EP1307211A1 - Immune mediators and related methods - Google Patents

Immune mediators and related methods

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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)
French (fr)
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EP1307211A4 (en
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/en
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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

The present invention relates to nucleic acids encoding single chain MHC class II molecules that form multimers via inter-chain multimerization domains, and methods of treating autoimmune disease using the same.

Description

IMMUNE MEDIATORS AND RELATED METHODS
CROSS-REFERENCES TO RELATED APPLICATIONS This application is related to U.S. Serial No. 60/191,274, filed March 22, 2000; U.S. Serial No. 60/204,249, filed May 15, 2000; and U.S. Serial No. 60/264,003, filed January 23, 2001. Each ofthe aforementioned applications are herein incorporated by reference in their entirety.
This application is also related to U.S. Serial No. 09/261,811, filed March 3, 1999; which is a continuation of U.S. Serial No. 08/657,581, filed June 7, 1996, now abandoned; which is a continuation in part of U.S. Serial No. 08/480,002, filed June 7, 1995, now abandoned; U.S. Serial No 09/184,692, filed November 2, 1998, now abandoned; U.S. Serial No. 08/483,241, filed June 1, 1995; U.S. Serial No. 08/482,133, filed June 7, 1995; and U.S. Provisional Application No. 60/005,964, filed October 27, 1995.
BACKGROUND OF THE INVENTION 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. The major function of MHC glycoproteins appears to be the binding and presentation of processed antigen in the form of short antigenic peptides.
In addition to binding foreign or "non-self 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. Over 30 autoimmune diseases are presently known, 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.
There is therefore cunently a great interest in developing pharmaceuticals based on the growing understanding ofthe structure and function ofthe major histocompatibility complex (MHC) antigens. Identification of synthetic autoantigenic peptides, and demonstration that these peptides bind selectively to MHC molecules associated with disease and that stimulates T cells would help to implicate a particular peptide or peptide:MHC complex in susceptibility to an autoimmune disease. In particular, the development of single chain MHC class II complexes would be particularly useful in treatment of a number of diseases associated with antigen presentation by MHC molecules. Furthermore, the development of single chain, multimeric complexes would be of interest (see, e.g., WO 93/10220, WO 98/05684, WO 97/35991, WO 98/03552, WO
99/13095, WO 98/06749, WO 99/09064, and U.S. Patent 5,869,270).
SUMMARY OF THE INVENTION The present invention provides recombinant nucleic acid constructs that encode single chain, recombinant MHC class II molecules comprising a βl domain and an αl domain that may or may not be further linked to an antigenic peptide. In one embodiment, the single chain polypeptide is a βl domain and an αl domain. In another embodiment, the single chain polypeptide is a βl domain-β2 domain (a β chain) and an αl domain- α2 domain (an α 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. As shown below, 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 Cλ, or constant regions from Ig heavy chains, e.g., CHI, hinge, CH2, or CH3). In another embodiment, a leucine zipper domain forms a non-covalent linkage. One of skill will recognize that any of a number of polypeptide sequences can be used for this purpose. The single chain molecules ofthe invention thus can be multimers wherein each single chain molecule is from a different MHC class II allele. In addition, each single chain molecule in the multimer can be bound to a different antigen.
In one embodiment, 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. The in vivo activity of the recombinant single chain I- AS fusion proteins in the ΕAΕ model using susceptible
S JL mice shows that treatment with the recombinant single chain I- AS proteins prevents mortality and significantly reduces paralysis induced by myelin homogenate. Histological examination of sections from ammal spinal cord reveals that these treatments also reduce the inflammatory lesions. These results demonstrate that the single chain MHC class II molecules have therapeutic benefit as antigen-specific drugs for the treatment of autoimmune diseases.
In another embodiment, novel linkers are provided for forming single chain MHC class II molecules. These linkers can be used with the multimer constructs described above. In another embodiment, 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 β chain and a recombinant α 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 Cλ, or constant regions from Ig heavy chains, e.g., CHI, hinge, CH2, or CH3). Such a heterodimer can also be "dimerized" or "multimerized" by the use of additional fusion domains, such as leucine zipper domains or immunoglobulin domains (see Figure 10).
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Schematic structure ofthe recombinant single chain I- AS .MBP.βlαl (monomer) and I-AS.MBP.βlβ2αlα2.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. A. I-AS.MBP.βlαl, monomer, B. I-AS.MBP.Ck, dimer, and C. APC+ antigenic peptide. The positive (anti-CD3) and the negative (HS-1 cell alone) controls are also shown in each panel.
Figure 3. Diagram ofthe EAE model and standard for EAE scoring.
Figure 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.βlαl . This recombinant molecule does not carry the antigenic peptide. Panel D: Treated with the recombinant I-AS.MBP.βlαl, a monomer form.
Figure 5 shows a schematic representation of a βl-αl single chain MHC class II peptide complex that is a dimer with two peptide specificities.
Figure 6 shows a schematic representation of a βlβ2-αlα2 single chain MHC class π peptide complex that is a dimer with two peptide specificities. Figure 7 shows a schematic representation of a β 1 β2-αl α2 single chain
MHC class II peptide complex that is a tetramer with four peptide specificities.
Figure 8 shows a schematic representation of a βlβ2-αlα2 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 βl-αl single chain MHC class II peptide complex; diagram of recombinant βlβ2 chains fused to recombinant αlα2 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.
Figure 12. Sequence comparison of mouse CO561 single chain molecules.
DETAILED DESCRIPTION OF THE INVENTION Introduction 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. Typically, the constructs comprise a first DNA segment encoding a βl domain of a selected MHC class II molecule; a second DNA segment encoding a αl 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. The 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
DNA polysegment. In another embodiment, the present invention also provides recombinant components of an MHC class II heterodimer, which comprise a fusion domain. One recombinant component comprises a βl domain, or optionally a βl domain-β2 domain (i.e., a β chain). One recombinant component comprises an αl domain, or optionally an αl domain- α2 domain (i.e., an α 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. In one embodiment, 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. In one embodiment, 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)). In one embodiment, the molecules are covalently linked using heterobifunctional protein cross-linkers. As shown below, one means for carrying this out is through use of 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. Microbiol. Immunol. 211:121-128 (1996); Klemm βt al, Annu. Rev. Immunol. 16:569-592 (1998); Ho et al, Nature 382:822-826 (1996)). One of skill will recognize that any of a number of polypeptide sequences can be used for this purpose.
In addition, 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. In one example, the multimeric, single chain class II molecules have chains from different DR2 alleles, e.g., DRB5*0101 and DRB1*1501. In another embodiment, 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. In addition, other antigens associated with autoimmune disease, such as acetylcholine receptor and type II collagen, can be linked to the single chain molecules ofthe invention..
In a further embodiment, 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.
Definitions
Prior to setting forth the invention, it may be helpful to an understanding thereof to provide definitions of certain terms to be used hereinafter:
Single chain MHC class II molecule: As used herein this term 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 βl domain and an MHC class II αl domain, optionally βl β2 αl α2 domains or any combination thereof in any order. Such molecules are also known as "fused heterodimers." Optionally, 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. Such MHC domains would include the extracellular portion ofthe two polypeptide chains of Class II MHC. This would include the α chain (αl and α2 domains) and β chain (βl and β2 domains) of Class II MHC. This would include βl and αl, βl, β2 and αl, α2, αl or α2 independent ofthe other, or αl and α2 in tandem (αlα2). It would also include βl or β2 independent ofthe other, or βl and β2 in tandem (βlβ2). This would also include any suitable combination ofthe αl, α2, βl, and β2 domains. 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,
Nature 331:171-173, 1988); 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-
Orbea et al, Ann. Rev. Imm. 7:377-405, 1989). In addition, 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 (MHC) is a family of highly polymorphic proteins, divided into two classes, Class I and Class II, which are membrane-associated and present antigen to T lymphocytes (T cells). MHC Class I and Class II molecules are distinguished by the types of cells on which they are expressed, and by the subsets of T cells which recognize them. Class I MHC molecules (e.g., HLA- A, -B and -C molecules in the human system) are expressed on almost all nucleated cells and are recognized by cytotoxic T lymphocytes (CTL), which then destroy the antigen- bearing cells. 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 (TH). TH 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).
Two distinct antigen processing pathways are associated with the two MHC classes. 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. After the antigenic material is proteolytically processed by the MHC-bearing 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.
The MHC of humans (also refened to as human leukocyte antigens (HLA)) on chromosome 6 has three loci, HLA- A, HLA-B and HLA-C, the first two of which have a large number of alleles encoding alloantigens. 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, β2-microglobulin. The heavy chain consists of two distinct extracellular regions, the membrane distal, peptide binding region formed by the αl and α2 domains, and the membrane proximal, CD8-binding region derived from the α3 domain. β2- 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 α and one β chain in noncovalent association. The two chains are similar to each other, with the α chain having a molecular weight of 32-34 kD and the β 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 α and β chain that comprise the class II molecule have been subdivided into two domains of about 90 amino acids each, called αl, α2, and βl, β2, respectively. The α2 and β2 domains each contain a disulfide-linked loop. The peptide-binding region ofthe class II molecule is formed by the interaction of the αl and βl domains. This interaction results in an open-ended, antigenic peptide- binding groove made up of two α helices, and an eight-stranded β-pleated sheet platform. The α and β chains of Class II molecules are encoded by different MHC genes and are polymorphic (see Addas et al, Cellular and Molecular Immunology, 2d Ed.
(1994), which is incorporated by reference in its entirety). Within the present invention, a prefened α chain is DRA*0101 and a prefened β chain is DRβl* 1501.
MHC Class II alleles
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. 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.
Uses of single chain MHC class II molecules
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. In addition, 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. Previous methods for producing desirable MHC Class II histocompatibility proteins have provided material that contains a mixture of antigenic peptides (Buus et al, Science 242:1045-1047, 1988; and Rudensky et al, Nature 353:622-627, 1991), which can be only partially loaded with a defined antigenic peptide (Watts and McConnel, Pro.
Natl. Acad. Sci. USA 83:9660-64, 1986; and Ceppellini et al, Nature 339:392-94, 1989).
Various methods have been developed to produce heterodimers that do not present endogenous antigens (Stern and Wiley, Cell 68:465-77, 1992; Ljunggren et al, Nature 346:476-80, 1990; and Schumacher et al, Cell 62:563-67, 1990) that can be loaded with a peptide of choice. WO 95/23814 and Kozono et al. have described production of soluble murine Class II molecules, I-Edk and I-Ad, each with a peptide attached by a linker to the
N terminus ofthe β chain. Ignatowicz et al. (J. Immunol. 154:38-62, 1995) have expressed membrane-bound I-Ad with peptide attached. These methods incorporate the use of both membrane-bound heterodimer and soluble heterodimer.
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. Producing the claimed MHC:peptide complexes by recombinant methodology results in specific, high yield protein production, where the final product contains only the properly configured MHC:peptide complex of choice. As used herein, 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 α or β chain of MHC through a peptide linkage, and the C terminal ofthe linked α or β chain may be attached to the N terminal portion of another α or β 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.
The amino acid sequence of 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
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. In one embodiment, 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 α and β segments to properly configure the binding groove, and also allows for maximum positioning ofthe peptide in the groove. In another embodiment, the linker comprises a CD4 binding site, as described below in the Example section (see also Table 1). In another embodiment, longer linkers between the chains contain flexible residues (e.g. alanine or glycine) and polar residues (e.g. serine and threonine). To inhibit the continuation of secondary structure across the linker, prolines can be added to bracket the linkers. These prolines are known to inhibit the formation of alpha helices and beta sheets. In another embodiment, 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 αl and βl are assembled to form the peptide binding groove. Linkers joining segments ofthe α and β 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,
Nature 368:215-221, 1994) dictates the length of linkers joining antigenic peptide, α chain segments and β chain segments. The recombinant portions ofthe molecules ofthe invention can also be directly linked, without additional amino acids linkers.
Identification of autoantigens
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 moleculeφeptide 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. For example, 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. The stimulator cells are pulsed or primed with whole GAD or an appropriate antigenic peptide. For example, stimulator cells from the PBMNCs of LDDM patients can be stimulated with antigenic GAD peptides then combined with PBMNCs or responder cells. After seven or 14 days, 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. The 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). Once such a peptide fragment has been identified, 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. Dissecting the functional role of individual peptides and peptide clusters in the interaction of a peptide ligand with an MHC molecule, and also in subsequent T cell recognition and reactivity, is a difficult undertaking due to the degeneracy of peptide binding to the MHC. Changes in T cell recognition or in the ability of an altered peptide to associate with MHC can be used to establish that a particular amino acid or group of amino acids comprises part of an MHC or T cell determinant. The interactions of altered peptides can be further assessed by competition with the parental peptide for presentation to a T cell, or through development of direct peptide-MHC binding assays. Changes to a peptide that do not involve MHC binding could well affect T cell recognition. For example, in a peptide, specific MHC contact points might only occur within a central core of a few consecutive or individual amino acids, whereas those amino acids involved in T cell recognition may include a completely different subset of residues.
In a prefened method, 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. 10:835-73, 1992; Hammer et al, Cell 74:197-203, 1993; Evavold et al, Immunol. Today 14:602-9, 1993; Hammer et al, Proc. Natl. Acad. Sci. USA 91:4456-60, 1994; and Reich et al, J. Immunol. 154:2279-88, 1994). One method would involve generating a panel of altered peptides wherein individual or groups of amino acid residues are substituted with conservative, semi-conservative or non- conservative residues. 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. Additional structural and conformational information regarding each residue and the peptide as a whole can be gained, for example, by synthesizing a series of analogs wherein each residue is substituted with a D- amino acid such as D-alanine (D-Ala scan) (Galantino et al, in Smith, J. and Rivier, J. (eds.), Peptides Chemistry and Biology (Proceedings ofthe Twelfth American Peptide Symposium), ESCOM, Leiden, 1992, pp. 404-05). 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 Ν- or C- terminus to determine the shortest active peptide, or between the Ν- 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.
