EP1307211A1 - Mediateur immunitaire et methodes associees - Google Patents

Mediateur immunitaire et methodes associees

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Publication number
EP1307211A1
EP1307211A1 EP01934857A EP01934857A EP1307211A1 EP 1307211 A1 EP1307211 A1 EP 1307211A1 EP 01934857 A EP01934857 A EP 01934857A EP 01934857 A EP01934857 A EP 01934857A EP 1307211 A1 EP1307211 A1 EP 1307211A1
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EP
European Patent Office
Prior art keywords
gly
glu
thr
val
ser
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EP01934857A
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German (de)
English (en)
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EP1307211A4 (fr
Inventor
Darrick Carter
Shirley Zhu
Subhashini Arimilli
Aijun Wang
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Corixa Corp
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Corixa Corp
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Publication of EP1307211A1 publication Critical patent/EP1307211A1/fr
Publication of EP1307211A4 publication Critical patent/EP1307211A4/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/605—MHC molecules or ligands thereof
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • T cells unlike B cells, do not directly recognize antigens. Instead, an accessory cell must first process an antigen and present it in association with an MHC molecule in order to elicit a T cell-mediated immunological response.
  • MHC glycoproteins appears to be the binding and presentation of processed antigen in the form of short antigenic peptides.
  • MHC molecules can also bind "self peptides. If T lymphocytes then respond to cells presenting "self or autoantigenic peptides, a condition of autoimmunity results.
  • autoimmune diseases including myasthenia gravis (MG), multiple sclerosis (MS), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), insulin- dependent diabetes mellitus (IDDM) , etc. Characteristic of these diseases is an attack by the immune system on the tissues ofthe host. In non-diseased individuals, such attack does not occur because the immune system recognizes these tissues as "self.”
  • Autoimmunity occurs when a specific adaptive immune response is mounted against self tissue antigens.
  • MHC major histocompatibility complex
  • the present invention provides recombinant nucleic acid constructs that encode single chain, recombinant MHC class II molecules comprising a ⁇ l domain and an ⁇ l domain that may or may not be further linked to an antigenic peptide.
  • the single chain polypeptide is a ⁇ l domain and an ⁇ l domain.
  • the single chain polypeptide is a ⁇ l domain- ⁇ 2 domain (a ⁇ chain) and an ⁇ l domain- ⁇ 2 domain (an ⁇ chain).
  • the single chain constructs ofthe invention can be further dimerized or multimerized by inter-chain fusion.
  • the fusion sequence (also refened to as a dimerization or multimerization sequence) can be any sequence that allows for covalent or non-covalent linkages between the molecules ofthe invention.
  • a prefened means for carrying this out is through use of segments from immunoglobulin family proteins (e.g., antibodies, MHC molecules, T cell receptors and the like) that have cysteine residues capable of forming interchain disulfide bonds (e.g., constant regions from Ig light chains, e.g. CK or C ⁇ , or constant regions from Ig heavy chains, e.g., CHI, hinge, CH2, or CH3).
  • a leucine zipper domain forms a non-covalent linkage.
  • the single chain molecules ofthe invention thus can be multimers wherein each single chain molecule is from a different MHC class II allele.
  • each single chain molecule in the multimer can be bound to a different antigen.
  • monomeric and dimeric forms of recombinant single chain mouse I-AS-peptide complexes, fused to an antigenic MBP 90-101 peptide with flexible linkers were constructed.
  • the recombinant single chain I- AS proteins share structural similarity to that of crystallized native human MHC class II protein as determined by protein modeling.
  • the recombinant single chain proteins were expressed in E. coli and in an insect expression system and purified by affinity chromatography and FPLC.
  • the purified single chain recombinant I- AS proteins showed in vitro biological activity as assayed using an antigen-specific mouse T cell clone.
  • novel linkers are provided for forming single chain MHC class II molecules. These linkers can be used with the multimer constructs described above.
  • the constructs ofthe invention are optimized for prokaryotic expression, using codons adjusted for E. coli codon bias.
  • the present invention also provides MHC class II heterodimers, wherein a recombinant ⁇ chain and a recombinant ⁇ chain are covalently linked using polypeptide fusion segments, e.g., from immunoglobulin family proteins (e.g., antibodies, MHC molecules, T cell receptors and the like) that have cysteine residues capable of forming interchain disulfide bonds (e.g., constant regions from Ig light chains, e.g. CK or C ⁇ , or constant regions from Ig heavy chains, e.g., CHI, hinge, CH2, or CH3).
  • immunoglobulin family proteins e.g., antibodies, MHC molecules, T cell receptors and the like
  • cysteine residues capable of forming interchain disulfide bonds e.g., constant regions from Ig light chains, e.g. CK or C ⁇ , or constant regions from Ig heavy chains, e.g., CHI, hinge, CH2, or CH3
  • FIG. 1 Schematic structure ofthe recombinant single chain I- AS .MBP. ⁇ l ⁇ l (monomer) and I-AS.MBP. ⁇ l ⁇ 2 ⁇ l ⁇ 2.CK (dimer) proteins.
  • Figure 2. In vitro biological activities ofthe recombinant I- AS proteins compared with APC+ antigenic peptide in the mouse T cell clone, HS-1.
  • the positive (anti-CD3) and the negative (HS-1 cell alone) controls are also shown in each panel.
  • Figure 3 Diagram of the EAE model and standard for EAE scoring.
  • FIG. 4 The EAE model studies.
  • the recombinant I- AS proteins were admimstered to SJL mice on day 1, 4, 7, and II by i.v. injection after inducing the disease with myelin emulsified in CFA. The animals were evaluated for neurological dysfunction.
  • Panel A Untreated, injected with same amount of PBS solution.
  • Panel B Treated with the recombinant I-AS.MBP.Ck protein, a dimer form.
  • Panel C Treated with the recombinant I-AS. ⁇ l ⁇ l . This recombinant molecule does not carry the antigenic peptide.
  • Panel D Treated with the recombinant I-AS.MBP. ⁇ l ⁇ l, a monomer form.
  • Figure 5 shows a schematic representation of a ⁇ l- ⁇ l single chain MHC class II peptide complex that is a dimer with two peptide specificities.
  • Figure 6 shows a schematic representation of a ⁇ l ⁇ 2- ⁇ l ⁇ 2 single chain MHC class ⁇ peptide complex that is a dimer with two peptide specificities.
  • Figure 7 shows a schematic representation of a ⁇ 1 ⁇ 2- ⁇ l ⁇ 2 single chain
  • MHC class II peptide complex that is a tetramer with four peptide specificities.
  • Figure 8 shows a schematic representation of a ⁇ l ⁇ 2- ⁇ l ⁇ 2 single chain MHC class II peptide complex that is a tetramer with two peptide specificities and two different MHC class II alleles.
  • Figure 9 shows the effect of different recombinant MHC class II molecules on the development of EAD (day 60+).
  • Figure 10 Diagram of ⁇ l- ⁇ l single chain MHC class II peptide complex; diagram of recombinant ⁇ l ⁇ 2 chains fused to recombinant ⁇ l ⁇ 2 chains via a fusion domain from an immunoglobulin; and diagram of multimerized MHC class II molecules.
  • Figure 11 Sequence comparison of mouse CO608 single chain molecules.
  • the present invention provides recombinant DNA constructs that encode single chain MHC class II molecules that may or may not be further linked to an antigenic peptide.
  • the constructs comprise a first DNA segment encoding a ⁇ l domain of a selected MHC class II molecule; a second DNA segment encoding a ⁇ l domain of the selected MHC class II molecule; and a first linker DNA segment connecting in-frame the first and second DNA segments; wherein linkage ofthe first DNA segment to the second DNA segment by the first linker DNA segment results in a fused first DNA-first linker-second DNA polysegment.
  • constructs ofthe invention may also comprise a third DNA segment encoding an antigenic peptide capable of associating with a peptide binding groove ofthe selected MHC class II molecule and a second linker DNA segment connecting in-frame the third DNA segment to the fused first DNA-first linker-second
  • the present invention also provides recombinant components of an MHC class II heterodimer, which comprise a fusion domain.
  • One recombinant component comprises a ⁇ l domain, or optionally a ⁇ l domain- ⁇ 2 domain (i.e., a ⁇ chain).
  • One recombinant component comprises an ⁇ l domain, or optionally an ⁇ l domain- ⁇ 2 domain (i.e., an ⁇ chain).
  • the two recombinant chains are linked, either covalently, e.g., via a disulfide bond, or non covalently, using the fusion domain.
  • Such molecules can also be made into multimers using additional fusion or multimerization domains.
  • the invention provides the following recombinant components of an MHC class II heterodimer: pCB220, which is an IAS.MBP.alpha chain fused to an IgG2a CHI and truncated hinge region; pCB223, which is an IAS.MBP.alpha chain fused to an IgG2a CHI .H.CH2.CH3; and pCB229, which is an lAS.MBP.beta chain fused to a mouse CK domain.
  • These recombinant components can be fused via the fusion domain to form a MHC class II heterodimer molecule that is covalently linked via a disulfide bond at the fusion domain.
  • the present invention provides single chain MHC class II molecules that comprise an additional polypeptide sequence that allows for inter-chain dimerization of the single chain molecules ofthe invention.
  • the additional polypeptide allows multimerization ofthe single chain MHC class II molecules, to produce, e.g., dimers and tetramers.
  • the sequence can be any sequence that allows for covalent or non-covalent linkages between the molecules ofthe invention.
  • the single chain molecules are covalently linked using chemical methods known to those of skill in the art, e.g., photoaffmity methods or homo-bifunctional protein cross-linkers (see, e.g., Hermanson et al, Bioconjugate Techniques, (1996)).
  • the molecules are covalently linked using heterobifunctional protein cross-linkers.
  • segments form immunoglobulin family proteins (e.g., antibodies, MHC molecules, T cell receptors and the like) that have cysteine residues capable of forming interchain disulfide bonds.
  • An example shown below is the use ofthe constant region o the kappa chain of an antibody (CK), from either a heavy or a light chain.
  • Other dimerization sequences include a leucine zipper, a STAT protein N-terminal domain, or the FK506 binding protein (see, e.g., O'Shea, Science 254: 539 (1991), Barahmand-Pour et al, Curr. Top.
  • the multimeric, single chain class II molecules ofthe invention comprise at least two different MHC class II alleles that are associated with an autoimmune disease state, and/or at least two different autoantigenic peptides that are associated with a particular autoimmune disease state.
  • the multimeric, single chain class II molecules have chains from different DR2 alleles, e.g., DRB5*0101 and DRB1*1501.
  • the autoantigenic peptides are peptides associated with multiple sclerosis, e.g., MBP (e.g., amino acid residues 83-102Y83), PLP (e.g., amino acid residues 40-60, 89-106, 95-117, and 185-206); and MOG.
  • other antigens associated with autoimmune disease such as acetylcholine receptor and type II collagen, can be linked to the single chain molecules ofthe invention..
  • the single chain class II molecules ofthe invention have novel linkers, as described herein.
  • the mammalian MHC class II single chain constructs ofthe invention may also be constructed to use prefened prokaryotic codons, for expression, e.g., in E. coli, using codon preference tables and methods known to those of skill in the art.
  • Single chain MHC class II molecule refers to a fusion protein such as the recombinant single chain MHC class II complex ofthe invention, which optionally also is fused to a peptide to form a single chain MHC class:peptide complex.
  • the fusion proteins ofthe invention can also be multimers, having two, four or more single chain molecules linked covalently or non-covalently through multimerization domains in the single chain molecule.
  • a single chain molecule ofthe invention typically comprises at least an MHC class LT ⁇ l domain and an MHC class II ⁇ l domain, optionally ⁇ l ⁇ 2 ⁇ l ⁇ 2 domains or any combination thereof in any order.
  • the single chain molecules are soluble, that is, they lack the naturally occurring cytoplasmic and transmembrane MHC class JJ domains.
  • a domain of a selected MHC molecule A portion of an MHC domain which is sufficient to form, either alone, or in combination with another portion of an MHC domain, a peptide binding site which is capable of presenting an antigenic peptide in such a fashion that it is recognized by a T cell receptor.
  • MHC domains would include the extracellular portion ofthe two polypeptide chains of Class II MHC. This would include the ⁇ chain ( ⁇ l and ⁇ 2 domains) and ⁇ chain ( ⁇ l and ⁇ 2 domains) of Class II MHC.
  • the domains can be directly linked, or can be linked via an amino acid linker.
