WO2009090651A2 - Ligands des molécules hla-b2705 du complexe majeur d'histocompatibilité, utilisés à des fins de diagnostic et de traitement - Google Patents

Ligands des molécules hla-b2705 du complexe majeur d'histocompatibilité, utilisés à des fins de diagnostic et de traitement

Info

Publication number
WO2009090651A2
WO2009090651A2 PCT/IL2009/000067 IL2009000067W WO2009090651A2 WO 2009090651 A2 WO2009090651 A2 WO 2009090651A2 IL 2009000067 W IL2009000067 W IL 2009000067W WO 2009090651 A2 WO2009090651 A2 WO 2009090651A2
Authority
WO
WIPO (PCT)
Prior art keywords
peptide
peptides
hla
cells
protein complex
Prior art date
Application number
PCT/IL2009/000067
Other languages
English (en)
Other versions
WO2009090651A3 (fr
Inventor
Lilach Halevi
Arie Admon
Eilon Barnea
Ilan Beer
Original Assignee
Technion Research And Development Foundation Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Technion Research And Development Foundation Ltd. filed Critical Technion Research And Development Foundation Ltd.
Publication of WO2009090651A2 publication Critical patent/WO2009090651A2/fr
Publication of WO2009090651A3 publication Critical patent/WO2009090651A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to multimeric HLA-B2705-peptide complexes, and their use in therapy and diagnosis of cancer and autoimmune disorders.
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • MHC-displayed peptides can include normal peptides, as well as peptide antigens derived from the infectious agent.
  • Li autoimmune disease states tissues display normal peptides, but at least some of them elicit a specific T cell response, resulting in uncontrolled and harmful inflammation.
  • MHC class I molecules are composed of a polymorphic alpha chain non-covalently associated with a conserved ⁇ 2 -microglobulin subunit. Their peptide binding specificity is haplotype specific, and is defined by the binding "pocket", formed by two of the three extracellular domains of the alpha chain i.e. domains ⁇ l and ⁇ 2 which fold into a groove-like structure. Peptides of about eight to ten residues can be accommodated by the binding pocket, with longer peptides bulging out in the middle.
  • the term "peptidome” has been used to describe the pool of peptides which specifically interacts with a particular MHC haplotype, and can encompass thousands of different sequences.
  • Antigen display by MHC class I molecules on antigen presenting cells involves a pathway in which transporter proteins, encoded within the MHC locus, bind and shuttle peptides into the lumen of the endoplasmic reticulum. Peptides having the appropriate motifs are recognized and bound by the resident MHC class I alpha chains, which then associate with ⁇ 2 -microglobulin to form integral membrane proteins. At the cell surface, the MHC-peptide complex is presented to the appropriately rearranged T cell receptor (TCR) on a CD8 + T cell.
  • TCR T cell receptor
  • TCR recognition of MHC-peptide is specific for both peptide and MHC haplotype, and adhesion between T cell and APC involves multiple TCR/MHC-peptide contacts, as well as the T cell coreceptor CD8.
  • Cytotoxic T cells are activated CD8 + T cells which lyse APCs displaying antigen.
  • T cells Under normal circumstances, T cells undergo selection and tolerance processes which prevent their recognition and binding of self antigens. hi various autoimmune diseases however, CD8 + T cells recognize self antigens as foreign and attack the cells presenting them.
  • SpA Spondyloarthropathies
  • AS ankylosing spondylitis
  • ReA reactive arthritis
  • psoriatic spondylitis enteropathic arthritis
  • peripheral psoriatic arthritis spondylitis associated with inflammatory bowel disease
  • acute anterior uveitis undifferentiated spondyloarthropathy.
  • HLA-B27 is present in about 10% of European populations, it is found in about 95% of AS patients and about 80% of ReA patients.
  • HLA-B27 related SpA has been hypothesized to involve the presentation of an arthritogenic peptide, possibly derived from an infectious agent, particularly Salmonella or Mycoplasma species.
  • an infectious agent particularly Salmonella or Mycoplasma species.
  • B*2705 is the dominant subtype and is highly associated with SpA.
  • Other prevalent and disease associated subtypes are B*2702 and B*2704, while B*2703, B*2706, B*2707, and B*2709 have a lesser extent of disease association.
  • HLA-B27 11-mer spanning HLA-B27 residues 169- 179 is a natural HLA-B27 ligand with homology to proteins from Gram-negative bacteria (Alvarez et al. (2001) J. Biol. Chem. 276(35):32729-37). Herberts et al. disclose that presentation by HLA-B27 requires two N-terminal basic amino acid residues, and that B27 presented peptides usearginine in the second position as an anchor residue, and frequently have arginine or lysine in the first position (Herberts et al. (2006) J. Immunol 176(5):2697-701).
  • Boisgerault et al. disclose that peptides presented by B*2705 and B*2703 subtypes differ only at a single residue in pocket A of the peptide binding groove.
  • the sequence RRYLENGKETL is reportedly presented naturally as a major endogenous peptide by B*2705 and B*2702 disease-associated subtypes and not by B*2703 (Boisgerault F. et al (1996) J Clin Invest 98(12):2764-70).
  • Stodulkova et al. disclose that HLA-B27 bound peptides from AS patients have an increased frequency of GIu and a decreased frequency of GIn, as compared to healthy controls (Stodulkova et al. (2006) Immunol Lett.
  • Rammensee et al. disclose a database of MHC ligands and peptide motifs, of which about 200 peptides of various origin are specific for the HLA-B *2705 subtype (Rammensee et al. (1999) Immunogenetics 50(3-4):213-9).
  • Brusic et al. disclose an MHC peptide database, of which about 450 peptides are specific for different HLA-B27 subtypes (Brusic et al. (1997) Nucleic Acids Res. 26(1):368-71).
  • Lopez de Castro et al. disclose a statistical assessment of residue usage among constitutive peptide repertoires of multiple HLA-B27 subtypes (Lopez de Castro et al (2004) Tissue Antigens 63(5):424-45).
  • Binding motifs of MHCs haplotypes were first examined by Edman sequencing of unfractionated peptide mixtures eluted from MHC molecules extracted from cell membranes (FaIk et al. (1991) Nature 351(6324):290-6; Pamer et al. (1991) Nature 353(6347):852-5). Tandem mass spectrometry has been used to analyze individual peptides following their separation by reversed phase chromatography (Hunt et al. (1992) Science 255(5049): 1261-3; Cox et al. (1994) Science, 264(5159):716-9; Storkus et al. (1993) J. Immunother. 14(2):94-103). A method of identifying peptides originating from a particular cell type, which bind
  • MHC molecules of a particular haplotype is disclosed in US patent application Publication No. 2005/0053918 and in Barnea et al., (2002) Eur. J. Immunol. 32(1):213- 22.
  • the disclosed method is based on analysis of soluble HLA (sHLA) molecules engineered so as to be secreted into the cell growth medium.
  • sHLA soluble HLA
  • US 2005/0053918 describes recovery of sHLA-peptide complexes from human cancer cell lines transformed to express HLA-A2, HLA-B7 and HLA-Cw4 molecules.
  • the bound peptides are purified by immunoaffinity chromatography and sequenced by electrospray tandem mass spectrometry.
  • Multimeric protein complexes comprising peptide loaded MHC class I molecules have been described.
  • US 5,635,363 discloses a multimeric binding complex comprising at least two monomers, wherein each monomer comprises a soluble form of a class I ⁇ chain; ⁇ 2 -microglobulin and peptide bound in the alpha chain groove.
  • the monomers have specific attachment sites engineered on either of the class I chains, and are reportedly multimerized by binding to a multivalent entity via the attachment sites.
  • use of the BirA enzyme recognition sequence enables biotinylation of the monomers, which are then attached to streptavidin to form tetramers.
  • the invention is reportedly useful to detect and/or separate antigen specific T cells, and has relevance for disease conditions associated with T cell activation and/or a specific MHC haplotype.
  • US 5,635,363 further discloses that the multimeric complexes may be used to inhibit immune function by inducing anergy of specific T cells, or by ablation of reactive T cells.
  • US 6,248,564 discloses a multimeric MHC class I conjugate comprising a mutant ⁇ 2-microglobulin subunit into which a new cysteine residue is engineered, for conjugation of a compound thereto via the cysteine sulfhydryl group and a functional group of the compound.
  • the compound may be, for example, a ligand for a multivalent binding molecule or an antibody directed against a tumor antigen. Further disclosed are biotinylated tetramers of the invention loaded with peptide.
  • the multimeric complex of the invention may reportedly be used to to elicit a cytotoxic T cell response in order to destroy undesired tissues.
  • US 6,811,785 discloses a multimeric complex of at least two covalently linked chimeric molecules, wherein each chimera comprises an immunoglobulin constant region element and two MHC class I extracellular domains associated with peptide.
  • US 7,202,349 discloses a multimeric protein complex comprising subunits of an MHC class I molecule in fusion with DsRed, a fluorescent self-multimerizing tetrameric protein, and a bound peptide antigen. The complex is reportedly useful for labeling and sorting T cells.
  • US 2005/0287611 discloses use of a multivalent MHC binding molecule bound with peptide in a bridging assay to enumerate the number of antigen specific CTLs in a sample.
  • WO 99/58557 discloses a disulfide bonded homodimer of the HLA-B27 heavy chain, which is shown to bind an HIV gag HLA-B27 epitope.
  • the dimer may reportedly be used for treating or preventing onset of a spondylarthropathy or for tolerizing a human to the native homodimer of the HLA-B27 heavy chains.
  • Appel et al. describe formation of HLA-B27 tetramers loaded with peptides from
  • Epstein Barr virus or from Chlamydia (Appel et al. (2004) Arthritis Res 6(6):R521-34).
  • the current invention provides a method of immunomodulating T cells, wherein the method utilizes a multimeric protein complex comprising specifically a plurality of soluble fragments derived from the human leukocyte antigen subtype HLA-B2705 molecule and a plurality of peptide ligands specific therefor.
  • the multimeric protein complex is further useful for the identification of specifically reactive T cell subsets which play a role in various autoimmune disease states, and for the development of pharmaceutical compositions which target specifically such reactive T cells.
  • the multimeric protein complex is further useful for the development of pharmaceutical compositions which target cancer cells expressing cancer specific antigens which are displayed or presented by HLA-B2705.
  • the invention is based, in part, on the unexpected discovery that peptides which are naturally presented by HLA-B2705 include those which are derived from: (i) proteins selectively expressed in cartilage and bone cells; (ii) proteins of arthritogenic bacteria; (iii) human proteins which have substantial homology with proteins of arthritogenic bacteria, and (iv) tumor associated antigens specifically expressed in cancerous cells.
  • the peptides associated with cartilage and bone cells, and those associated with arthritogenic bacteria have potential for the development of therapeutic strategies for spondyloarthropathies, and other autoimmune disorders which have a high rate of association with the HLA-B2705 allele.
  • Peptides derived from cancer associated markers have potential for the development of anti-cancer vaccines.
  • the invention provides a method of immunomodulating T cells, the method comprising contacting T cells from a subject in need thereof, with a multimeric protein complex, the multimeric protein complex comprising a plurality of soluble fragments of HLA-B2705 molecules, and a plurality of peptides, wherein each peptide is a ligand for HLA-B2705; thereby modulating said T cells.
  • a "plurality of peptides" refers to either multiple copies of the same peptide i.e. a single amino acid sequence, or a multiplicity of different peptides i.e. different amino acid sequences, wherein each of the different peptides may be present as a single copy or in multiple copies.
  • the method of immunomodulating T cells comprises down regulating T cells of a subject having an autoimmune or chronic inflammatory disorder.
  • the method of immunomodulating T cells comprises stimulating T cells of a subject having cancer.
  • the contacting is in vivo, ex vivo or in vitro.
  • the plurality of peptides comprises at least one peptide selected from the group consisting of: SEQ ID NOS.: 1- 1278.
  • the plurality comprises at least one peptide selected the group consisting of: (i) a peptide derived from a human protein selectively expressed in cartilage and/or bone, wherein the peptide is selected from the group consisting of: SEQ ID NOS.: 43; 203; 236; 256; 272; 274; 278; 316; 358; 424; 506; 515; 528; 534; 611; 709; 714; 774; 875; 1011; 1019; 1030; 1035; 1077; 1157, and combinations thereof; (ii) a peptide derived from a protein expressed in Salmonella sp., wherein the peptide is selected from the group consisting of: SEQ ID NOS.: 502; 626; 843; 913; 932; 933; 945; 1040; 1062; 1071; 1103; 1108; 1120; 1151; 1162; 1183; 1194
  • the plurality comprises multiple copies of a peptide derived from a human protein selectively expressed in cartilage and/or bone, wherein the peptide is selected from the group consisting of: selected from the group consisting of: SEQ ID NOS.: 43; 203; 236; 256; 272; 274; 278; 316; 358; 424; 506; 515; 528; 534; 611; 709; 714; 774; 875; 1011; 1019; 1030; 1035; 1077; 1157, and combinations thereof.
  • the plurality comprises multiple copies of a peptide derived from a protein expressed in Salmonella sp., wherein the peptide is selected from the group consisting of: SEQ ID NOS.: 502; 626; 843; 913; 932; 933; 945; 1040; 1062; 1071; 1103; 1108; 1120; 1151; 1162; 1183; 1194; 1203; 1208; 1209; 1224; 1235; 1238; 1247; 1248; 1269, and combinations thereof.
  • the plurality comprises multiple copies of a peptide derived from a protein expressed in Ureaplasma sp., wherein the peptide is selected from the group consisting of: SEQ ID NOS.: 938; 1036; 1088, and combinations thereof.
  • the plurality comprises multiple copies of a peptide derived from a human protein, wherein the peptide is selected from the group consisting of: SEQ ID NOS.: 77; 221; 241; 246; 247; 299; 378; 520; 563; 825; 910; 1110, and combinations thereof.
  • the autoimmune or chronic inflammatory disorder is selected from ankylosing spondylitis, reactive arthritis, psoriatic spondylitis, enteropathic arthritis, peripheral psoriatic arthritis, spondylitis associated with inflammatory bowel disease, acute anterior uveitis and undifferentiated spondyloarthropathy.
