the overlapping sequences which include the putative segments of 8-18 amino acids of the allergenic protein can be synthesized on the method of Geysen, H.M. , et al. J. Immun . Meth . (1987) 102:274.
  • the synthesized radio labeled peptides are tested by incubating them individually (on the plates) with purified MHC proteins which have been formulated into lipid membrane bilayers as above.
  • the relevant allergenic peptide subunits, as they are relatively short, can readily by synthesized using standard automated methods for peptide synthesis.
  • they can be made recombinantly using isolated or synthetic DNA sequences; though this is not the most efficient approach for peptides of this length.
  • a set of labeled test peptides is prepared, and those which bind to MHC in planar lipid membranes containing MHC proteins are shown to contain the agretope.
  • the identified peptides are then prepared by conventional solid phase synthesis and the subset which contain epitopes for the disease-associated helper T cell clones i ⁇ determined by incubation of the candidate peptides with antigen-presenting cells (APC) (or with isolated MHC complex) and spleen or lymph node T cells from mice immunized with the full length protein. Successful candidates will stimulate T cell proliferation in this system.
  • APC antigen-presenting cells
  • This second, smaller, subset represents the suitable peptide component.
  • the elements of the complex can be associated by standard means known in the art.
  • the allergenic peptides can be associated noncovalently with the antigen binding sites of the MHC protein by, for example, mixing the two components. Excess peptide can be removed any of a number of standard procedures, such as ultrafiltration or dialysis.
  • the peptides can also be covalently bound using standard procedures by, for example, photo affinity labelling, (see e.g., Hall et al., Biochemistry 24:5702-5711 (1985).
  • the complexes of the invention can be assayed using an in vitro system or using an in vivo model.
  • the complex is incubated with peripheral blood T cells from subjects immunized with, or showing an allergic response to, the protein or antigen responsible for the condition associated with the peptide of the complex.
  • the successful complexes will induce eliminate syngeneic T cells or induce anergy and prevent proliferation of the T cells even upon stimulation with additional allergen.
  • T cells that proliferate in response to the isolated epitope or to the full length allergen in the presence of APC are cloned.
  • the clones are injected into histocompatible animals which have not been immunized in order to induce the allergic response.
  • Symptoms related to the relevant complex should ameliorate or eliminate the symptoms of the disease. Either of the types of complexes, i.e., with or without the effector component, may be used. In one mode the treatment is two-fold.
  • the individual is treated with the complex of MHC-encoded antigen-presenting glycoprotein containing an allergenic peptide to down-regulate the immune system.
  • Further down-regulation is achieved by treatment with the three component complex with includes the MHC-encoded antigen-presenting glycoprotein, an allergenic peptide which is specific for the allergic response being treated, and an effector component.
  • panels of complexes may be used for treatment. For example, if it is suspected that more than one peptide of an allergen is involved in the response, and/or if it is suspected that more than one antigen is involved, the individual may be treated with several complexes, with or without effector components.
  • the MHC haplotyes which are involved in the presentation of the allergen are identified.
  • Identification of the allele is accomplished by determining the strong positive association of a specific subtype with the disease. This may be accomplished in a number of ways, all of which are known to those skilled in the art. E.g., ⁇ ubtyping may be accomplished by mixed lymphocyte response (MLR) typing and by primed lymphocyte testing (PLT) . Both methods are described in Weir and Blackwell, eds., Handbook of Experimental Immunology, which is incorporated herein by reference. It may also be accomplished by analyzing DNA restriction fragment length polymorphism (RFLP) using DNA probes that are specific for the MHC locus being examined. E.g., Nepom (1986), Annals N.
  • the most complete identification of subtypes conferring disease susceptibility is accomplished by sequencing of genomic DNA of the locus, or cDNA to mRNA encoded within the locus.
  • the DNA which is sequenced includes the section encoding the hypervariable regions of the MHC encoded polypeptide.