  • Linker DNA segment A segment of DNA encoding from about 1 to about 50, preferably from about 5 to about 25 amino acids, which forms a flexible link between two DNA segments. This flexible link allows the two DNA segments to attain a proper configuration, such as an MHC peptide binding groove, or allows a peptide to properly bind into such a groove.
  • Antigenic peptide The immunological properties of MHC histocompatibility proteins are largely defined by the antigenic peptide that is bound to them.
  • An antigenic peptide is one which contains an epitope (an amino acid sequence) recognized by immune cells, e.g., T cells, and is capable of stimulating an MHC-mediated immune response.
  • Antigenic peptides for a number of autoimmune diseases are known. For example, in experimentally induced autoimmune diseases, antigens involved in pathogenesis have been characterized: in arthritis in rat and mouse, native type JJ collagen is identified in collagen-induced arthritis, and mycobacterial heat shock protein in adjuvant arthritis (Stuart et al., Ann. Rev. Immunol. 2:199-218, 1984; and van Eden et al,
  • thyroglobulin has been identified in experimental allergic thyroiditis (EAT) in mice (Marion et al, J. Exp. Med. 152:1115-1120, 1988); acetyl- choline receptor (AChR) in experimental allergic myasthenia gravis (EAMG) (Lindstrom et al, Adv. Immunol. 42:233-284, 1988) ; and myelin basic protein (MBP) and proteolipid protein (PLP) in experimental allergic encephalomyelitis (EAE) in mouse and rat (Acha-
  • target antigens have been identified in humans: type II collagen in human rheumatoid arthritis (Holoshitz et al, Lancet ii:305-309, 1986), acetylcholine receptor in myasthenia gravis (Lindstrom et al, Adv. Immunol. 42.:233-284, 1988), and MBP, PLP, and MOG in multiple sclerosis in humans.
  • MHC The major histocompatibility complex
  • Class I and Class II are membrane-associated and present antigen to T lymphocytes (T cells).
  • T lymphocytes T cells
  • Class I MHC molecules e.g., HLA- A, -B and -C molecules in the human system
  • CTL cytotoxic T lymphocytes
  • Class II MHC molecules (HLA-DP, -DQ and -DR, for example, in humans) are expressed primarily on the surface of antigen-presenting cells, such as B lymphocytes, dendritic cells, macrophages, and the like. Class II MHC is recognized by CD4 + T helper lymphocytes (T H ). T H cells induce proliferation of both B and T lymphocytes, thus amplifying the immune response to the particular antigenic peptide that is displayed (Takahashi, Microbiol. Immunol, 37:1-9, 1993).
  • Intracellular antigens synthesized inside ofthe cell, such as from viral or newly synthesized cellular proteins, for example, are processed and presented by Class I MHC.
  • Exogenous antigens taken up by the antigen-presenting cell (APC) from outside ofthe cell through endocytosis, are processed and presented by Class II MHC.
  • APC antigen-presenting cell
  • the resulting antigenic peptide forms a complex with the antigen binding groove ofthe MHC molecule through various noncovalent associations.
  • the MHC-peptide complex on the cell surface is recognized by a specific T cell receptor on a cytotoxic or helper T cell.
  • HLA- A human leukocyte antigens (HLA)
  • HLA-B human leukocyte antigens (HLA)
  • HLA-C human leukocyte antigens
  • An adjacent region, known as HLA-D is subdivided into HLA-DR, HLA-DQ and HLA-DP.
  • the HLA region is now known as the human MHC region, and is equivalent to the H-2 region in mice.
  • HLA- A, - B and -C resemble mouse H-2K, -D, and -L and are the Class I MHC molecules.
  • HLA- DP, -DQ and -DR resemble mouse I-A and I-E and are the Class JJ molecules.
  • MHC glycoproteins of both classes have been isolated and characterized (see Fundamental Immunology, 2d Ed., Paul (ed.), (1989); and Roitt et al, Immunology, 2d Ed., (1989) , which are both incorporated herein by reference).
  • Human MHC Class I molecules consist of a polymorphic type I integral membrane glycoprotein heavy chain of about 46 kD, noncovalently associated with a 12 kD soluble subunit, ⁇ 2-microglobulin.
  • the heavy chain consists of two distinct extracellular regions, the membrane distal, peptide binding region formed by the ⁇ l and ⁇ 2 domains, and the membrane proximal, CD8-binding region derived from the ⁇ 3 domain.
  • ⁇ 2 - microglobulin is a single, compact immunoglobulin-like domain that lacks a membrane anchor, and exists either associated with the class I heavy chain or free in plasma (Germain and Margulies, Annu. Rev. Immunol. 11 :403-50, 1993).
  • Human MHC Class II is a heterodimeric integral membrane protein. Each dimer consists of one ⁇ and one ⁇ chain in noncovalent association. The two chains are similar to each other, with the ⁇ chain having a molecular weight of 32-34 kD and the ⁇ chain having a molecular weight of 29-32 kD. Both polypeptide chains contain N- linked oligosaccharide groups and have extracellular amino termini and intracellular carboxy termini.
  • the extracellular portions ofthe ⁇ and ⁇ chain that comprise the class II molecule have been subdivided into two domains of about 90 amino acids each, called ⁇ l, ⁇ 2, and ⁇ l, ⁇ 2, respectively.
  • the ⁇ 2 and ⁇ 2 domains each contain a disulfide-linked loop.
  • the peptide-binding region ofthe class II molecule is formed by the interaction of the ⁇ l and ⁇ l domains. This interaction results in an open-ended, antigenic peptide- binding groove made up of two ⁇ helices, and an eight-stranded ⁇ -pleated sheet platform.
  • the ⁇ and ⁇ chains of Class II molecules are encoded by different MHC genes and are polymorphic (see Addas et al, Cellular and Molecular Immunology, 2d Ed.
  • a prefened ⁇ chain is DRA*0101 and a prefened ⁇ chain is DR ⁇ l* 1501.
  • the single chain MHC class ILpeptide complexes ofthe present invention can incorporate cDNA from any allele that predisposes or increases the likelihood of susceptibility to a specific autoimmune disease.
  • Specific autoimmune diseases are conelated with specific MHC types.
  • Specific haplotypes have been associated with many ofthe autoimmune diseases. For example, HLA-DR2 + and HLA-DR3 + individuals are at a higher risk than the general population to develop systemic lupus erythematosus (SLE) (Reinertsen et al, N. Engl. J. Med. 299:515-18, 1970).
  • Myasthenia gravis has been linked to HLA-D (Safwenberg et al, Tissue Antigens 12:136-42,1978.
  • HLA-D/DR Susceptibility to rheumatoid arthritis is associated with HLA-D/DR in humans.
  • Methods for identifying which alleles, and subsequently which MHC-encoded polypeptides, are associated with an autoimmune disease are known in the art.
  • Exemplary alleles for JDDM include DR4, DQ8, DR3, DQ3.2.
  • Single chain MHC class II molecules and/or single chain MHC class ILpeptide complexes ofthe present invention can be used as antagonists to therapeutically block the binding of particular T cells and antigen-presenting cells.
  • the molecules can induce anergy, or proliferative nonreponsiveness, and possibly apoptosis, in targeted T cells, both in vivo and in vitro.
  • a single chain MHC class ILpeptide molecule directed toward a desired autoimmune disease contains the antigenic peptide implicated for that autoimmune disease properly positioned in the binding groove ofthe MHC molecule, without need for solublization of MHC or exogenous loading of an independently manufactured peptide.
  • the cunent invention offers the advantage of a recombinant single chain MHC class II molecule made up of two or more MHC domains joined together via a flexible linkage, and onto which is tethered (via an additional flexible linkage) an antigenic peptide which is able to bind to the peptide binding groove presented by the single chain MHC class TJ molecule.
  • Such a complex provides an MHC molecule which is soluble and, because the MHC class II components and conesponding antigenic peptide are permanently linked into a single chain configuration, there is no need for complex heterodimer truncation or formation. These complexes eliminate inefficient and nonspecific peptide loading.
  • a soluble MHC class II molecules is one that does not contain the naturally occurring membrane-associated MHC class II sequences.
  • the soluble MHC molecules ofthe present invention has never been membrane-associated. Further, the soluble MHC class II molecules do not contain an amino acid sequence that acts as a transmembrane domain or as a cytoplasmic domain.
  • the present invention therefore provides a single chain MHC class II molecule which optionally includes an antigenic peptide covalently attached to the amino terminal portion of an ⁇ or ⁇ chain of MHC through a peptide linkage, and the C terminal ofthe linked ⁇ or ⁇ chain may be attached to the N terminal portion of another ⁇ or ⁇ chain, there by creating a two, three, or four domain MHC molecule.
  • the invention further provides a multimerization domain to provide a multimeric single chain MHC class II molecules.
  • the invention further provides novel linkers, and multimeric MHC class TJ molecules that are bound to different antigenic peptides.
  • each of a number of Class I and Class JJ proteins are known, and the genes or cDNAs have been cloned. Thus, these nucleic acids can be used to express MHC polypeptides. If a desired MHC gene or cDNA is not available, cloning methods known to those skilled in the art may be used to isolate the genes. One such method that can be used is to purify the desired MHC polypeptide, obtain a partial amino acid sequence, synthesize a nucleotide probe based on the amino acid sequence, and use the probe to identify clones that harbor the desired gene from a cDNA or genomic library.
  • Linkers ofthe cunent invention may be from about 1 to about 50 amino acids in length, depending on the molecular model ofthe MHC or MHC:peptide complex.
  • flexible linkers are made of repeating Gly residues separated by one or more Ser residues to permit a random, flexible motion. In the case of Class II MHC complexes this flexibility accommodates positioning ofthe ⁇ and ⁇ segments to properly configure the binding groove, and also allows for maximum positioning ofthe peptide in the groove.
  • the linker comprises a CD4 binding site, as described below in the Example section (see also Table 1).
  • longer linkers between the chains contain flexible residues (e.g. alanine or glycine) and polar residues (e.g.
  • prolines can be added to bracket the linkers. These prolines are known to inhibit the formation of alpha helices and beta sheets.
  • flexible regions present in the human MHC and in the murine MHC could be used to make a linker by extending the region of interest and ligating the ends together. Finally, a combination of these types of linkers could also be used.
  • Linker position and length can be modeled based on the crystal structure of MHC Class II molecules (Brown et al, Nature 364:33-39, 1993), where ⁇ l and ⁇ l are assembled to form the peptide binding groove. Linkers joining segments ofthe ⁇ and ⁇ chains together are based on the geometry ofthe region in the hypothetical binding site and the distance between the C terminus and the N terminus ofthe relevant segments. Molecular modeling based on the X-ray crystal structure of Class II MHC (Stern et al,
  • the invention also provides methods for preparing responder T-cell clones that proliferate when combined with a selected antigenic peptide presented by a stimulator cell. Such clones can be used to identify and map antigenic peptides associated with autoimmune disease. These peptides can then be incorporated into the single chain MHC class II molecule ⁇ eptide complexes ofthe invention.
  • the method provides isolation and enrichment of non-adherent, CD56 " , CD8 " T cells that are reactive with a selected antigenic peptide. These cells are herein refened to as responder cells. Suitable responder cells can be isolated, for example, from peripheral blood mononuclear cells (PBMNC) obtained from patients prior to or after onset of an autoimmune disease of interest.
  • PBMNC peripheral blood mononuclear cells
  • PBMNCs can be obtained from prediabetic and new onset diabetic patients. These patients can be pre-screened for specific HLA markers, such as DR3- DR4 or DQ3.2, which have the highest association with susceptibility to IDDM. From the collected PBMNCs, a portion is kept to serve as stimulator cells. From the remainder, the desired autoreactive responder cells are purified and isolated by two rounds of plating, to remove adherent cells from the population, followed by removal of monocytes and B cells with nylon wool. Enrichment for non-adherent CD4 + T cells is completed by sequential plating ofthe cells onto plates coated with anti-CD8 and anti-CDS6 antibodies.
  • HLA markers such as DR3- DR4 or DQ3.2
  • the stimulator cells are pulsed or primed with whole GAD or an appropriate antigenic peptide.
  • stimulator cells from the PBMNCs of LDDM patients can be stimulated with antigenic GAD peptides then combined with PBMNCs or responder cells.
  • responder cell (T cell) clones are generated through limiting dilution and tested for antigen reactivity. These responder cell (T cell) clones can then be used, for example, to map epitopes which bind to MHC and are recognized by a particular T cell.
  • One such method uses overlapping peptide fragments ofthe autoantigen which are generated by tryptic digestion, or more preferably, overlapping peptides are synthesized using known peptide synthesis techniques.