  • the plurality comprises at least one peptide peptide derived from a tumor associated antigen, wherein the peptide is selected from the group consisting of: SEQ ID NOS.: 61; 88; 97;
  • the cancer is selected from melanoma, lymphoma, sarcoma, leukemia, cervical cancer, hepatic cancer, renal cancer, testicular cancer, ovarian cancer and colon cancer.
  • the peptides are derived from a protein selected from an oncogene product, a tumor antigen, a SEREX antigen, a protein overexpressed in cancer, an apoptosis-related protein and an angiogenesis-related protein.
  • the plurality comprises multiple copies of a peptide selected from the group consisting of: SEQ ID NOS.: 61; 88; 97; 99; 131; 132; 197; 198; 210; 231; 254; 280; 286; 288; 290; 304; 305; 323; 337; 340; 341; 353; 363; 369; 371; 382; 389; 397; 404; 432; 444; 448; 483; 488; 499; 500; 586; 627; 652; 710; 724; 728; 754; 756; 772; 844; 845; 873; 907; 921; 951; 997; 1010; 1016; 1026; 1038; 1043; 1074; 1097; 1125; 1131; 1139; 1146; 1154; 1184; 1189; 1190; 1242; 1244; 1253; 1274; 1175,
  • the contacting is carried out in vivo, ex vivo or in vitro.
  • the method comprises the steps of withdrawing the T cells from the body of the subject, contacting the cells with the multimeric protein complex ex vivo, and returning the cells to the body of the subject.
  • the invention provides a multimeric protein complex for immunomodulating T cells, wherein the multimeric protein complex comprises a plurality of soluble fragments of HLA-B2705 molecules, and a plurality of peptides, wherein each peptide is a ligand for HLA-B2705.
  • the invention provides use of a multimeric protein complex comprising a plurality of soluble fragments of HLA-B2705 molecules, and a plurality of peptides, wherein each peptide is a ligand for HLA-B2705, for the preparation of a medicament for immunomodulating T cells.
  • the invention provides a multimeric protein complex comprising a plurality of soluble fragments of HLA-B2705 molecules, and a plurality of peptides, wherein each peptide is a ligand for HLA-B2705, and wherein the plurality comprises at least one peptide selected from the group consisting of: (i) a peptide derived from a human protein selectively expressed in cartilage and/or bone, wherein the peptide is selected from the group consisting of: SEQ ID NOS.: 43; 203; 236; 256; 272; 274; 278; 316; 358; 424; 506; 515; 528; 534; 611; 709; 714; 774; 875; 1011; 1019; 1030; 1035; 1077; 1157, and combinations thereof; (ii) a peptide derived from a protein expressed in Salmonella sp., wherein the peptide is selected from the group consisting of: SEQ ID NO
  • the peptide is a bacterial arthritogenic peptide selected from the group consisting of: SEQ ID NOS.: 502; 626; 843; 913; 932; 933; 938; 945; 1036; 1040; 1062; 1071; 1088; 1103; 1108; 1120; 1151; 1162; 1183; 1194; 1203; 1208; 1209; 1224; 1235; 1238; 1247; 1248; 1269, and combinations thereof.
  • the plurality comprises multiple copies of a peptide selected from the group consisting of: SEQ ID NOS.: 502; 626; 843; 913; 932; 933; 945; 1040; 1062; 1071; 1103; 1108; 1120; 1151; 1162; 1183; 1194; 1203; 1208; 1209; 1224; 1235; 1238; 1247; 1248; 1269, and combinations thereof.
  • the peptide is a human arthritogenic peptide selected from the group consisting of: SEQ ID NOS.: 43; 77; 203; 221; 236; 241; 246; 247; 256; 272; 274; 278; 299; 316; 358; 378; 424; 506; 515; 520; 528; 534; 563; 611; 709; 714; 774; 825; 875; 910, 1110; 1011; 1019; 1030; 1035; 1077; 1157, and combinations thereof.
  • the plurality comprises multiple copies of a peptide selected from the group consisting of: SEQ ID NOS.: 43; 203; 236; 256; 272; 274; 278; 316; 358; 424; 506; 515; 528; 534; 611; 709; 714; 774; 875; 1011; 1019; 1030; 1035; 1077; 1157, and combinations thereof.
  • the plurality comprises multiple copies of a peptide selected from the group consisting of: SEQ ID NOS.: 938; 1036; 1088, and combinations thereof. In a particular embodiment, the plurality comprises multiple copies of a peptide selected from the group consisting of: SEQ ID NOS.: 77; 221; 241; 246; 247; 299; 378; 520; 563; 825; 910; 1110, and combinations thereof.
  • the multimeric protein complex has activity in down regulating of T cells, or in inducing tolerization of T cells, hi a particular embodiment, a pharmaceutical composition having activity against an autoimmune disorder comprises the multimeric soluble protein complex, and at least one pharmaceutically acceptable carrier.
  • the invention provides a multimeric protein complex comprising a plurality of soluble fragments of HLA-B2705 molecules, and a plurality of peptides, wherein each peptide is a ligand for HLA-B2705, and wherein the the plurality of peptides comprises at least one peptide selected from the group consisting of: SEQ ID NOS.: 61; 88; 97; 99; 131; 132; 197; 198; 210; 231; 254; 280;
  • the plurality comprises multiple copies of a peptide selected from the group consisting of: SEQ ID NOS.: 61; 88; 97; 99; 131; 132; 197; 198; 210; 231; 254; 280; 286; 288; 290; 304; 305; 323; 337; 340; 341; 353; 363; 369; 371; 382; 389; 397; 404; 432; 444; 448; 483; 488; 499; 500; 586; 627; 652; 710; 724; 728; 754; 756; 772; 844; 845; 873; 907; 921; 951; 997; 1010; 1016; 1026; 1038; 1043; 1074; 1097; 1125; 1131; 1139; 11
  • a pharmaceutical composition having activity against cancer cells comprises the multimeric soluble protein complex, and at least one pharmaceutically acceptable adjuvant, hi a particular embodiment, the pharmaceutical composition comprises the multimeric soluble protein complex conjugated to antigen presenting cells.
  • the cancer is selected from melanoma, lymphoma, sarcoma, leukemia, cervical cancer, hepatic cancer, renal cancer, testicular cancer, ovarian cancer and colon cancer.
  • the ratio of fragments of HLA-B2705 molecules to peptides is 1 : 1.
  • the multimeric protein complex comprises at least one linker molecule, hi particular embodiments, at least one fragment of an HLA-B2705 molecule, is in covalent association with at least one peptide, hi particular embodiments, the covalent association includes a linker molecule, hi particular embodiments, the multimeric protein complex is selected from a dimer, a trimer, a tetramer, a pentamer, a hexamer, a heptamer, an octamer, a nonamer, a decamer, an undecamer and a dodecamer. hi particular embodiments, the multimeric protein complex is a tetramer.
  • the plurality of peptides is a plurality of homogeneous peptides, or a plurality of heterogeneous peptides, hi particular embodiments, the plurality of heterogeneous peptides consists of 2-100, 2-50, 2-20, or 2-10 heterogeneous peptides, hi particular embodiments, the multimeric soluble protein complex further comprises at least one component selected from the group consisting of a radioactive moiety, an enzyme, a fluorescent moiety, a carbohydrate moiety, a lipid moiety, a solid matrix, a carrier, a toxin, a linker and combinations thereof, hi particular embodiments, the peptides are synthetically or recombinantly manufactured, hi particular embodiments, one or more of the peptides comprises a modification, hi particular embodiments, the modification comprises at least one non- hydrolyzable peptide bond.
  • the multimeric soluble protein complex has activity in down regulation of T cells
  • the multimeric soluble protein complex has activity in inducing tolerization of T cells.
  • a method of selectively enriching a population of T cells with CD8 + T cells specific for at least one HLA-B2705 binding peptide comprises contacting a population of T cells with the soluble multimeric protein complex of the invention, so as to cause CD8 + T cells specific for the at least one HLA-B2705 binding peptide to bind thereto, washing away the unbound T cells, and dissociating the bound T cells from the soluble multimeric protein complex.
  • a method of diagnosing or prognosing a cancer characterized by expression of at least one HLA-B2705 binding peptide comprises contacting a T cell sample from a subject with the multimeric soluble protein complex of the invention, and determining the extent of specific binding of the T cell sample as a diagnostic or prognostic indicator of the cancer.
  • a conjugate comprises the multimeric protein complex and an inert carrier, hi a particular embodiment, the carrier is particulate, hi a particular embodiment, the carrier is biodegradable, hi a particular embodiment, the carrier is non-immunogenic.
  • the carrier has a net negative charge, hi a particular embodiment, the carrier has no net charge, hi a particular embodiment, the carrier is covalently bound to the multimeric protein complex, hi a particular embodiment, the carrier is non-covalently bound to the multimeric protein complex, hi a particular embodiment, the carrier is a bead comprising a material selected from the group consisting of glass, silica, polyesters of hydroxylcarboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxylcarboxylic acids, and dicarboxylic acids, hi a particular embodiment, the carrier comprises a branched polymer, hi a particular embodiment, the ratio of multimeric protein complex to carrier is in the range from about 5:1 to 10000:1.
  • Figure 1 is a histogram of the distribution of length groups among 1089 identified HLA-B27 peptides from the Certain and Probable subsets. Peptide length (in amino acid residues) is shown on the X-axis and the number of peptides of that length group is shown on the Y-axis.
  • Figure 2 shows the abundance of lysine at the different positions in peptides of different length.
  • the position along the peptide (position 1 -position 11) is represented on the X-axis; the peptide length (L9-L11) is represented on the Y-axis, and the percentage of lysine relative to its natural abundance in human proteins is represented on the Z-axis.
  • Figure 3 shows the length extension of peptide variants at the amino and carboxyl termini.
  • the X-axis is the length extension, for example N-2 is a change of two amino acids in the N terminal (as between the peptides RRLRNHMAVAF and LRNHMAVAF), while C-3 is a change of three amino acids in the C terminal (as between the peptides ARLDHKFDLMY and ARLDHKFD).
  • the Y-axis shows the number of such length variants.
  • the invention provides a method of immunoregulating T cells, by contacting T cells from a subject in need thereof with a multimeric protein complex, the multimeric protein complex comprising a plurality of soluble fragments of HLA-B2705 molecules, and a plurality of peptides, wherein each peptide is a ligand for HLA-B2705.
  • Immunoregulation of T cells encompasses both down regulating and stimulating of T cells. Depending on the disease being treated, either down regulation or stimulation of T cells may be desired. For example, T cell down regulation may be sought for treating autoimmune disorders which are characterized by excess immune reactivity, whereas T cell stimulation may be sought for treating cancer by harnessing the host immune system to attack the cancerous tissue. Both types of immunomodulating effects may be elicited by the multimeric protein complex described herein.
  • peptides suitable for the method of the invention include SEQ ID NOS.: 1-1278, but the peptides can include any that are ligands for HLA-B2705.
  • the inventors have surprisingly found that peptides which are naturally presented by HLA-B2705.
  • B2705 and which are bound by soluble fragments of HLA-B2705 molecules include those which are derived from: (i) proteins selectively expressed in cartilage and/or bone cells; (ii) proteins of arthritogenic bacteria; (iii) human proteins which have substantial homology with proteins of arthritogenic bacteria, and (iv) tumor associated antigens expressed in cancerous cells.
  • the peptides disclosed herein for use in the invention refer to isolated peptides which are distinct from any full-length proteins or partial fragments thereof which contain such corresponding sequences.
  • Certain autoimmune disorders including spondyloarthropathies, have a high rate of association with the HLA-B2705 allele, and are hypothesized to be triggered by an arthritogenic peptide, possibly derived from host cartilage tissue or from a bacterial pathogen.
  • T cells binding to such peptide-MHC complexes through their T cell receptor are targets for down regulation using the multimeric protein complex described herein.
  • T cells reactive against peptides derived from cancer associated markers are targets for stimulation using the multimeric protein complex described herein.
  • the multimeric protein complex described herein is further useful for the detection, enrichment, characterization and separation of specific T cells, in diagnostic, prognostic and therapeutic methods. Specifically, it may be used for: (i) identifying antigen specific CD8 + T cell subsets in spondyloarthropathies and other disorders; (ii) preparing a therapeutic composition directed against specifically reactive CD8 + T cells, and (iii) preparing a vaccine directed against HLA-B27 positive cancers.
  • T cells include all lineages and subsets of T lymphocytes, including CD8 + T cells, CD4 + T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, suppressor T cells, natural killer T cells and ⁇ T cells.
  • the T cells may be those from spleen, thymus, peripheral blood, lymph node, joint, synovia or any other source.
  • Down regulating of T cells refers to inducing a change in T cells such that their immunologic activity is suppressed or eliminated.
  • Stimulating'Of T cells refers to inducing a change in T cells such that their immunologic activity is induced or augmented.
  • immunologic activity refers to all functions and activities carried out by T cells specifically as a result of being immune functioning cells. Accordingly, immunologic activity includes but is not limited to, recognition and binding of target cells, activation, proliferation, cytokine secretion (including of interleukins, tumor necrosis factor and interferons), signaling and lytic activity.
  • Multimeric protein complex refers to a multiple subunit assembly of identical or different proteins or polypeptides. That is, an assembly of multiple copies of the same protein or polypeptide (the "subunit") which are associated together in an orderly fashion to form a complex.
  • the term “monomer” is used interchangeably with the term “subunit” to refer to the constituent building blocks of the complex.
  • the subunits or monomers may themselves be comprised of more than one polypeptide chain.
  • a covalent or non-covalent assembly of a soluble HLA-B2705 alpha chain together with the ⁇ 2-microglobulin subunit can be considered a "subunit".