Analysis of single chain MHC class II moleculeφeptide complexes
The physical and biological properties ofthe single chain MHC class II molecule:peptide complexes may be assessed in a number of ways. 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.
FACs analysis can be used to determine proper folding ofthe single chain complex.
An ELISA (Enzyme-linked Immunosorbent Assay) can be used to measure concentration and confirm conect folding ofthe single chain MHC class II molecule:peptide complexes. 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. In an exemplary ELISA, an antibody that detects the recombinant MHC haplotype is coated onto wells of a microtiter plate. In a prefened embodiment, the antibody is L243, a monoclonal antibody that recognizes only conectly folded HLA-DR MHC dimers. One of skill in the art will recognize that other MHC Class II-specific antibodies are known and available. Alternatively, there are numerous routine techniques and methodologies in the field for producing antibodies (for example, Hunell, (ed)., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press Inc., Boca Raton, FL, 1982), if an appropriate antibody for a particular haplotype does not exist. 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. 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.
In the ELISA format, 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.
Other assay strategies can incorporate specific T-cell receptors to screen for their conesponding MHC-peptide complexes, which can be done either in vitro or in vivo. For example, an in vitro anergy assay determines if non-responsiveness has been induced in the T cells being tested. Briefly, an MHC molecule containing antigenic peptide in the peptide binding groove can be mixed with responder cells, preferably peripheral blood mononuclear cells (PBMN) (a heterogeneous population including B and
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.
Subsequently, these responder cells are combined with stimulator cells
(antigen presenting cells; APCs) that have been pulsed or primed with the same antigenic peptide. In a prefened embodiment, 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. 179:2017-22, 1994), or in vivo or in vitro primed or pulsed splenocytes. Stimulator cells from mammals immunized with, or having a demonstrable cellular immune response to, the antigenic peptide are particularly prefened. For certain assay formats, 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). Further, to assure that non-responsiveness represents anergy, the proliferation assay may be set upϊn duplicate, +/- recombinant IL-2 since it has been demonstrated that IL-2, can rescue anergized cells.
After an approximately 72 hour incubation, the activation of responder cells in response to the stimulator cells is measured. In a prefened embodiment, responder cell activation is determined by measuring proliferation using 3H-thymidine uptake (Crowley et al, J. Immunol. Meth. 133:55-66, 1990). Alternatively, 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.
Preferably, the single chain MHC class LT molecule:peptide complex induces non-responsiveness (for example, anergy) in the antigenic peptide-reactive responder cells. In addition to single chain MHC class II molecule :peptide complex recognition, responder cell activation requires the involvement of co-receptors on the stimulator cell (the APC) that have been stimulated with co-stimulatory molecules. 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 moleculeφeptide complex.
In a prefened embodiment, responder cells are obtained from a source manifesting an autoimmune disease or syndrome. Alternatively, autoantigen-reactive T cell clones or lines are prefened responder cells. In another prefened embodiment, stimulator cells are obtained from a source manifesting an autoimmune disease or syndrome. Alternatively, APC cell lines or clones that are able to appropriately process and/or present autoantigen to responder cells are prefened stimulator cells. In a particularly prefened embodiment, responder and stimulator cells are obtained from a source with diabetes or multiple sclerosis.
At this point, 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. For instance, 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.
Other methods to assess the biological activity ofthe single chain MHC class II molecule:peptide complexes are known in the art and can be used herein, such as using a microphysiometer, to measure production of acidic metabolites in T cells following interaction with antigenic peptide. Other assay methods include competition assays, comparing single chain MHC class II molecule omplex response with that to the normal antigen. Also measurement production of such indicators as cytokines or γ interferon can provide an indication of complex response.
Animal models of autoimmune disease
Similar assays and methods can be developed for and used in animal models. For instance, 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. These diseases include, but are not limited to, insulin-dependent diabetes mellitus, multiple sclerosis, myasthenia gravis, pernicious anemia, rheumatoid arthritis, and systemic lupus erythematosus.
For example, 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) . Alternatively, induced models of autoimmune disease, such as EAE, can be treated with pharmaceutical composition. Treatment in a preventive or intervention mode can be followed by monitoring the clinical symptoms of EAE.
Following is a description of several other animal models of HLA-DR- associated autoimmune disease which can be used to assay in vivo effects ofthe peptide. It will be obvious to one of skill in the art that other suitable animal models for autoimmune diseases can be utilized in a similar manner.
Systemic Lupus Erythematosus (SLE)
Ε\ hybrids of autoimmune New Zealand black (NZB) mice and the phenotypically normal New Zealand White (NZW) mouse strain develop severe systemic autoimmune disease, more fulminant than that found in the parental NZB strain. These 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.
147:1653 (1978)), which is incorporated hereby by reference.
In both the human and murine forms ofthe disease, a strong association with MHC gene products has been reported. 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-2d/u), a gene linked to the h-2u 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.
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.
Myasthenia Gravis fiVlG
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. In MG antibodies to the acetyl choline receptors (AcChoR) impair neuromuscular transmission by mediating loss of AcChoR in the postsynaptic membrane.
S JL/J female mice are a model system for human MG. In these animals, 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. For example, emulsified AcChoR, 15 μg in complete Freund adjuvant, is injected mtradermally among six sites on the back, the hind foot pads, and the base ofthe tail. Animals are reimmunized with this same regimen 4 weeks later.
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. In an exemplary assay, the standard reagent volume is 50 μl per well. Reagents are usually incubated in the wells for 2 hr at RT. Five μg of AcChoR diluted in bicarbonate buffer, pH 9.6, is added to each well. After incubation with
AcChoR, the plates are rinsed four times with a wash solution consisting of phosphate- buffer saline containing 0.05% Tween and 0.05% NaN3. Mouse sera are diluted in 0.01M PBS (pH 7.2), 1.5 mfr MgCl2, 2.0 mM 2-mercaptoethanol, 0.05% Tween-80, 0.05% NaN3 (p-Tween buffer) and incubated on the plate. After the plate is washed, beta- galactosidase-conjugated sheep anti-mouse antibody diluted in P-Tween buffer is added to each well. After a final washing, 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.
The effect of treatment with the invention on clinical EAMG can also be assessed by any method known to those of skill in the art. 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.
Rheumatoid Arthritis (RA In humans, susceptibility to rheumatoid arthritis is associated with HLA
D/DR. 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.
Proc. 43:1820 (1984)).
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
Wooley et al, J. Immunol. 134:2366 (1985), incorporated herein by reference.
In another model adjuvant arthritis in rats is an experimental model for human arthritis, and a prototype of autoimmune arthritis triggered by bacterial antigens
(Holoschitz et al, Prospects of Immunology (1986); Pearson, Arthritis Rheum. 7:80 (1964)). 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.
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.
Insulin Dependent Diabetes Mellitus (IDDM) IDDM is observed as a consequence ofthe selective destruction of insulin- secreting cells within the Islets of Langerhans ofthe pancreas. Involvement ofthe immune system in this disease is suggested by morphologic evidence of early infiltration ofthe Islets by mononuclear cells, by the detection of anti-islet cell antibodies, by the high frequency of HLA-DR3 and -DR4 alleles in IDDM populations, and by clinical associations between IDDM and various autoimmune diseases. An animal model for spontaneous IDDM and thyroiditis has been developed in the BB rat. As in humans, 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
(1985). In morphologic evaluation, 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.
In another model, the NOD mouse strain (H-2Kd Db) 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). Untreated animals develop profound glucose intolerance and ketosis and succumb within weeks ofthe onset ofthe disease. Seventy to ninety percent of female and 20-30% of male animals develop diabetes within the first six months of life. Breeding studies have defined at least two genetic loci responsible for disease susceptibility, one of which maps to the MHC. Characterization of NOD Class II antigens at both the serologic and molecular level suggest that the susceptibility to autoimmune disease is linked to I-AB (Acha-Orbea and McDevitt, Proc. Natl. Acad. Sci. USA 84:235 (1907)).
Treatment of Female NOD mice with complex is expected to lengthen the time before the onset of diabetes and/or to ameliorate or prevent the disease. Experimental Allergic Encephalomyelitis (EAE
Experimental allergic encephalomyelitis (EAE) is an induced autoimmune disease ofthe central nervous system which is a model for multiple sclerosis (MS). The disease can be induced in many species, including mice and rats.
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).
One ofthe genes mediating susceptibility is localized in the MHC class II region (Moore et al, J. Immunol. 124:1815-1820 (1980)). The best analyzed encephalitogenic protein is myelin basic protein (MBP), but other encephalitogenic antigens are found in the brain. The immunogenic epitopes have been mapped (see, Acha- Orbea et al, supra.). In the PL mouse strains (H-2U) two encephalitogenic peptides in MBP have been characterized: MBP peptide p35-47 (MBP 35-47), and acetylated NSF pi -9 (MBP 1-9).
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.
Methods of making the complexes of the invention
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. According to the invention, 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. For instance, a promoter or enhancer is operably linked to a coding sequence if it effects the transcription ofthe sequence. Generally, 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.
The 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 molecμles 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).
Plasmid vectors that contain replication sites and control sequences derived from a species compatible with the chosen host are used. For example, 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. Often, 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. In addition to a promoter sequence, 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. Commonly used 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
198:1056, 1977) and the tryptophan (trp) promoter system (Goeddel et al, Nucleic Acids Res. 8:4057-74, 1980) and the lambda-derived P promoter and N-gene ribosome binding site (Shimatake et al, Nature 292:128-32, 1981). Any available promoter system that functions in prokaryotes can be used.
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. For expression of single chain MHC class II molecule:peptide complexes in prokaryotic cells other than E. coli, 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. For example, 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). In one embodiment, for purification and refolding 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. In one embodiment, e.g., for large scale refolding, the sample can be ultrafiltered into a urea-borate buffer to which is then added 0.2 μM to 1 mM copper sulfate, preferably 0.2 to 20 μM, 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,
Proc. Natl. Acad. Sci. USA 77:39892, 1980; Takahara et al, J. Biol. Chem. 260: 2670-74, 1985). Once again, multiple polypeptides can be expressed in a single cell for periplasmic association. Eukaryotic signal sequences are also well known to those of skill in the art, and cause the MHCφeptide complexes ofthe invention to be secreted into the extracellular medium.
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. This can be accomplished by any of several methods known in the art, including cleavage by cyanogen bromide, a protease, or by Factor X, (see, e.g., Sambrook et al, supra.; Goeddel et al, Proc. Natl. Acad. Sci. USA 76:106-10, 1979; Nagai et al, Nature 309:810-12, 1984; Sung et al, Proc. Natl. Acad. Sci. USA 83:561-65, 1986). 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. These 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. Such expression systems are available from numerous sources, such as Invitrogen Inc. (San Diego, CA) and Pharmacia LKB Biotechnology Inc. (Piscataway, NJ). A method for obtaining recombinant proteins from E. coli which maintains the integrity of their N-termini has been described by Miller et al. Biotechnology 7:698- 704 (1989). In this system, 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.
Cleavage at the junction ofthe two moieties results in production of a protein having an intact authentic N-terminal reside.
The vectors containing the nucleic acids that code for the single chain MHC class II moleculeφeptide complexes are transformed into prokaryotic host cells for expression. "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.
After standard transfection or transformation methods are used to produce prokaryotic cell lines that express large quantities ofthe single chain MHC class II molecule:peptide complex polypeptide, 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. For intracellular expression, 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), or by use of ammonium sulfate fractionation, polyethylene glycol precipitation, or by ultrafiltration. Other means known in the art may be equally suitable.
Further purification ofthe 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.
For secretion of a polypeptide or protein of interest, 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). Very often the 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:
Kurjan & Herskowitz, Cell 3:933-943, 1982; Kurjan et al, U.S. Patent No. 4,546,082; Brake, EP 116, 201), the PH05 signal sequence (Beck et al, WO 86/00637) , the BARl secretory signal sequence (MacKay et al, U.S. Patent No. 4,613,572; MacKay, WO
87/002670), the SUC2 signal sequence (Carlsen et al, Molecular and Cellular Biology 3:
439-447 , 1983) , the a-1-antitrypsin signal sequence (Kurachi et al, Proc. Natl. Acad. Sci.
USA 78: 6826-6830, 1981) , the 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). Alternately, 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. For example, 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.
The choice of suitable promoters, terminators and secretory signals for all expression systems, is well within the level of ordinary skill in the art. Methods for expressing cloned genes in Saccharomyces cerevisiae are generally known in the art (see, "Gene Expression Technology," Methods in Enzymology, Vol. 185, Goeddel (ed.), Academic Press, San Diego, CA, 1990 and "Guide to Yeast Genetics and Molecular Biology, "Methods in Enzymology, Guthrie and Fink (eds.), Academic Press, San Diego, CA, 1991; which are incorporated herein by reference). 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.
Second Edition, Cold Spring Harbor, NY, 1989; which is incorporated herein by reference). As would be evident to one skilled in the art, one could express the proteins ofthe instant invention in other host cells such as avian, insect and plant cells using regulatory sequences, vectors and methods well established in the literature.
In yeast, suitable yeast vectors for use in the present invention include
YRp7 (Struhl etal, Proc. Natl. Acad. Sci. USA 76:1035-1039, 1978), YΕpl3 (Broach t al, Gene 8: 121-133, 1979), POT vectors (Kawasaki et al, U.S. Patent No. 4,931,373, which is incoφorated by reference herein), pJDB249 and pJDB219 (Beggs, Nature
275:104-108, 1978) and derivatives thereof. Prefened 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). Other promoters are the TPIl promoter (Kawasaki, U.S. Patent No. 4,599,311, 1986) and the ADH2-4C promoter (Russell et al, Nature 304: 652- 654, 1983; Irani and Kilgore, U.S. Patent Application Serial No. 07/784,653, CA 1,304,020 and EP 284 044, which are incorporated herein by reference). The expression units may also include a transcriptional terminator such as the TPIl terminator (Alber and Kawasaki, ibid.).