  • peptide fragments are then tested for their ability to stimulate the responder T cell clones or lines (for example, Ota et al, Nature 346:183-187, 1990).
  • synthetic antigenic peptides can be specifically designed, for example, to enhance the binding affinity for MHC and to out-compete any naturally processed peptides.
  • Such synthetic peptides when combined into a single chain MHC class II molecule:peptide complex, would allow manipulation ofthe immune system in vivo, in order to tolerize or anergize disease- associated activated T cells, thereby ameliorating the autoimmune disease.
  • residues that alter T cell recognition are determined by substituting amino acids for each position in the peptide in question, and by assessing whether such change in residues alters the peptide 's ability to associate with MHC (Allen et al, Nature 327:713-15, 1987; Sette et al, Nature 328:395-99, 1987; O'Sullivan et al, J. Immunol. 147:2663-69, 1991; Evavold et al, J. Immunol. 148:347-53, 1992; Jorgensen et al., Annu. Rev. Immunol.
  • a prefened variant of this method is an alanine scan (Ala scan) where a series of synthetic peptides are synthesized wherein each individual amino acid is substituted with L-alanine (L-Ala scan).
  • Alanine is the amino acid of choice because it is found in all positions (buried and exposed), in secondary structure, it does not impose steric hindrances, or add additional hydrogen bonds or hydrophobic side chains. Alanine substitutions can be done independently or in clusters depending on the information desired. Where the information pertains to specific residues involved in binding, each residue in the peptide under investigation can be converted to alanine and the binding affinity compared to the unsubstituted peptide.
  • Essential residues can be identified, and nonessential residues targeted for modification, deletion or replacement by other residues that may enhance a desired quality (Cunningham and Wells, Science 244:1081- 1085, 1989; Cunningham and Wells, Natl. Acad. Sci. USA, 88:3407-3411, 1991; Ehrlich et al, J. Biol. Chem. 267:11606-11, 1992; Zhang et al, Proc. Natl. Acad. Sci. USA 90:4446-50, 1993; see also "Molecular Design and Modeling: Concepts and Applications Part A Proteins, Peptides, and Enzymes," Methods in Enzymology, Vol. 202, Langone (ed.), Academic Press, San Diego, CN 1991).
  • Truncated peptides can be generated from the altered or unaltered peptides by synthesizing peptides wherein amino acid residues are truncated from the ⁇ - or C- terminus to determine the shortest active peptide, or between the ⁇ - and C-terminus to determine the shortest active sequence. Such peptides could be specifically developed to stimulate a response when joined to a particular MHC to form a peptide ligand to induce anergy in appropriate T cells in vivo or in vitro.
  • Mass spectral analysis methods such as electrospray and Matrix-Assisted Laser Desorption/Ionization Time Of Flight mass spectrometry (MALDI TOF) analysis are routinely used in the art to provide such information as molecular weight and confirm disulfide bond formation.
  • MALDI TOF Matrix-Assisted Laser Desorption/Ionization Time Of Flight mass spectrometry
  • FACs analysis can be used to determine proper folding ofthe single chain complex.
  • An ELISA Enzyme-linked Immunosorbent Assay
  • This assay can be used with either whole cells; solubilized
  • MHC removed from the cell surface; or free single chain MHC class II molecule.-peptide complexes ofthe cunent invention.
  • an antibody that detects the recombinant MHC haplotype is coated onto wells of a microtiter plate.
  • the antibody is L243, a monoclonal antibody that recognizes only conectly folded HLA-DR MHC dimers.
  • MHC Class II-specific antibodies are known and available.
  • Anti-MHC Class II antibodies can also be used to purify Class II molecules through techniques such as affinity chromatography, or as a marker reagent to detect the presence of Class II molecules on cells or in solution. Such antibodies are also useful for Western analysis or immunoblotting, particularly of purified cell-secreted material.
  • Polyclonal, affinity purified polyclonal, monoclonal and single chain antibodies are suitable for use in this regard.
  • proteolytic and recombinant fragments and epitope binding domains can be used herein. Chimeric, humanized, veneered, CDR- replaced, reshaped or other recombinant whole or partial antibodies are also suitable.
  • bound MHC molecules can be detected using an antibody or other binding moiety capable of binding MHC molecules.
  • This binding moiety or antibody may be tagged with a detectable label, or may be detected using a detectably labeled secondary antibody or binding reagent.
  • Detectable labels or tags are known in the art, and include fluorescent, colorimetric and radiolabels, for instance.
  • an in vitro anergy assay determines if non-responsiveness has been induced in the T cells being tested.
  • responder cells preferably peripheral blood mononuclear cells (PBMN) (a heterogeneous population including B and
  • T lymphocytes T lymphocytes, monocytes and dendritic cells
  • PBMNC lymphocytes freshly isolated T lymphocytes, in vivo primed splenocytes, cultured T cells, or established T cell lines or clones.
  • Responder cells from mammals immunized with, or having a demonstrable cellular immune response to, the antigenic peptide are particularly prefened.
  • the stimulator cells are antigenic peptide-presenting cells, such as PBMNCs, PBMNCs that have been depleted of lymphocytes, appropriate antigenic peptide-presenting cell lines or clones (such as EBV-fransforrned B cells), EBV transformed autologous and non-autologous PMNCs, genetically engineered antigen presenting cells, such as mouse L cells or bare lymphocyte cells BLS-1, in particular, DRB 0401, DRB1*0404 and DRB1*0301 (Kovats et al, J. Exp. Med.
  • Stimulator cells from mammals immunized with, or having a demonstrable cellular immune response to, the antigenic peptide are particularly prefened.
  • it is prefened to inhibit the proliferation of stimulator cells prior to mixing with responder cells. This inhibition may be achieved by exposure to gamma inadiation or to an anti-mitotic agent, such as mitomycin C, for instance.
  • Appropriate negative controls are also included (nothing; syngeneic APC; experimental peptide; APC + Peptide; MHC:peptide complex; control peptide +/- APC).
  • the proliferation assay may be set up ⁇ n duplicate, +/- recombinant IL-2 since it has been demonstrated that IL-2, can rescue anergized cells.
  • responder cell activation is determined by measuring proliferation using 3 H-thymidine uptake (Crowley et al, J. Immunol. Meth. 133:55-66, 1990).
  • responder cell activation can be measured by the production of cytokines, such as IL-2, or by determining the presence of responder cell-specific, and particularly T cell-specific, activation markers. Cytokine production can be assayed by testing the ability ofthe stimulator + responder cell culture supernatant to stimulate growth of cytokine-dependent cells.
  • Responder cell- or T cell-specific activation markers may be detected using antibodies specific for such markers.
  • the single chain MHC class LT molecule:peptide complex induces non-responsiveness (for example, anergy) in the antigenic peptide-reactive responder cells.
  • responder cell activation requires the involvement of co-receptors on the stimulator cell (the APC) that have been stimulated with co-stimulatory molecules.
  • responder cells By blocking or eliminating stimulation of such co-receptors (for instance, by exposing responder cells to purified single chain MHC class II molecule :peptide complex, by blocking with anti-receptor or anti-ligand antibodies, or by "knocking out” the gene(s) encoding such receptors), responder cells can be rendered non-responsive to antigen or to single chain MHC class LT molecule ⁇ eptide complex.
  • responder cells are obtained from a source manifesting an autoimmune disease or syndrome.
  • autoantigen-reactive T cell clones or lines are prefened responder cells.
  • stimulator cells are obtained from a source manifesting an autoimmune disease or syndrome.
  • APC cell lines or clones that are able to appropriately process and/or present autoantigen to responder cells are prefened stimulator cells.
  • responder and stimulator cells are obtained from a source with diabetes or multiple sclerosis.
  • the responder T cells can be selectively amplified and/or stimulated, thereby producing a subset of T cells that are specific for the antigenic peptide.
  • antigenic peptide-reactive responder cells may be selected by flow cytometry, and particularly by fluorescence activated cell sorting. This subset of responder cells can be maintained by repetitive stimulation with APCs presenting the same antigenic peptide. Alternatively, responder cell clones or lines can be established from this responder cell subset. Further, this subset of responder cells can be used to map epitopes ofthe antigenic peptide and the protein from which it is derived.
  • Similar assays and methods can be developed for and used in animal models.
  • the therapeutic effect of a pharmaceutical composition ofthe single chain molecule or multimer or a polynucleotide encoding the single chain molecule or multimer can be tested in vivo in a number of animal models of HLA-DR-associated autoimmune disease.
  • diseases include, but are not limited to, insulin-dependent diabetes mellitus, multiple sclerosis, myasthenia gravis, pernicious anemia, rheumatoid arthritis, and systemic lupus erythematosus.
  • NOD mice are a spontaneous model of IDDM.
  • Treatment with the pharmaceutical compositions prior to or after onset of disease can be monitored by assay of urine glucose levels in the NOD mouse, as well as by in vitro T cell proliferation assays to assess reactivity to known autoantigens (see, e.g., Kaufman et al, Nature 366:69-72 (1993)) for example) .
  • induced models of autoimmune disease such as EAE
  • Treatment in a preventive or intervention mode can be followed by monitoring the clinical symptoms of EAE.
  • SLE Systemic Lupus Erythematosus
  • NZB mice autoimmune New Zealand black mice
  • NZW mice phenotypically normal New Zealand White mice
  • SLE glomerulonephritis
  • mice manifest several immune abnormalities, including antibodies to nuclear antigens and subsequent development of a fatal, immune complex-mediated glomerulonephritis with female predominance, remarkably similar to SLE in humans (Knight et al, J. Exp. Med.
  • HLA-DR2 and HLA-DR3 individuals are at a higher risk than the general population to develop SLE (Reinertsen et al, N. Engl. J. Med. 299:515 (1970)), while in NZB/W F ! mice (H-2 d/u ), a gene linked to the h-2 u haplotype derived from the NZW parent contributes to the development ofthe lupus-like nephritis.
  • the effect ofthe invention can be measured by survival rates and by the progress of development ofthe symptoms, such as protenuria and appearance of anti- DNA antibodies.
  • Proteinuria can be measured by any method known to those of skill in the art, e.g. colorimetrically by the use of Uristix (Miles Laboratories, Inc., Elkhart, IN), giving an approximation of proteinuria as follows: trace, 10 mg/dl; 1+, 30 mg/dl; 100 mg/dl; 3+, 300 mg/dl; and 4+, 1000 mg/dl.
  • NZB/W V ⁇ mice The presence of anti-DNA specific antibodies in NZB/W V ⁇ mice can be determined by using a modification of a linked immunosorbent assay (ELISA) described by Zouali et al, J. Immunol. Methods 90:105 (1986)) which is incorporated herein by reference.
  • ELISA linked immunosorbent assay
  • Myasthenia gravis is one of several human autoimmune diseases linked to HLA-D (Safenberg, et al, Tissue Antigens 12:136 (1978); McDevitt et al, Arth. Rheum. 20:59 (1977)) which are incorporated herein by reference.
  • HLA-D Safenberg, et al, Tissue Antigens 12:136 (1978); McDevitt et al, Arth. Rheum. 20:59 (1977)
  • MG antibodies to the acetyl choline receptors (AcChoR) impair neuromuscular transmission by mediating loss of AcChoR in the postsynaptic membrane.
  • mice are a model system for human MG.
  • experimental autoimmune myasthenia gravis (EAMG) can be induced by immunizing the mice with soluble AcChoR protein from another species. Susceptibility to EAMG is linked in part to the MHC and has been mapped to the region within H-2 (Chnstadoss et al, J. Immunol. 123:2540 (1979)).
  • AcChoR protein can purified from Torpedo californica and assayed according to the method of Waldor et al, Proc. Natl. Acad. Sci. USA 80:2713 (1983), incorporated by reference.
  • emulsified AcChoR 15 ⁇ g in complete Freund adjuvant
  • emulsified AcChoR 15 ⁇ g in complete Freund adjuvant
  • Evaluation can be made by measurement of anti- AcChoR antibodies by any method known to those of skill in the art, e.g., a microtiter ELISA assay as described in Waldor et al, supra.
  • the standard reagent volume is 50 ⁇ l per well. Reagents are usually incubated in the wells for 2 hr at RT. Five ⁇ g of AcChoR diluted in bicarbonate buffer, pH 9.6, is added to each well. After incubation with
  • the plates are rinsed four times with a wash solution consisting of phosphate- buffer saline containing 0.05% Tween and 0.05% NaN 3 .
  • Mouse sera are diluted in 0.01M PBS (pH 7.2), 1.5 mfr MgCl 2 , 2.0 mM 2-mercaptoethanol, 0.05% Tween-80, 0.05% NaN 3 (p-Tween buffer) and incubated on the plate.