  • the multimeric protein complex has a spatial orientation such that the functional domains (i.e.
  • peptide binding grooves of the subunits are not hindered one by the other with respect to their ability to bind peptide.
  • the subunits are in covalent association with each other, or are associated via a common multivalent linker to which each subunit is covalently linked.
  • a "multimeric protein complex comprising a plurality of soluble fragments of HLA-B2705 molecules” has as its subunit a soluble fragment of an HLA-B2705 molecule, and an assembly of multiple copies thereof forms the complex.
  • Soluble fragment of an HLA-B2705 molecule refers to any derivative or truncated mutant of an HLA-B2705 molecule which is soluble due to the fact that the alpha chain thereof substantially lacks the transmembrane domain. It also encompasses secreted forms of HLA-B2705 molecule, including those which are fusion proteins with heterologous secretory elements. It does not encompass HLA-B2705 molecules which are rendered soluble by treatment with detergents or other reagents.
  • the fragment of an HLA-B2705 molecule may comprise a truncated HLA-B2705 alpha chain together with the ⁇ 2-microglobulin subunit, or may comprise a truncated HLA-B2705 alpha chain on its own.
  • Multimerization “oligomerization” and “dimerization” are used interchangeably herein to refer to the formation of a multimeric protein complex containing at least two soluble fragments of HLA-B2705 molecules, as described above.
  • the formation of such a complex involves the binding, association or clustering of two or more HLA-B2705 molecules, and is mediated by a common divalent or multivalent oligomerizing agent, functional group or moiety.
  • Multimerizing agent “oligomerizing agent” and “dimerizing agent” are used interchangeably to refer to a divalent or multivalent compound, functional group or moiety which brings together two or more HLA-B2705 protein molecules in a multimeric complex.
  • HLA-B2705 binding peptides “HLA-B2705 ligands” and “HLA-B2705 restricted epitopes” are used interchangeably herein to refer to peptides of varying length and amino acid sequence which specifically interact with and bind within the binding pocket of the native HLA-B2705 allele.
  • HLA-B2705 ligands are "displayed” or “presented” at the cell surface, and may be recognized by specific T cell receptors on CD8 + T cells.
  • “Plurality” refers to a multiplicity (i.e. two or more) of units, subunits or peptides which are spatially or chemically associated within the complex of the invention.
  • the units comprising the plurality may be homogeneous or heterogeneous.
  • the soluble fragments of HLA-B2705 molecules are preferably a homogeneous plurality i.e. a multiplicity of the same molecule, while the HLA-B2705 ligands i.e. binding peptides, may be either a homogeneous or a heterogeneous plurality i.e. a multiplicity of the same peptide or of different peptides, respectively.
  • a "plurality of peptides” refers to either multiple copies of the same peptide i.e. a single amino acid sequence, or a multiplicity of different peptides i.e. different amino acid sequences, wherein each of the different peptides may be present as a single copy or in multiple copies.
  • Covalent association refers to an intermolecular association or bond which involves the sharing of electrons in the bonding orbitals of two atoms.
  • Non-covalent association refers to intermolecular interaction among two or more separate molecules which does not involve a covalent bond.
  • Intermolecular interaction is dependent upon a variety of factors, including, for example, the polarity of the involved molecules, and the charge (positive or negative), if any, of the involved molecules.
  • Non-covalent associations are selected from ionic interactions, hydrophobic interactions, dipole- dipole interactions, van der Waal's forces, and combinations thereof.
  • “Selectively expressed” refers to the specific expression of proteins, factors or regulatory elements in a particular tissue or organ, and/or at a particular stage of development and/or in response to a particular stimulus.
  • HLA-B2705 binding peptides HLA-B2705 binding peptides
  • HLA-(HLA-1) The total set of peptides disclosed herein are collectively referred to as "HLA-
  • HLA-B2705 binding peptides "HLA-B2705 ligands” or “HLA-B2705 restricted epitopes”; subsets thereof are referred to as "HLA-B2705 arthritogenic peptides”, “cartilage specific HLA-B2705 peptides”, “HLA-B2705 cancer associated peptides”, "HLA-
  • HLA-B2705 bacterially derived peptides B2705 bacterially derived peptides
  • the peptides disclosed herein were not previously known to be HLA-B2705 ligands. They may correspond to fragments of known proteins, but do not include any of the full-length parent proteins.
  • HLA-B2705 ligands Such HLA-B2705
  • B2705 ligands are provided in soluble isolated form, and are "loaded” into the binding sites on the fragments of HLA-B2705 molecules which form the soluble multimeric protein complex of the invention.
  • HLA-B2705 binding peptides The peptides are most frequently nine amino acids in length, but range from six to 14 amino acids in length, as shown in Figure 1. Some of these exemplary peptides correspond to overlapping sequences which vary, for the most part, in their C termini, as shown in Figure 3. Some overlapping peptide sequences appear to be derived from the same protein, possibly reflecting a common processing pathway for presentation by HLA-B2705.
  • Peptides for use in the multimeric complex of the invention include the novel sequences SEQ ID NOS.: 1-1278 which are shown herein for the first time to be naturally presented HLA-B2705 ligands.
  • the subset of sequences comprising SEQ ID NOS.: 1-1278 are shown herein for the first time to be naturally presented HLA-B2705 ligands.
  • SEQ ID NO: 256 (VRNPSVVVK) is derived from collagen type VI alpha 3.
  • the multimeric protein complex of the invention may be used to determine which of these peptides are targets of the autoimmune inflammation at the joints, spine and retina during spondyloarthropathies. Those peptides specifically identified to play a role in autoimmune T cell responses may be used in a method of down regulating T cells, as described herein.
  • the method involves contacting T cells with a multimeric protein complex comprising a plurality of soluble HLA-B2705 molecules or fragments thereof, and a plurality of the peptides identified.
  • Other peptides suitable for use in the multimeric complex of the invention include those which are shown herein to be naturally presented HLA-B2705 ligands in cells following infection with the arthritogenic bacteria Salmonella sp.
  • SEQ ID NO: 1062 KRHFPSLSI
  • SEQ ID NO: 1108 TRSKAIGIAR
  • Other peptides suitable for use in the multimeric complex of the invention include
  • SEQ ID NOS.: 333; 522; 790, and 893 which are peptides derived from the HLA- B2705 molecule itself:
  • peptides suitable for use in the multimeric complex of the invention include SEQ ID NOS.: 77; 221; 241; 246; 247; 299; 378; 520; 563; 825; 910, and 1110. These peptides are derived from human proteins, and display substantial homology to bacterial peptide sequences.
  • substantially homology means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 65 percent sequence identity, preferably at least 80 or 90 percent sequence identity, more preferably at least 95 percent sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • Optimal alignment of sequences for comparison can be conducted, e.g. by the local homology algorithm of Smith and Waterman (1981) Adv Appl Math 2,482; by the homology alignment algorithm of Needleman and Wunsch (1970) J MoI Biol 48,443; by the search for similarity method of Pearson and Lipman (1988) Proc Natl Acad Sci USA 85,2444; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection.
  • BLAST Altschul et al (1990) J MoI Biol 215,403-410.
  • Software for performing BLAST analyses is publicly available through the National Center or Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • default program parameters can be used to perform the sequence comparison, although customized parameters can also be used.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc Natl Acad Sci USA 89, 10915).
  • amino acids may be grouped in classes according to the following properties: hydrophobic sidechains: norleucine, met, ala, val, leu, ile; neutral hydrophilic side chains: cys, ser, thr; acidic side chains: asp, glu; basic side chains: asn, gin, his, lys, arg; residues influencing chain orientation: gly, pro; and aromatic side chains: trp, tyr, phe.
  • Conservative substitutions involve substitutions between amino acids in the same class. Non-conservative substitutions constitute exchanging a member of one of these classes for a member of another.
  • peptides suitable for use in the multimeric complex of the invention include SEQ ID NOS.: 61; 88; 97; 99; 131; 132; 197; 198; 210; 231; 254; 280; 286; 288; 290; 304; 305; 323; 337; 340; 341; 353; 363; 369; 371; 382; 389; 397; 404; 432; 444; 448; 483; 488; 499; 500; 586; 627; 652; 710; 724; 728; 754; 756; 772; 844; 845; 873; 907; 921; 951; 997; 1010; 1016; 1026; 1038; 1043; 1074; 1097; 1125; 1131; 1139; 1146; 1154; 1184; 1189; 1190; 1242; 1244; 1253; 1274, and 1175, which are HLA-B2705 ligands derived from tumor
  • the ability of the multimeric complex to present a plurality of peptides increases the efficiency of binding to T cells bearing TCR having antigenic specificity for the presented peptide(s).
  • the multimeric complex When compared to the binding of singly presented peptide(s) to a T cell, the multimeric complex will have greatly increased stability, in which the half- life of binding (tm) is increased by at least about 10-fold, more usually by about 20- fold, and may be increased by as much as about 50-fold.
  • a plurality of HLA-B2705 binding peptides may be provided by peptides covalently linked to each other to form polymers i.e. oligopeptides, or can be provided as a plurality without linkage to each other, e.g. as an admixture. Peptides of the plurality moreover, may be covalently linked to the multimeric protein complex, for example in a 1:1 ratio with respect to the HLA-B2705 molecules therein.
  • the aforementioned covalent linkages may be direct, or constructed via a linker group, a substituted amino acid or a modified amino acid.
  • the plurality may be composed of the same peptide, providing a plurality of homogeneous peptides. When the same peptide is linked to itself, a homopolymer is provided, enabling presentation of repeating epitopic units. Alternately, the plurality may be composed of different peptides, providing a plurality of heterogeneous peptides.
  • a plurality of heterogeneous peptides may consist of 2-100 heterogeneous peptides, and more specifically, may consist of 2-50, 2-20, or 2-10 heterogeneous peptides. Higher numbers of heterogeneous peptides are also contemplated, depending on the number of different HLA-B2705-peptide targets sought for targeting
  • heteropolymers When a plurality of heterogeneous peptides is provided in covalent linkage, heteropolymers are formed.
  • a plurality of heterogeneous peptides forms a "cocktail" of HLA-B2705 ligands, which may be advantageous for example, for a therapeutic composition directed against a number of different arthritogenic peptides.
  • Heterogeous peptides may correspond to different epitopes on the same protein, different epitopes on different proteins, different HLA restriction specificities, and/or a peptide that contains T helper epitopes, hi addition to covalent linkages, noncovalent linkages capable of forming intermolecular and intrastructural bonds are included.
  • Linkages for homo- or heteropolymers or for coupling to carriers or HLA-B2705 molecules can be provided in a variety of ways.
  • cysteine residues can be added at both the N- and C- termini, where the peptides are covalently bonded via controlled oxidation of the cysteine residues.
  • heterobifunctional agents that generate a disulfide link at one functional group end and a peptide link at the other, including N-succidimidyl-3-(2-pyridyl-dithio) proprionate (SPDP).
  • This reagent creates a disulfide linkage between itself and a cysteine residue in one protein and an amide linkage through the amino on a lysine or other free amino group in the other.
  • disulfide/amide forming agents are known.
  • Other bifunctional coupling agents form a thioether rather than a disulfide linkage, many of which include reactive esters of 6-maleimidocaproic acid, 2 bromoacetic acid, 2-iodoacetic acid, 4-(N-maleimido- methyl)cyclohexane-l-carboxylic acid and the like.
  • the carboxyl groups can be activated by combining them with succinimide or l-hydroxy-2-nitro-4-sulfonic acid, sodium salt.
  • SMCC succinimidyl-4-(n- maleimidomethyl)cyclohexane-l-carboxylate
  • SMCC succinimidyl-4-(n- maleimidomethyl)cyclohexane-l-carboxylate
  • a suitable linkage does not substantially interfere with either of the linked groups to function as described, e.g. as an HLA-B2705 ligand or a cytotoxic T cell determinant/stimulant.
  • the ratio of HLA-B2705 molecule to peptide may conveniently be 1:1, as occurs for example, when a single HLA-B2705 molecule accommodates one peptide in its binding groove. Other ratios are also envisioned, for example when an oligopeptide is associated with a single HLA-B2705 molecule.
  • Multimeric protein complexes comprising HLA-B2705 molecules
  • the invention utilizes a stable multimeric complex composed of a plurality of soluble HLA-B2705 protein molecules and a plurality of HLA-B2705 peptide ligands.
  • the complex may be constructed in various forms, differing for example, with respect to the multiplicity of the HLA-B2705 protein molecules, the moiety which serves to oligomerize the HLA-B2705 protein molecules into a multimeric complex, and the composition of the HLA-B2705 protein molecules, which may differ, for example, by the inclusion or exclusion of the ⁇ 2-microglobulin subunit.
  • Such varying forms and techniques for their preparation are described, for example, in US 5,635,363; Appel et al. (2004) Arthritis Res. 6:R521-534; US 6,248,564; US 7,202,349 and WO 99/58557.
  • the multimeric complex is constructed from a subunit corresponding to a mutant soluble form of the HLA-B2705 alpha chain from which the transmembrane domain is substantially deleted.
  • both the cytoplasmic and transmembrane domains are completely deleted, although a short portion of the transmembrane domain, for example, not more than about 5 amino acids, can be included.
  • Truncation of the HLA- B2705 alpha chain protein may be accomplished by proteolytic cleavage of a full length polypeptide, or by expression of a genetically engineered truncated form.