Yeast cells, particularly cells ofthe genus Pichia or Saccharomyces, 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 MFαl gene
(Brake, ibid.; Kurjan et al, U.S. Patent No. 4,546,082) . Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g.,
Kawasaki, U.S. Patent No. 4,599,311; Kingsman et α/., U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092, which are incorporated herein by reference) and alcohol dehydrogenase genes. See also U.S. Patent Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454, which are incorporated herein by reference. Transformation systems for other yeasts, including Hansenula polymorpha, Schizosaccharomycespom.be,
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.
Other fungal cells are also suitable as host cells. For example, 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
Acremonium 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. A variety of 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, for example, 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
Chemical Co., St. Louis, MO). 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 incoφorated 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)ρropyl]-N,N,N- trimethylammonium chloride and dioleoyl phosphatidylethanolamme; GIBCO-BRL, Gaithersburg, MD) using the manufacturer-supplied directions, may also be used. 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 incoφorated 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.
In general, 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 incoφorated 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) can also be used. 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. (Peptides Structure and Function, Pierce Chemical Company, Rockford, IL, pp 369-72, 1983) or by reverse-phase chromatography as described, for example, by Andreu and Merrifield (Eur. J. Biochem. 164: 585-90, 1987), or by HPLC as described, for example, by Kofod et al. (Int. J. Peptide and Protein Res. 32.: 436-40, 1988). Additional purification can be achieved by additional conventional purification means, such as liquid chromatography, gradient centrifugation, and gel electrophoresis, among others. Methods of protein purification are known in the art (see generally, Scopes, R., Protein Purification, Springer- Verlag, NY, 1982, which is incoφorated by reference herein) and can be applied to the purification ofthe recombinant polypeptides described herein. Single chain MHC class II molecule:peptide complexes of at least about 50% purity are prefened, at least about 70-80% purity more prefened, and about
95-99% or more pμrity most prefened, particularly for pharmaceutical uses. Once purified, either partially or to homogeneity, as desired, the single chain MHC class II molecule :peptide complexes may then be used diagnostically or therapeutically, as further described below.
Methods of using single chain MHC class II molecule:peptide complexes
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. The patient can then be treated with complexes ofthe invention. Such methods will generally include administering single chain MHC class II molecule:peptide complex in an amount sufficient to lengthen the time period before onset ofthe autoimmune disease and/or to ameliorate or prevent that disease. Single chain MHC class II molecule:peptide complexes ofthe present invention are therefore contemplated to be advantageous for use in both therapeutic and diagnostic applications related to auto immune diseases. Kits can also be supplied for therapeutic or diagnostic uses. Thus, 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. Generally, 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.
Within one aspect ofthe present invention, single chain MHC class II molecule:peptide complexes are utilized to prepare antibodies for diagnostic or therapeutic uses. As used herein, the term "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 incoφorate 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)
Methods for preparing polyclonal and monoclonal antibodies have been well described in the literature (see, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989; and Hunell, J. G. R., Ed., Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC Press, Ind., Boca Raton, FL, 1982, which is incoφorated herein by reference) . As would be evident to one of ordinary skill in the art, 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. A variety of 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
Laboratory Press, 1988. Representative examples of such assays include: concunent immunoelectrophoresis, radio-immunoassays, radio-immunoprecipitations, enzyme- linked immuno-sorbent assays, dot blot assays, inhibition or competition assays, and sandwich assays. Additional techniques for the preparation of monoclonal antibodies may be utilized to construct and express recombinant monoclonal antibodies. Briefly, mRNA is isolated from a B cell population and used to create heavy and light chain immunoglobulin cDNA expression libraries in a suitable vector such as the λΙMMUNOZAP(H) and λIMMUNOZAP(L) vectors, which may be obtained from
Stratagene Cloning Systems (La Jolla, CA). These vectors are then screened individually or are co-expressed to form Fab fragments or antibodies (Huse et al, Science 246 1275- 81, 1989; Sasfry et al, Proc. Natl. Acad. Sci. USA 86: 5728-32, 1989). Positive plaques are subsequently converted to a non-lytic plasmid which allows high level expression of monoclonal antibody fragments in E. coli.
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. Using recombinant DNA techniques, 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 ΕP publications 173,494 and 239,400, which are incoφorated herein by reference.
In another aspect ofthe invention, 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. Once the single chain MHC class II molecule:peptide complex-specific T cells are isolated and cloned using techniques generally available to the skilled artisan, 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, lagomoφh, 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.
The coupling of antibodies to solid supports and their use in purification of proteins is well known in the literature (see, for example, Methods in Molecular Biology.
Vol. 1, Walker (Ed.), Humana Press, New Jersey, 1984, which is incoφorated by reference herein in its entirety). Antibodies ofthe present invention may be used as a marker reagent to detect the presence of MHC heterodimeπpeptide 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.
Pharmaceutical 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.,
Remington 's Pharmaceutical Sciences ed., 1989). 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.
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.
The compound of choice, alone or in combination with other suitable components, can be made into aerosol formulations (t'.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.
Formulations suitable for parenteral administration, such as, for example, by mtraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, 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. In the practice of this invention, 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. In one embodiment, 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 μg/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 moleculeφeptide 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.
USA 91: 10795-99, 1994.
Within another aspect ofthe invention, a pharmaceutical composition is provided 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.
Pharmaceutical 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. Preferably the amount ofthe single chain MHC class II molecule:peptide complex administered will be within the range of 20-80 μg/kg. Compounds having significantly enhanced half-lives may be administered at lower doses or less frequently.
Adjuvants An immunostimulant 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. Certain 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.
The 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 Coφoration (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 Gypsophϊla or Chenopodium quinoa saponins . 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, β-escin, or digitonin. Alternatively 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. The saponins may also be formulated in the presence of cholesterol to form particulate structures such as liposomes or ISCOMs. Furthermore, 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 CarbopolR to increase viscosity, or may be formulated in a dry powder form with a powder excipient such as lactose.
In one embodiment, 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. Preferably 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 incoφorated herein by reference in their entireties, and polyoxyethylene ether adjuvants such as those described in WO 99/52549 Al.
Other prefened adjuvants include adjuvant molecules ofthe general formula (I): HO(CH2CH2O)n-A-R, wherein, n is 1-50, A is a bond or -C(O)-, R is Cι-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 C1-50, preferably C4-C20 alkyl and most preferably C12 alkyl, and A is a bond. The 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-
8-steoryl ether, polyoxyethylene-4-lauryl ether, polyoxyethylene-35-lauryl ether, and polyoxyethylene-23-lauryl ether. Polyoxyethylene ethers such as polyoxyethylene lauryl ether are described in the Merck index (12th edition: entry 7717). These adjuvant molecules are described in WO 99/52549.
The polyoxyethylene ether according to the general formula (I) above may, if desired, be combined with another adjuvant. For example, a prefened adjuvant combination is preferably with CpG as described in the pending UK patent application
GB 9820956.2.
Liposome-. Nanocapsule-, and Microparticle-Mediated Delivery In certain embodiments, the inventors contemplate the use of liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, for the introduction ofthe compositions ofthe present invention into the subjects. In particular, the 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. The formation and use of liposomes is 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). Recently, 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 incoφorated herein by reference in its entirety). Further, various methods of liposome and liposome-like preparations as potential drug carriers have been reviewed (Takakura, 1998; Chandran et al, 1997; Margalit, 1995; U. S. Patent 5,567,434; U. S. Patent 5,552,157; U. S. Patent 5,565,213; U. S. Patent 5,738,868 and U. S. Patent 5,795,587, each specifically incoφorated 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. In addition, several successful clinical trials examining the effectiveness of liposome- mediated drug delivery have been completed (Lopez-Berestein et al, 1985a; 1985b;
Coune, 1988; Sculier et al, 1988). Furthermore, several studies suggest that the use of liposomes is not associated with autoimmune responses, toxicity or gonadal localization after systemic delivery (Mori & Fukatsu, 1992).
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 μm. 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.
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. At low ratios 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.
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.
In addition to liposome characteristics, an important determinant in entrapping compounds is the physicochemical properties ofthe compound itself. Polar compounds are trapped in the aqueous spaces and nonpolar compounds bind to the lipid bilayer ofthe vesicle. Polar compounds are released through permeation or when the bilayer is broken, but nonpolar compounds remain affiliated with the bilayer unless it is disrupted by temperature or exposure to lipoproteins. Both types show maximum efflux rates at the phase transition temperature.
Liposomes interact with cells via four different mechanisms: endocytosis by phagocytic cells ofthe reticuloendothelial system such as macrophages and neufrophils; adsoφtion 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.
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.
On the other hand, 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.
These include the blood, liver, spleen, bone manow, and lymphoid organs.
Targeting is generally not a limitation in terms ofthe present invention.
However, should specific targeting be desired, methods are available for this to be accomplished. 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 (glycoprotein or glycolipid cell-surface components that play a role in cell-cell recognition, interaction and adhesion) may also be used as recognition sites as they have potential in directing liposomes to particular cell types. Mostly, it is contemplated that intravenous injection of liposomal preparations would be used, but other routes of administration are also conceivable.
Alternatively, 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). To avoid side effects due to intracellular polymeric overloading, such ulfrafine particles (sized around 0.1 μm) should be designed using polymers able to be degraded in vivo. 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 incoφorated herein by reference in its entirety).
All publications and patent applications cited in this specification are herein incoφorated by reference as if each individual publication or patent application were specifically and individually indicated to be incoφorated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for pmposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light ofthe teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope ofthe appended claims.
The following example is provided by way of illustration only and not by way of limitation. Those of skill in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.
EXAMPLES Example 1 : Construction of DNA sequences encoding human single chain MHC class II φeptide complexes. HLA-DR4 molecules(CO563 and CO564\ DNA constructs encoding gp39 βl/αl human molecules were prepared from a cDNA encoding the gp39 peptide fused to the βl/αl domains of HLA-DR4 according to standard techniques. For the production of "empty" βl/ l DR4 molecules, a cDNA encoding the fused βl/αl domains of HLA-DR4 was prepared using cloned α and β chains from DR4. Amino acid sequence ofthe gp39-βl/αl HLA-DR4 human single chain molecule (linkers are shown in bold)
MGDTGRSFTLASSETGVGASGGGGSGGGGDTRPRFLEQVKHECH FFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNSQKDLLEQ KRAAVDTYCRHNYGVGESFTVQRRGGKEEHVIIQAEFYLNPDQSGEFMFDFDG DEIFHVDMAKJ^TVWRLEEFGRFASFEAQGALANIAVDKANLEMTKRSNYTPIT N* aal - aa4: leader sequence aa5 - aal 8: gp39 peptide aal 9 - aa28: linker aa29 - aal22: HLA-DR4 βl domain aal23 - aal24: linker aal 25 - aa 208: HLA-DR4 αl domain
Amino acid sequence ofthe "empty" βl/αl HLA-DR4 human single chain molecule (linkers in bold): MGDTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYRAVTEL GRPDAEYWNSQRDLLEQKRAAVDTYCRHNYGVGESFTVQRRGGΓKEEHVIIQAE FYLNPDQSGEFMFDFDGDEIFHVDMAKKETVWPVLEEFGPVFASFEAQGALANIAV DKANLEΓMTKRSNYTPITN* aa 1 - aa95: HLA-DR4 βl domain aa96- aa97: linker aa98- aal 81 : HLA-DR4 αl domain
For the production of recombinant proteins, the bacteria (pLysS) were grown in LB (containing ampicillin (50 μg / ml) and chloramphenicol (5 μg / ml)) at 37°C until OD 600 = 0.5. IPTG was added at the final concentration of 0.5 mM final and the bacteria were further incubated for 3 hours at 37°C with shaking. The bacteria were centrifuged at 4°C, 4000xg for 20 min and the pellet was frozen at -80°C. The following day, 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 2nd linker (between βl and αl) 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:
5 ' pC ACC AGGAGGAGAGCCGCCC ACGCCGGTCTCGCTGG Primer 2: 5 ' pGACC ACCTGGATCTGGGGAC ACCCGACCACGTTTC
PCR reaction (100 μl) was made ofthe following components: 2 μl CO567 (80 ng) as template, 2 μl each of primer 1 and primer 2 (10 μM), 2 μl of dNTP mix (20 mM each), 10 μl of 10X pfu buffer, and 80 μl of sterile water. After all the components were mixed, 2 μl 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 μl 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.
Next day, about a dozen single colony from the transformation were randomly picked for overnight culture in 5 ml LB (+Carb) 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.
To express the recombinant proteins, a clone with confirmed DNA sequence was used to transform BL21(DE3)CodonPlus-RIL (Stratagene) by standard transformation protocol and plated on LB (Carb+Cam) plates overnight at 37°C. Next morning, a single colony as picked to inoculate 100 ml LB (+Carb +Cam) and the culture was grown till OD reach between 0.8-1.0, and stored overnight at 4°C. Next day, the culture was pellet down and used to inoculate into 2xYT (+Carb+Cam) at ratio of 25 ml culture per liter new media. These large cultures were grown at 37°C till OD=0.5-0.6, and IPTG was added to induce recombinant protein at 37°C for 3 h. The induced cultures were pellet and stored at -80°C till purification.