  • beta- galactosidase-conjugated sheep anti-mouse antibody diluted in P-Tween buffer is added to each well.
  • the enzyme substrate, p-nitrophenylgalctopyranoside is added to the plate, and the degree of substrate catalysis is determined from the absorbance at 405 nm after 1 hr.
  • Anti- AcChoR antibodies are expected to be present in the mice immunized with AcChoR as compared to nonimmunized mice. Treatment with complex is expected to significantly reduce the titer of anti- AcChoR antibodies in the immunized mice.
  • Myasthenia symptoms include a characteristic hunched posture with drooping ofthe head and neck, exaggerated arching ofthe back, splayed limbs, abnormal walking, and difficulty in righting. Mild symptoms are present after a standard stress test, and should be ameliorated by administration of complex.
  • RA Rheumatoid Arthritis
  • mice The immune response in mice to native type II collagen has been used to establish an experimental model for arthritis with a number of histological and pathological features resembling human RA.
  • Susceptibility to collagen-induced arthritis (CIA) in mice has been mapped to the H-21 region, particularly the I-A subregion (Huse et al, Fed.
  • mice from a susceptible strain DEA-1 can be caused to have CIA by treatment ofthe mice with native type II collagen, using the technique described in
  • adjuvant arthritis in rats is an experimental model for human arthritis, and a prototype of autoimmune arthritis triggered by bacterial antigens
  • the disease is the result of a cell-mediated immune response, as evidenced by its transmissibility by a clone of T cells which were reactive against the adjuvant (MT); the target self-antigen in the disease, based upon studies with the same cloned cells, appears to be part(s) of a proteoglycan molecule of cartilage.
  • MT adjuvant
  • Adjuvant disease in rats is produced as described by Pearson supra, i.e., by a single injection of Freund's adjuvant (killed tubercle bacilli or chemical fractions of it, mineral oil, and an emulsifying agent) given into several depot sites, preferably intracutaneously or into a paw or the base ofthe tail.
  • the adjuvant is given in the absence of other antigens.
  • the effect ofthe invention treatment on manifestations ofthe disease can be monitored by any method known to those of skill in the art.
  • These manifestations are histopathological, and include an acute and subacute synovitis with proliferation of synovial lining cells, predominantly a mononuclear infiltration ofthe articular and particular tissues, the invasion of bone and articular cartilage by connective tissue pannus, and periosteal new bone formation, especially adjacent to affected joints. In severe or chronic cases, destructive changes occur, as do fibrous or bony ankylosis.
  • These histopathological symptoms are expected to appear in control animals at about 12 days after sensitization to the Freund's adjuvant.
  • IDDM Insulin Dependent Diabetes Mellitus
  • the rat disease is controlled in part by the genes encoding the MHC antigens, is characterized by islet infiltration, and is associated with the presence of anti-islet antibodies.
  • the I-E equivalent class II MHC antigens appear to be involved in manifestation ofthe autoimmune diseases in the BB rat. Biotard et al, Proc. Natl. Acad. Sci. USA 82:6627
  • insulitis is characterized by the presence of mononuclear inflammatory cells- within the islets.
  • Thyroiditis is characterized by focal interstitial lymphocytic infiltrate within the thyroid gland, as a minimum criterion. Most severe cases show diffuse extensive lymphocytic infiltrates, disruption of acini, fibrosis, and focal Hurthle call change. See Biotard et al. supra. Treatment ofthe BB rats with the invention is expected to ameliorate or prevent the manifestation ofthe clinical and morphological symptoms associated with IDDM and thyroiditis.
  • the NOD mouse strain (H-2K d D b ) is a murine model for autoimmune IDDM.
  • the disease in these animals is characterized by anti-islet cell antibodies, severe insulitis, and evidence for autoimmune destruction ofthe beta-cells (Kanazawa, et al, Diabetolooia 27:113 (1984)).
  • the disease can be passively transfened with lymphocytes and prevented by treatment with cyclosporin-A (Ikehara et al, Proc. Natl. Acad. Sci. USA 82:7743 (1985)); Mori et al, Diabetolooia 29:244 (1986).
  • EAE Experimental Allergic Encephalomyelitis
  • EAE Experimental allergic encephalomyelitis
  • MS multiple sclerosis
  • the disease is characterized by the acute onset of paralysis. Perivascular infiltration by mononuclear cells in the CNS is observed in both mice and rats. Methods of inducing the disease, as well as symptomology, are reviewed in Aranson, The Autoimmune Diseases (Rose and Mackay, eds., 1985), and in Acha-Orbea et al, Ann. Rev. Imm. 7:377-405 (1989).
  • MBP myelin basic protein
  • the effect ofthe invention on ameliorating disease symptoms in individuals in which EAE has been induced can be measured by survival rates, and by the progress ofthe development of symptoms.
  • Expression systems suitable for production of appropriate recombinant single chain MHC class II molecule.-peptide complexes are available and known in the art.
  • Various prokaryotic, fungal, and eukaryotic host cells are suitable for expression of recombinatn, single chain MHC class II molecule:peptide complexes, as well as for individual recombinant alpha and beta MHC class II chains.
  • Prokaryotes that are useful as host cells, according to the present invention, most frequently are represented by various strains of Escherichia coli. However, other microbial strains can also be used, such as bacilli, for example Bacillus subtilis, various species of Pseudomonas, or other bacterial strains.
  • the single chain MHC class II molecule:peptide complexes are expressed from recombinantly engineered nucleotide sequences that encode the single chain MHC class II molecule:peptide polypeptides by operably linking the engineered nucleic acid coding sequence to signals that direct gene expression in prokaryotes.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it effects the transcription ofthe sequence.
  • operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • genes encoding the single chain MHC class TI molecule:peptide complexes may be inserted into an "expression vector,” “cloning vector,” or “vector,” terms which are used interchangeably herein and usually refer to plasmids or other nucleic acid molec ⁇ les that are able to replicate in a chosen host cell.
  • Expression vectors may replicate autonomously, or they can replicate by being inserted into the genome of the host cell, by methods well known in the art. Vectors that replicate autonomously will have an origin of replication or autonomous replicating sequence (ARS) that is functional in the chosen host cell(s).
  • ARS autonomous replicating sequence
  • Plasmid vectors that contain replication sites and control sequences derived from a species compatible with the chosen host are used.
  • E. coli is typically transformed using derivatives of pBR322, a plasmid derived from E. coli species by Bolivar et al, Gene 2:95-113, 1977.
  • a vector it is desirable for a vector to be usable in more than one host cell, e.g., in E. coli for cloning and construction, and in a Bacillus cell for expression.
  • the expression vectors typically contain a transcription unit or expression cassette that contains all the elements required for the expression ofthe DNA encoding the MHC molecule in the host cells.
  • a typical expression cassette contains a promoter operably linked to the DNA sequence encoding a single chain MHC class II molecule:peptide complex and a ribosome binding site.
  • the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • the expression cassette can also contain a transcription termination region downstream ofthe structural gene to provide for efficient termination. The termination region may be obtained from the same gene as the promoter sequence or may be obtained from a different gene.
  • prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, include such commonly used promoters as the beta- lactamase (penicillinase) and lactose (lac) promoter systems (Change et al, Nature
  • Either constitutive or regulated promoters can be used in the present invention.
  • Regulated promoters can be advantageous because the host cells can be grown to high densities before expression ofthe single chain MHC class II molecule:peptide complexes is induced. High level expression of heterologous proteins slows cell growth in some situations.
  • Regulated promoters especially suitable for use in E. coli include the bacteriophage lambda PL promoter, the hybrid trp-lac promoter (Amann et al, Gene 25:167-78 1983; and the bacteriophage T7 promoter.
  • a promoter that functions in the particular prokaryotic species is required.
  • Such promoters can be obtained from genes that have been cloned from the species, or heterologous promoters can be used.
  • the hybrid trp-lac promoter functions in Bacillus in addition to E. coli.
  • a ribosome binding site (RBS) is also necessary for expression of single chain MHC class II molecule :peptide complexes in prokaryotes.
  • An RBS in E. coli for example, consists of a nucleotide sequence 3-9 nucleotides in length located 3-11 nucleotides upstream ofthe initiation codon (Shine and Dalgarno, Nature, 254:34-40, 1975; Steitz, In Biological regulation and development: Gene expression (ed., Goldberger), vol. 1, p. 349, 1979).
  • Translational coupling may be used to enhance expression.
  • the strategy uses a short upstream open reading frame derived from a highly expressed gene native to the translational system, which is placed downstream ofthe promoter, and a ribosome binding site followed after a few amino acid codons by a termination codon. Just prior to the termination codon is a second ribosome binding site, and following the termination codon is a start codon for the initiation of translation.
  • the system dissolves secondary structure in the RNA, allowing for the efficient initiation of translation. See Squires, et. al., J. Biol. Chem. 263:16297-16302, 1988.
  • the single chain MHC class II molecule:peptide complexes can be expressed intracellularly, or can be secreted from the cell. Intracellular expression often results in high yields. However, some ofthe protein may be in the form of insoluble inclusion bodies. Although some ofthe intracellularly produced MHC polypeptides of the present invention may be active upon being harvested following cell lysis, the amount of soluble, active MHC polypeptide may be increased by performing refolding procedures using methods known to those of skill in the art (see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual Second Edition, Cold Spring Harbor, NY, 1989.; Marsion et al, Bio/Technology 2:800-804, 1985; Schoner et al, Bio/Technology 3:151-54, 1985).
  • the cell pellet is lysed and refolded in urea-borate-DTT buffer followed by urea-borate buffer and reverse phase HPLC purification using either silica gel based Vydac (Hewlett Packard, Wilmington, DE) or polymer based Poros-R2 (PerSeptive Biosystems) resins, with bead size varying based on the scale ofthe culture and is described in further detail below.
  • silica gel based Vydac Hewlett Packard, Wilmington, DE
  • polymer based Poros-R2 PerSeptive Biosystems
  • the sample can be ultrafiltered into a urea-borate buffer to which is then added 0.2 ⁇ M to 1 mM copper sulfate, preferably 0.2 to 20 ⁇ M, after which folding occurs immediately.
  • More than one MHC:peptide complex may be expressed in a single prokaryotic cell by placing multiple transcriptional cassettes in a single expression vector, or by utilizing different selectable markers for each ofthe expression vectors which are employed in the cloning strategy.
  • a second approach for expressing the MHC:peptide complexes ofthe invention is to cause the polypeptides to be secreted from the cell, either into the periplasm or into the extracellular medium.
  • the DNA sequence encoding the MHC polypeptide is linked to a cleavable signal peptide sequence.
  • the signal sequence directs translocation ofthe MHC:peptide complex through the cell membrane.
  • An example of a suitable vector for use in E. coli that contains a promoter-signal sequence unit is pTAlS29, which has the E. coli phoA promoter and signal sequence see, e.g., Sambrook et al, supra; Oka et al, Proc. Natl. Acad. Sci. USA 82:7212-16, 1985; Talmadge et al,
  • the MHC:peptide complexes ofthe invention can also be produced as fusion proteins. This approach often results in high yields, because normal prokaryotic control sequences direct transcription and translation. In E. coli, lacZ fusions are often used to express heterologous proteins. Suitable vectors are readily available, such as the pUR, pEX, and pMRlOO series (see, e.g., Sambrook et al, supra). For certain applications, it may be desirable to cleave the non-MHC amino acids from the fusion protein after purification.
  • Cleavage sites can be engineered into the gene for the fusion protein at the desired point of cleavage.
  • Foreign genes such as single chain MHC class II molecule:peptide complexes, can be expressed in E. coli as fusions with binding partners, such as glutathione-S-transferase (GST), maltose binding protein, or thioredoxin.
  • GST glutathione-S-transferase
  • thioredoxin thioredoxin
  • binding partners are highly translated and can be used to overcome inefficient initiation of translation of eukaryotic messages in E. coli. Fusion to such binding partner can result in high-level expression, and the binding partner is easily purified and then excised from the protein of interest.
  • GST glutathione-S-transferase
  • thioredoxin thioredoxin.
  • Such binding partners are highly translated and can be used to overcome inefficient initiation of translation of eukaryotic messages in E. coli. Fusion to such binding partner can result in high-level expression, and the binding partner is easily purified and then excised from the protein of
  • the gene of interest is produced as a C-terminal fusion to the first 76 residues ofthe yeast ubiquitin gene containing a peptidase cleavage site.