  • the transmembrane domain of the native HLA-B2705 molecule is encoded by exon 5 of the corresponding gene.
  • the soluble form of the mutant HLA- B2705 alpha chain preferably includes the complete ⁇ l, ⁇ 2 and ⁇ 3 domains.
  • the aforementioned deletion may extend as much as about 10 amino acids into the ⁇ 3 domain, but preferably none of the amino acids of the ⁇ 3 domain will be deleted. The deletion will be such that it does not interfere with the ability of the ⁇ 3 domain to fold into a disulfide bonded structure, or with the ability of the ⁇ l and ⁇ 2 domain to form the binding groove. Alignment of amino acids into domains may be determined according to Kabat et al. (Kabat et al. (1991) Sequences of Proteins of Immunological Interest).
  • the subunits of the multimeric complex may optionally include the non-variant ⁇ 2-microglobulin subunit of HLA class I molecules.
  • ⁇ 2-microglobulin may be used in its native form since it naturally lacks a transmembrane domain.
  • the ⁇ 3 domain of the alpha chain and the ⁇ 2-microglobulin do not form part of the HLA class I binding groove, but they are postulated to be important for stabilizing the overall structure of HLA class I molecules. Accordingly, it may be preferable that the subunits retain these components.
  • DNA sequences may be introduced at the termini of the DNA sequences encoding the truncated HLA-B2705 alpha chain and/or ⁇ 2-microglobulin subunit. Such insertions may be used according to the needs of the construction, providing for convenient restriction sites, addition of processing signals, ease of manipulation, improvement in levels of expression, or the like.
  • the DNA sequence encoding the HLA-B2705 alpha chain may further include a sequence which encodes a domain which promotes secretion of the product in soluble form.
  • An example of such a domain is the murine MHC class I variant QlO, as described in Kuipers et al (Kuipers et al. (2002) Clin. Exp. Rheumatol. 20(4):455-62).
  • the polypeptides may be separately produced and allowed to associate in vitro to form a stable heteroduplex complex, or both polypeptides may be expressed in a single cell.
  • a single molecule having both the truncated HLA-B2705 alpha chain and the ⁇ 2-microglobulin subunit i.e. a heterodimer, may be created by as a fusion of the two subunits joined by a peptide linker, e.g. 15 to 25 amino acids in length.
  • the soluble heterodimer may be produced by isolation of a native heterodimer and cleavage with an appropriate protease, e.g. papain, to produce a soluble product.
  • appropriate DNA sequences are respectively inserted into appropriate expression vectors, each employing either the native transcriptional initiation region or an exogenous transcriptional initiation region, i.e. a promoter other than the promoter which is associated with the gene in the normally occurring chromosome.
  • the promoter may be introduced by recombinant methods in vitro, or as the result of homologous integration of the sequence into a chromosome.
  • transcriptional initiation regions are known for a wide variety of expression hosts, where the expression hosts may involve prokaryotes or eukaryotes, particularly E. coli,
  • B. subtilis mammalian cells, such as CHO cells, COS cells, monkey kidney cells, lymphoid cells, particularly human cell lines, and the like. Generally a selectable marker operative in the expression host will be present.
  • Expression cassettes may be used for expression.
  • An example of such a cassette comprises a transcription initiation region, the DNA sequence encoding the truncatedHLA-B2705 alpha chain, and a transcriptional termination region, optionally having a signal for attachment of a poly A sequence.
  • Suitable restriction sites may be engineered into the termini of the HLA-B2705 alpha subunit, such that different subunits may be put into the cassette for expression. Restriction sites may be engineered by various means, e.g. via polymerase chain reaction or site directed mutagenesis.
  • HLA-B2705 subunits Following expression of the HLA-B2705 subunits in a suitable host cell, they are refolded in vitro.
  • the HLA-B2705 subunit is composed of a truncated alpha chain and ⁇ 2-microglobulin, the two polypeptides are provided together in the folding reaction, so as to form a stable heterodimer complex with intrachain disulfide bonds.
  • the peptide of interest may be included in the initial folding reaction, or may be added to the folded subunit in a later step, particularly if the peptide is found to cause aggregation during the folding procedure.
  • Conditions that permit folding and association of the subunits and peptide are known in the art (see for example, Altaian et al. (1993) Proc. Natl. Acad. Sci. 90:10330-10334, and Garboczi et al. (1992) Proc. Natl. Acad. Sci. 89(8):3429-3433).
  • the HLA-B2705 protein molecules produced as described above constitute the subunits of the multimeric protein complex of the invention, and are multimerized. It is possible to form the multimer by binding the subunits to a multivalent entity (i.e. an oligomerization agent) through specific attachment sites on the truncated HLA-B2705 alpha chain or the ⁇ 2-microglobulin.
  • the multimer may also be formed by chemical cross-linking of the subunits.
  • a number of reagents capable of cross-linking proteins are known in the art, including for example, azidobenzoyl hydrazide, N-[4-(p-azidosalicylamino)butyl]-3'-[2'-pyridyldithio]propionamide), bis- sulfosuccinimidyl suberate, dimethyladipimidate, disuccinimidyltartrate, N-. gamma.
  • An attachment site for binding to a multivalent entity may be naturally occurring, or may be introduced through genetic engineering.
  • the site will be a specific binding pair member or one that is modified to provide a specific binding pair member, where the complementary pair has a multiplicity of specific binding sites.
  • Binding to the complementary binding member can be mediated by a chemical reaction, epitope- receptor binding or hapten-receptor binding where a hapten is linked to the subunit chain.
  • one of the subunits is fused to an amino acid sequence providing a recognition site for a modifying enzyme.
  • the recognition sequence will usually be fused proximal to the C-terminus of one of the HLA-B2705 subunits to avoid potential hindrance at the peptide binding site.
  • an expression cassette will include the sequence encoding the recognition site.
  • Modifying enzymes of interest include BirA, various glycosylases, farnesyl protein transferase, protein kinases and the like.
  • the subunit may be reacted with the modifying enzyme at any convenient time, usually after its formation.
  • the group introduced by the modifying enzyme e.g. biotin, sugar, phosphate, farnesyl, etc. provides a complementary binding pair member, or a unique site for further modification, such as chemical cross-linking, biotinylation, etc. that -will provide a complementary binding pair member.
  • One method of multimer formation involves the introduction onto the monomer of the recognition sequence for the enzyme BirA, which catalyzes biotinylation of the protein substrate, following the enzymatic reaction, the biotinylated monomer is bound to streptavidin or avidin, to which biotin binds with extremely high affinity. Streptavidin has a valency of 4, providing a tetramer. Other methods may be used to produce the subunits of the multimeric complex, which involve incorporation of different oligomerization agents into the subunits.
  • the subunit may be a fusion protein in which the extracellular domain of the HLA-B2705 alpha chain is fused to immunoglobulin Fab and Fc constant regions or portions thereof; dimerization of subunits via disulfide bonding between the Fc constant regions enables formation of a divalent antibody-like molecule bearing two HLA- B2705 binding domains.
  • additional immunoglobulin regions e.g. hinge regions, IgM constant regions, J-chain protein and combinations thereof, enables formation of multimeric complexes of various valencies and geometries including decavalent pentamers. Exemplary methods and multimeric structures are disclosed for example, in US 2005/0003431 with respect to HLA class II multimers.
  • An alternative strategy is to introduce an unpaired cysteine residue to the subunit, thereby introducing a unique and chemically reactive site for binding an oligomerizing agent.
  • the attachment site may also be a naturally occurring or introduced epitope, where the multivalent binding partner will be an antibody, e.g. IgG, IgM, etc.
  • the cysteine may be introduced into the ⁇ 2-microglobulin, as described in US 6,248,564.
  • the multivalent complex may be free in solution, or may be attached to an insoluble support.
  • suitable insoluble supports include beads, e.g. magnetic beads, membranes and microtiter plates. These are typically made of glass, plastic (e.g. polystyrene), polysaccharides, nylon or nitrocellulose. Attachment to an insoluble support is useful when the complex is to be used for separation of T cells.
  • the multimeric complex may be labeled, so as to be directly detectable, or to be used in conjunction with secondary labeled immunoreagents which will specifically bind the complex.
  • the label will have a light detectable characteristic.
  • Preferred labels are fluorophors, such as fluorescein isothiocyanate (FITC), rhodamine, Texas Red, phycoerythrin and allophycocyanin.
  • Other labels of interest may include dyes, enzymes, chemiluminescers, particles, radioisotopes, or other directly or indirectly detectable agents.
  • the multimeric complex will have the labeling group.
  • a second stage label may be used, e.g. labeled antibody directed to one of the peptide constituents, and the like.
  • Modified HLA-B2705 binding peptides The invention further encompasses use of HLA-B2705 binding peptides which comprise a modification, on the condition that such modified peptide(s) retain the HLA- B2705 and T cell receptor binding properties attributed to the HLA-B2705 binding peptides disclosed herein.
  • modified peptides may be considered to be functional variants of the HLA-B2705 binding peptides.
  • a "functional variant" of an HLA-B2705 binding peptide is a peptide which contains one or more modifications to the primary amino acid sequence of an HLA-B2705 binding peptide and retains its HLA-B2705 and T cell receptor binding properties.
  • Modifications are desired, for example, to 1) enhance a property of the peptide, such as increased stability within the multimeric complex and/or more efficient presentation to T cells; 2) provide a novel activity or property to the peptide, such as addition of an antigenic epitope or addition of a detectable moiety; 3) provide a different amino acid sequence that produces the same or similar T cell stimulatory properties, or 4) improve the pharmacological characteristics of the multimeric complex.
  • Modifications to HLA-B2705 binding peptides can be made to nucleic acids which encode the peptides, and can include deletions, point mutations, truncations, amino acid substitutions and additions of amino acids.
  • modifications can be made directly to the peptide, such as by glycosylation, side chain oxidation, phosphorylation, cleavage, addition of a linker molecule, addition of a detectable moiety, such as biotin, addition of a fatty acid, amino acid substitution and the like.
  • Variants also can be selected from libraries of peptides, which can be random peptides or peptides based on the sequence of the HLA-B2705 peptides including substitutions at one or more positions.
  • a peptide library can be used in competition assays with soluble multimeric protein complexes of the invention.
  • Peptides which compete for binding of the HLA-B2705 peptide to the HLA-B2705 molecules on the complex can be sequenced and used in other assays (e.g., CD8 lymphocyte proliferation) to determine suitability as HLA-B2705 peptide functional variants.
  • Amino acid substitution to produce HLA-B2705 peptide functional variants preferably involves conservative amino acid substitution i.e., replacing one amino acid residue with another that is biologically and/or chemically similar, e.g., a hydrophobic residue for another, or a polar residue for another.
  • conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) Met, He, Leu, VaI; (b) Phe, Tyr, Trp; (c) Lys, Arg, His; (d) Ala, GIy; (e) Ser, Thr; (f) GIn, Asn; and (g) GIu, Asp.
  • the peptides of the present invention can be prepared by any suitable means, such as synthetically using standard peptide synthesis chemistry or by using recombinant DNA technology.
  • the HLA-B2705 binding peptides may be modified so as to enhance their CTL inducing activity, such that the modified peptides have CTL inducing activity greater than a peptide of the wild-type sequence.
  • peptide modifications may serve to increase their binding affinity to HLA-B2705, and thus increase the clinical efficacy of the multimeric complex described herein.
  • any substitutions, additions or deletions between epitopic and/or conformationally important residues will employ amino acids or moieties chosen to avoid stearic and charge interference that might disrupt binding.
  • Modifications also encompass fusion proteins comprising all or part of a HLA-
  • B2705 binding peptide amino acid sequence with a related or unrelated protein or polypeptide For example, a fusion protein comprising an HLA-B2705 binding peptide and an endosomal targeting signal can be constructed to increase the efficiency of targeting of the peptide to the HLA class I peptide presentation pathway.
  • Additional amino acids can be added to the termini of an HLA-B2705 binding peptide to provide for ease of linking peptides one to another, or for coupling the peptide to the HLA-B2705 molecules of the multimeric complex. Addition of amino acids may also be used for modifying the physical or chemical properties of the peptide and/or multimeric complex. Suitable amino acids, such as tyrosine, cysteine, lysine, glutamic or aspartic acid, can be introduced at the C- or N-terminus of the peptide.
  • the peptide of the present invention can differ from the natural sequence by being modified for example, by N-terminal acylation, N-terminal amidation, or C- terminal amidation. In some instances these modifications may provide sites for linking to a support or other molecule, thereby providing a linker function.
  • Modifications also encompass HLA-B2705 binding peptides having one or more non-natural amino acids, introduced so as to render HLA-B2705 binding peptides non- hydrolyzable or less susceptible to hydrolysis.
  • HLA-B2705 binding peptides may select HLA-B2705 binding peptides from a library of non-hydrolyzable peptides, such as peptides containing one or more D-amino acids or peptides containing one or more non- hydrolyzable peptide bonds linking amino acids.