Table 1 provides a listing of various constructs made according to the invention. TABLE I
Construct peptide upstream Knker downstream linker
C0523 yes GGGG GG
C0543 none none GG
C0563 yes ASGGGSGGG GG
C0567 yes ASGGGSGGG TSGGGGSGGGGSSS
C0580 yes ASGGGSGGG GSPGGGGSGGGPGS
C0581 yes ASGGGSGGG GSPPGGPPGS
C0582 yes ASGGGSGGG GSPGGGGPGS
C0583 yes ASGGGSGGG TSGGGGS
C0584 yes ASGGGSGGG SGGSGGS
C0585 yes ASGGGSGGG FDAPSPLP
C0586 none none TSGGGGSGGGGSSS
C0587 none none GSPGGGGSGGGPGS
C0588 none none GSPPGGPPGS
C0589 none none GSPGGGGPGS
C0590 none none TSGGGGS
C0591 none none SGGSGGS
C0592 none none FDAPSPLP
C0593 yes ASGGGSGGG VYPEVTV
C0594 none none VYPEVTV
C0595 yes ASGGGSGGG GGGG
C0596 yes ASGGGSGGG GGGGS
C0597 yes ASGGGSGGG GGGSGG Example 3: Four classes of novel linkers for MHC class II single chain molecules
Inspection of circular dichroism spectra of purified, refolded single chain constructs indicated that novel linkers could be used for the constracts. The atomic structures ofthe various murine and human MHC class II molecules, as determined by X- ray crystallography, indicated that these molecules have a high degree of structural similarity. The circular dichroism results were consistent with two folded molecules of clearly different secondary structure. Careful inspection ofthe structures reveals that the human MHC has a longer distance between chains than the equivalent murine molecule.
This results led to proposing longer linkers between the chains which would contain flexible residues (e.g. alanine or glycine) and polar residues (e.g. serine and threonine). These constracts make up the first class of linkers. To inhibit the continuation of secondary stracture across the linker, prolines were added to bracket the linkers. These prolines are known to inhibit the formation of alpha helices and beta sheets. These linkers make up the second class of linkers disclosed here. Next, 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. These are the third class of linkers. Finally, a combination of these types of linkers could also be used. These are the fourth class of linkers.
Example 4: Human MHC class II single chain molecule with murine linkers
Another linker has been suggested based on a combination of murine and human MHC class II single chain molecules. This fusion would incoφorate 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):
MGDTGRSFTLASSETGVGASGGGGSGGGGDTRPRFLEQVKHECHFFNGTERVRF LDRYFYHQEEYVRFDSDVGEYRAVTELGRPDAEYWNSQKDLLEQKRAAVDTYC RHNYGVGESFTVLRRLGGEDDEADHHVIIQAEFYLNPDQSGEFMFDFDGDEIFH VDMAKKETVWI^LEEFGRFASFEAQGALAMAVDKANLEΓMTKRSNYTPITN* Further MHC class II hybrid single chain molecules may be designed by fusing other portions ofthe alpha chain and the beta chain together using linkers as described elsewhere. 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). Other, 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.
The following describes the concept of different linkers. Peptide is attached to a linker 1 (LI) which is attached to N-terminus of βl domain that is linked to L2. L2 is linked to L3, which in turn is linked to N-terminus of αl chain of MHC class II. Here L2 represents the human CD4 binding sequences. It should be noted that L2 could also be directly linked to N-terminus of αl 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.
RNGQEEKAGVVSTGLI, RNGQETKAGVVSTGLI, YNQQEEKAGGVSTGLI,
FRNGQEEKAGWSTGLI, FRNGQETKAGWSTGLI, FYNQQEEKAGGVSTGLI, andLNGQEEKAGMVSTGLI.
Example 6: 1-As MBP.βlβ2αlα2.Cκ constract and activity
The "full anergix" single chain molecule, mouse I- As MBP.βlβ2αlα2.Cκ, 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.βlβ2αlα2.Cκ is shown in Figure 1.
The amino acid sequence ofthe I-As MBP.βlβ2αlα2.Cκ shown in Figure 1 is as follows: METDTLLLWVLLLWVPGSTGDFKNIVTPRTPPPASGGGGSGGGGDSERHFVFQF KGECYFTNGTQRIRSVDRYIYNREEYLRFDSDVGEYRAVTELGRPDPEYYNKQY LEQTRAELDTVCRHNYEGVETHTSLRRLEQPNVVISLSRTEALNHHNTLVCSVTD FYPAKΓKVRWFRNGQEETVGVSSTQLΓRNGDWTFQVLVMLEMTPRRGEVYTCH VEHPSLKSPITVEWTSGGGGSGGGGSGGGGSGGGGSSSEDDIEADHVGVYGTTV YQSPGDIGQYTHEFDGDEWFYVDLDKKETIWMLPEFGQLTSFDPQGGLQNIATG KYTLGILTKRSNSTPATNEAPQATVFPKSPVLLGQPNTLICFVDNΓFPPVΓNITWLR NSKSVTDGVYETSFLVNRDHSFHKLSYLTFIPSDDDIYDCKVEHWGLEEPVLKHW ASGGGGSGGGGADAAPTVSFFPPSSEQLTSGGASVVCFLNL^YPKOINVKWKIDG
SERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVK SFNRNEC
The in vitro activity of recombinant I-As MBP.βlβ2αlα2.Cκ 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.βlβ2αlα2.Cκ and plate bound anti-CD28 antibody. T cell activation was assayed by 3H-thymidine incoφoration 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.βlβ2αlα2.Cκ reduces the incidence and severity of
EAE. EAE was induced according to standard methodology according to the myelin model. Ten μg of recombinant I-As MBP.βlβ2αlα2.Cκ was given intravenously in 100 μl 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.βlβ2αlα2.Cκ significantly reduced the incidence of myelin- induced EAE in SJL mice. In one experiment, 11 out of 20 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%). Similarly, in another experiment, 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%).
Histological examination from SJL mice in the EAE model studies: spinal cords were removed, fixed in formalin solution and embedded in paraffin. Sections were cut, stained with hematoxylin, eosin and graded for inflammatory lesions. A. Section of spinal cord from untreated mouse, score = 2.5; B. Section of spinal cord from mouse treated with the recombinant I-AS.MBP.Ck, score = 0; C. Section of spinal cord from mouse treated with the recombinant I-AS.βlαl without antigenic peptide fusion, score = 2.0; D. Section of spinal cord from mouse treated with the recombinant I-AS.MBP.βl αl , score = 0.5. The histology score for each section is marked.
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. The results indicate that treatment with the recombinant IAs fusion proteins prevents mortality and significantly reduces paralysis induced by myelin homogenate in CFA. In conclusion, these studies suggest that the recombinant MHC class II fusion protein has therapeutic benefit as antigen-specific drugs for the treatment of autoimmune diseases.
Example 8: Synthesis of mouse model equivalent of CO608 Four forms ofthe murine MHC Class II IAs βlαl 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 (same as m608 except lacking four amino acids, GSER, after the methionine as compared to m608), B. 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). To make mCO608, the upstream linker of mouse CO521 (GGGS) was replaced with the human CO608 linker (ASGGGGSGGG) and the downstream linker of mouse CO521 (GG) was replaced with the downstream linker of CO608 (TSGGGGS), using PCR according to standard methodology. mCO608-A and m6O8-B were made from mCO608 using PCR according to standard methodology.
All publications and patent applications cited in this specification are herein incoφorated by reference as if each individual publication or patent application were specifically and individually indicated to be incoφorated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for puφoses of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light ofthe teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope ofthe appended claims.
SEQUENCE LISTING
<210> CO602
<211> 558
<212> DNA
<213> Homo sapiens atgggggac cccgaccacg tttcttggag caggttaaac atgagtgtca tttcttcaac 60 gggacggagc gggtgcggtt cctggacaga tacttctatc accaagagga gtacgtgcgc 120 ttcgacagcg acgtggggga gtaccgggcg gtgacggagc tggggcggcc tgatgccgag 180 tactggaaca gccagaagga cctcctggag cagaagcggg ccgcggtgga cacctactgc 240 agacacaact acggggttgg tgagagcttc acagtgctgc gccgactcgg aggtgaagat 300 gacgaggcag atcaccatgt gatcatccag gccgagttct atctgaatcc tgaccaatca 360 ggcgagttta tgtttgactt tgatggtgat gagattttcc atgtggatat ggcaaagaag 420 gagacggtct ggcggcttga agaatttgga cgatttgcca gctttgaggc tcaaggtgca 480 ttggccaaca tagctgtgga caaagccaac ctggaaatca tgacaaagcg ctccaactat 540 actccgatca ccaattaa 558
<210> CO601
<211> 558
<212> DNA
<213> Homo sapiens atgggggaca cccgaccacg tttcttggag caggttaaac atgagtgtca tttcttcaac 60 gggacggagc gggtgcggtt cctggacaga tacttctatc accaagagga gtacgtgcgc 120 ttcgacagcg acgtggggga gtaccgggcg gtgacggagc tggggcggcc tgatgccgag 180 tactggaaca gccagaagga cctcctggag cagaagcggg ccgcggtgga cacctactgc 240 agacacaact acggggttgg tgagagcttc acagtgcagc ggcgaggagg tggaagcggc 300 ggaatcaaag aagaacatgt gatcatccag gccgagttct atctgaatcc tgaccaatca 360 ggcgagttta tgtttgactt tgatggtgat gagattttcc atgtggatat ggcaaagaag 420 gagacggtct ggcggcttga agaatttgga cgatttgcca gctttgaggc tcaaggtgca 480 ttggccaaca tagctgtgga caaagccaac ctggaaatca tgacaaagcg ctccaactat 540 actccgatca ccaattaa 558
<210> CO600
<211> 555
<212> DNA
<213> Homo sapiens atgggggaca cccgaccacg tttcttggag caggttaaac atgagtgtca tttcttcaac 60 gggacggagc gggtgcggtt cctggacaga tacttctatc accaagagga gtacgtgcgc 120 ttcgacagcg acgtggggga gtaccgggcg gtgacggagc tggggcggcc tgatgccgag 180 tactggaaca gccagaagga cctcctggag cagaagcggg ccgcggtgga cacctactgc 240 agacacaact acggggttgg tgagagcttc acagtgcagc ggcgaggagg tggaggcagc 300 atcaaagaag aacatgtgat catccaggcc gagttctatc tgaatcctga ccaatcaggc 360 gagtttatgt ttgactttga tggtgatgag attttccatg tggatatggc aaagaaggag 420 acggtctggc ggcttgaaga atttggacga tttgccagct ttgaggctca aggtgcattg 480 gccaacatag ctgtggacaa agccaacctg gaaatcatga caaagcgctc caactatact 540 ccgatcacca attaa 555
<210> C0599
<211> 552
<212> DNA
<213> Homo sapiens atgggggaca cccgaccacg tttcttggag caggttaaac atgagtgtca tttcttcaac 60 gggacggagc gggtgcggtt cctggacaga tacttctatc accaagagga gtacgtgcgc 120 ttcgacagcg acgtggggga gtaccgggcg gtgacggagc tggggcggcc tgatgccgag 180 tactggaaca gccagaagga cctcctggag cagaagcggg ccgcggtgga cacctactgc 240 agacacaact acggggttgg tgagagcttc acagtgcagc ggcgaggagg tggaggcatc 300 aaagaagaac atgtgatcat ccaggccgag ttctatctga atcctgacca atcaggcgag 360 tttatgtttg actttgatgg tgatgagatt ttccatgtgg atatggcaaa gaaggagacg 420 gtctggcggc ttgaagaatt tggacgattt gccagctttg aggctcaagg tgcattggcc 480 aacatagctg tggacaaagc caacctggaa atcatgacaa agcgctccaa ctatactccg 540 atcaccaatt aa 552
<210> C0594
<211> 561
<212> DNA
<213> Homo sapiens atgggggaca cccgaccacg tttcttggag caggttaaac atgagtgtca tttcttcaac 60 gggacggagc gggtgcggtt cctggacaga tacttctatc accaagagga gtacgtgcgc 120 ttcgacagcg acgtggggga gtaccgggcg gtgacggagc tggggcggcc tgatgccgag 180 tactggaaca gccagaagga cctcctggag cagaagcggg ccgcggtgga cacctactgc 240 agacacaact acggggttgg tgagagcttc acagtgcagc ggcgagtcta ccctgaggta 300 actgtcatca aagaagaaca tgtgatcatc caggccgagt tctatctgaa tcctgaccaa 360 tcaggcgagt ttatgtttga ctttgatggt gatgagattt tccatgtgga tatggcaaag 420 aaggagacgg tctggcggct tgaagaattt ggacgatttg ccagctttga ggctcaaggt 480 gcattggcca acatagctgt ggacaaagcc aacctggaaa tcatgacaaa gcgctccaac 540 tatactccga tcaccaatta a 561
<210> C0592
<211> 564
<212> DNA
<213> Homo sapiens atgggggaca cccgaccacg tttcttggag caggttaaac atgagtgtca tttcttcaac 60 • gggacggagc gggtgcggtt cctggacaga tacttctatc accaagagga gtacgtgcgc 120 ttcgacagcg acgtggggga gtaccgggcg gtgacggagc tggggcggcc tgatgccgag 180 tactggaaca gccagaagga cctcctggag cagaagcggg ccgcggtgga cacctactgc 240 agacacaact acggggttgg tgagagcttc acagtgcagc ggcgattcga cgcacctagc 300 ccactcccaa tcaaagaaga acatgtgatc atccaggccg agttctatct gaatcctgac 360 caatcaggcg agtttatgtt tgactttgat ggtgatgaga ttttccatgt ggatatggca 420 aagaaggaga cggtctggcg gcttgaagaa tttggacgat ttgccagctt tgaggctcaa 480 ggtgcattgg ccaacatagc tgtggacaaa gccaacctgg aaatcatgac aaagcgctcc 540 aactatactc cgatcaccaa ttaa 564
<210> C0591 <211> 561 <212> DNA
<213> Homo sapiens atgggggaca cccgaccacg tttcttggag caggttaaac atgagtgtca tttcttcaac 60 gggacggagc gggtgcggtt cctggacaga tacttctatc accaagagga gtacgtgcgc 120 ttcgacagcg acgtggggga gtaccgggcg gtgacggagc tggggcggcc tgatgccgag 180 tactggaaca gccagaagga cctcctggag cagaagcggg ccgcggtgga cacctactgc 240 agacacaact acggggttgg tgagagcttc acagtgcagc ggcgaagtgg cggtagtggc 300 ggtagtatca aagaagaaca tgtgatcatc caggccgagt tctatctgaa tcctgaccaa 360 tcaggcgagt ttatgtttga ctttgatggt gatgagattt tccatgtgga tatggcaaag 420 aaggagacgg tctggcggct tgaagaattt ggacgatttg ccagctttga ggctcaaggt 480 gcattggcca acatagctgt ggacaaagcc aacctggaaa tcatgacaaa gcgctccaac 540 tatactccga tcaccaatta a 561