  • transformation refers to the introduction of vectors containing the nucleic acids of interest directly into host cells by well known methods. The particular procedure used to introduce the genetic material into the host cell for expression ofthe single chain
  • MHC class II molecule :peptide complex is not particularly critical. Any ofthe well known procedures for introducing foreign nucleotide sequences into host cells may be used. It is only necessary that the, particular host cell utilized be capable of expressing the gene.
  • Transformation methods which vary depending on the type ofthe prokaryotic host cell, include electroporation; transfection employing calcium chloride, rubidium chloride calcium phosphate, or other substances; microprojectile bombardment; infection (where the vector is an infectious agent) ; and other methods. See, generally, Sambrook et al, supra, and Ausubel et al, (eds.) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987. Reference to cells into which the nucleic acids described above have been introduced is meant to also include the progeny of such cells. Transformed prokaryotic cells that contain expression vectors for single chain MHC class II molecule :peptide complexes are also included in the invention.
  • the polypeptide is then purified using standard techniques. See, e.g., Colley et al, J. Chem. 64:17619-22, 1989; and Methods in Enzymology, "Guide to Protein Purification", Deutscher, ed., Vol. 182 (1990).
  • the recombinant cells are grown and the single chain MHC class II molecule:peptide complex is expressed.
  • the purification protocol will depend upon whether single chain MHC class II molecule:peptide complex is expressed intracellularly, into the periplasm, or secreted from the cell.
  • the cells are harvested, lysed, and the polypeptide is recovered from the cell lysate (Sambrook et al, supra). Periplasmic MHC polypeptide is released from the periplasm by standard techniques (Sambrook et al, supra). If the MHC polypeptide is secreted from the cells, the culture medium is harvested for purification ofthe secreted protein. The medium is typically clarified by centrifugation or filtration to remove cells and cell debris.
  • the MHC polypeptides can be concentrated by adsorption to any suitable resin (such as, for example, CDP-Sepharose, Asialoprothrombin-Sepharose 4B, or Q
  • Sepharose Sepharose
  • ammonium sulfate fractionation polyethylene glycol precipitation
  • ultrafiltration Other means known in the art may be equally suitable.
  • MHC polypeptides can be accomplished by standard techniques, for example, affinity chromatography, ion exchange chromatography, sizing chromatography, reverse phase HPLC, or other protein purification techniques used to obtain homogeneity.
  • the purified proteins are then used to produce pharmaceutical compositions.
  • recombinant nucleic acid constructs ofthe invention may include sequences that encode signal sequences or other sequences that direct secretion.
  • Secretory signal sequences also called leader sequences, prepro sequences and/or pre sequences, are amino acid sequences that play a role in secretion of mature polypeptides or proteins from a cell.
  • Such sequences are characterized by a core of hydrophobic amino acids and are typically (but not exclusively) found at the amino termini of newly synthesized proteins.
  • the secretory signal sequence may be that ofthe protein of interest, or may be derived from another secreted protein (e.g., t-PA, a prefened mammalian secretory leader) or synthesized de novo.
  • the secretory signal sequence is joined to the DNA sequence encoding a protein ofthe present invention in the conect reading frame.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al, U.S. Patent No. 5,037,743; Holland et al, U.S. Patent No. 5,143,830).
  • secretory peptide is cleaved from the mature protein during secretion.
  • Such secretory peptides contain processing sites that allow cleavage ofthe secretory peptide from the mature protein as it passes through the secretory pathway.
  • An example of such a processing site is a dibasic cleavage site, such as that recognized by the Saccharomyces cerevisiae KEX2 gene or a Lys-Arg processing site.
  • Processing sites may be encoded within the secretory peptide or may be added to the peptide by, for example, in vitro mutagenesis.
  • Secretory signals include the factor signal sequence (prepro sequence:
  • a-2 plasmin inhibitor signal sequence (Tone et al, J. Biochem. (Tokyo) 102: 1033-1042, 1987) and the tissue plasminogen activator signal sequence (Pennica et al, Nature 301: 214-221, 1983).
  • a secretory signal sequence may be synthesized according to the rules established, for example, by von Heinje (European Journal of Biochemistry 133: 17-21, 1983; Journal of Molecular Biology 184: 99-105, 1985; Nucleic Acids Research 14: 4683-4690; 1986).
  • Another signal sequence is the synthetic signal LaC212 spx (1-47). ERLE described in WO 90/10075.
  • Secretory signal sequences may be used singly or may be combined.
  • a first secretory signal sequence may be used in combination with a sequence encoding the third domain of barrier (described in U.S. Patent No. 5,037,243, which is incorporated by reference herein in its entirety).
  • the third domain of barrier may be positioned in proper reading frame 3' ofthe DNA segment of interest or 5' to the DNA segment and in proper reading frame with both the secretory signal sequence and a DNA segment of interest.
  • Proteins ofthe present invention can also be expressed in filamentous fungi, for example, strains ofthe fungi Aspergillus (McKnight et al, U.S. Patent No. 4,935,349, which is incorporated herein by reference). Expression of cloned genes in cultured mammalian cells and inE. coli, for example, is discussed in detail in Sambrook et al. (Molecular Cloning: A Laboratory Manual.
  • suitable yeast vectors for use in the present invention include
  • YRp7 (Struhl etal, Proc. Natl. Acad. Sci. USA 76:1035-1039, 1978), Y ⁇ pl3 (Broach t al, Gene 8: 121-133, 1979), POT vectors (Kawasaki et al, U.S. Patent No. 4,931,373, which is inco ⁇ orated by reference herein), pJDB249 and pJDB219 (Beggs, Nature
  • promoters for use in yeast include promoters from yeast glycolytic genes (Hitzeman et al, J. Biol. Chem. 255: 12073-12080, 1980; Alber and Kawasaki, J. Mol Appl. Genet. 1: 419-434, 1982; Kawasaki, U.S. Patent No. 4,599,311) or alcohol dehydrogenase genes (Young et al, in Genetic Engineering of Microorganisms for Chemicals, Hollaender et al, (eds.), p. 355, 1982; Ammerer, Meth. Enzymol. 101 : 192-201, 1983).
  • the expression units may also include a transcriptional terminator such as the TPIl terminator (Alber and Kawasaki, ibid.).
  • Yeast cells are a prefened host for use in producing compound ofthe cunent invention.
  • Methods for transforming yeast cells with exogenous DNA and producing recombinant proteins therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311 ; Kawasaki et al, U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al, U.S. Patent No. 5,037,743; and Munay et al, U.S. Patent No. 4,845,075, which are incorporated herein by reference.
  • Transformed cells are selected by phenotype determined by a selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine) .
  • a prefened vector system for use in yeast is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • a prefened secretory signal sequence for use in yeast is that ofthe S. cerevisiae MF ⁇ l gene
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g.,
  • Kluyveromyces lactis, Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichia guillermondii and.
  • Candida maltosa are known in the art. See, for example, Gleeson et al, J. Gen. Microbiol. 132:3459-65, 1986; Gregg, U.S. Patent No. 4,882,279; and Stroman et al, U.S. Patent No. 4,879,231.
  • fungal cells are also suitable as host cells.
  • Aspergillus cells may be utilized according to the methods of McKnight et al, U.S. Patent No. 4,935,349, which is incorporated herein by reference. Methods for transforming
  • chrysogenum are disclosed by Sumino et al, U.S. Patent No. 5,162,228, which is incorporated herein by reference. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533, which is incorporated herein by reference.
  • Host cells containing DNA constructs ofthe present invention are then cultured to produce the heterologous proteins.
  • the cells are cultured according to standard methods in a culture medium containing nutrients required for growth ofthe particular host cells.
  • suitable media are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals and growth factors.
  • the growth medium will generally select for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient which is complemented by a selectable marker on the DNA construct or co-transfected with the DNA construct.
  • Yeast cells are preferably cultured in a chemically defined medium, comprising a non-amino acid nitrogen source, inorganic salts, vitamins and essential amino acid supplements.
  • the pH ofthe medium is preferably maintained at a pH greater than 2 and less than 8, preferably at pH 6.5.
  • Methods for maintaining a stable pH include buffering and constant pH control, preferably through the addition of sodium hydroxide.
  • Prefened buffering agents include succinic acid and Bis-Tris (Sigma
  • Yeast cells having a defect in a gene required for asparagine-linked glycosylation are preferably grown in a medium containing an osmotic stabilizer.
  • a prefened osmotic stabilizer is sorbitol supplemented into the medium at a concentration between 0.1 M and 1.5 M, preferably at 0.5 M or 1.0 M.
  • Cultured mammalian cells are generally cultured in commercially available serum-containing or serum-free media. Selection of a medium appropriate for the particular host cell used is within the level of ordinary skill in the art.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al, Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603, 1981; Graham and Van der Eb, Virology 52:456, 1973) , electroporation (Neumann et al, EMBO J. 1 :841-45, 1982) and DEAE-dextran mediated 'transfection (Ausubel et al, (eds), Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1987), which are inco ⁇ orated herein by reference.
  • Cationic lipid transfection using commercially available reagents including the Boehringer Mannheim TRANSFECTION-REAGENT (N-[l- (2,3- dioleoyloxy)propyl] -N,N,N-trimethyl ammomummethylsulfate; Boehringer Mannheim, Indianapolis, IN) or LIPOFECTIN reagent (N-[l-(2,3-dioleoyloxy) ⁇ ropyl]-N,N,N- trimethylammonium chloride and dioleoyl phosphatidylethanolamme; GIBCO-BRL, Gaithersburg, MD) using the manufacturer-supplied directions, may also be used.
  • Boehringer Mannheim TRANSFECTION-REAGENT N-[l- (2,3- dioleoyloxy)propyl] -N,N,N-trimethyl ammomummethylsulfate
  • LIPOFECTIN reagent N-[l-(2,3-dioleo
  • a prefened mammalian expression plasmid is Zem229R (deposited under the terms ofthe Budapest Treaty with American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD on September 28, 1993 as anE. coli HBIOI transformant and assigned Accession Number 69447) .
  • the production of recombinant proteins in cultured mammalian cells is disclosed, for example, by Levinson et al, U.S. Patent No. 4,713,339; Hagen et al, U.S. Patent No. 4,784,950; Palmiter et al, U.S. Patent No. 4,579,821; and Ringold, U.S. Patent No. 4,656,134, which are inco ⁇ orated herein by reference.
  • Prefened cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), DG44, and 293 (ATCC No. CRL 1573; Graham et al, J. Gen. Virol. 36:59-72, 1977) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland.
  • strong transcription promoters are prefened, such as promoters from S V-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288.
  • Other suitable promoters include those from metallothionein genes (U.S. Patents Nos. 4,579,821 and 4,601,978, which are inco ⁇ orated herein by reference) and the adenovirus major late promoter.
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly refened to as
  • transfectants Cells that have been cultured in the presence ofthe selective agent and are able to pass the gene of interest to their progeny are refened to as “stable transfectants.”
  • a prefened selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin-type drag, such as G-418 or the like. Selection systems may also be used to increase the expression level ofthe gene of interest, a process refened to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level ofthe selective agent and then increasing the amount of selective agent to select for cells that produce high levels ofthe products ofthe introduced genes.
  • a prefened amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • Other drug resistance genes e.g. hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • the soluble, fused MHC:peptide complexes ofthe present invention can be purified by first isolating the polypeptides from the cells followed by conventional purification methods, such as by ion-exchange and partition chromatography as described by, for example, Coy et al.
  • the single chain MHC class II molecule :peptide complexes may then be used diagnostically or therapeutically, as further described below.
  • the single chain MHC class II molecule:peptide complexes ofthe present invention may be used within methods for down-regulating parts ofthe immune system that are reactive in autoimmune diseases.
  • the single chain MHC class II molecule:peptide complexes ofthe present invention are contemplated to be advantageous for use as immunotherapeutics to induce immunological tolerance or nonresponsiveness (anergy) in patients predisposed to mount or already mounting an immune response those particular autoantigens.
  • a patient having or predisposed to a particular autoimmune disease is identified and MHC type is determined by methods known in the art.
  • the patient's T cells can be examined in vitro to determine autoantigenic peptide(s) recognized by the patient's autoreactive T cells using complexes and methods described herein.
  • kits can also be supplied for therapeutic or diagnostic uses.
  • the subject composition ofthe present invention may be provided, usually in a lyophilized form, in a container.
  • the single chain MHC class II molecule:peptide complex is included in the kits with instructions for use, and optionally with buffers, stabilizers, biocides, and inert proteins.