  • a library of non-hydrolyzable peptides such as peptides containing one or more D-amino acids or peptides containing one or more non- hydrolyzable peptide bonds linking amino acids.
  • peptides may be labeled and incubated with cell extracts or purified proteases and then isolated to determine which peptide bonds are susceptible to proteolysis.
  • potentially susceptible peptide bonds can be identified by comparing the amino acid sequence of an HLA-B2705 binding peptide with the known cleavage site specificity of a panel of proteases. Based on the results of such assays, individual peptide bonds which are susceptible to proteolysis can be replaced with non-hydrolyzable peptide bonds by in vitro synthesis of the peptide.
  • Non-hydrolyzable peptide bonds are known in the art, along with procedures for synthesis of peptides containing such bonds.
  • Non-hydrolyzable bonds include, but are not limited to, -psi[CH 2 NH] ⁇ reduced amide peptide bonds, -psi[COCH 2 ] ⁇ ketomethylene peptide bonds, -psi [CH(CN)NH]-- (cyanomethylene)amino peptide bonds, -psi [CHiCH(OH)] hydroxyethylene peptide bonds, -psi [CH 2 O]-- peptide bonds, and -psi[CH 2 S] ⁇ thiomethylene peptide bonds.
  • Modifications also encompass peptides that have conjugated thereto a substance, such as a radioactive moiety, an enzyme, a fluorescent moiety, a solid matrix, a carrier, and a CTL epitope.
  • a substance such as a radioactive moiety, an enzyme, a fluorescent moiety, a solid matrix, a carrier, and a CTL epitope.
  • the substance can be conjugated to the peptide at any suitable position, including the N and C termini and points in between, depending on the availability of appropriate reactive groups in the side chains of the constituent amino acids of the peptide of interest. Additionally, the substance can be conjugated directly to the peptide or indirectly by way of a linker.
  • Radiolabels include 3 H, 14 C, 32 P, 35 S, 125 I, and other suitable radioactive moieties for use in various radioimmunoassays and the like.
  • Fluorescent moieties include fluorescein, rhodamine, and other suitable fluorescent labels for use in fluorescent assays and the like.
  • Enzymes include alkaline phosphatase, Bir A and other suitable enzymes useful for any suitable purpose, including as a marker in an assay procedure.
  • Preferred solid matrices are glass, plastic, or other suitable surfaces, including various resins such as Sephadex ® , chromatography media and the like.
  • Carriers include immunogenic lipids, proteins, and other suitable compounds, such as a liposome or bovine serum albumin.
  • the invention further encompasses use of mimetic analogs i.e. non-peptide analogs, for example, to provide a stabilized structure or one less susceptible to biodegradation.
  • Mimetic analogs can be prepared based on a selected HLA-B2705 binding peptide by replacement of one or more residues by nonpeptide moieties.
  • the nonpeptide moieties permit the peptide to retain its natural conformation, or stabilize a preferred, e.g., bioactive, confirmation.
  • Such peptides can be tested in molecular or cell-based binding assays to assess the effect of the substitution(s) on conformation and/or activity.
  • One example of methods for preparation of nonpeptide mimetic analogs from peptides is described in Nachman et al. (1995) Regul. Pept. 57:359-370.
  • the peptides of the invention can be prepared using any suitable means. Because of their relatively short size (generally less than 20 amino acids), the peptides can be synthesized in solution or on a solid support in accordance with conventional peptide synthesis techniques. Various automatic synthesizers are commercially available (for example, from Applied Biosystems) and can be used in accordance with known protocols (see, for example, Stewart and Young (1984) Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co.; Tarn et al. (1983) J. Am. Chem. Soc. 105:6442;
  • recombinant DNA technology may be employed, wherein a nucleotide sequence that encodes a peptide of interest is inserted into an expression vector, transformed or transfected into a suitable host cell and cultivated under conditions suitable for expression.
  • a nucleotide sequence that encodes a peptide of interest is inserted into an expression vector, transformed or transfected into a suitable host cell and cultivated under conditions suitable for expression.
  • These procedures are generally known in the art, as described generally in Sambrook et al. (2001) Molecular Cloning, A Laboratory Manual 3rd ed. Cold Spring Harbor Press, Cold Spring Harbor, N. Y., and Ausubel et al., eds. (1987) Current Protocols in Molecular Biology John Wiley and Sons, Inc., N. Y., and U.S. Patent Nos. 4,237,224, 4,273,875, 4,431,739, 4,363,877 and 4,428,941.
  • the coding sequence for peptides of the length contemplated herein can be synthesized by chemical techniques, for example, the phosphotriester method of Matteucci et al., J. Am. Chem. Soc, 103, 3185 (1981), modification can be made simply by substituting the appropriate base(s) for those encoding the native peptide sequence.
  • the coding sequence can then be provided with appropriate linkers and ligated into expression vectors commonly available in the art, and the vectors used to transform suitable hosts to produce the desired fusion protein.
  • the coding sequence will be provided with operably linked start and stop codons, promoter and terminator regions and usually a replication system to provide an expression vector for expression in a suitable cellular host.
  • promoter sequences compatible with bacterial hosts are provided in plasmids containing convenient restriction sites for insertion of the desired coding sequence.
  • the resulting expression vectors are transformed into suitable bacterial hosts.
  • Yeast or mammalian cell hosts may also be used, employing suitable vectors and control sequences.
  • peptides produced by recombinant methods as a fusion to proteins that are one of a specific binding pair enable purification of the fusion protein by means of affinity reagents, followed by proteolytic cleavage, usually at an engineered site to yield the desired peptide (see for example Driscoll et al. (1993) J. MoI. Bio. 232:342- 350).
  • the peptides may also be isolated from natural sources and purified by known techniques, including, for example, chromatography on ion exchange materials, separation by size, immunoaffinity chromatography and electrophoresis.
  • Uses of the multimeric protein complex The present invention provides for the production of multimeric complexes of soluble HLA-B2705 proteins in association with specific HLA-B2705 ligands, for which no counterparts had previously existed. These multimeric complexes may be contacted with T cells in solution, in vivo or in vitro, to identify, isolate, detect, separate, enrich, tolerize, anergize, down regulate, stimulate or activate T cells which are reactive to the defined HLA-B2705-peptide complex.
  • the multivalency of the complexes is particularly advantageous for increasing the efficiency of binding to T cells.
  • the multimeric complexes may be used to identify specifically reactive T cell populations in patients suffering from various autoimmune disorders including spondyloarthropathies. Such analyses are conveniently conducted as binding assays, using a panel or array of multimeric complexes, wherein each complex is loaded with a different HLA-B2705 presented peptide. Following identification of specifically reactive T cell subsets, and the peptides inducing the reactivity, the multimeric complexes loaded with the specific peptide(s) of interest may be used in the preparation of a therapeutic composition having activity in down regulating the reactive T cells.
  • Such therapeutic compositions are envisioned for in vivo, ex vivo or in vitro use. For example, they may be administered to patients, or, patients' cells may be withdrawn, treated with the compositions, and returned to the patient or used for other purposes.
  • T cells from a physiologic sample, or from an in vitro culture of patient's cells are used in binding assays.
  • the physiologic sample is usually blood or lymph fluid, but may be another source such as brain, spinal fluid, lymph node, neoplasms, spleen, liver, kidney, pancreas, tonsil, thymus, joints, synovia, and the like.
  • the sample may be used as obtained, or may be treated, such as by dilution, concentration, removal of unwanted cells, for example by centrifugation, Ficoll-Hypaque absorption, panning or affinity separation using antibodies specific for cell surface markers.
  • the multimeric complex is added to a suspension comprising T cells of interest, and incubated under conditions which enable binding to the cognate T cell receptor(s) on the target population.
  • a labeling reagent is included in the reaction mixture.
  • Appropriate conditions of the binding asay, for example, time, temperature, medium, labeling reagents, washing conditions, and methods of detecting and quantifying the binding may be readily determined by one of average skill in the art.
  • the cell suspension may be washed prior to incubation with the second stage reagent.
  • the second stage reagent may be added directly into the reaction mix.
  • Flow cyometry may also be used for the separation of a labeled subset of T cells from a complex mixture of cells.
  • Alternative means of separation utilize the multimeric complex bound directly or indirectly to an insoluble support, e.g. column, microtiter plate, magnetic beads, etc.
  • the cell sample is added to the binding complex, the latter of which is bound to the support by any convenient means. After incubation, the insoluble support is washed to remove non-bound components. The desired cells are then eluted from the binding complex.
  • an insoluble support e.g. column, microtiter plate, magnetic beads, etc.
  • the cell sample is added to the binding complex, the latter of which is bound to the support by any convenient means. After incubation, the insoluble support is washed to remove non-bound components.
  • the desired cells are then eluted from the binding complex.
  • magnetic particles to separate cell subsets from complex mixtures is described in Miltenyi et al. (1990) Cytometry 11 :23
  • Detecting and/or quantitating specific T cells in a sample or fraction thereof may be accomplished by a variety of specific assays.
  • the assay will measure the binding between the sample and the subject multimeric binding complexes, for example, a sandwich assay is performed by first attaching the multimeric complex to an insoluble surface or support.
  • the multimeric complex may be bound to the surface by any convenient means, depending upon the nature of the surface, either directly or through specific antibodies. They may be bound to the plates covalently or non- covalently.
  • suitable insoluble supports include beads, membranes and microtiter plates, typically made of glass, plastic, polysaccharides, nylon or nitrocellulose. Microtiter plates are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples.
  • the non-specific binding sites on the insoluble support i.e. those not occupied by the multimeric binding complex, are generally blocked with non-interfering proteins such as bovine serum albumin, casein, gelatin, and the like.
  • Samples, fractions or aliquots thereof are then added to separately assayable supports (for example, separate wells of a microtiter plate) containing support-bound multimeric complex.
  • each sample and standard will be added to multiple wells so that mean values can be obtained for each.
  • the insoluble support is generally washed of non-bound components and a solution containing specific second receptor is applied.
  • the receptor may be any compound that binds patient T cells with sufficient specificity such that they can be distinguished from other components present.
  • Such second receptors are usually antibodies (monoclonal or polyclonal) specific for common T cell antigens.
  • T cell specific antibodies may be labeled to facilitate direct or indirect quantification of binding.
  • labels that permit direct measurement include radiolabels, such as 3 H or 125 I, fluorescers, dyes, beads, chemilumninescers, colloidal particles, and the like.
  • labels which permit indirect measurement of binding include enzymes where the substrate may provide for a colored or fluorescent product.
  • suitable enzymes for use in conjugates include horseradish peroxidase, alkaline phosphatase, malate dehydrogenase and the like. Where not commercially available, such antibody-enzyme conjugates are readily produced by techniques known to those skilled in the art.
  • the second receptor may be unlabeled, hi this case, a labeled second receptor-specific compound is employed which binds to the bound second receptor.
  • a labeled second receptor-specific compound can be labeled, in any manner as described above. It is possible to select such compounds such that multiple compounds bind each molecule of bound second receptor. Examples of second receptor/second receptor-specific molecule pairs include antibody/anti-antibody and avidin (or streptavidin)/biotin. Since the resultant signal is thus amplified, this technique may be advantageous where only a small number of cells are present. An example is the use of a labeled antibody specific to the second receptor.
  • the second receptor is a rabbit anti-allotypic antibody
  • an antibody directed against the constant region of rabbit antibodies provides a suitable second receptor specific molecule.
  • the antiimmunoglobulin will usually come from any source other than human, such as mouse, or bovine antibody.
  • the insoluble support is again washed free of non-specifically bound second receptor, and the signal produced by the bound conjugate is detected by conventional means.
  • an enzyme conjugate an appropriate enzyme substrate is provided so a detectable product is formed.
  • a peroxidase is the enzyme conjugate
  • a preferred substrate combination is H 2 O 2 and O-phenylenediamine which yields a colored product under appropriate reaction conditions.
  • the product may be measured by spectrophotometric light absorbance at 490-495 nm.
  • Appropriate substrates for other enzyme conjugates, reaction conditions and means of detecting reaction products are known to those skilled in the art.
  • the number of bound T cells detected will be compared to control samples e.g. T cells from an individual that does not express HLA-B2705.
  • the above methods and assays employing multimeric complexes of the invention may be used to screen a large panel of different peptides, such as the set of naturally presented HLA- B2705 ligands disclosed herein, and/or large numbers of clinical samples. Screening of large sample numbers is preferably carried out using automated machinery, as is known in the art.