<210> CO590 <211> 561 <212 > DNA <213 > Homo sapiens atgggggaca cccgaccacg tttcttggag caggttaaac atgagtgtca tttcttcaac 60 gggacggagc gggtgcggtt cctggacaga tacttctatc accaagagga gtacgtgcgc 120 ttcgacagcg acgtggggga gtaccgggcg gtgacggagc tggggcggcc tgatgccgag 180 tactggaaca gccagaagga cctcctggag cagaagcggg ccgcggtgga cacctactgc 240 agacacaact acggggttgg tgagagcttc acagtgcagc ggcgaactag tggtggcggt 300 ggcagcatca aagaagaaca tgtgatcatc caggccgagt tctatctgaa tcctgaccaa 360 tcaggcgagt ttatgtttga ctttgatggt gatgagattt tccatgtgga tatggcaaag 420 aaggagacgg tctggcggct tgaagaattt ggacgatttg ccagctttga ggctcaaggt 480 gcattggcca acatagctgt ggacaaagcc aacctggaaa tcatgacaaa gcgctccaac 540 tatactccga tcaccaatta a 561
<210> C0589
<211> 570
<212> DNA
<213> Homo sapiens atgggggaca cccgaccacg tttcttggag caggttaaac atgagtgtca tttcttcaac 60 gggacggagc gggtgcggtt cctggacaga tacttctatc accaagagga gtacgtgcgc 120 ttcgacagcg acgtggggga gtaccgggcg gtgacggagc tggggcggcc tgatgccgag 180 tactggaaca gccagaagga cctcctggag cagaagcggg ccgcggtgga cacctactgc 240 agacacaact acggggttgg tgagagcttc acagtgcagc ggcgaggctc tcctggaggt 300 ggaggtcctg gatctatcaa agaagaacat gtgatcatcc aggccgagtt ctatctgaat 360 cctgaccaat caggcgagtt tatgtttgac tttgatggtg atgagatttt ccatgtggat 420 atggcaaaga aggagacggt ctggcggctt gaagaatttg gacgatttgc cagctttgag 480 gctcaaggtg cattggccaa catagctgtg gacaaagcca acctggaaat catgacaaag 540 cgctccaact atactccgat caccaattaa 570
<210> C0588
<211> 570
<212> DNA
<213> Homo sapiens atgggggaca cccgaccacg tttcttggag caggttaaac atgagtgtca tttcttcaac 60 gggacggagc gggtgcggtt cctggacaga tacttctatc accaagagga gtacgtgcgc 120 ttcgacagcg acgtggggga gtaccgggcg gtgacggagc tggggcggcc tgatgccgag 180 tactggaaca gccagaagga cctcctggag cagaagcggg ccgcggtgga cacctactgc 240 agacacaact acggggttgg tgagagcttc acagtgcagc ggcgaggctc tcctcctggt 300 ggaccacctg gatctatcaa agaagaacat gtgatcatcc aggccgagtt ctatctgaat 360 cctgaccaat caggcgagtt tatgtttgac tttgatggtg atgagatttt ccatgtggat 420 atggcaaaga aggagacggt ctggcggctt gaagaatttg gacgatttgc cagctttgag 480 gctcaaggtg cattggccaa catagctgtg gacaaagcca acctggaaat catgacaaag 540 cgctccaact atactccgat caccaattaa 570
<210> C0587
<211> 582 <212> DNA
<213> Homo sapiens atgggggaca cccgaccacg tttcttggag caggttaaac atgagtgtca tttcttcaac 60 gggacggagc gggtgcggtt cctggacaga tacttctatc accaagagga gtacgtgcgc 120 ttcgacagcg acgtggggga gtaccgggcg gtgacggagc tggggcggcc tgatgccgag 180 tactggaaca gccagaagga cctcctggag cagaagcggg ccgcggtgga cacctactgc 240 agacacaact acggggttgg tgagagcttc acagtgcagc ggcgagggag tccaggcggt 300 ggcgggagcg gcggtggtcc tgggagtatc aaagaagaac atgtgatcat ccaggccgag 360 ttctatctga atcctgacca atcaggcgag tttatgtttg actttgatgg tgatgagatt 420 ttccatgtgg atatggcaaa gaaggagacg gtctggcggc ttgaagaatt tggacgattt 480 gccagctttg aggctcaagg tgcattggcc aacatagctg tggacaaagc caacctggaa 540 atcatgacaa agcgctccaa ctatactccg atcaccaatt aa 582
<210> C0586 <211> 582
<212> DNA
<213> Homo sapiens ' atgggggaca cccgaccacg tttcttggag caggttaaac atgagtgtca tttcttcaac 60 gggacggagc gggtgcggtt cctggacaga tacttctatc accaagagga gtacgtgcgc 120 ttcgacagcg acgtggggga gtaccgggcg gtgacggagc tggggcggcc tgatgccgag 180 tactggaaca gccagaagga cctcctggag cagaagcggg ccgcggtgga cacctactgc 240 agacacaact acggggttgg tgagagcttc acagtgcagc ggcgaactag tggtggcggt 300 ggcagcggcg gtggtggttc ctcgagtatc aaagaagaac atgtgatcat ccaggccgag 360 ttctatctga atcctgacca atcaggcgag tttatgtttg actttgatgg tgatgagatt 420 ttccatgtgg atatggcaaa gaaggagacg gtctggcggc ttgaagaatt tggacgattt 480 gccagctttg aggctcaagg tgcattggcc aacatagctg tggacaaagc caacctggaa 540 atcatgacaa agcgctccaa ctatactccg atcaccaatt aa 582
<210> C0602
<211> 186 <212> PRT <213> Homo sapiens
Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys
5 10 15
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe 20 25 30
Tyr His Gin Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr 35 40 45 Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 50 55 60
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 Leu Arg Arg Leu 85 90 95
Gly Gly Glu Asp Asp Glu Ala Asp His His Val He He Gin Ala Glu 100 105 110
Phe Tyr Leu Asn Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp
115 120 125 Gly Asp Glu He Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp 130 135 140
Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala 145 150 155 160
Leu Ala Asn He Ala Val Asp Lys Ala Asn Leu Glu He Met Thr Lys 165 170 175
Arg Ser Asn Tyr Thr Pro He Thr Asn 180 185
<210> C0601 <211> 186 <212> PRT <213> Homo sapiens
Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys 5 10 15
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe 20 25 30 Tyr His Gin Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr 35 40 45
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 50 55 60
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 Gly 85 90 95
Gly Gly Ser Gly Gly He Lys Glu Glu His Val He He Gin Ala Glu 100 105 110 Phe Tyr Leu Asn Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp 115 120 125
Gly Asp Glu He Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp 130 135 140
Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala 145 150 155 160
Leu Ala Asn He Ala Val Asp Lys Ala Asn Leu Glu He Met Thr Lys 165 170 175
Arg Ser Asn Tyr Thr Pro He Thr Asn 180 185
<210> CO600
<211> 185
<212> PRT
<213> Homo sapiens
Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys
5 10 15
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe 20 25 30
Tyr His Gin Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr 35 40 45 Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 50 55 60
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 Gly 85 90 95 Gly Gly Gly Ser He Lys Glu Glu His Val He He Gin Ala Glu Phe 100 105 110
Tyr Leu Asn Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly 115 120 125
Asp Glu He Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg 130 135 140
Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu 145 150 ' 155 160
Ala Asn He Ala Val Asp Lys Ala Asn Leu Glu He Met Thr Lys Arg 165 170 175 Ser Asn Tyr Thr Pro He Thr Asn 180
<210> C0599 <211> 184 <212> PRT <213> Homo sapiens
Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys 5 10 15
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe 20 25 30 Tyr His Gin Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr 35 40 45
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 50 55 60
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 Gly 85 90 95
Gly Gly Gly He Lys Glu Glu His Val He He Gin Ala Glu Phe Tyr 100 105 110 Leu Asn Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp 115 120 125
Glu He Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu 130 135 140 Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala 145 150 155 160
Asn He Ala Val Asp Lys Ala Asn Leu Glu He Met Thr Lys Arg Ser 165 170 175
Asn Tyr Thr Pro He Thr Asn 180
<210> C0594 <211> 187 <212> PRT <213> Homo sapiens
Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys
5 10 15 His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe
20 25 30
Tyr His Gin Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr 35 40 45
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 50 55 60
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 Val 85 90 95 Tyr Pro Glu Val Thr Val He Lys Glu Glu His Val He He Gin Ala 100 105 110
Glu Phe ' Tyr Leu Asn Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe 115 120 125
Asp Gly Asp Glu He Phe His Val Asp Met Ala Lys Lys Glu Thr Val 130 135 140
Trp Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly 145 150 155 160
Ala Leu Ala Asn He Ala Val Asp Lys Ala Asn Leu Glu He Met Thr 165 170 175 Lys Arg Ser Asn Tyr Thr Pro He Thr Asn 180 185
<210> C0592 <211> 188 < 12> PRT <213> Homo sapiens
Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys 10 15
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe 20 25 30
Tyr His Gin Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr 35 40 45
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 50 55 60
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 Phe
85 90 95
Asp Ala Pro Ser Pro Leu Pro He Lys Glu Glu His Val He He Gin 100 105 110
Ala Glu Phe Tyr Leu Asn Pro Asp Gin Ser Gly Glu Phe Met Phe Asp 115 120 125
Phe Asp Gly Asp Glu He Phe His Val Asp Met Ala Lys Lys Glu Thr 130 135 140
Val Trp Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin
145 150 155 160 Gly Ala Leu Ala Asn He Ala Val Asp Lys Ala Asn Leu Glu He Met
165 170 175
Thr Lys Arg Ser Asn Tyr Thr Pro He Thr Asn 180 185
<210> C0591 <211> 187 <212> PRT <213> Homo sapiens
Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys
5 10 15 His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe
20 25 30
Tyr His Gin Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr 35 40 45
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 50 55 60
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 Ser 85 90 95 Gly Gly Ser Gly Gly Ser He Lys Glu Glu His Val He He Gin Ala 100 105 110
Glu Phe Tyr Leu Asn Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe 115 120 125
Asp Gly Asp Glu He Phe His Val Asp Met Ala Lys Lys Glu Thr Val 130 135 140 Trp Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly 145 150 155 160
Ala Leu Ala Asn He Ala Val Asp Lys Ala Asn Leu Glu He Met Thr 165 170 175
Lys Arg Ser Asn Tyr Thr Pro He Thr Asn 180 185
<210> CO590 <211> 187 <212> PRT <213> Homo sapiens Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys
5 10 15
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe 20 25 30
Tyr His Gin Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr 35 40 45
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 50 55 60
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
85 90 95
Ser Gly Gly Gly Gly Ser He Lys Glu Glu His Val He He Gin Ala 100 105 110
Glu Phe Tyr Leu Asn Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe 115 120 125
Asp Gly Asp Glu He Phe His Val Asp Met Ala Lys Lys Glu Thr Val 130 135 140
Trp Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly 145 150 155 160 Ala Leu Ala Asn He Ala Val Asp Lys Ala Asn Leu Glu He Met Thr
165 170 175
Lys Arg Ser Asn Tyr Thr Pro He Thr Asn 180 185 <210> C0589
<211> 190
<212 > PRT
<213 > Homo sapiens
Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys
5 10 15
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe 20 25 30
Tyr His Gin Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr 35 40 45
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 50 55 60 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 Gly 85 90 95
Ser Pro Gly Gly Gly Gly Pro Gly Ser He Lys Glu Glu His Val He 100 105 110
He Gin Ala Glu Phe Tyr Leu Asn Pro Asp Gin Ser Gly Glu Phe Met 115 120 125
Phe Asp Phe Asp Gly Asp Glu He Phe His Val Asp Met Ala Lys Lys 130 135 140 Glu Thr Val Trp Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu 145 150 155 160
Ala Gin Gly Ala Leu Ala Asn He Ala Val Asp Lys Ala Asn Leu Glu 165 170 175
He Met Thr Lys Arg Ser Asn Tyr Thr Pro He Thr Asn 180 185
<210> C0588 <211> 190 <212> PRT <213> Homo sapiens Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys
5 10 15
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe 20 25 30
Tyr His Gin Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr 35 40 45
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 50 55 60
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 Gly 85 90 95
Ser Pro Pro Gly Gly Pro Pro Gly Ser He Lys Glu Glu His Val He 100 105 110
He Gin Ala Glu Phe Tyr Leu Asn Pro Asp Gin Ser Gly Glu Phe Met 115 120 125 Phe Asp Phe Asp Gly Asp Glu He Phe His Val Asp Met Ala Lys Lys 130 135 140
Glu Thr Val Trp Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu 145 150 155 160
Ala Gin Gly Ala Leu Ala Asn He Ala Val Asp Lys Ala Asn Leu Glu 165 170 175
He Met Thr Lys Arg Ser Asn Tyr Thr Pro He Thr Asn 180 185
<210> C0587 <211> 194 <212> PRT
<213> Homo sapiens
Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys
5 10 15
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe 20 25 30
Tyr His Gin Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr 35 40 45
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 50 55 60 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 Gly 85 90 95
Ser Pro Gly Gly Gly Gly Ser Gly Gly Gly Pro Gly Ser He Lys Glu 100 105 110
Glu His Val He He Gin Ala Glu Phe Tyr Leu Asn Pro Asp Gin Ser 115 120 125
Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu He Phe His Val Asp 130 135 140 Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe Gly Arg Phe 145 150 155 160
Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala Asn He Ala Val Asp Lys 165 170 175
Ala Asn Leu Glu He Met Thr Lys Arg Ser Asn Tyr Thr Pro He Thr 180 185 190 Asn