  • Techniques for identifying specifically desired DNA with a probe, for amplification of the desired region are known in the art, and include, for example, the polymerase chain reaction (PCR) technique.
  • the polypeptide encoded within the allele is also identifiable, i.e., the polypeptide sequence may be deduced from the sequence of DNA within the allele encoding it.
  • the MHC antigen complexes of the invention used for diagnosis and/or therapy are derived from the effective portion 01 the MHC antigen associated with the disease state.
  • the MHC molecule with the appropriate allergenic peptide, identified by the methods described above. This is typically done by incubating the purified MHC with excess peptide at a low or high pH so as to open the antigen binding pocket (see, Harding et al. , Proc . Nat . Acad . Sci . USA 88:2740-2744 (1991).
  • the peptide is thus noncovalently linked with the antigen binding pocket of the MHC component.
  • the allergenic peptide can also be covalently bound using standard procedures such as photo-affinity labelling, (see e.g., Hall et al., Biochemistry 24:5702-5711 (1985).
  • the MHC molecules of the invention can be assayed using an in vitro system or using an in vivo model.
  • the molecule is incubated with peripheral blood T cells from subjects immunized with, or showing an allergic response to, the protein or allergen responsible for the condition associated with the peptide of the complex.
  • the successful molecules will eliminate or induce anergy in syngeneic T cells as measured in the assays described above.
  • T cells that proliferate in response to the isolated epitope or to the full length antigen in the presence of antigen presenting cells are cloned.
  • the clones are injected into histocompatible animals which have not been immunized in order to induce the disease. Symptoms related to the relevant complex should ameliorate or eliminate the symptoms of the disease.
  • the detectably labeled MHC complex is given in a dose which is diagnostically effective.
  • diagnostically effective means that the amount of detectably labeled protein is administered in sufficient quantity to enable detection of cells having the T cell receptor for which the MHC component is specific.
  • concentration of detectably labeled protein which is administered should be sufficient such that the binding to those cells having the receptor is detectable compared to the background signal.
  • the detectably labeled complex be rapidly cleared from the circulatory system in order to give the best target-to-background signal ratio.
  • the dosage of detectably labeled complex for in vivo diagnosis will vary depending on such factors as age, sex and extent of disease of the individual.
  • radioisotopes are typically used.
  • the type of detection instrument available is a major factor in selecting the radioisotope used.
  • the radioisotope chosen must have a type of decay which is detectable for a given type of instrument.
  • Still another important factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the host is minimized.
  • a radioisotope used for in vivo imaging will lack a particle emission, but produce a large number of photons in the 140-250 keV range, which may be readily detected by conventional gamma cameras.
  • radioisotopes may be bound to the complex either directly or indirectly by using an intermediate functional group.
  • Intermediate functional groups which often are used to bind radioisotopes which exist as metallic ions to proteins are the bi-functional chelating agents such as diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the MHC complexes of the invention can also be labeled with a paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR) .
  • MRI magnetic resonance imaging
  • ESR electron spin resonance
  • any conventional method for visualizing diagnostic imaging can be utilized.
  • gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI.
  • the complexes of the present invention can be used to monitor the course of amelioration of an allergic response in an individual. Thus, by measuring the increase or decrease in the number of targeted T cells, it is possible to determine whether a particular therapeutic regimen aimed at ameliorating the disorder is effective.
  • compositions comprising the claimed complexes can be prepared.
  • Pharmaceutical compositions comprising the complexes are useful for, e.g., parenteral administration, i.e., subcutaneously, intramuscularly or intravenously.
  • parenteral administration i.e., subcutaneously, intramuscularly or intravenously.
  • new drug delivery approaches are being developed.
  • the pharmaceutical compositions of the present invention are suitable for administration using these new methods, as well. See, Langer, Science 249:1527-1533 (1990), which is incorporated herein by reference.
  • compositions for parenteral administration will commonly comprise a solution of the MHC complex or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.