  • the multimeric complexes may further be used in a method of stimulating T cells, and in the production of a vaccine, as explained below. These applications are relevant to therapy of autoimmune disorders and of cancers expressing tumor associated antigens, and fragments of which are displayed by HLA-B2705.
  • the multimeric complexes can be applied to development of diagnostic and therapeutic methods and compositions for management of spondyloarthropathies which have a high rate of association with HLA-B2705.
  • Such disorders include ankylosing spondylitis, reactive arthritis, psoriatic spondylitis, enteropathic arthritis, peripheral psoriatic arthritis, spondylitis associated with inflammatory bowel disease, acute anterior uveitis and undifferentiated spondyloarthropathy.
  • the clinical applications envisioned are based on the prior identification of T cell subsets specifically reactive against particular HLA-B2705 ligand(s) in patients, which can be carried out using the subject multimeric complexes, as described above.
  • Therapeutic strategies directed to neutralizing the problematic immune response include anergizing T cells reactive to a defined HLA-B2705-peptide complex, inducing tolerization to a particular HLA- B2705-peptide complex, and killing (also termed "ablation") reactive T cells.
  • Such strategies can utilize a therapeutic composition which comprises the multimeric complex described herein, since it is capable of specifically targeting T cells which attack a particular HLA-B2705-peptide complex. It is known in the art that high doses of an antigen may have a T cell tolerizing or anergizing effect, rather than a T cell stimulating effect.
  • a pharmaceutical composition comprising the subject multimeric complex is administered to an individual in need thereof, in a manner that will maximize the binding of the complex to the targeted T cell.
  • Such binding may induce T cell anergy or tolerization, particularly when co-stimulator molecules necessary for T cell activation are not present.
  • Induction of anergy or tolerization may be assessed in isolated T cells, following treatment with the therapeutic composition, by analysis of their ability or failure to activate, proliferate or secrete cytokines, when presented with the complex for which they are specific.
  • a therapeutic pharmaceutical comprising the subject multimeric complex conjugated to a toxin moiety
  • cytotoxic agents are known and have been used in conjunction with specific binding molecules.
  • Such toxic compounds include ricin, abrin, diphtheria toxin, maytansinoids, cisplatin, and the like.
  • the toxin contains two subunits, only the cytotoxic subunit may be used, e.g. the alpha chain of ricin.
  • the toxin is conjugated to the multimeric complex, generally by means of a cross-linker, or other conjugation that includes a disulfide bond.
  • Toxin conjugates are disclosed in U.S. Pat. Nos. 5,208,020; 4,863,726; 4,916,213; and 5,165,923. The toxin conjugate is administered so as to specifically eliminate the target T cells without exerting significant toxicity against other cells.
  • the multimeric complexes may further be used in the development of an immunostimulatory pharmaceutical composition directed against cancer cells which display tumor specific peptide ligands on HLA-B2705 molecules.
  • a composition may also be considered a "vaccine”, which refers to a composition useful for stimulating a specific immune response in a vertebrate.
  • the composition described herein explicitly includes both immunotherapeutic vaccines, i.e. a vaccine administered to treat and/or prevent further progression of a cancer in a host already diagnosed with the cancer, and prophylactic vaccines i.e. a vaccine administered to prevent or delay cancer development in a susceptible host.
  • a pharmaceutical composition for use against cancer comprises the multimeric protein complex, and a plurality of peptides selected from the group consisting of: SEQ ID NOS.: 61; 88; 97; 99; 131; 132; 197; 198; 210; 231; 254; 280; 286; 288; 290; 304; 305; 323; 337; 340; 341; 353; 363; 369; 371; 382; 389; 397; 404; 432; 444; 448; 483; 488; 499; 500; 586; 627; 652; 710; 724; 728; 754; 756; 772; 844; 845; 873; 907; 921; 951; 997; 1010; 1016; 1026; 1038; 1043; 1074; 1097; 1125; 1131; 1139; 1146; 1154; 1184; 1189; 1190; 1242; 1244; 1253; 12
  • Tumor associated antigens are tumor associated antigens, which are naturally presented in HLA-B2705 molecules.
  • Other HLA-B2705 displayed peptides which are derived from tumor associated antigens may also be used.
  • Tumor associated antigens fall into different classifications and include, but are not limited to oncogene products, SEREX antigens i.e. those obtained by serological identification of antigens by recombinant cDNA expression cloning, proteins overexpressed in cancer, apoptosis-related proteins and angiogenesis-related proteins.
  • Cancers of interest include, but are not limited to melanoma, lymphoma, sarcoma, leukemia, cervical cancer, hepatic cancer, renal cancer, testicular cancer, ovarian cancer and colon cancer.
  • compositions include but are not limited to vegetable oils or emulsions thereof, surface active substances, e.g., hexadecylamin, octadecyl amino acid esters, octadecylamine, lysolecithin, dimethyl-dioctadecylammonium bromide, N,N-dicoctadecyl-N'-N'bis (2-hydroxyethyl-propane diamine), methoxyhexadecylglycerol, and pluronic polyols; polyamines, e.g., pyran, dextransulfate, poly IC, carbopol; peptides, e.g., muramyl dipeptide, dimethylglycine, tuftsin; immune stimulating complexes; oil emulsions (including, but not limited to, oil- in-water emulsions having oil droplets in the submicron range, such as
  • the multimeric complexes of this invention can also be incorporated into liposomes, cochleates, biodegradable polymers such as poly-lactide, poly-glycolide and poly-lactide-co-glycolides, or ISCOMS (immunostimulating complexes), and supplementary active ingredients may also be employed.
  • the vaccine may also comprise the multimeric complex conjugated to the surface of antigen presenting cells or to a non-immunogeneic carrier. Peptide loaded HLA class I molecules conjugated to APCs are described, for example, in US 6,248,564.
  • Cells derived from the subject with cancer may be conjugated in vitro to the multimeric complexes comprising the tumor peptide antigens of interest.
  • the cells used are preferably APCs which can provide the requisite costimulatory signals for inducing an effective T cell response.
  • Sources of APCs include T and B cells from peripheral blood lymphocytes, macrophages/monocytes, and dendritic cells.
  • the conjugates are then introducted back into the subject.
  • Such an immunization strategy allows the control of epitope density and circumvents a variety of problems associated with classical vaccination strategies. For example, unlike traditional peptide vaccines, the peptides embedded in the subject multimeric complexes are protected from rapid enzymatic degradation or intracellular processing.
  • the vaccines can be administered to a human or animal by a variety of routes, including but not limited to parenteral, intradermal, transdermal (such as by the use of slow release polymers), intramuscular, intraperitoneal, intravenous, subcutaneous, oral and intranasal routes of administration, according to protocols well known in the art.
  • the vaccine compositions of the invention are administered in a dose which is suitable to elicit an immune response in said subject.
  • the particular dosage of the multimeric complex will depend upon the age, weight and medical condition of the subject to be treated, as well as on the identity of the antigen and the method of administration. Suitable doses will be readily determined by the skilled artisan.
  • a preferred dose for human intramuscular, subcutaneous and oral vaccination is between about 50 ⁇ g to about 100 mg, preferably between about 200 ⁇ g to about 40 mg, and more preferably between about 500 ⁇ g to about 10 mg. Adjustment and manipulation of established dosage ranges used with traditional carrier antigens for adaptation to the present vaccine is well within the ability of those skilled in the art.
  • a “therapeutically effective amount” is an amount that when administered to a patient is sufficient to inhibit, preferably to eradicate, or, in other embodiments, to prevent or delay the progression of a T cell mediated pathology.
  • a “therapeutically effective amount” further refers to an amount which, when administered to a subject, results in a substantial increase in the immune response of the subject to the administered immunogen, as described herein.
  • the multimeric complexes for parenteral administration, they are prepared in an injectable dosage form (solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle.
  • a pharmaceutically acceptable parenteral vehicle examples include water, saline, Ringer's solution, dextrose solution, and Hanks' solution.
  • Non-aqueous vehicles such as fixed oils and ethyl oleate may also be used.
  • the vehicle may contain small amounts of additives, such as substances that enhance isotonicity and chemical stability, e.g. buffers and preservatives.
  • Such vehicles are inherently non-toxic and non- therapeutic.
  • the multimeric complexes are preferably formulated in purified form substantially free of aggregates and other proteins at concentrations of about 1-50 mg/ml. Suitable pharmaceutical vehicles and their formulations are described, for example, in Remington's Pharmaceutical Sciences.
  • the invention also provides a conjugate in which a single carrier bears a multiplicity of multimeric protein complexes. Compared to the multimeric protein complex, a conjugate will have even greater avidity for T cells expressing the cognate TCRs, and accordingly, greater biological activity.
  • the multimeric complexes used to form the conjugate are as described above.
  • the carrier may be selected from a variety of particles, beads, branched polymers, dendrimers, or liposomes.
  • the carrier must be capable of being conjugated, either directly or indirectly, to a multiplicity of multimeric complexes and, therefore, preferably comprises a multiplicity of reactive groups near the surface which can be used in conjugation reactions.
  • the carrier may have a surface to which conjugating moieties may be adsorbed without chemical bond formation.
  • the carrier is particulate, and generally spherical, ellipsoidal, rod-shaped, globular, or polyhedral in shape.
  • the carrier is preferably composed of a material which is biodegradable and non-immunogenic. It is further preferred that the carrier have a net neutral or negative charge, in order to reduce non-specific binding to cell surfaces which, in general, bear a net negative charge.
  • the carrier is microscopic or nanoscopic in size, both to enhance solubility, and to avoid possible complications caused by aggregation in vivo.
  • the carrier is a bead, comprising a material selected from glass, silica, polyesters of hydroxylcarboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxylcarboxylic acids, and dicarboxylic acids
  • the carrier is a branched polymer.
  • Branched polymers are advantageous, for example over linear polymers, since they provide numerous termini which can be functionalized and conjugated to a multiplicity of multimers, either directly or indirectly through conjugating moieties.
  • the ratio of multimeric protein complex to carrier is in the range from about 5:1 to 10000:1.
  • Human chondrosarcoma cells (SW1353) were a generous gift by Dr. Lubna Nasir.
  • Human immortalized chondrocytes C-20/A4 were a generous gift by Dr. Mary Goldring.
  • HeLa, HEK 293T and chondrocyte cells were grown in DMEM with 3 to 10% FCS, O.lmg/ml streptomycin and lOOU/ml penicillin.
  • Retroviral transfection of soluble HLA-B27 Retroviral transfection of soluble HLA-B27
  • the RSV.neo B27/Q10 plasmid was a generous gift from Dr. Jane Goodall (17).
  • the B27/Q10 cDNA was excised and ligated into the pLNCX retroviral vector (BD Biosciences).
  • the pLNCX-B27Q10 was used together with pCL (RetroMax System) to produce retroviral particles in 293 T cells and transfect target cells (according to the manufacturer's instructions, Imgenex).
  • Isolated resistant clones were screened for soluble HLA-B27 (sHLA-B27) secretion using sandwich-ELISA with the monoclonal antibodies HB149 anti (b2m) and W6/32 (anti-native HLA).
  • Soluble HLA-B27 molecules secreted from the cells with their bound peptides were immunoaffinity purified from the conditioned growth medium with the W6/32 monoclonal antibody cross-linked to protein A Sepharose (Amersham), as in (18) after pre-clearing of the IgG from the FCS-containing growth medium with protein A
  • HLA peptides were labeled with stable isotopes at specific residues using iTRAQ according to the manufacturer's protocol (Applied Biosystems) and stable isotope labeling in tissue culture (SILAC) (22) as in (23) using Leu-D3 (+3 amu).
  • Peptides were selected as correct only if they contained the amino acids arginine or glutamine at their second position and their observed and calculated mass deviated by less than 0.005 amu. Confidence in sequence assignment was higher for peptides detected at multiple charge states, chemical or metabolic isotope modified states or in extended or shortened forms, with Mascot score higher than 10, Sequest score of at least 2 and Pep-Miner score of 85 and higher. When the MS/MS spectra fitted more than one peptide (45 cases) only the highest scoring peptides are listed. The different scores and verification criteria for exemplary peptides are listed in Table 1.
  • Certain peptides for refinement of the motif was selected with at least three positive MS verifications, unlikely to be tryptic (with arginine or lysine at both their C-termini and at the position preceding their first amino acid) by comparing the abundance of each amino acid at each peptide positions to its abundance in the human proteome (http://www.ebi.ac.uk).
  • Statistical significance was defined using the chi square statistical test with Yates' and with Bonferroni's corrections (the latter multiplies the p-value by 20, the number of possible amino acids at a position), as described by (24).
  • HeLa cells transfected with HLA-B27/Q10, were infected with Salmonella enterica sp. typhimurium (strains SL1344 and ATCC 14028, generous gift from Dr. Sima Yaron) transformed with the green fluorescent protein (pGFP, Clontech) and grown to at multiplicity of infection (MOI) of 30-50.
  • the cells were centrifuged for 5 min at 25Og at room temperature to enhance adherence of bacteria to cells, and incubated for one hour. Infection rate were checked by incubating the infected cells in DMEM without penicillin and streptomycin but with 50 ug/mL gentamycin for fifteen minutes to kill extracellular bacteria.