<210> C0586 <211> 194 <212> PRT <213> Homo sapiens
Met Gly Asp Thr Arg Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys 5 10 15
His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe 20 25 30 Tyr His Gin Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr 35 40 45
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser 50 55 60
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 85 90 95
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser He Lys Glu 100 105 110 Glu His Val He He Gin Ala Glu Phe Tyr Leu Asn Pro Asp Gin Ser 115 120 125
Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu He Phe His Val Asp 130 135 140
Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe Gly Arg Phe 145 150 155 160
Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala Asn He Ala Val Asp Lys 165 170 175
Ala Asn Leu Glu He Met Thr Lys Arg Ser Asn Tyr Thr Pro He Thr 180 185 190 Asn
<210> C0598 <211> 639 <212> DNA <213> Homo sapiens atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60 ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120 catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180 caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240 ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300 gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgctg 360 cgccgactcg gaggtgaaga tgacgaggca gatcaccatg tgatcatcca ggccgagttc 420 tatctgaatc ctgaccaatc aggcgagttt atgtttgact ttgatggtga tgagattttc 480 catgtggata tggcaaagaa ggagacggtc tggcggcttg aagaatttgg acgatttgcc 540 agctttgagg ctcaaggtgc attggccaac atagctgtgg acaaagccaa cctggaaatc 600 atgacaaagc gctccaacta tactccgatc accaattaa 639
<210> C0597
<211> 639
<212> DNA
<213> Homo sapiens atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60 ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120 catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180 caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240 ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300 gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360 cggcgaggag gtggaagcgg cggaatcaaa gaagaacatg tgatcatcca ggccgagttc 420 tatctgaatc ctgaccaatc aggcgagttt atgtttgact ttgatggtga tgagattttc 480 catgtggata tggcaaagaa ggagacggtc tggcggcttg aagaatttgg acgatttgcc 540 agctttgagg ctcaaggtgc attggccaac atagctgtgg acaaagccaa cctggaaatc 600 atgacaaagc gctccaacta tactccgatc accaattaa 639
<210> C0596
<211> 636
<212> DNA
<213> Homo sapiens atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60 ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120 catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180 caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240 ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300 gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360 cggcgaggag gtggaggcag catcaaagaa gaacatgtga tcatccaggc cgagttctat 420 ctgaatcctg accaatcagg cgagtttatg tttgactttg atggtgatga gattttccat 480 gtggatatgg caaagaagga gacggtctgg cggcttgaag aatttggacg atttgccagc 540 tttgaggctc aaggtgcatt ggccaacata gctgtggaca aagccaacct ggaaatcatg 600 acaaagcgct ccaactatac tccgatcacc aattaa 636
<210> C0595
<211> 633
<212> DNA
<213> Homo sapiens atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60 ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120 catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180 caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240 ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300 gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360 cggcgaggag gtggaggcat caaagaagaa catgtgatca tccaggccga gttctatctg 420 aatcctgacc aatcaggcga gtttatgttt gactttgatg gtgatgagat tttccatgtg 480 gatatggcaa agaaggagac ggtctggcgg cttgaagaat ttggacgatt tgccagcttt 540 gaggctcaag gtgcattggc caacatagct gtggacaaag ccaacctgga aatcatgaca 600 aagcgctcca actatactcc gatcaccaat taa 633
<210> C0593 <211> 642 <212> DNA <213> Homo sapiens atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60 ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120 catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180 caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240 ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300 gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360 cggcgagtct accctgaggt aactgtcatc aaagaagaac atgtgatcat ccaggccgag 420 ttctatctga atcctgacca atcaggcgag tttatgtttg actttgatgg tgatgagatt 480 ttccatgtgg atatggcaaa gaaggagacg gtctggcggc ttgaagaatt tggacgattt 540 gccagctttg aggctcaagg tgcattggcc aacatagctg tggacaaagc caacctggaa 600 atcatgacaa agcgctccaa ctatactccg atcaccaatt aa 642
<210> C0585
<211> 645
<212> DNA
<213> Homo sapiens atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60 ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120 catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180 caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240 ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300 gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360 cggcgattcg acgcacctag cccactccca atcaaagaag aacatgtgat catccaggcc 420 gagttctatc tgaatcctga ccaatcaggc gagtttatgt ttgactttga tggtgatgag 480 attttccatg tggatatggc aaagaaggag acggtctggc ggcttgaaga atttggacga 540 tttgccagct ttgaggctca aggtgcattg gccaacatag ctgtggacaa agccaacctg 600 gaaatcatga caaagcgctc caactatact ccgatcacca attaa 645
<210> C0584 <211> 642 <212> DNA
<213> Homo sapiens atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60 ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120 'catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180 caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240 ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300 gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360 cggcgaagtg gcggtagtgg cggtagtatc aaagaagaac atgtgatcat ccaggccgag 420 ttctatctga atcctgacca atcaggcgag tttatgtttg actttgatgg tgatgagatt 480 ttccatgtgg atatggcaaa gaaggagacg gtctggcggc ttgaagaatt tggacgattt 540 gccagctttg aggctcaagg tgcattggcc aacatagctg tggacaaagc caacctggaa 600 atcatgacaa agcgctccaa ctatactccg atcaccaatt aa 642 <210> C0583 <211> 642 <212> DNA <213> Homo sapiens atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60 ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120 catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180 caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240 ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300 gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360 cggcgaacta gtggtggcgg tggcagcatc aaagaagaac atgtgatcat ccaggccgag 420 ttctatctga atcctgacca atcaggcgag tttatgtttg actttgatgg tgatgagatt 480 ttccatgtgg atatggcaaa gaaggagacg gtctggcggc ttgaagaatt tggacgattt 540 gccagctttg aggctcaagg tgcattggcc aacatagctg tggacaaagc caacctggaa 600 atcatgacaa agcgctccaa ctatactccg atcaccaatt aa 642
<210> C0582 <211> 651
<212> DNA
<213> Homo sapiens atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60 ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120 catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180 caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240 ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300 gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360 cggcgaggct ctcctggagg tggaggtcct ggatctatca aagaagaaca tgtgatcatc 420 caggccgagt tctatctgaa tcctgaccaa tcaggcgagt ttatgtttga ctttgatggt 480 gatgagattt tccatgtgga tatggcaaag aaggagacgg tctggcggct tgaagaattt 540 ggacgatttg ccagctttga ggctcaaggt gcattggcca acatagctgt ggacaaagcc 600 aacctggaaa tcatgacaaa gcgctccaac tatactccga tcaccaatta a 651
<210> C0581 <211> 651 <212> DNA <213> Homo sapiens atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60 ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120 catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180 caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240 ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300 gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360 cggcgaggct ctcctcctgg tggaccacct ggatctatca aagaagaaca tgtgatcatc 420 caggccgagt tctatctgaa tcctgaccaa tcaggcgagt ttatgtttga ctttgatggt 480 gatgagattt tccatgtgga tatggcaaag aaggagacgg tctggcggct tgaagaattt 540 ggacgatttg ccagctttga ggctcaaggt gcattggcca acatagctgt ggacaaagcc 600 aacctggaaa tcatgacaaa gcgctccaac tatactccga tcaccaatta a 651
<210> CO580 <211> 663 <212> DNA <213> Homo sapiens atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60 ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120 catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180 caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240 ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300 gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360 cggcgaggga gtccaggcgg tggcgggagc ggcggtggtc ctgggagtat caaagaagaa 420 catgtgatca tccaggccga gttctatctg aatcctgacc aatcaggcga gtttatgttt 480 gactttgatg gtgatgagat tttccatgtg gatatggcaa agaaggagac ggtctggcgg 540 cttgaagaat ttggacgatt tgccagcttt gaggctcaag gtgcattggc caacatagct 600 gtggacaaag ccaacctgga aatcatgaca aagcgctcca actatactcc gatcaccaat 660 taa 663
<210> C0567 <211> 663 <212> DNA <213> Homo sapiens atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggcgctagc 60 ggagggggcg gaagcggcgg agggggggac acccgaccac gtttcttgga gcaggttaaa 120 catgagtgtc atttcttcaa cgggacggag cgggtgcggt tcctggacag atacttctat 180 caccaagagg agtacgtgcg cttcgacagc gacgtggggg agtaccgggc ggtgacggag 240 ctggggcggc ctgatgccga gtactggaac agccagaagg acctcctgga gcagaagcgg 300 gccgcggtgg acacctactg cagacacaac tacggggttg gtgagagctt cacagtgcag 360 cggcgaacta gtggtggcgg tggcagcggc ggtggtggtt cctcgagtat caaagaagaa 420 catgtgatca tccaggccga gttctatctg aatcctgacc aatcaggcga gtttatgttt 480 gactttgatg gtgatgagat tttccatgtg gatatggcaa agaaggagac ggtctggcgg 540 cttgaagaat ttggacgatt tgccagcttt gaggctcaag gtgcattggc caacatagct 600 gtggacaaag ccaacctgga aatcatgaca aagcgctcca actatactcc gatcaccaat 660 taa 663
<210> C0598 <211> 213 <212> PRT <213> Homo sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly 5 10 15
Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg 20 25 30 Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys His Phe Phe Asn Gly 35 40 45
Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gin Glu Glu 50 55 60
Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu 65 70 75 80
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin Lys Asp Leu Leu 85 90 95
Glu Gin Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly 100 105 110 Val Gly Glu Ser Phe Thr Val Leu Arg Arg Leu Gly Gly Glu Asp Asp
115 120 125
Glu Ala Asp His His Val He He Gin Ala Glu Phe Tyr Leu Asn Pro 130 135 140
Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu He Phe 145 150 155 160 His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe
165 170 175
Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala Asn He Ala 180 185 190
Val Asp Lys Ala Asn Leu Glu He Met Thr Lys Arg Ser Asn Tyr Thr 195 200 205
Pro He Thr Asn 2io
<210> C0597 <211> 213 <212> PRT
<213> Homo sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15
Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg 20 25 30
Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys His Phe Phe Asn Gly 35 40 45
Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gin Glu Glu 50- 55 60 Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu 65 70 75 80
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin Lys Asp Leu Leu 85 90 95
Glu Gin Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly 100 105 110
Val Gly Glu Ser Phe Thr Val Gin Arg Arg Gly Gly Gly Ser Gly Gly 115 120 125
He Lys Glu Glu His Val He He Gin Ala Glu Phe Tyr Leu Asn Pro 130 135 140 Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu He Phe 145 150 155 160
His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg. Leu Glu Glu Phe 165 170 175 Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala Asn He Ala 180 185 190
Val Asp Lys Ala Asn Leu Glu He Met Thr Lys Arg Ser Asn Tyr Thr 195 200 205
Pro He Thr Asn 210
<210> C0596 <211> 212 <212> PRT <213> Homo sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15 Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg
20 25 30
Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys His Phe Phe Asn Gly 35 40 45
Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gin Glu Glu 50 55 60
Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu 65 70 75 80
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin Lys Asp Leu Leu 85 90 95 Glu Gin Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly 100 105 110