  • these optional materials will be present at less than about 5% by weight, based on the amount of single chain MHC class II molecule:peptide complex, and will usually be present in a total amount of at least about 0.001% by weight, based on the single chain MHC class II molecule:peptide complex concentration. It may be desirable to include an inert extender or excipient to dilute the active ingredients, where the excipient may be present in from about 1 to 99% weight of the total composition.
  • single chain MHC class II molecule:peptide complexes are utilized to prepare antibodies for diagnostic or therapeutic uses.
  • antibodies includes polyclonal antibodies, monoclonal antibodies, antigen-binding fragments thereof such as F(ab') 2 and Fab fragments, as well as recombinantly produced binding partners. These binding partners inco ⁇ orate the variable or CDR regions from a gene which encodes a specifically binding antibody. The affinity of a monoclonal antibody or binding partner may be readily determined by one of ordinary skill in the art (see, Scatchard, Ann. NY Acad. Sci. 51: 660-72, 1949)
  • polyclonal antibodies may be generated from a variety of warm-blooded animals, such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, or rats, for example.
  • the immunogenicity ofthe single chain MHC class II molecule:peptide complexes may be increased through the use of an adjuvant, such as Freund's complete or incomplete adjuvant.
  • an adjuvant such as Freund's complete or incomplete adjuvant.
  • assays known to those skilled in the art may be utilized to detect antibodies which specifically bind to a single chain MHC class II molecule:peptide complex. Exemplary assays are described in detail in Antibodies: A Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor
  • Antibodies ofthe present invention may be produced by immunizing an animal selected from a wide variety of warm-blooded animals, such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, and rats, with a recombinant single chain MHC class II molecule :peptide complex. Serum from such animals are a source of polyclonal antibodies. Alternatively antibody producing cells obtained from the immunized animals are immortalized and screened. As the generation of human monoclonal antibodies to a human antigen, such as a single chain MHC class II molecule:peptide complex, may be difficult with conventional immortalization techniques, it may be desirable to first make non-human antibodies.
  • the antigen binding regions ofthe non-human antibody is transfened to the conesponding site of a human antibody coding region to produce a substantially human antibody molecules.
  • Such methods are generally known in the art and are described in, for example, U.S. Patent No. 4,816,397, and ⁇ P publications 173,494 and 239,400, which are inco ⁇ orated herein by reference.
  • the single chain MHC class II molecule :peptide complexes can be used to clone T cells which have specific receptors for the single chain MHC class II molecule:peptide complex.
  • the T cells or membrane preparations thereof can be used to immunize animals to produce antibodies to the single chain MHC class II molecule:peptide complex receptors on T cells.
  • the antibodies can be polyclonal or monoclonal. If polyclonal, the antibodies can be murine, lagomo ⁇ h, equine, ovine, or from a variety of other mammals.
  • Monoclonal antibodies will typically be murine in origin, produced according to known techniques, or human, as described above, or combinations thereof, as in chimeric or humanized antibodies.
  • the anti- single chain MHC class II molecule:peptide complex receptor antibodies thus obtained can then be administered to patients to reduce or eliminate T cell subpopulations that display such receptor.
  • This T-cell population recognizes and participates in the immunological destruction of cells bearing the autoantigenic peptide in an individual predisposed to or already suffering from a disease, such as an autoimmune disease related to the autoantigenic peptide.
  • Antibodies ofthe present invention may be used as a marker reagent to detect the presence of MHC heterodime ⁇ peptide complexes on cells or in solution. Such antibodies are also useful for Western analysis or immunoblotting, particularly of purified cell-secreted material. Polyclonal, affinity purified polyclonal, monoclonal and single chain antibodies are suitable for use in this regard. In addition, proteolytic and recombinant fragments and epitope binding domains can be used herein. Chimeric, humanized, veneered, CDR-replaced, reshaped or other recombinant whole or partial antibodies are also suitable.
  • compositions Pharmaceutically acceptable carriers are determined in part by the particular composition being administered (e.g., nucleic acid, protein) as by the particular method used to administer the composition. Accordingly, there are a wide variety of suitable formulations of pharmaceutical compositions ofthe present invention (see, e.g.,
  • Administration can be in any convenient manner, e.g., by injection, oral administration, inhalation, or transdermal application.
  • Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount ofthe packaged nucleic acid or polypeptide suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount ofthe active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions.
  • liquid solutions such as an effective amount ofthe packaged nucleic acid or polypeptide suspended in diluents, such as water, saline or PEG 400
  • capsules, sachets or tablets each containing a predetermined amount ofthe active ingredient, as liquids, solids, granules or gelatin
  • suspensions in an appropriate liquid and (d) suitable emulsions.
  • Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, macrocrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceutically compatible carriers.
  • Lozenge forms can comprise the active ingredient in a flavor, e.g., sucrose, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • a flavor e.g., sucrose
  • an inert base such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.
  • Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood ofthe intended recipient, and aqueous and nonaqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally.
  • Parenteral administration and intravenous administration are the prefened methods of administration.
  • the formulations of commends can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets ofthe kind previously described.
  • Cells transduced by nucleic acids for ex vivo therapy can also be administered intravenously or parenterally as described above.
  • the polypeptides ofthe invention are admimstered prophylactically or to an individual already suffering from the disease.
  • the compositions are administered to a patient iii an amount sufficient to elicit an effective immune response.
  • An amount adequate to accomplish this is defined as “therapeutically effective dose” or “immunogenically effective dose.”
  • Amounts effective for this use will depend on, e.g., the peptide composition, the manner of administration, the stage and severity ofthe disease being treated, the weight and general state of health ofthe patient, and the judgment ofthe prescribing physician, but generally range for the initial immunization dose (that is for therapeutic or prophylactic administration) from about 0.01 mg to about 50 mg per 70 kilogram patient, more commonly from about 0.5-1 mg to about 10-15 mg per 70 kg of body weight.
  • Boosting dosages are typically from about 0.01 mg to about 50 mg of peptide, more commonly about 0.5-1 mg to about 10-15 mg, using a boosting regimen over weeks to months depending upon the patient's response and condition.
  • a suitable protocol would include injection at time 0, 2, 6, 8, 10 and 14 weeks, followed by booster injections at 24 and 28 weeks.
  • Booster injections can be from one, two, three, four, five or more.
  • Initial and booster injection amounts and timing are determined based on the judgment ofthe physician and the antigen being administered.
  • the initial and booster dose is 1.3 mg, 4 mg, or 13 mg, administered via intramuscular injection, with at least one and up to 3 booster injections at 8 week intervals, or at least one and up to 4 booster injections at 6 week intervals.
  • the therapeutic methods ofthe present invention may involve oral tolerance (Weiner et al, Nature 376: 177-80, 1995), or intravenous tolerance, for example.
  • Tolerance can be induced in mammals, although conditions for inducing such tolerance will vary according to a variety of factors.
  • To induce immunological tolerance in an adult susceptible to or already suffering from an autoantigen-related disease such as IDDM the precise amounts and frequency of administration will also vary. For instance for adults about 20-80 ⁇ g/kg can be administered by a variety of routes, such as parenterally, orally, by aerosols, intradermal injection, and the like. For neonates, tolerance can be induced by parenteral injection or more conveniently by oral administration in an appropriate formulation. The precise amount administrated, and the mode and frequency of dosages, will vary.
  • the single chain MHC class II molecule:peptide complexes will typically be more tolerogenic when administered in a soluble form, rather than in an aggregated or particulate form. Persistence of a single chain MHC class II molecule ⁇ eptide complex of the invention is generally needed to maintain tolerance in an adult, and thus may require more frequent administration ofthe complex, or its administration in a form which extends the half-life ofthe complex. See for example, Sun et al., Proc. Natl. Acad. Sci.
  • a pharmaceutical composition which comprises a single chain MHC class II molecule:peptide complex ofthe present invention contained in a pharmaceutically acceptable carrier or vehicle for parenteral, topical, oral, or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment, according to conventional methods.
  • the composition may typically be in a form suited for systemic injection or infusion and may, as such, be formulated with sterile water or an isotonic saline or glucose solution.
  • Formulations may further include one or more diluents, fillers, emulsifiers, preservatives, buffers, excipients, and the like, and may be provided in such forms as liquids, powders, emulsions, suppositories, liposomes, transdermal patches and tablets, for example.
  • compositions ofthe present invention are administered at daily to weekly intervals.
  • An "effective amount" of such a pharmaceutical composition is an amount that provides a clinically significant decrease in a deleterious T cell-mediated immune response to an autoantigen, for example, those associated with IDDM, or provides other pharmacologically beneficial effects. Such amounts will depend, in part, on the particular condition to be treated, age, weight, and general health ofthe patient, and other factors evident to those skilled in the art.
  • the amount ofthe single chain MHC class II molecule:peptide complex administered will be within the range of 20-80 ⁇ g/kg. Compounds having significantly enhanced half-lives may be administered at lower doses or less frequently.
  • Adjuvants refers to essentially any substance that enhances or potentiates an immune response (antibody and/or cell-mediated) to an exogenous antigen.
  • One prefened type of immunostimulant comprises an adjuvant.
  • Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins.
  • adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, PA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A.
  • Cytokines such as GM-CSF, interleukin-2, -7, -12, and other like growth factors, may also be used as adjuvants.
  • compositions may also include a Mycobacterium species CWS adjuvant, as described above.
  • the effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against the immunogenic peptide.
  • Certain adjuvants for eliciting a predominantly Thl -type response include, for example, a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, together with an aluminum salt.
  • MPL ® adjuvants are available from Corixa Co ⁇ oration (Seattle, WA; see, for example, US Patent Nos. 4,436,727; 4,877,611; 4,866,034 and 4,912,094).
  • CpG-containing oligonucleotides in which the CpG dinucleotide is unmethylated also induce a predominantly Thl response.
  • Such oligonucleotides are well known and are described, for example, in WO 96/02555, WO 99/33488 and U.S. Patent Nos. 6,008,200 and 5,856,462.
  • Immunostimulatory DNA sequences are also described, for example, by Sato et al, Science 273:352, 1996.
  • Another prefened adjuvant comprises a saponin, such as Quil A, or derivatives thereof, including QS21 and QS7 (Aquila Biopharmaceuticals Inc., Framingham, MA); Escin;
  • Digitonin; or Gypsoph ⁇ la or Chenopodium quinoa saponins are known in the art.
  • Other prefened formulations include more than one saponin in the adjuvant combinations ofthe present invention, for example combinations of at least two ofthe following group comprising QS21, QS7, Quil A, ⁇ -escin, or digitonin.
  • the saponin formulations may be combined with vaccine vehicles composed of chitosan or other polycationic polymers, polylactide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc.
  • vaccine vehicles composed of chitosan or other polycationic polymers, polylactide and polylactide-co-glycolide particles, poly-N-acetyl glucosamine-based polymer matrix, particles composed of polysaccharides or chemically modified polysaccharides, liposomes and lipid-based particles, particles composed of glycerol monoesters, etc.
  • the saponins may also be formulated in the presence of cholesterol to form particulate structures such as liposomes or ISCOMs.
  • the saponins may be formulated together with a polyoxyethylene ether or ester, in either a non-particulate solution or suspension, or in a particulate structure such as a paucilamelar liposome or ISCOM.
  • the saponins may also be formulated with excipients such as Carbopol R to increase viscosity, or may be formulated in a dry powder form with a powder excipient such as lactose.
  • the adjuvant system includes the combination of a monophosphoryl lipid A and a saponin derivative, such as the combination of QS21 and
  • 3D-MPL ® adjuvant as described in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol, as described in WO 96/33739.
  • Other formulations comprise an oil-in-water emulsion and tocopherol.
  • Another adjuvant formulation employs QS21, 3D-MPL ® adjuvant and tocopherol in an oil-in-water emulsion is described in WO 95/17210.
  • Another enhanced adjuvant system involves the combination of a CpG- containing oligonucleotide and a saponin derivative particularly the combination of CpG and QS21 as disclosed in WO 00/09159.
  • the formulation additionally comprises an oil in water emulsion and tocopherol.
  • Additional illustrative adjuvants for use in the pharmaceutical compositions ofthe invention include Montanide ISA 720 (Seppic, France), SAF (Chiron, California, United States), ISCOMS (CSL), MF-59 (Chiron), the SBAS series of adjuvants (e.g., SBAS-2 or SBAS-4, available from SmithKline Beecham, Rixensart, Belgium), Detox (Enhanzyn ® ) (Corixa, Hamilton, MT), RC-529 (Corixa, Hamilton, MT) and other aminoalkyl glucosaminide 4-phosphates (AGPs), such as those described in pending U.S. Patent Application Serial Nos. 08/853,826 and 09/074,720, the disclosures of which are inco ⁇ orated herein by reference in their entireties, and polyoxyethylene ether adjuvants such as those described in WO 99/52549 Al.