  • the concentration of the MHC complex in these formulations can vary widely, i.e., from less than about 1 ⁇ g/ml, usually at least about 0.1 mg/ml to as much as 10 - 100 mg/ml by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • a typical pharmaceutical composition for intramuscular injection could be made up to contain 1 ml sterile buffered water, and 0.1 mg of MHC complex.
  • a typical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 10 mg of MHC complex.
  • parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington ' s Pharmaceutical Science , 17th Ed., Mack Publishing Company, Easton, Pennsylvania (1985) , which is incorporated herein by reference.
  • methods suitable for increasing serum half-life of the complexes include treatment to remove carbohydrates which are involved in the elimination of the complexes from the bloodstream.
  • substantially all of the carbohydrate moieties are removed by the treatment.
  • substantially all of the carbohydrate moieties are removed if at least about 75%, preferably about 90%, and most preferably about 99% of the carbohydrate moieties are removed.
  • Conjugation to soluble macromolecules such as proteins, polysaccharides, or synthetic polymers, such as polyethylene glycol
  • Other methods include protection of the complexes in vesicles composed of substances such as proteins, lipids (for example, liposomes) , carbohydrates, or synthetic polymers.
  • the complexes of the invention are conveniently administered after being incorporated in lipid monolayers or bilayers.
  • liposomes are used for this purpose but any form of lipid membrane, such as planar lipid membranes or the cell membrane of a cell (e.g., a red blood cell) may be used.
  • the complexes are also conveniently incorporated into micelles.
  • Liposomes can be prepared according to standard methods, as described below. However, if the transmembrane region is deleted, the complex can be administered in a manner conventionally used for peptide-containing pharmaceuticals.
  • Liposomes of the present invention typically contain the MHC-peptide complexes positioned on the surface of the liposome in such a manner that the complexes are available for interaction with the T cell receptor.
  • the transmembrane region is usually first incorporated into the membrane at the time of forming the membrane.
  • the liposomes can be used to target desired drugs (e.g. toxins or chemotherapeutic agents) to particular autoreactive T cells.
  • desired drugs e.g. toxins or chemotherapeutic agents
  • the complexes embedded in the liposome may be used to induce anergy in the targeted cells.
  • Liposome charge is an important determinant in liposome clearance from the blood, with negatively charged liposomes being taken up more rapidly by the reticuloendothelial system (Juliano, Biochem . Biophys . Res . Commun . 63:651 (1975)) and thus having shorter half-lives in the bloodstream. Liposomes with prolonged circulation half- lives are typically desirable for therapeutic and diagnostic uses. For instance, liposomes which can be maintained from 8, 12, or up to 24 hours in the bloodstream are particularly preferred.
  • the liposomes are prepared with about 5- 15 mole percent negatively charged phospholipids, such as phosphatidylglycerol, phosphatidylserine or phosphatidylinositol.
  • Added negatively charged phospholipids, such as phosphatidylglycerol also serve to prevent spontaneous liposome aggregating, and thus minimize the risk of undersized liposomal aggregate formation.
  • Membrane- rigidifying agents such as sphingomyelin or a saturated neutral phospholipid, at a concentration of at least about 50 mole percent, and 5-15 mole percent of monosialylganylioside, may provide increased circulation of the liposome preparation in the bloodstream, as generally described in U.S. Pat. No. 4, 837,028.
  • the liposome suspension may include lipid-protective agents which protect lipids against free- radical and lipid-peroxidative damages on storage.
  • Lipophilic free-radical quenchers such as ⁇ tocopherol and water-soluble iron-specific chelators, such as ferrioxianine, are preferred.
  • One method produces multilamellar vesicles of heterogeneous sizes.
  • the vesicle-forming lipids are dissolved in a suitable organic solvent or solvent system and dried under vacuum or an inert gas to form a thin lipid film.
  • the film may be redissolved in a suitable solvent, such as tertiary butanol, and then lyophilized to form a more homogeneous lipid mixture which is in a more easily hydrated powder like form.