Val Gly Glu Ser Phe Thr Val Gin Arg Arg Gly Gly Gly Gly Ser He 115 120 125
Lys Glu Glu His Val He He Gin Ala Glu Phe Tyr Leu Asn Pro Asp 130 135 140
Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu He Phe His 145 150 155 160
Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe Gly 165 170 175 Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala Asn He Ala Val 180 185 190
Asp Lys Ala Asn Leu Glu He Met Thr Lys Arg Ser Asn Tyr Thr Pro 195 200 205
He Thr Asn 210 <210> C0595 <211> 211 <212> PRT
<213> Homo sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15
Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg 20 25 30
Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys His Phe Phe Asn Gly 35 40 45 Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gin Glu Glu 50 55 60
Tyr 'Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu 65 70 75 80
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin Lys Asp Leu Leu 85 90 95
Glu Gin Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly 100 105 110
Val Gly Glu Ser Phe Thr Val Gin Arg Arg Gly Gly Gly Gly He Lys 115 120 125 Glu Glu His Val He He Gin Ala Glu Phe Tyr Leu Asn Pro Asp Gin 130 135 140
Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu He Phe His Val 145 150 155 160
Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu Phe Gly Arg 165 170 175
Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala Asn He Ala Val Asp 180 185 190
Lys Ala Asn Leu Glu He Met Thr Lys Arg Ser Asn Tyr Thr Pro He 195 200 205 Thr Asn 210
<210> C0593 <211> 214 <212> PRT <213> Homo sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly 5 10 15
Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg 20 25 30 Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys His Phe Phe Asn Gly 35 40 45
Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gin Glu Glu 50 55 60
Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu 65 70 75 80 Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin Lys Asp Leu Leu
85 90 95
Glu Gin Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly 100 105 110
Val Gly Glu Ser Phe Thr Val Gin Arg Arg Val Tyr Pro Glu Val Thr 115 120 125
Val He Lys Glu Glu His Val He He Gin Ala Glu Phe Tyr Leu Asn 130 135 140
Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu He
145 150 155 160 Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu
165 170 175
Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala Asn He 180 185 190
Ala Val Asp Lys Ala Asn Leu Glu He Met Thr Lys Arg Ser Asn Tyr 195 200 205
Thr Pro He Thr Asn 2io
<210> C0585 <211> 215 <212> PRT
<213> Homo sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15
Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg 20 25 30
Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys His Phe Phe Asn Gly 35 40 45
Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gin Glu Glu 50 55 60 Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu 65 70 • 75 80
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin Lys Asp Leu Leu 85 90 95 Glu Gin Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly 100 105 110
Val Gly Glu Ser Phe Thr Val Gin Arg Arg Phe Asp Ala Pro Ser Pro 115 120 125
Leu Pro He Lys Glu Glu His Val He He Gin Ala Glu Phe Tyr Leu 130 135 140
Asn Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu 145 150 155 160
He Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu 165 170 175
Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala Asn 180 185 190 He Ala Val Asp Lys Ala Asn Leu Glu He Met Thr Lys Arg Ser Asn 195 200 205
Tyr Thr Pro He Thr Asn 210
<210> C0584 <211> 214 <212> PRT <213> Homo sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15 Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg
20 25 30
Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys His Phe Phe Asn Gly 35 40 45
Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gin Glu Glu 50 55 60
Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu 65 70 75 80
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin Lys Asp Leu Leu 85 90 95 Glu Gin Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly 100 105 110
Val Gly Glu Ser Phe Thr Val Gin Arg Arg Ser Gly Gly Ser Gly Gly 115 120 125
Ser He Lys Glu Glu His Val He He Gin Ala Glu Phe Tyr Leu Asn 130 135 140
Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu He 145 150 155 160
Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu 165 170 175
Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala Asn He 180 185 190
Ala Val Asp Lys Ala Asn Leu Glu He Met Thr Lys Arg Ser Asn Tyr 195 200 205
Thr Pro He Thr Asn 210
<210> C0583
<211> 214
<212> PRT
<213> Homo sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15
Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg 20 25 30
Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys His Phe Phe Asn Gly 35 40 45 Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gin Glu Glu 50 55 60
Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu 65 70 75 80
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin Lys Asp Leu Leu 85 90 95
Glu Gin Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly 100 105 110
Val Gly Glu Ser Phe Thr Val Gin Arg Arg Thr Ser Gly Gly Gly Gly 115 120 125 Ser_ He Lys Glu Glu His Val He He Gin Ala Glu Phe Tyr Leu Asn "l30 ' 135 140
Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu He 145 150 155 160
Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu 165 170 175
Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala Asn He 180 185 190
Ala Val Asp Lys Ala Asn Leu Glu He Met Thr Lys Arg Ser Asn Tyr 195 200 205 Thr Pro He Thr Asn 210
<210> C0582
<211> 217
<212> PRT
<213> Homo sapiens Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15
Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg 20 25 30
Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys His Phe Phe Asn Gly 35 40 45
Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gin Glu Glu 50 55 60
Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu 65 70 75 80 Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin Lys Asp Leu Leu
85 90 95
Glu Gin Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly 100 105 110
Val Gly Glu Ser Phe Thr Val Gin Arg Arg Gly Ser Pro Gly Gly Gly 115 120 125
Gly Pro Gly Ser He Lys Glu Glu His Val He He Gin Ala Glu Phe 130 135 140
Tyr Leu Asn Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly 145 150 155 160 Asp Glu He Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg
165 170 175
Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu 180 185 190
Ala Asn He Ala Val Asp Lys Ala Asn Leu Glu He Met Thr Lys Arg 195 200 205
Ser Asn Tyr Thr Pro He Thr Asn 210 215
<210> C0581 <211> 217 <212> PRT
<213> Homo sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15 Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg 20 25 30
Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys His Phe Phe Asn Gly 35 40 45
Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gin Glu Glu 50 55 60
Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu 65 70 75 80
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin Lys Asp Leu Leu 85 90 95
Glu Gin Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly 100 105 110 Val Gly Glu Ser Phe Thr Val Gin Arg Arg Gly Ser Pro Pro Gly Gly 115 120 125
Pro Pro Gly Ser He Lys Glu Glu His Val He He Gin Ala Glu Phe 130 135 140
Tyr Leu Asn Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly 145 150 155 160
Asp Glu He Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg 165 170 175
Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu 180 185 190 Ala Asn He Ala Val Asp Lys Ala Asn Leu Glu He Met Thr Lys Arg 195 200 205
Ser Asn Tyr Thr Pro He Thr Asn 210 215
<210> CO580 <211> 221 <212> PRT <213> Homo sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15 Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg
20 25 30
Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys His Phe Phe Asn Gly 35 40 45
Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gin Glu Glu 50 55 60
Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu 65 70 75 80
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin Lys Asp Leu Leu 85 90 95
Glu Gin Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly 100 105 110
Val Gly Glu Ser Phe Thr Val Gin Arg Arg Gly Ser Pro Gly Gly Gly 115 120 125
Gly Ser Gly Gly Gly Pro Gly Ser He Lys Glu Glu His Val He He 130 135 140 Gin Ala Glu Phe Tyr Leu Asn Pro Asp Gin Ser Gly Glu Phe Met Phe 145 150 155 160
Asp Phe Asp Gly Asp Glu He Phe His Val Asp Met Ala Lys Lys Glu 165 170 175
Thr Val Trp Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala 180 185 190
Gin Gly Ala Leu Ala Asn He Ala Val Asp Lys Ala Asn Leu Glu He 195 200 205
Met Thr Lys Arg Ser Asn Tyr Thr Pro He Thr Asn 210 215 220
<210> C0567
<211> 221
<212> PRT
<213> Homo sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15
Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg 20 25 30
Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys His Phe Phe Asn Gly 35 40 45 Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gin Glu Glu 50 55 60
Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu
65 70 75 80
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin Lys Asp Leu Leu
85 90 95
Glu Gin Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly 100 105 110
Val Gly Glu Ser Phe Thr Val Gin Arg Arg Thr Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Ser Ser He Lys Glu Glu His Val He He 130 135 140
Gin Ala Glu Phe Tyr Leu Asn Pro Asp Gin Ser Gly Glu Phe Met Phe 145 150 155 160
Asp Phe Asp Gly Asp Glu He Phe His Val Asp Met Ala Lys Lys Glu 165 170 175 Thr Val Trp Arg Leu Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala 180 185 190
Gin Gly Ala Leu Ala Asn He Ala Val Asp Lys Ala Asn Leu Glu He 195 200 205
Met Thr Lys Arg Ser Asn Tyr Thr Pro He Thr Asn 210 215 220
I-As MBP.βlβ2αlα2.CK nucleotide sequence gcggccgcca ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca 60 ggttccactg gtgacttcaa gaacattgtg acacctcgaa caccacctcc agctagcgga 120 gggggcggaa gcggcggagg gggagactcc gaaaggcatt tcgtgttcca gttcaagggc 180 gagtgctact tcaccaacgg gacgcagcgc atacgatctg tggacagata catctacaac 240 cgggaggagt acctgcgctt cgacagcgac gtgggcgagt accgcgcggt gaccgagctg 300 gggcggccag accccgagta ctacaataag cagtacctgg agcaaacgcg ggccgagctg 360 gacacggtgt gcagacacaa ctacgagggg gtggagaccc acacctccct gcggcggctt 420 gaacagccca atgtcgtcat ctccctgtcc aggacagagg ccctcaacca ccacaacact 480 ctggtctgct cagtgacaga tttctaccca gccaagatca aagtgcgctg gttccggaat 540 ggccaggagg agacggtggg ggtctcatcc acacagctta ttaggaatgg ggactggacc 600 ttccaggtcc tggtcatgct ggagatgacc cctcggcggg gagaggtcta cacctgccac 660 gtggagcatc cgagcctgaa gagccccatc actgtggagt ggactagtgg tggcggtggc 720 agcggcggtg gtggttccgg tggcggcggt tctggcggtg gcggttcctc gagtgaagac 780 gacattgagg ccgaccacgt aggcgtctat ggtacaactg tatatcagtc tcctggagac 840 attggccagt acacacatga atttgatggt gatgagtggt tctatgtgga cttggataag 900 aaggagacta tctggatgct tcctgagttt ggccaattga caagctttga cccccaaggt 960 ggactgcaaa acatagctac aggaaaatac accttgggaa tcttgactaa gaggtcaaat 1020 tccaccccag ctaccaatga ggctcctcaa gcgactgtgt tccccaagtc ccctgtgctg 1080 ctgggtcagc ccaacaccct catctgcttt gtggacaaca tcttccctcc tgtgatcaac 1140 atcacatggc tcagaaatag taagtcagtc acagacggcg tttatgagac cagcttcctt 1200 gtcaaccgtg accattcctt ccacaagctg tcttatctca ccttcatccc ttctgacgat 1260 gatatttatg actgcaaggt ggagcactgg ggcctggagg agccggttct gaaacactgg 1320 gctagcggag ggggcggaag cggcggaggg ggagctgatg ctgcaccaac tgtatccatc 1380 ttcccaccat ccagtgagca gttaacatct ggaggtgcct cagtcgtgtg cttcttgaac 1440 aacttctacc ccaaagacat caatgtcaag tggaagattg atggcagtga acgacaaaat 1500 ggcgtcctga acagttggac tgatcaggac agcaaagaca gcacctacag catgagcagc 1560 accctcacgt tgaccaagga cgagtatgaa cgacataaca gctatacctg tgaggccact 1620 cacaagacat caacttcacc cattgtcaag agcttcaaca ggaatgagtg ttagggtacc 1680
I-As MBP.βlβ2αlα2.CK amino acid sequence
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
5 10 15 Gly Ser Thr Gly Asp Phe Lys Asn He Val Thr Pro Arg Thr Pro Pro
20 25 30 Pro Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser Glu Arg 35 40 45
His Phe Val Phe Gin Phe Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr 50 55 60
Gin Arg He Arg 'Ser Val Asp Arg Tyr He Tyr Asn Arg Glu Glu Tyr 65 70 75 80
Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu 85 90 95
Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys Gin Tyr Leu Glu Gin Thr 100 105 110
Arg Ala Glu Leu Asp Thr Val Cys Arg His Asn Tyr Glu Gly Val Glu 115 120 125 Thr His Thr Ser Leu Arg Arg Leu Glu Gin Pro Asn Val Val He Ser 130 135 140