  • SBAS series of adjuvants e.g., SBAS-2 or SBAS
  • prefened adjuvants include adjuvant molecules ofthe general formula (I): HO(CH 2 CH 2 O) n -A-R, wherein, n is 1-50, A is a bond or -C(O)-, R is C ⁇ -50 alkyl or Phenyl C1-50 alkyl.
  • One embodiment ofthe present invention consists of a vaccine formulation comprising a polyoxyethylene ether of general formula (I), wherein n is between 1 and 50, preferably 4-24, most preferably 9; the R component is C 1 - 50 , preferably C 4 -C 20 alkyl and most preferably C 12 alkyl, and A is a bond.
  • concentration ofthe polyoxyethylene ethers should be in the range 0.1-20%, preferably from 0.1-10%, and most preferably in the range 0.1-1%.
  • Prefened polyoxyethylene ethers are selected from the following group: poly oxy ethylene-9-lauryl ether, poly oxy ethylene-9-steoryl ether, poly oxy ethylene-
  • polyoxyethylene ether according to the general formula (I) above may, if desired, be combined with another adjuvant.
  • a prefened adjuvant combination is preferably with CpG as described in the pending UK patent application
  • compositions ofthe present invention may be formulated for delivery either encapsulated in a lipid particle, a liposome, a vesicle, a nanosphere, or a nanoparticle or the like.
  • Such formulations may be prefened for the introduction of pharmaceutically-acceptable formulations ofthe compositions disclosed herein.
  • liposomes are generally known to those of skill in the art (see, for example, Couvreur et al, 1977; Couvreur, 1988; Lasic, 1998; which describes the use of liposomes and nanocapsules in the targeted antibiotic therapy for intracellular bacterial infections and diseases).
  • liposomes were developed with improved serum stability and circulation half-times (Gabizon & Papahadjopoulos, 1988; Allen and Choun, 1987; U. S. Patent 5,741 ,516, specifically inco ⁇ orated herein by reference in its entirety).
  • Liposomes have been used effectively to introduce genes, drugs (Heath & Martin, 1986; Heath et al, 1986; Balazsovits et al, 1989; Fresta & Puglisi, 1996), radiotherapeutic agents (Pikul et al, 1987), enzymes (Imaizumi et al, 1990a; Imaizumi et al, 1990b), viruses (Faller & Baltimore, 1984), transcription factors and allosteric effectors (Nicolau & Gersonde, 1979) into a variety of cultured cell lines and animals.
  • drugs Heath & Martin, 1986; Heath et al, 1986; Balazsovits et al, 1989; Fresta & Puglisi, 1996)
  • radiotherapeutic agents Pieris et al, 1987
  • enzymes Imaizumi et al, 1990a; Imaizumi et al, 1990b
  • viruses Faller & Baltimore, 1984
  • transcription factors and allosteric effectors Nicol
  • Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multilamellar concentric bilayer vesicles (also termed multilamellar vesicles (MLVs).
  • MLVs generally have diameters of from 25 nm to 4 ⁇ m. Sonication of MLVs results in the formation of small unilamellar vesicles (SUVs) with diameters in the range of 200 to 500 A, containing an aqueous solution in the core.
  • SUVs small unilamellar vesicles
  • Liposomes bear resemblance to cellular membranes and are contemplated for use in connection with the present invention as carriers for the compositions. They are widely suitable as both water- and lipid-soluble substances can be entrapped, i.e., in the aqueous spaces and within the bilayer itself, respectively. It is possible that the drug- bearing liposomes may even be employed for site-specific delivery of active agents by selectively modifying the liposomal formulation. In addition to the teachings of Couvreur et al. (1977, 1988), the following information may be utilized in generating liposomal formulations. Phospholipids can form a variety of structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water.
  • the liposome is the prefened structure.
  • the physical characteristics of liposomes depend on pH, ionic strength and the presence of divalent cations. Liposomes can show low permeability to ionic and polar substances, but at elevated temperatures undergo a phase transition which markedly alters their permeability.
  • the phase transition involves a change from a closely packed, ordered structure, known as the gel state, to a loosely packed, less-ordered structure, known as the fluid state. This occurs at a characteristic phase-transition temperature and results in an increase in permeability to ions, sugars and drugs.
  • lipids In addition to temperature, exposure to proteins can alter the permeability of liposomes. Certain soluble proteins, such as cytochrome c, bind, deform and penetrate the bilayer, thereby causing changes in permeability. Cholesterol inhibits this penetration of proteins, apparently by packing the phospholipids more tightly. It is contemplated that the most useful liposome formations for antibiotic and inhibitor delivery will contain cholesterol.
  • the ability to trap solutes varies between different types of liposomes. For example, MLVs are moderately efficient at trapping solutes, but SUVs are extremely inefficient. SUVs offer the advantage of homogeneity and reproducibility in size distribution. However, a compromise between size and trapping efficiency is offered by large unilamellar vesicles (LUVs). These are prepared by ether evaporation and are three to four times more efficient at solute entrapment than MLVs.
  • LUVs large unilamellar vesicles
  • Liposomes interact with cells via four different mechanisms: endocytosis by phagocytic cells ofthe reticuloendothelial system such as macrophages and damrophils; adso ⁇ tion to the cell surface, either by nonspecific weak hydrophobic or electrostatic forces, or by specific interactions with cell-surface components; fusion with the plasma cell membrane by insertion ofthe lipid bilayer ofthe liposome into the plasma membrane, with simultaneous release of liposomal contents into the cytoplasm; and by transfer of liposomal lipids to cellular or subcellular membranes, or vice versa, without any association ofthe liposome contents. It often is difficult to determine which mechanism is operative and more than one may operate at the same time.
  • liposomes The fate and disposition of intravenously injected liposomes depend on their physical properties, such as size, fluidity, and surface charge. They may persist in tissues for hours or days, depending on their composition, and half lives in the blood range from minutes to several hours. Larger liposomes, such as MLVs and LUVs, are taken up rapidly by phagocytic cells ofthe reticuloendothelial system, but physiology of the circulatory system restrains the exit of such large species at most sites. They can exit only in places where large openings or pores exist in the capillary endothelium, such as the sinusoids ofthe liver or spleen. Thus, these organs are the predominant site of uptake.
  • SUVs show a broader tissue distribution but still are sequestered highly in the liver and spleen. In general, this in vivo behavior limits the potential targeting of liposomes to only those organs and tissues accessible to their large size.
  • Targeting is generally not a limitation in terms ofthe present invention.
  • Antibodies may be used to bind to the liposome surface and to direct the antibody and its drug contents to specific receptors located on a particular cell-type surface.
  • Carbohydrate determinants may also be used as recognition sites as they have potential in directing liposomes to particular cell types.
  • intravenous injection of liposomal preparations would be used, but other routes of administration are also conceivable.
  • the invention provides for pharmaceutically-acceptable nanocapsule formulations ofthe compositions ofthe present invention.
  • Nanocapsules can generally entrap compounds in a stable and reproducible way (Henry-Michelland et al, 1987; Quintanar-Guenero et al, 1998; Douglas et al, 1987).
  • ulfrafine particles sized around 0.1 ⁇ m
  • Biodegradable polyalkyl- cyanoacrylate nanoparticles that meet these requirements are contemplated for use in the present invention.
  • Such particles may be are easily made, as described (Couvreur et al, 1980; 1988; zur Muhlen et al, 1998; Zambaux et al. 1998; Pinto-Alphandry et al, 1995 and U. S. Patent 5, 145,684, specifically inco ⁇ orated herein by reference in its entirety).
  • HLA-DR4 molecules (CO563 and CO564 ⁇ DNA constructs encoding gp39 ⁇ l/ ⁇ l human molecules were prepared from a cDNA encoding the gp39 peptide fused to the ⁇ l/ ⁇ l domains of HLA-DR4 according to standard techniques.
  • a cDNA encoding the fused ⁇ l/ ⁇ l domains of HLA-DR4 was prepared using cloned ⁇ and ⁇ chains from DR4. Amino acid sequence ofthe gp39- ⁇ l/ ⁇ l HLA-DR4 human single chain molecule (linkers are shown in bold)
  • aal - aa4 leader sequence aa5 - aal 8: gp39 peptide aal 9 - aa28: linker aa29 - aal22: HLA-DR4 ⁇ l domain aal23 - aal24: linker aal 25 - aa 208: HLA-DR4 ⁇ l domain
  • the pellet was resuspended in 40 ml of lysis buffer (50 mM Tris-HCl pH 8, 50 mM NaCl, 2 mM EDTA, 1 protease inhibitor cocktail tablet, 1% Triton XI 00 and 1% deoxycholate), and incubated for 1 hour at 4°C under continuous agitation with a magnetic stiner.
  • the sample was then homogenized using a French Press with a 16,000 psi setting, and centrifuged at 4°C, 9000g for 20 min.
  • the pellet was then resuspended in 30 ml of lysis buffer without Triton and deoxycholate and centrifuged at 4°C, 9000 g for 20 min.
  • the new pellet was resuspended in 10 ml of 20 mM ethanolamine/6 M urea pH 10, and eventually frozen at -80°C.
  • the recombinant protein was then purified by FPLC ion-exchange chromatography using Source 30Q anion-exchange media in an XK26/20 column using a step gradient going from 1 mM to 1 M NaCl in 20 mM ethanolamine/6M urea pH 10. Fractions were analyzed by SDS/PAGE and those conesponding to the proteins of interest are pooled and dialyzed against PBS IX.
  • Example 2 Production of additional single chain constructs Additional constructs with different composition and length ofthe 2 nd linker (between ⁇ l and ⁇ l) were engineered by using standard techniques using CO567 as the template. Specifically, PCR primers were designed to replace the old sequence in CO567 with the new sequence. For example, to make CO581, the primers were designed with the following sequences (note these primers were phosphorylated at 5'). Primer 1:
  • PCR reaction 100 ⁇ l was made ofthe following components: 2 ⁇ l CO567 (80 ng) as template, 2 ⁇ l each of primer 1 and primer 2 (10 ⁇ M), 2 ⁇ l of dNTP mix (20 mM each), 10 ⁇ l of 10X pfu buffer, and 80 ⁇ l of sterile water. After all the components were mixed, 2 ⁇ l of Turbo pfu (5U total ) was added, mixed and put on PCR machine. The PCR cycles has a pre-denaturation at 95°C for 30 sec, then 10 cycles of 95°C for 30°C, 60°C for 1 min, and 72°C for 7 min. Then another 22 cycles of 95°C for 30°C, 65°C for 1 min and 72°C for 7 min, followed by a final 10 min at 72°C.
  • the PCR mixture was digested with 2 ⁇ l of Dpnl (lOU) for 2h at 37°C. Then the PCR product at ⁇ 6 kb was purified from agarose gel after electrophoresis. The purified PCR product was ligated by T4 DNA ligase for 1 h at room temperature then used to transform into NovaBlue (Novagen)competent cells by standard protocol. Cells were plated on LB (+Carb) and grow overnight at 37°C.
  • Plasmids from these culture were purified with Wizard Miniprep kit, and analyzed by Xho I digestion. A few plasmids that passed the Xho I digestion were further confirmed by DNA sequencing.
  • Table 1 provides a listing of various constructs made according to the invention. TABLE I
  • linker residues from the functional murine single chain MHC class II molecule and the alpha and beta chains ofthe human molecule.
  • the protein sequence ofthe single chain molecule is provided below (the linker residues from the mouse constract are in bold):
  • a properly folded molecule may be obtained by putting appropriate linkers between portions ofthe human MHC class II which are proximal to each other as determined by visual inspection ofthe atomic coordinates of residues ofthe native MHC available in the publicly accessible protein structure database. These structures would predict possible fusion proteins which covalently attach any part ofthe beta chain between residues 82 to 123 or between residues 148 to 164 to portions ofthe alpha chain such as the N-terminal residues, residues 79 to 84, or 92 to 106.
  • the numbering system of residues in this example conesponds to those found in the coordinates ofthe stracture described in: DESSEN, et al. Immunity 7:473 (1997).
  • homologous residues could be used to create equivalent constructs for genotypic and allelic variants of these molecules e.g. equivalent residues in DR2 or such.
  • DNAs for such hybrids would be prepared and expressed in a recombinant expression system by someone skilled in the art and could be assayed for stracture and function in appropriate assays.