  • This film is covered with an aqueous solution of the targeted drug and the targeting component and allowed to hydrate, typically over a 15-60 minute period with agitation.
  • the size distribution of the resulting multilamellar vesicles can be shifted toward smaller sizes by hydrating the lipids under more vigorous agitation conditions or by adding solubilizing detergents such as deoxycholate.
  • the hydration medium contains the targeted drug at a concentration which is desired in the interior volume of the liposomes in the final liposome suspension.
  • the drug solution contains between 10-100 mg/ml of the complexes in a buffered saline solution.
  • the liposomes may be sized to achieve a desired size range and relatively narrow distribution of liposome sizes.
  • One preferred size range is about 0.2-0.4 microns, which allows the liposome suspension to be sterilized by filtration through a conventional filter, typically a 0.22 micron filter.
  • the filter sterilization method can be carried out on a high through-put basis if the liposomes have been sized down to about 0.2-0.4 microqs.
  • Extrusion of liposome through a small-pore polycarbonate membrane or an asymmetric ceramic membrane is also an effective method for reducing liposome sizes to a relatively well-defined size distribution.
  • the suspension is cycled through the membrane one or more times until the desired liposome size distribution is achieved.
  • the liposomes may be extruded through successively smaller-pore membranes, to achieve a gradual reduction in liposome size.
  • the initial sized liposome suspension may contain up to 50% or more complex in a free (nonencapsulated) form.
  • the liposomes in the suspension are pelleted by high-speed centrifugation leaving free compound and very small liposomes in the supernatant.
  • Another method involves concentrating the suspension by ultrafiltration, then resuspending the concentrated liposomes in a replacement medium.
  • gel filtration can be used to separate large liposome particles from solute molecules.
  • the liposome suspension is brought to a desired concentration for use in intravenous administration. This may involve resuspending the liposomes in a suitable volume of injection medium, where the liposomes have been concentrated, for example by centrifugation or ultrafiltration, or concentrating the suspension, where the drug removal step has increased total suspension volume. The suspension is then sterilized by filtration as described above.
  • the liposomes comprising the MHC-peptide complex may be administered parenterally or locally in a dose which varies according to, e.g., the manner of administration, the drug being delivered, the particular disease being treated, etc.
  • Micelles are commonly used in the art to increase solubility of molecules having nonpolar regions. One of skill will thus recognize that micelles are useful in compositions of the present invention.
  • Micelles comprising the complexes of the invention are prepared according to methods well known in the art (see, e.g., Remington ' s Pharmaceutical Sciences , supra , Chap. 20) .
  • Micelles comprising the complexes of the present invention are typically prepared using standard surfactants or detergents. Micelles are formed by surfactants (molecules that contain a hydrophobic portion and one or more ionic or otherwise strongly hydrophilic groups) in aqueous solution.
  • critical micelle concentration As the concentration of a solid surfactant increases, its monolayers adsorbed at the air/water or glass/water interfaces become so tightly packed that further occupancy requires excessive compression of the surfactant molecules already in the two monolayers. Further increments in the amount of dissolved surfactant beyond that concentration cause amounts equivalent to the new molecules to aggregate into micelles. This process begins at a characteristic concentration called "critical micelle concentration”.
  • the shape of micelles formed in dilute surfactant solutions is approximately spherical.
  • the polar head groups of the surfactant molecules are arranged in an outer spherical shell whereas their hydrocarbon chains are oriented toward the center, forming a spherical core for the micelle.
  • the hydrocarbon chains are randomly coiled and entangled and the micellar interior has a nonpolar, liquid-like character.
  • the micelles of polyoxyethylated nonionic detergents the polyoxyethlene moieties are oriented outward and permeated by water. This arrangement is energetically favorable since the hydrophilic head groups are in contact with water and the hydrocarbon moieties are removed from the aqueous medium and partly shielded from contact with water by the polar head groups.
  • the hydrocarbon tails of the surfactant molecules located in the interior of the micelle, interact with one another by weak van der Waals forces.