  • the cells were washed again with PBS and transferred to DMEM containing 10 ug/mL gentamycin. Infection efficacy was followed both by fluorescent microscopy and by lysing the cells and counting the surviving intracellular bacteria by plating. When efficacy of infection was over one (sometimes up to twelve) colony-forming units (CFU) per cell, growth media of infected cells containing the sHLA-B27 molecules was collected at 4-6 hr, 22-24 hr and 48-50 hr post infection.
  • CFU colony-forming units
  • HeLa B2705/Q10 cells were infected with Ureaplasma urealyticum using filtered conditioned medium of small cell lung cancer (69AR) cells infected with the mycoplasma. Infection was verified by PCR amplification of a conserved 16S rRNA region of 270bp using the EZ-PCR mycoplasma test kit (Biological Industries, Beit).
  • Sequence alignment was performed for human HLA-B27 peptides with the twelve bacteria species known to be associated with B27 dependent reactive arthritis (25) (NCBI database) i.e. Salmonella typhimurium, S. enteritidis, S. Paratyphi, Shigella flexneri, S. sonnei, S. dysenteriae, Chlamydia trachomatis, Yersinia enterocolitica, Y. pseudotuberculosis, Campylobacter jejuni, Ureaplasma urealyticum and Clostridium difficile.
  • the bacterial derived HLA-B27 peptides were aligned also with all the other arthritogenic bacteria and the human sequences using the BlastP program.
  • HLA-B27 peptidome Soluble HLA-B27 preparations, collected from about 10 s cells, yielded sufficient amounts of peptides for multiple analyses by ⁇ LC-MS/MS.
  • HLA-B27 peptides derived from human, Salmonella and Ureaplasma proteins were identified (selected examples in Table 1; complete list in Sequence Listing). Many of the peptides were detected as multiple forms; unlabeled and isotopically or chemically labeled (165 peptides), natural length variants (87 peptides) and different charge-state (180 peptides).
  • the peptides are divided into three groups according to their level of identification confidence.
  • the Certain subset consists of peptides (about 500 in number) with three or four MS verifications on top of the Pep-Miner score; the Probable subset includes peptides (about 550 in number) with one or two such verifications, and the Possible subset consists of peptides (about 150 in number) that conform to the B27 motif with no added verifications except for the Pep-Miner score.
  • the Possible group is included due to the importance of the B2707 allele.
  • some peptides are possibly related to cancer or apoptosis (Table 6).
  • ARLEKVHSLF 1 86 1.48 19 Ch 7661952 - T cell carcinoma Ag3
  • a Cancer and apoptosis related peptides are underlined; peptides used for motif calculations are in bold.
  • b Detected variants of the same sequence: iT — iTRAQ labeled variant, L3 — heavy leucine labeled variant, Ch - Charge variant, Lg - length variant.
  • the HLA-B27 motif is typed in bold in Table 2.
  • the motif analyses were preformed for the three main peptide lengths (9, 10 and 11 amino acids (aa)) of this subset (Table 2).
  • the four N-terminal and five C-terminal amino acids (4N+5C) were used as separate parts of the motif, while allowing larger divergence at the middle positions.
  • the middle amino acids overrepresented significantly in longer peptides (P 5+6 ), were aspartate and glutamate, also typical in P 4 .
  • An example for the 4N+5C motif is shown for lysine (Figure 2) that is significantly over-represented at fixed distances from both ends of the peptides, regardless of their lengths.
  • Another characteristic of longer peptides is their tendency to have arginine in their N termini. This was statistically significant only in 11-mers and 12-mers and not in shorter peptides, possibly due to the N-terminal arginine stabilizing effect (28).
  • Figure 1 depicts a histogram of the distribution of the different length groups in both the Certain and Probable peptide lists. Even though most peptides fit the canonical consensus length of 8-10 aa, seven heptamers and 191 longer peptides (11-15-mers) were also observed. As many as 79 peptides were detected at more than one length, adding one to four amino acids to one of the termini. Most variations were at the C- termini and less at the N-termini ( Figure 3, same as HLA-A2 (18)). This finding supports either a higher efficiency in the N terminal trimming process or stronger constraints imposed by the N-terminus binding motif of the B27.
  • HLA-B2705 peptidomes of the two cultures of chondrocytic cell lines (SW1353 chondrosarcoma and C-20/A4 immortalized chondrocytes) and the HeLa cells serving as arthritogenic peptide candidates (Table 3).
  • aThe upper peptide marked in bold is the detected human peptide, while the bottom sequence was found by alignment with the proteome of arthritogenic bacteria.
  • HLA-B2705 peptides derived from Salmonella typhimurium and Ureaplasma urealyticum were identified from the HeLa cells expressing the soluble HLA-B2705 following infection with bacteria.
  • Table 5 lists six identified HLA-B2705 binding Salmonella typhimurium peptides and three from Ureaplasma, all from the Probable peptide subset. Some of these bacterial antigens are also shared with other arthritogenic bacteria, as shown in Table 5.
  • Some of the Salmonella and Ureaplasma HLA-B2705 peptides identified here are homologous to human protein sequences.
  • peptide KRHFPSLSI (SEQ ID NO: 1062) is homologous to the human sequence FRLFPSLSI from the Calcium-activated potassium channel (KCNTl), changed only in P 1+3 .
  • Table 5 S. Typhimurium and U. urealyticum peptides presented on HLA-B27.
  • HLA-B27 peptides as cancer markers
  • the HLA-B2705 peptidome obtained from cancer cells was evaluated using SW1353 (sarcoma) and HeLa (carcinoma) cell lines, each transfected with a solube version of HLA-B2705.
  • HLA-B2705 Peptides originating from tumor associated proteins, that are not expected to be presented by healthy cells, could become vaccine candidates for HLA-B2705 positive cancer patients.
  • a large number of novel peptide ligands of HLA-B2705 were identified and may be grouped into different antigen groups, including those derived from testis tumor, oncogenes, tumor suppressors, SEREX antigens and other cancer related proteins, as detailed in Table 6. Table 6. HLA-B2705 peptide ligands according to cancer-related antigen groups.
  • the large HLA-B2705 peptidome established here by direct biochemical analysis, contains about 1200 naturally processed peptides. It is by far the largest to- date, thus facilitating better statistical definition of the HLA-B2705 binding motif and serving as a valuable source for selection of candidate peptides for immunotherapy.
  • the SYPPEITHI database (26) includes already close to 200 HLA-B*2705 peptides. Our dataset enlarges the HLA-B2705 peptidome by more than seven fold.
  • the validity of the identifications described here is relatively high due to the use of high accuracy mass spectrometry, data clustering of multiple MS/MS spectra belonging to individual peptides by Pep-Miner (20), identification with different search engines, validation with MS/MS of different charge states, rugged-end peptides, and the presence of the SILAC and iTRAQ labels.
  • the isotope and chemical labeling resolved sequence ambiguities by helping to differentiate between the isobaric amino acids such leucine/isoleucine (using the heavy Leu).
  • the iTRAQ labeling improved the quality of the MS/MS data, probably by enhancing the mobile proton effect, and by the mass added to the amino termini, which increased the number of b-series MS/MS fragments detected by the ion-traps.
  • the HLA-B2705 consensus binding motif defined here with the Certain peptides subset refines the existing other three motifs (21, 24, 26) (summarized in Table 2).
  • Our motif favors the negative amino acids in the middle positions (P 4 in nonamers, P 4+5 in decamers, P 4+6 in dodecamers), and the positive arginine to be statistically under- represented in these positions.
  • HLA-B2705 peptides from cartilage/bone related proteins are the targets of the autoimmune T cells, such peptides are possibly present among the HLA-B2705 peptidome of chondrocyte cells.
  • Such peptides can be identified by direct biochemical analysis of the peptidome of cartilage cells, as was done here. Identification of HLA peptides directly from the affected tissues is still a demanding task that should be attempted in the future.
  • HLA-B2705 peptides previously identified biochemically were obtained from human transformed B-lymphoid cells (24). Most significantly, many of the published cartilage specific HLA peptides that were identified by software prediction and by T cells based assays (4, 35) were not detected in this study. Furthermore, the peptides recovered from soluble HLA are likely to be high affinity binders since they were recovered from the sHLA molecules after being bound to it for hours in the conditioned medium and throughout the affinity purification. High affinity peptides are prime candidates for eliciting T cell responses. Twenty four identified peptides are derived from cartilage or bone related proteins.
  • FRFAVPTKF (SEQ ID NO: 709) derived from Osteoprotegerin, a regulator of bone resorption and osteoclast development, possibly an important target for pharmacological intervention in arthritis (36), as well as SRSEVDMLK (SEQ ID NO: 534) and ARDL YDAGVKR (SEQ ID NO: 274) from Annexin II which functions in pathological mineralization of articular cartilage (reviewed in (37)).
  • SRSEVDMLK SEQ ID NO: 53
  • ARDL YDAGVKR SEQ ID NO: 274
  • three peptides were identified from the ADAM family, chondrocyte derived metalloproteinase enzymes, involved in joint pathology (38).
  • Another interesting peptide is FRLDTPLYF (SEQ ID NO: 1077) from the proteoglycan Leprecan.
  • Proteoglycans are the primary building blocks of the extracellular matrix of articular cartilage. Proteoglycans are thought to be important contributors to the inflammation in ankylosing spondylitis (reviewed in (39)) and the Leprecan gene expression was shown to be different in osteoarthritic (OA) cartilage. Additionally, three peptides from Collagen type VI were observed. The potential of this collagen to contribute many different HLA-B2705 ligands was already noted by predictions (35). Nonamer peptides were tested by Atagunduz et al.
  • DRASFIKNL from Collagen VI was found to be immunogenic but was not observed in this study as a natural HLA ligand.
  • Collagen type VI is found in substantial amounts in the extracellular matrix of the human joint and intervertebral disc.
  • Antibodies against it were found in AS patients' and not in controls' blood, and were shown to cross-react with an antigen from Klebsiella pneumonia (40).
  • Another peptide was identified from Bone morphogenic protein (BMP) receptors. Overexpression of BMPs (and TGF ⁇ ) was already suggested to induce pathological ossification, or promote disc degeneration (41). These are all HLA-B2705 antigens presented on chondrocytic cells and potentially involved in cartilage pathology.
  • Human HLA-B2705 antigens homologous to arthritogenic bacteria include three peptides related to cartilage/bone which share 6-8 aa with a sequence from arthritogenic bacteria. Most significant among these is the natural peptide RRGLLA WISR (SEQ ID NO: 203) from chondroitin sulfate GaI-NAcT- 1, a protein which plays a critical role in chondroitin sulfate biosynthesis in cartilage. It shares 8 aa with a FhuB of Y. enterocolitica and Y. pseudotuberculosis sequence, and 7aa with IgaA of S. typhimurium and S. paratyphi.
  • RRGLLA WISR SEQ ID NO: 203
  • chondroitin sulfate GaI-NAcT- 1 a protein which plays a critical role in chondroitin sulfate biosynthesis in cartilage. It shares 8 aa with a FhuB of Y. enter
  • peptides not derived from cartilage/bone proteins, have significant homologies to bacterial sequences. They include three peptides perfectly matching protein sequences from arthritogenic bacteria and other peptides with one conservative or non-conservative amino acid difference in their sequence relative to the homologous bacterial peptide. Some of the peptides were homologous to a sequence common to many of the arthritogenic bacteria. For example, a change in P 1 in the human peptide SRHPFPGPGL (SEQ ID NO: 241) observed here yields the sequence YRHPFPGPGL common to eight of the arthritogenic bacteria species.
  • HLA-B2705 peptides were observed. Such peptides were also proposed as inducing factors of SpA.
  • the peptide RRYLENGKETL detected here was previously tested (42) and reported not to activate patients' T cells.
  • the very similar HLA-B2705 peptide LRRYLENGK did activate, the overlapping peptide found by us RRYLENGKET should be tested for its immunogenicity, as some CTLs are known to distinguish between very similar natural ligands on B2705 (43).
  • the HLA-B2705 self-peptide RRKSSGGKGGSY suggested to be mimetic and arthritogenic (3) was not observed here, possibly due to the use of different cell types in this study.
  • Biochemical evidence for presentation of bacterial immunodominant epitope derived from the S. typhimurium GroEL molecule on the mouse class Ib molecule Qa-I and its recognition by CD8+ T cells was already demonstrated (44).
  • biochemical evidence for presentation of Chlamydia trachomatis peptides on HLA- B2705 was published, but the peptides originated from a protein transfected into the human cells (6).
  • our study is the first report of bacterial peptide presentation following infection with viable intracellular bacteria. This study also provides biochemical indication for class I presentation of bacterial peptides in non- phagocytic cells.
  • peptide KRHFPSLSI (SEQ ID NO: 1062) derived from the Salmonella RcsC system that is restricted to enterobacteria and is important for systemic infection (46).
  • This nonamer sequence common to all the other arthritogenic species of Salmonella and Shigella, shares 7aa with a predicted human B27 derived from the Calcium dependent potassium channel.
  • Such bacterial peptides also carry the potential to become immunotherapeutics.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Toxicology (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Cette invention concerne des complexes HLA-B2705-peptide multimères, et leur utilisation dans le diagnostic et le traitement du cancer et d'autres troubles autoimmuns.
PCT/IL2009/000067 2008-01-15 2009-01-15 Ligands des molécules hla-b2705 du complexe majeur d'histocompatibilité, utilisés à des fins de diagnostic et de traitement WO2009090651A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US2105108P 2008-01-15 2008-01-15
US61/021,051 2008-01-15