  • Example 5 Use of CD4 binding site(s of MHC class II molecules as linkers for the production of bioactive recombinant MHC class ILpeptide complexes
  • HLA class II molecules present antigenic peptides to the T cell receptor of the CD4+ T lymphocytes and interact with CD4 during the antigen recognition process.
  • Native MHC class Il-peptide complexes have been shown to bind to MHC class II restricted and antigen specific TCRs on a particular T cell and induce T cell nonresponsiveness. It is proposed that the CD4 binding site is important in the docking of MHC class Il-peptide complex with the TCR and induce nonresponsiveness. Since the binding of CD4 to MHC class Il-peptide is important in antigen presentation and/or induction of T cell nonresponsiveness, it is proposed that recombinant MHC class Il- peptide molecules (truncated or whole) containing CD4 binding site will be biologically active. Furthermore, a polypeptide fragment from MHC class II which binds the CD4, when used as a linker in preparation of MHC class Il-peptide truncated molecules, provides resulting recombinant molecules that will be biologically active.
  • L2 is attached to a linker 1 (LI) which is attached to N-terminus of ⁇ l domain that is linked to L2.
  • L2 is linked to L3, which in turn is linked to N-terminus of ⁇ l chain of MHC class II.
  • L2 represents the human CD4 binding sequences. It should be noted that L2 could also be directly linked to N-terminus of ⁇ l domain by completely deleting L3.
  • Specific examples of LI and L3 are given in a examples.
  • the sequences of L2 are given below. These sequences are applicable to most ofthe DR-Peptide molecules.
  • the "full anergix" single chain molecule mouse I- As MBP. ⁇ l ⁇ 2 ⁇ l ⁇ 2.C ⁇ , was generated by overlap PCR using standard methodology. The molecules was expressed in 293T cells and baculoviral cells according to standard methodology, and purified according to standard methodology using affinity chromatography using goat- anti mouse antibodies. The structure of I-As MBP. ⁇ l ⁇ 2 ⁇ l ⁇ 2.C ⁇ is shown in Figure 1.
  • the in vitro activity of recombinant I-As MBP. ⁇ l ⁇ 2 ⁇ l ⁇ 2.C ⁇ was tested using MBP90-101 specific IAs restricted mouse T cell clone HS1.
  • This clone was prepared by the immunization of SJL mice with the MBP90-101 peptide, followed by cloning out CD4+ T cells by limited dilution techniques. These cells were maintained by stimulation every 10 days with inadiated SJF splenocytes and PBP90-101 peptide.
  • the T cells are activated by a combination of soluble recombinant I-As MBP. ⁇ l ⁇ 2 ⁇ l ⁇ 2.C ⁇ and plate bound anti-CD28 antibody. T cell activation was assayed by 3 H-thymidine inco ⁇ oration according to standard methodology. Figure 2 shows the results of this assay.
  • MS is a T cell dependent autoimmune disease caused by localized demyelination in the central nervous system.
  • Experimental autoimmune encephalomyelitis is a accepted animal model of MS. The following results demonstrate that administration of I-As MBP. ⁇ l ⁇ 2 ⁇ l ⁇ 2.C ⁇ reduces the incidence and severity of
  • EAE EAE was induced according to standard methodology according to the myelin model. Ten ⁇ g of recombinant I-As MBP. ⁇ l ⁇ 2 ⁇ l ⁇ 2.C ⁇ was given intravenously in 100 ⁇ l of PBS at days 1, 4, 7, and 11 after disease induction. 12 days after immunization, animals are observed daily for the onset of neurological dysfunction. Disease is graded by trained technicians according to standard methods (see Figure 3). Mice are followed for up to 60-70 days. The data shown in Figure 4 demonstrate that administration ofthe recombinant I-As MBP. ⁇ l ⁇ 2 ⁇ l ⁇ 2.C ⁇ significantly reduced the incidence of myelin- induced EAE in SJL mice.
  • mice developed EAE in the untreated group (55%), whereas only 2 out of 21 animal showed sign ofthe disease in the treated group (16.6%).
  • 12 out of 16 mice developed EAE in the untreated group (75%), while only 2 out of 16 developed EAE in the treated group (12.5%).
  • Example 7 Functional human anergix molecules optimized for E. coli expression with E. coli codons
  • Two human single chain MHC class II molecules (CO528-AC and
  • CO608-AC have been optimized for E. coli expression using "artificial codons," e.g., prefened E. coli codons encoding the mammalian protein.
  • CO528-AC and CO608-AC were made according to standard PCR overlap technology.
  • Example 7 Recombinant MHC class II IAs.MBPJg multimeric complexes
  • Recombinant MHC class II (IAs)-peptide-Ig fusion complexes were constructed by fusing the mlgG leader, MBP 90-101, or MBP1-14 (as a control) to IAs single chain (Mb2ala2), and mlgG.Ck, mlgG.CHl.H, mIgG.CHl.H.CH2, or mlgG. CHI. H.CH2.CH3 with flexible linkers, according to standard methodology.
  • the recombinant IAs fusion proteins were expressed in both mammalian and insect cells and detected by western analysis and ELISA.
  • the overexpressed and secreted recombinant IAs fusion proteins from both human 293 cell cultures or from insect culture medium were purified by affinity chromatography.
  • the purified dimeric and tetrameric recombinant IAs proteins have in vitro biological activity as assayed using an antigen- specific mouse T cell clone.
  • the in vivo activity ofthe recombinant IAs fusion proteins were studied with the experimental autoimmune encephalomyelitis (EAE) model using susceptible SJL mice. In these EAE studies, recombinant IAs fusion protein was delivered on days 1, 4, 7, and 11 by IN. injections after induction ofthe disease with myelin. The animals were then examined for neurological dysfunction.
  • EAE experimental autoimmune encephalomyelitis
  • Example 8 Synthesis of mouse model equivalent of CO608 Four forms ofthe murine MHC Class II IAs ⁇ l ⁇ l with MBP peptide linkers analogous to CO608 human were made. These constructs can be used, e.g., as murine clinical control for human 608.
  • mCO608 mouse CO608, lacking the first five amino acids, GDSER, as compared to native beta 1 domain
  • MCO608-A asame as m608 except lacking four amino acids, GSER, after the methionine as compared to m608
  • megaterium-mCO608-A (expressed in Bacillus megaterium), and mCO608-B (same as m608 except lacking first four amino acids, GSER, after the methionine as compared to m608; also lacking second amino acid, D, as compared to native beta 1 domain).
  • GSER first four amino acids
  • D second amino acid
  • mCO608-A and m6O8-B were made from mCO608 using PCR according to standard methodology.
  • Gin Lys Asp Leu Leu Glu Gin Lys Arg Ala Ala Val Asp Thr Tyr Cys 65 70 75 80 Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gin Arg Arg Thr

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Abstract

L'invention concerne des acides nucléiques codant pour des molécules de classe II du CMH qui forment des multimères via des domaines de multimérisation entre chaînes, ainsi que des méthodes de traitement de maladie auto-immune les utilisant.
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WO2001094418A2 (fr) * 2000-06-05 2001-12-13 Corixa Corporation Nouvelles peptides de tete permettant d'augmenter la secretion d'une proteine recombinee dans une cellule hote
EP1749889A1 (fr) * 2000-06-05 2007-02-07 Corixa Corporation Peptides leader permettant d'augmenter la sécrétion d'une protéine recombinée dans une cellule hôte
NZ533587A (en) * 2001-11-16 2005-11-25 Pharmexa As Immunogenic mimetics of multimer proteins with promiscuous T cell epitope inserts
US8314210B2 (en) 2002-07-12 2012-11-20 Dana-Farber Cancer Institute, Inc. Compositions and methods for the generation of MHC class II compounds by peptide exchange
US20050287631A1 (en) * 2003-11-25 2005-12-29 Martin Kroenke Compositions and methods related to a dimeric MHC class I and II-Like molecule (dsMHCI and dsMHCII)
US7576183B2 (en) * 2003-12-24 2009-08-18 Los Alamos National Security, Llc Structure-based receptor MIMICS targeted against bacterial superantigen toxins
WO2008116468A2 (fr) 2007-03-26 2008-10-02 Dako Denmark A/S Complexes peptidiques du cmh et leurs utilisations dans des maladies infectieuses
EP2167537A2 (fr) 2007-07-03 2010-03-31 Dako Denmark A/S Procedes compiles pour analyser et trier des echantillons
EP2197908A2 (fr) 2007-09-27 2010-06-23 Dako Denmark A/S Multimères cmh dans le diagnostic, le vaccin et le traitement de la tuberculose
US10968269B1 (en) 2008-02-28 2021-04-06 Agilent Technologies, Inc. MHC multimers in borrelia diagnostics and disease
US10722562B2 (en) 2008-07-23 2020-07-28 Immudex Aps Combinatorial analysis and repair
GB0817244D0 (en) 2008-09-20 2008-10-29 Univ Cardiff Use of a protein kinase inhibitor to detect immune cells, such as T cells
WO2010037402A1 (fr) 2008-10-02 2010-04-08 Dako Denmark A/S Vaccins moléculaires contre les maladies infectieuses
US11992518B2 (en) 2008-10-02 2024-05-28 Agilent Technologies, Inc. Molecular vaccines for infectious disease

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999009064A1 (fr) * 1997-08-19 1999-02-25 Mount Sinai School Of Medicine Of New York University ; Element de la classe ii du complexe majeur d'histocompatibilite portant un epitope/molecules chimeres d'immunoglobuline
WO1999013095A2 (fr) * 1997-09-11 1999-03-18 The Johns Hopkins University School Of Medicine Utilisation de molecules polyvalentes du type complexe majeur d'histocompatibilite (cmh)/immoglobuline (ig) chargees en peptides chimeres pour deceler, activer ou supprimer les reponses immunes dependant des cellules t specifiques de l'antigene
WO1999014236A1 (fr) * 1997-09-16 1999-03-25 Oregon Health Sciences University Molecules de cmh recombinees utiles pour la manipulation de cellules t specifiques a un antigene

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5194425A (en) * 1988-06-23 1993-03-16 Anergen, Inc. Mhc-mediated toxic conjugates useful in ameliorating autoimmunity
US6015884A (en) * 1996-03-28 2000-01-18 The Johns Hopkins University Soluble divalent and multivalent heterodimeric analogs of proteins

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999009064A1 (fr) * 1997-08-19 1999-02-25 Mount Sinai School Of Medicine Of New York University ; Element de la classe ii du complexe majeur d'histocompatibilite portant un epitope/molecules chimeres d'immunoglobuline
WO1999013095A2 (fr) * 1997-09-11 1999-03-18 The Johns Hopkins University School Of Medicine Utilisation de molecules polyvalentes du type complexe majeur d'histocompatibilite (cmh)/immoglobuline (ig) chargees en peptides chimeres pour deceler, activer ou supprimer les reponses immunes dependant des cellules t specifiques de l'antigene
WO1999014236A1 (fr) * 1997-09-16 1999-03-25 Oregon Health Sciences University Molecules de cmh recombinees utiles pour la manipulation de cellules t specifiques a un antigene

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BURROWS G G ET AL: "Two-domain MHC class II molecules form stable complexes with myelin basic protein 69-89 peptide that detect and inhibit rat encephalitogenic T cells and treat experimental autoimmune encephalomyelitis" JOURNAL OF IMMUNOLOGY, THE WILLIAMS AND WILKINS CO. BALTIMORE, US, vol. 161, 1998, pages 5987-5996, XP002216664 ISSN: 0022-1767 *
CASARES ET AL: "ENGINEERING AND CHARACTERIZATION OF A MURINE MHC CLASS II-IMMUNOGLOBULIN CHIMERA EXPRESSING AN IMMUNODOMINANT CD4 T VIRAL EPITOPE" PROTEIN ENGINEERING, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 10, no. 11, 1997, pages 1295-1301, XP002097124 ISSN: 0269-2139 *
GREER J M ET AL: "Immunogenic and encephalitogenic epitope clusters of myelin proteolipid protein." JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 1 JAN 1996, vol. 156, no. 1, 1 January 1996 (1996-01-01), pages 371-379, XP002304260 ISSN: 0022-1767 *
RHODE P R ET AL: "SINGLE-CHAIN MHC CLASS II MOLECULES INDUCE T CELL ACTIVATION AND APOPTOSIS" JOURNAL OF IMMUNOLOGY, THE WILLIAMS AND WILKINS CO. BALTIMORE, US, vol. 157, no. 11, 1996, pages 4885-4891, XP000652225 ISSN: 0022-1767 *
See also references of WO0170245A1 *

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