  • the size of a micelle or its aggregation number is governed largely by geometric factors.
  • the radius of the hydrocarbon core cannot exceed the length of the extended hydrocarbon chain of the surfactant molecule. Therefore, increasing the chain length or ascending homologous series increases the aggregation number of spherical micelles.
  • the maximum aggregation numbers consistent with spherical shape are approximately 27, 39, 54, 72, and 92 for c 8 ' C 10 ' c i 2 ' C 14 and C 16 ' respectively.
  • the micelles increase in size. Under these conditions, the micelles are too large to remain spherical and become ellipsoidal, cylindrical or finally lamellar in shape.
  • Suitable surfactants include sodium laureate, sodium oleate, sodium lauryl sulfate, octaoxyethylene glycol monododecyl ether, octoxynol 9 and PLURONIC F-127 ® (Wyandotte Chemicals Corp.).
  • Preferred surfactants are nonionic polyoxyethylene and polyoxypropylene detergents compatible with IV injection such as PLURONIC F-127 ® , n-octyl-/3-D-glucopyranoside, and the like.
  • phospholipids such as those described for use in the production of liposomes, may also be used for micelle formation.
  • the MHC subunits of the present invention comprise a lipophilic transmembrane region and a relatively hydrophilic extracellular domain
  • mixed micelles are formed in the presence of common surfactants or phospholipids and the subunits.
  • the mixed micelles of the present invention may comprise any combination of the subunits, phospholipids and/or surfactants.
  • the micelles may comprise subunits and detergent, subunits in combination with both phospholipids and detergent, or subunits and phospholipid.
  • the MHC complexes of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with immunoglobulins and commonly used lyophilization and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilization and reconstitution can lead to varying degrees of activity loss and that use levels may have to be adjusted to compensate.
  • compositions containing the present MHC complexes or a cocktail thereof can be administered for the prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already affected by the particular disease, in an amount sufficient to cure or at least partially arrest the disease process and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.”
  • Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from about 0.01 to about 1000 mg of MHC complex per dose, with dosages of from about 10 to about 100 mg per patient being more commonly used.
  • compositions containing the MHC complexes or a cocktail thereof are administered to a patient not already in a disease state to enhance the patient's resistance. Such an amount is defined to be a "prophylactically effective dose.”
  • the precise amounts again depend upon the patient's state of health and general level of immunity, but generally range from 0.01 to 1000 mg per dose, especially about 10 to about 100 mg per patient.
  • compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • pharmaceutical formulations should provide a quantity of the MHC complexes of this invention sufficient to effectively treat the patient.
  • Kits can also be supplied for therapeutic or diagnostic uses.
  • the subject composition of the present invention may be provided, usually in a lyophilized form in a container.
  • the complexes which may be conjugated to a label or toxin, or unconjugated, are included in the kits with buffers, such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g., serum albumin, or the like, and a set of instructions for use.
  • buffers such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g., serum albumin, or the like
  • these materials will be present in less than about 5% wt. based on the amount of active MHC complex and usually present in total amount of at least about 0.001% wt. based again on the complex concentration.
  • an inert extender or excipient to dilute the active ingredients, where the excipient may be present in from about 1 to 99% wt. of the total composition.
  • an antibody capable of binding to the MHC complex is employed in an assay, this will usually be present in a separate vial.
  • the antibody is typically conjugated to a label and formulated in an analogous manner with the formulations described above.
  • Amb a V-specific T-cell clones were isolated from several DR2.2 + , highly Amb a V + subjects who had received short ragweed Rx and were known to have high levels of IgE and IgG antibodies to Amb a V. Two black patients, who had very high levels of IgE antibody prior to Rx and IgG antibody levels in excess of 20 ⁇ g/ml during Rx, were included. The cloning procedure was a modification of that used by O'Hehir et al. (1987) .