Publications (2)

Publication Number Publication Date
WO2009090651A2 true WO2009090651A2 (fr) 2009-07-23
WO2009090651A3 WO2009090651A3 (fr) 2009-12-03

Family

ID=40885729

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IL2009/000067 WO2009090651A2 (fr) 2008-01-15 2009-01-15 Ligands des molécules hla-b2705 du complexe majeur d'histocompatibilité, utilisés à des fins de diagnostic et de traitement

Country Status (1)

Country Link
WO (1) WO2009090651A2 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102481333A (zh) * 2009-09-03 2012-05-30 东丽株式会社 免疫诱导剂
EP2480563A2 (fr) * 2009-09-24 2012-08-01 The Regents of the University of California Peptides du type ligand spécifiques du cancer de la vessie
WO2013009690A2 (fr) 2011-07-09 2013-01-17 The Regents Of The University Of California Ligands de ciblage de cellules souches leucémiques et méthodes d'utilisation
US8540988B2 (en) 2006-02-13 2013-09-24 Alethia Biotherapeutics Inc. Antibodies that bind polypeptides involved in the process of bone remodeling
US8741289B2 (en) 2009-10-06 2014-06-03 Alethia Biotherapeutics Inc. Siglec 15 antibodies in treating bone loss-related disease
WO2014176235A3 (fr) * 2013-04-26 2015-04-30 Enzo Biochem, Inc. Traitements sensibilisants pour maladie auto-immune
US9493562B2 (en) 2012-07-19 2016-11-15 Alethia Biotherapeutics Inc. Anti-Siglec-15 antibodies
WO2016199140A1 (fr) 2015-06-08 2016-12-15 Adicet Bio Inc. Anticorps de type récepteur de cellules t ayant une spécificité fine
JPWO2017150595A1 (ja) * 2016-03-02 2018-12-27 東レ株式会社 免疫誘導剤
US10709774B2 (en) 2013-04-26 2020-07-14 Enzo Biochem, Inc. Immunomodulatory pharmaceutical compositions
GB2607829A (en) * 2018-08-14 2022-12-14 Univ Texas Single molecule sequencing peptides bound to the major histocompatibility complex

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998012221A1 (fr) * 1996-09-18 1998-03-26 Gerhild Wildner Peptides servant d'agent diagnostique et d'agent therapeutique pour les maladies auto-immunes
WO1999058557A2 (fr) * 1998-05-11 1999-11-18 Isis Innovation Limited Nouvelle molecule et methode diagnostique
US20050053918A1 (en) * 2001-05-16 2005-03-10 Technion Research & Development Foundation Ltd. Method of identifying peptides capable of binding to MHC molecules, peptides identified thereby and their uses

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998012221A1 (fr) * 1996-09-18 1998-03-26 Gerhild Wildner Peptides servant d'agent diagnostique et d'agent therapeutique pour les maladies auto-immunes
WO1999058557A2 (fr) * 1998-05-11 1999-11-18 Isis Innovation Limited Nouvelle molecule et methode diagnostique
US20050053918A1 (en) * 2001-05-16 2005-03-10 Technion Research & Development Foundation Ltd. Method of identifying peptides capable of binding to MHC molecules, peptides identified thereby and their uses

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GIQUEL BENOIT ET AL: "Two HLA-B27 alleles differently associated with spondylarthritis, B*2709 and B*2705, display similar intracellular trafficking and oligomer formation" ARTHRITIS & RHEUMATISM, vol. 56, no. 7, July 2007 (2007-07), pages 2232-2243, XP002547408 ISSN: 0004-3591 *

Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9067984B2 (en) 2006-02-13 2015-06-30 Alethia Biotherapeutics Inc. Methods of impairing osteoclast differentiation using antibodies that bind Siglec-15
US9040246B2 (en) 2006-02-13 2015-05-26 Alethia Biotherapeutics Inc. Methods of making antibodies that bind polypeptides involved in the process of bone remodeling
US9695419B2 (en) 2006-02-13 2017-07-04 Daiichi Sankyo Company, Limited Polynucleotides and polypeptide sequences involved in the process of bone remodeling
US8540988B2 (en) 2006-02-13 2013-09-24 Alethia Biotherapeutics Inc. Antibodies that bind polypeptides involved in the process of bone remodeling
CN102481333A (zh) * 2009-09-03 2012-05-30 东丽株式会社 免疫诱导剂
EP2474315A1 (fr) * 2009-09-03 2012-07-11 Toray Industries, Inc. Inducteur de la réponse immunitaire
US9289465B2 (en) 2009-09-03 2016-03-22 Toray Industries, Inc. Immunity-inducing agent
EP2474315A4 (fr) * 2009-09-03 2013-04-17 Toray Industries Inducteur de la réponse immunitaire
US10251942B2 (en) 2009-09-03 2019-04-09 Toray Industries, Inc. Immunity-inducing agent
US10335365B2 (en) 2009-09-24 2019-07-02 The Regents ot the Univershy of California Bladder cancer specific ligand peptides
EP2480563A4 (fr) * 2009-09-24 2014-04-23 Univ California Peptides du type ligand spécifiques du cancer de la vessie
US8946379B2 (en) 2009-09-24 2015-02-03 The Regents Of The University Of California Bladder cancer specific ligand peptides
JP2013505941A (ja) * 2009-09-24 2013-02-21 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 膀胱がん特異的なリガンドペプチド
US9539340B2 (en) 2009-09-24 2017-01-10 The Regents Of The University Of California Bladder cancer specific ligand peptides
US20120230994A1 (en) * 2009-09-24 2012-09-13 The Regents Of The University of California, Office of Technology Bladder cancer specific ligand peptides
TWI494566B (zh) * 2009-09-24 2015-08-01 Univ California 膀胱癌特異性配體肽
EP2480563A2 (fr) * 2009-09-24 2012-08-01 The Regents of the University of California Peptides du type ligand spécifiques du cancer de la vessie
US8741289B2 (en) 2009-10-06 2014-06-03 Alethia Biotherapeutics Inc. Siglec 15 antibodies in treating bone loss-related disease
US8900579B2 (en) 2009-10-06 2014-12-02 Alethia Biotherapuetics Inc. Siglec-15 antibodies in treating bone loss-related disease
USRE47672E1 (en) 2009-10-06 2019-10-29 Daiichi Sankyo Company, Limited Methods of impairing osteoclast differentiation using antibodies that bind siglec-15
US9388242B2 (en) 2009-10-06 2016-07-12 Alethia Biotherapeutics Inc. Nucleic acids encoding anti-Siglec-15 antibodies
US9617337B2 (en) 2009-10-06 2017-04-11 Daiichi Sankyo Company, Limited Siglec-15 antibodies in treating bone loss-related disease
EP2729486B1 (fr) * 2011-07-09 2017-12-13 The Regents of The University of California Ligands de ciblage de cellules souches leucémiques et méthodes d'utilisation
US10100083B2 (en) 2011-07-09 2018-10-16 The Regents Of The University Of California Leukemia stem cell targeting ligands and methods of use
WO2013009690A2 (fr) 2011-07-09 2013-01-17 The Regents Of The University Of California Ligands de ciblage de cellules souches leucémiques et méthodes d'utilisation
US9493562B2 (en) 2012-07-19 2016-11-15 Alethia Biotherapeutics Inc. Anti-Siglec-15 antibodies
WO2014176235A3 (fr) * 2013-04-26 2015-04-30 Enzo Biochem, Inc. Traitements sensibilisants pour maladie auto-immune
US10709774B2 (en) 2013-04-26 2020-07-14 Enzo Biochem, Inc. Immunomodulatory pharmaceutical compositions
US11173195B2 (en) 2013-04-26 2021-11-16 Enzo Biochem, Inc. Immunomodulatory pharmaceutical compositions including a synthetic peptide and transforming growth factor beta
WO2016199140A1 (fr) 2015-06-08 2016-12-15 Adicet Bio Inc. Anticorps de type récepteur de cellules t ayant une spécificité fine
JPWO2017150595A1 (ja) * 2016-03-02 2018-12-27 東レ株式会社 免疫誘導剤
EP3424518A4 (fr) * 2016-03-02 2019-08-14 Toray Industries, Inc. Inducteur d'immunité
RU2755542C2 (ru) * 2016-03-02 2021-09-17 Торэй Индастриз, Инк. Индуктор иммунитета
US11318185B2 (en) * 2016-03-02 2022-05-03 Toray Industries, Inc. Immunogenic peptides of SCD1 protein
JP7147168B2 (ja) 2016-03-02 2022-10-05 東レ株式会社 免疫誘導剤
GB2607829A (en) * 2018-08-14 2022-12-14 Univ Texas Single molecule sequencing peptides bound to the major histocompatibility complex
GB2607829B (en) * 2018-08-14 2023-08-30 Univ Texas Single molecule sequencing peptides bound to the major histocompatibility complex

Also Published As

Publication number Publication date
WO2009090651A3 (fr) 2009-12-03

Similar Documents

Publication Publication Date Title
WO2009090651A2 (fr) Ligands des molécules hla-b2705 du complexe majeur d'histocompatibilité, utilisés à des fins de diagnostic et de traitement
Newcomb et al. Characterization of endogenous peptides bound to purified HLA-DR molecules and their absence from invariant chain-associated alpha beta dimers.
KR930002835B1 (ko) 자기면역 개선에 유용한 mhc-중재 독성 포합체
EP0631502B1 (fr) Regulation de l'activite de lymphocytes a l'aide de peptides hla
JP2022522404A (ja) Ｔ細胞調節抗原提示ポリペプチド及びその使用方法
Geluk et al. Binding of a major T cell epitope of mycobacteria to a specific pocket within HLA‐DRw17 (DR3) molecules
AU675416B2 (en) Antigen associated with type I diabetes mellitus
US7408029B2 (en) Peptide epitopes recognized by disease promoting CD4+ T lymphocytes
WO1988005784A1 (fr) Inhibition de lymphocytes par des peptides hla
JPH06502916A (ja) 糖尿病自己抗体の検出方法
KR100769338B1 (ko) 류마티스성 관절염（ｒａ） 항원성 펩타이드
Wu et al. T cell recognition of MHC class II-associated peptides is independent of peptide affinity for MHC and sodium dodecyl sulfate stability of the peptide/MHC complex. Effects of conservative amino acid substitutions at anchor position 1 of influenza matrix protein19-31.
Myers et al. PART III. Autoimmunity An Altered Peptide Ligand of Type II Collagen Suppresses Autoimmune Arthritis
JPH09508617A (ja) 表面膜タンパク質および免疫応答に対するそれらの影響
Hudrisier et al. Relative implication of peptide residues in binding to major histocompatibility complex class I H-2Db: application to the design of high-affinity, allele-specific peptides
WO1997024140A1 (fr) Interaction de proteines hla avec des membres de la famille hsp70 de proteines
JP2002503094A (ja) 活性化レセプタに会合される新しいポリペプチド及びその生物学的応用
Jensen Peptide binding and antigen presentation by class II histocompatibility glycoproteins
US20090023630A1 (en) Methods and Means for Use in Diagnostics and Treatment of Diabetes
US20200172596A1 (en) Beta-2-glycoprotein 1 derived peptide and use thereof for treating antiphospholipid syndrome
KR0167016B1 (ko) 항원 특이적인 활성화된 t 림프구, 이것의 검출 및 사용방법
WO2002002620A2 (fr) Acide nucleique, polypeptide de crmp-5 et utilisations de ceux-ci
WO2005054292A1 (fr) Compositions et procedes relatifs a une molecule dimerique de type du cmh de classe i et ii (dsmhc i et dsmhc ii)
Plappert Analysis of disease-related T cell receptors and their antigens in GABAA receptor encephalitis
AU4483700A (en) Peptide epitopes recognized by disease promoting cd4+ t lymphocytes

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09702550

Country of ref document: EP

Kind code of ref document: A2

122 Ep: pct application non-entry in european phase

Ref document number: 09702550

Country of ref document: EP

Kind code of ref document: A2