  • PBMC peripheral blood mononuclear cells
  • Amb a V in culture medium (RPMI 1640 containing 1% penicillin, 1% streptomycin, 10% fetal calf serum, and 1% L-glutamine)
  • T-cell blasts were isolated 7 days later, and blast cells (1 x 10 5 cells/ml) were restimulated with Amb a V (1 ⁇ g/ml) together with irradiated (5000 rads) , autologous PBMC (1 x 10 6 cells/ml) as antigen- presenting cells (APCs) in culture medium.
  • APCs antigen- presenting cells
  • recombinant IL-2 (10 units/ml) and fresh culture medium were added for the remaining 5 days of culture.
  • T- cell blasts were cloned (0.3-1 cell/well) in 96-well round- bottomed plates in the presence of Amb a V (1 ⁇ g/ml) and autologous APCs (1 x 10 5 cells/well) in culture medium supplemented with recombinant IL-2 (20 units/ml) .
  • Proliferation assays A total of 1 x 10 4 cloned T cells per well were incubated with 1 x 10 5 autologous or allogeneic APCs, with or without .Amb a V (0.5 ⁇ g/ml, unless otherwise indicated), for 3 days.
  • the allogeneic APCs included 13 HTCs, DR2.2 (2 lines), 2.12 (2 lines) , 2.21 (2 lines) , 2.22, 4.4, 4.10, 4.14, Wll, w8 and 9, as well as a number of selected patients' cells (see
  • Amb a V-specific T cells (2 x 10 6 ) were cultured with 3 ⁇ g of 6 differentDR2:peptide complexes for 18 hours.

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Abstract

La présente invention se rapporte à des complexes essentiellement composés d'un constituant isolé du complexe d'histocompatibilité majeure (MCH) et d'un peptide allergénique associé au site de liaison d'antigène du constituant de MHC. Ces complexes permettent de traiter des réponses immunitaires délétères, telles que les réactions allergiques.
PCT/US1994/001919 1993-02-25 1994-02-24 Traitements de reactions allergiques au moyen de complexes peptidiques de mhc WO1994018998A1 (fr)

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US5869270A (en) * 1996-01-31 1999-02-09 Sunol Molecular Corporation Single chain MHC complexes and uses thereof
US6232445B1 (en) 1997-10-29 2001-05-15 Sunol Molecular Corporation Soluble MHC complexes and methods of use thereof
US7074904B2 (en) 1994-07-29 2006-07-11 Altor Bioscience Corporation MHC complexes and uses thereof
WO2022250811A3 (fr) * 2021-04-20 2023-06-08 Georgia Tech Research Corporation Nanoparticules pour la programmation de cellules spécifiques d'un antigène et leurs utilisations

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EUR. J. IMMUNOL., Vol. 21, issued 1991, S. HUANG et al., "Class II Major Histocompatibillty Complex Restriction of Human T Cell Responses to Short Ragweed Allergen, Amb a V", see pages 1469-1473. *
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Cited By (7)

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Publication number Priority date Publication date Assignee Title
US7074904B2 (en) 1994-07-29 2006-07-11 Altor Bioscience Corporation MHC complexes and uses thereof
US5869270A (en) * 1996-01-31 1999-02-09 Sunol Molecular Corporation Single chain MHC complexes and uses thereof
US6309645B1 (en) 1996-01-31 2001-10-30 Sunol Molecular Corporation MHC molecules and uses thereof
US7141656B2 (en) 1996-01-31 2006-11-28 Altor Bioscience Corporation MHC complexes and uses thereof
US6232445B1 (en) 1997-10-29 2001-05-15 Sunol Molecular Corporation Soluble MHC complexes and methods of use thereof
US7074905B2 (en) 1997-10-29 2006-07-11 Altor Bioscience Corporation Soluble MHC complexes and methods of use thereof
WO2022250811A3 (fr) * 2021-04-20 2023-06-08 Georgia Tech Research Corporation Nanoparticules pour la programmation de cellules spécifiques d'un antigène et leurs utilisations

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