Leu Ser Arg Thr Glu Ala Leu Asn His His Asn Thr Leu Val Cys Ser 145 150 155 160
Val Thr Asp Phe Tyr Pro Ala Lys He Lys Val Arg Trp Phe Arg Asn 165 170 175
Gly Gin Glu Glu Thr Val Gly Val Ser Ser Thr Gin Leu He Arg Asn 180 185 190
Gly Asp Trp Thr Phe Gin Val Leu Val Met Leu Glu Met Thr Pro Arg 195 200 205 Arg Gly Glu Val Tyr Thr Cys His Val Glu His Pro Ser Leu Lys Ser 210 215 220
Pro He Thr Val Glu Trp Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser Glu Asp 245 250 255
Asp He Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr Val Tyr Gin 260 265 270
Ser Pro Gly Asp He Gly Gin Tyr Thr His Glu Phe Asp Gly Asp Glu 275 280 285 Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr He Trp Met Leu Pro 290 295 300
Glu Phe Gly Gin Leu Thr Ser Phe Asp Pro Gin Gly Gly Leu Gin Asn 305 310 315 320
He Ala Thr Gly Lys Tyr Thr Leu Gly He Leu Thr Lys Arg Ser Asn 325 330 335
Ser Thr Pro Ala Thr Asn Glu Ala Pro Gin Ala Thr Val Phe Pro Lys 340 345 350
Ser Pro Val Leu Leu Gly Gin Pro Asn Thr Leu He Cys Phe Val Asp 355 360 365
Asn He Phe Pro Pro Val He Asn He Thr Trp Leu Arg Asn Ser Lys 370 375 380
Ser Val Thr Asp Gly Val Tyr Glu Thr Ser Phe Leu Val Asn Arg Asp 385 390 395 400
His Ser Phe His Lys Leu Ser Tyr Leu Thr Phe He Pro .Ser Asp Asp 405 410 415 Asp He Tyr Asp Cys Lys Val Glu His Trp Gly Leu Glu Glu Pro Val 420 425 430
Leu Lys His Trp Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ala 435 440 445
Asp Ala Ala Pro Thr Val Ser He Phe Pro Pro Ser Ser Glu Gin Leu 450 455 460
Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro 465 470 475 480
Lys Asp He Asn Val Lys Trp Lys He Asp Gly Ser Glu Arg Gin Asn 485 490 495 Gly Val Leu Asn Ser Trp Thr Asp Gin Asp Ser Lys Asp Ser Thr Tyr 500 505 510
Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His 515 520 525
Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro He
530 535 540
Val Lys Ser Phe Asn Arg Asn Glu Cys 545 550
<210> C0528-AC <211> 600 <212> DNA
<213> Homo sapiens atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggtggtggc eo ggttcgcgtc cacgtttctt ggaacaggtt aaacatgagt gtcatttttt caatgggacg 120 gaacgcgtgc gttttctgga tcgttacttt tatcaccaag aggaatacgt acgcttcgac 180 agcgatgtgg gcgaatatcg tgcggtcacg gaactgggtc gtcctgatgc cgaatactgg 240 aacagtcaga aggacttact ggagcagaaa cgtgcagcgg tggataccta ttgccgccac 300 aattacggcg ttggtgaaag cttcacagtc cagcgtcgcg gtggcatcaa agaagagcat 360 gtgattatcc aggcggaatt ctatctgaat ccggatcaat cgggcgaatt catgtttgac 420 ttcgatggtg atgagatttt ccatgttgat atggcaaaga aagaaacggt ctggcgctta 480 gaggaatttg gccgctttgc ctcgttcgaa gctcaaggcg cattggccaa cattgctgtg 540 gataaagcga acctggaaat catgacaaaa cgctccaact atactccgat taccaattaa 600 <210> CO608-AC <211> 642 <212> DNA <213> Homo sapiens atgggggaca ccggtcgcag cttcaccctg gcctccagcg agaccggcgt gggtgcttct 60 ggcgggggcg gttcgggcgg tgggggtgac acccgtccac gtttcttgga acaggttaaa 120 catgagtgtc attttttcaa tgggacggaa cgcgtgcgtt ttctggatcg ttacttttat 180 caccaagagg aatacgtacg cttcgacagc gatgtgggcg aatatcgtgc ggtcacggaa 240 ctgggtcgtc ctgatgccga atactggaac agtcagaagg acttactgga gcagaaacgt 300 gcagcggtgg atacctattg ccgccacaat tacggcgttg gtgaaagctt cacagtccag 360 cgtcgcacta gtggtggcgg tggctctatc aaagaagagc atgtgattat ccaggcggaa 420 ttctatctga atccggatca atcgggcgaa ttcatgtttg acttcgatgg tgatgagatt 480 ttccatgttg atatggcaaa gaaagaaacg gtctggcgct tagaggaatt tggccgcttt 540 gcctcgttcg aagctcaagg cgcattggcc aacattgctg tggataaagc gaacctggaa 600 atcatgacaa aacgctccaa ctatactccg attaccaatt aa 642
<210> C0528
<211> 600
<212> DNA
<213> Homo sapiens atgggggaca ccggaagatc gttcacactc gcatcatcag agacaggagt aggaggagga 60 ggatcgcgac cacgtttctt ggagcaggtt aaacatgagt gtcatttctt caacgggacg 120 gagcgggtgc ggttcctgga cagatacttc tatcaccaag aggagtacgt gcgcttcgac 180 agcgacgtgg gggagtaccg ggcggtgacg gagctggggc ggcctgatgc cgagtactgg 240 aacagccaga gggacctcct ggagcagaag cgggccgcgg tggacaccta ctgcagacac 300 aactacgggg ttggtgagag cttcacagtg cagcggcgag gaggtatcaa agaagaacat 360 gtgatcatcc aggccgagtt ctatctgaat cctgaccaat caggcgagtt tatgtttgac 420 tttgatggtg atgagatttt ccatgtggat atggcaaaga aggagacggt ctggcggctt 480 gaagaatttg gacgatttgc cagctttgag gctcaaggtg cattggccaa catagctgtg 540 gacaaagcca acctggaaat catgacaaag cgctccaact atactccgat caccaattaa 600
<210> CO608 <211> 630 <212> DNA <213> Homo sapiens
ATGGGGGACACCCGCAGCTTCACCCTGGCCTCCAGCGAGACCGGCGTGGGCGCTAGCGGAGGGGGCGGAAGC GGCGGAGGGGGGCCACGTTTCTTGGAGCAGGTTAAACATGAGTGTCATTTCTTCAACGGGACGGAGCGGGTG CGGTTCCTGGACAGATACTTCTATCACCAAGAGGAGTACGTGCGCTTCGACAGCGACGTGGGGGAGTACCGG GCGGTGACGGAGCTGGGGCGGCCTGATGCCGAGTACTGGAACAGCCAGAAGGACCTCCTGGAGCAGAAGCGG GCCGCGGTGGACACCTACTGCAGACACAACTACGGGGTTGGTGAGAGCTTCACAGTGCAGCGGCGAACTAGT GGTGGCGGTGGCAGCATCAAAGAAGAACATGTGATCATCCAGGCCGAGTTCTATCTGAATCCTGACCAATCA GGCGAGTTTATGTTTGACTTTGATGGTGATGAGATTTTCCATGTGGATATGGCAAAGAAGGAGACGGTCTGG CGGCTTGAAGAATTTGGACGATTTGCCAGCTTTGAGGCTCAAGGTGCATTGGCCAACATAGCTGTGGACAAA GCCAACCTGGAAATCATGACAAAGCGCTCCAACTATACTCCGATCACCAATTAA
<210> CO 608 variation <211> 642 <212> DNA--CO608
<213> Homo sapiens
ATGGGGGACACCGGTCGCAGCTTCACCCTGGCCTCCAGCGAGACCGGCGTGGGCGCTAGCGGAGGGGGCGGA AGCGGCGGAGGGGGGGACACCCGACCACGTTTCTTGGAGCAGGTTAAACATGAGTGTCATTTCTTCAACGGG ACGGAGCGGGTGCGGTTCCTGGACAGATACTTCTATCACCAAGAGGAGTACGTGCGCTTCGACAGCGACGTG GGGGAGTACCGGGCGGTGACGGAGCTGGGGCGGCCTGATGCCGAGTACTGGAACAGCCAGAAGGACCTCCTG GAGCAGAAGCGGGCCGCGGTGGACACCTACTGCAGACACAACTACGGGGTTGGTGAGAGCTTCACAGTGCAG CGGCGAACTAGTGGTGGCGGTGGCAGCATCAAAGAAGAACATGTGATCATCCAGGCCGAGTTCTATCTGAAT CCTGACCAATCAGGCGAGTTTATGTTTGACTTTGATGGTGATGAGATTTTCCATGTGGATATGGCAAAGAAG GAGACGGTCTGGCGGCTTGAAGAATTTGGACGATTTGCCAGCTTTGAGGCTCAAGGTGCATTGGCCAACATA GCTGTGGACAAAGCCAACCTGGAAATCATGACAAAGCGCTCCAACTATACTCCGATCACCAATTAA
<210> C0528-AC <211> 200 <212> PRT <213> Homo sapiens Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15
Val Gly Gly Gly Gly Ser Arg Pro Arg Phe Leu Glu Gin Val Lys His 20 25 30
Glu Cys His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg 35 40 45
Tyr Phe Tyr His Gin Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly 50 55 60
Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp 65 70 75 80 Asn Ser Gin Lys Asp Leu Leu Glu Gin Lys Arg Ala Ala Val Asp Thr
85 90 95
Tyr Cys Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gin Arg 100 105 110
Arg Gly Gly He Lys Glu Glu His Val He He Gin Ala Glu Phe. Tyr 115 120 125
Leu Asn Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp 130 135 140
Glu He Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu 145 150 155 160 Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala
165 170 175
Asn He Ala Val Asp Lys Ala Asn Leu Glu He Met Thr Lys Arg Ser 180 185 190
Asn Tyr Thr Pro He Thr Asn 195
<210> C0608-AC <211> 214 <212> PRT <213> Homo sapiens Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15
Val Gly Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Thr Arg 20 25 30
Pro Arg Phe Leu Glu Gin Val Lys His Glu Cys His Phe Phe Asn Gly 35 40 45 Thr Glu Arg Val Arg Phe Leu Asp Arg Tyr Phe Tyr His Gin Glu Glu 50 55 60
Tyr Val Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu 65 70 75 80
Leu Gly Arg Pro Asp Ala Glu Tyr Trp Asn Ser Gin Lys Asp Leu Leu 85 90 95
Glu Gin Lys Arg Ala Ala Val Asp Thr Tyr Cys Arg His Asn Tyr Gly 100 105 110
Val Gly Glu Ser Phe Thr Val Gin Arg Arg Thr Ser Gly Gly Gly Gly 115 120 125 Ser He Lys Glu Glu His Val He He Gin Ala Glu Phe Tyr Leu Asn 130 135 140
Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp Glu He 145' 150 155 160
Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu Glu Glu 165 170' 175
Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala Asn He 180 185 190
Ala Val Asp Lys Ala Asn Leu Glu He Met Thr Lys Arg Ser Asn Tyr 195 200 205 Thr Pro He Thr Asn 210
<210> CO608 <211> 209 <212> PRT <213> Homo sapiens
MGDTRSFTLASSETGVGASGGGGSGGGGPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDVGEYR AVTELGRPDAEY NSQKDLLEQKRAAVDTYCRHNYGVGESFTVQRRTSGGGGSIKEEHVIIQAEFYLNPDQS GEFMFDFDGDEIFHVDMAKKETV RLEEFGRFASFΞAQGALANIAVDKANLEIMTKRSNYTPITN
<210> C0608 -variation <211> 211 <212> PRT <213> Homo sapiens MGDTGRSFTLASSETGVGASGGGGSGGGGDTRPRFLEQVKHECHFFNGTERVRFLDRYFYHQEEYVRFDSDV GEYRAVTELGRPDAEY NSQKDLLEQ RAAVDTYCRHNYGVGESFTVQRRTSGGGGSIKEEHVIIQAEFYLN PDQSGEFMFDFDGDEIFHVDMAKKETV RLΞEFGRFASFEAQGALANIADKANLΞIMT RSNYTPIT
<210> C0528
<211> 200
<212> PRT
<213> Homo sapiens
Met Gly Asp Thr Gly Arg Ser Phe Thr Leu Ala Ser Ser Glu Thr Gly
5 10 15
Val Gly Gly Gly Gly Ser Arg Pro Arg Phe Leu Glu Gin Val Lys His 20 25 30
Glu Cys His Phe Phe Asn Gly Thr Glu Arg Val Arg Phe Leu Asp Arg 35 40 45 Tyr Phe Tyr His Gin Glu Glu Tyr Val Arg Phe Asp Ser Asp Val Gly 50 55 60
Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Ala Glu Tyr Trp 65 70 75 80
Asn Ser Gin Arg Asp Leu Leu Glu Gin Lys Arg Ala Ala Val Asp Thr 85 90 95
Tyr Cys Arg His Asn Tyr Gly Val Gly Glu Ser Phe Thr Val Gin Arg 100 105 110
Arg Gly Gly He Lys Glu Glu His Val He He Gin Ala Glu Phe Tyr 115 120 125 Leu Asn Pro Asp Gin Ser Gly Glu Phe Met Phe Asp Phe Asp Gly Asp 130 135 140
Glu He Phe His Val Asp Met Ala Lys Lys Glu Thr Val Trp Arg Leu 145 150 155 160
Glu Glu Phe Gly Arg Phe Ala Ser Phe Glu Ala Gin Gly Ala Leu Ala 165 170 175
Asn He Ala Val Asp Lys Ala Asn Leu Glu He Met Thr Lys Arg Ser 180 185 190
Asn Tyr Thr Pro He Thr Asn 195
<210> IAS MBP1-14 CHI . CH2. CH3 <211> 2346 <212> DNA <213> murine gcggccgcca ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca 60 ggttccactg gtgacatggc gtcacagaag agaccctccc agaggcacgg atccaaggct 120 agcggagggg gcggaagcgg cggaggggga gactccgaaa ggcatttcgt gttccagttc 180 aagggcgagt gctacttcac caacgggacg cagcgcatac gatctgtgga cagatacatc 240 tacaaccggg aggagtacct gcgcttcgac agcgacgtgg gcgagtaccg cgcggtgacc 300 gagctggggc ggccagaccc cgagtactac aataagcagt acctggagca aacgcgggcc 360 gagctggaca cggtgtgcag acacaactac gagggggtgg agacccacac ctccctgcgg 420 cggcttgaac agcccaatgt cgtcatctcc ctgtccagga cagaggccct caaccaccac 480 aacactctgg tctgctcagt gacagatttc tacccagcca agatcaaagt gcgctggttc 540 cggaatggcc aggaggagac ggtgggggtc tcatccacac agcttattag gaatggggac 600 tggaccttcc aggtcctggt catgctggag atgacccctc ggcggggaga ggtctacacc 660 tgccacgtgg agcatccgag cctgaagagc cccatcactg tggagtggac tagtggtggc 720 ggtggcagcg gcggtggtgg ttccggtggc ggcggttctg gcggtggcgg ttcctcgagt 780 gaagacgaca ttgaggccga ccacgtaggc gtctatggta caactgtata tcagtctcct 840 ggagacattg gccagtacac acatgaattt gatggtgatg agtggttcta tgtggacttg 900 gataagaagg agactatctg gatgcttcct gagtttggcc aattgacaag ctttgacccc 960 caaggtggac tgcaaaacat agctacagga aaatacacct tgggaatctt gactaagagg 1020 tcaaattcca ccccagctac caatgaggct cctcaagcga ctgtgttccc caagtcccct 1080 gtgctgctgg gtcagcccaa caccctcatc tgctttgtgg acaacatctt ccctcctgtg 1140 atcaacatca catggctcag aaatagtaag tcagtcacag acggcgttta tgagaccagc 1200 ttccttgtca accgtgacca ttccttccac aagctgtctt atctcacctt catcccttct 1260 gacgatgata tttatgactg caaggtggag cactggggcc tggaggagcc ggttctgaaa 1320 cactgggcta gcggaggggg cggaagcggc ggagggggag ccaaaacgac acccccatct 1380 gtctatccac tggcccctgg atctgctgcc caaactaact ccatggtgac cctgggatgc 1440 ctggtcaagg gctatttccc tgagccagtg acagtgacct ggaactctgg atccctgtcc 1500 agcggtgtgc acaccttccc agctgtcctg cagtctgacc tctacactct gagcagctca 1560 gtgactgtcc cctccagcac ctggcccagc gagaccgtca cctgcaacgt tgcccacccg 1620 gccagcagca ccaaggtgga caagaaaatt gtgcccaggg attgtggttg taagccttgc 1680 atatgtacag tcccagaagt atcatctgtc ttcatcttcc ccccaaagcc caaggatgtg 1740 ctcaccatta ctctgactcc taaggtcacg tgtgttgtgg tagacatcag caaggatgat 1800 cccgaggtcc agttcagctg gtttgtagat gatgtggagg tgcacacagc tcagacgcaa 1860 ccccgggagg agcagttcaa cagcactttc cgctcagtca gtgaacttcc catcatgcac 1920 caggactggc tcaatggcaa ggagttcaaa tgcagggtca acagtgcagc tttccctgcc 1980 cccatcgaga aaaccatctc caaaaccaaa ggcagaccga aggctccaca ggtgtacacc 2040 attccacctc ccaaggagca gatggccaag gataaagtca gtctgacctg catgataaca 2100 gacttcttcc ctgaagacat tactgtggag tggcagtgga atgggcagcc agcggagaac 2160 tacaagaaca ctcagcccat catggacaca gatggctctt acttcgtcta cagcaagctc 2220 aatgtgcaga agagcaactg ggaggcagga aatactttca cctgctctgt gttacatgag 2280 ggcctgcaca accaccatac tgagaagagc ctctcccact ctcctggtaa atgatctggt 2340 acctgc 2346
<210> IAS MBP 1-14 CH1.H <211> 1701 <212> DNA <213> murine gcggccgcca ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca 60 ggttccactg gtgacatggc gtcacagaag agaccctccc agaggcacgg atccaaggct 120 agcggagggg gcggaagcgg cggaggggga gactccgaaa ggcatttcgt gttccagttc 180 aagggcgagt gctacttcac caacgggacg cagcgcatac gatctgtgga cagatacatc 240 tacaaccggg aggagtacct gcgcttcgac agcgacgtgg gcgagtaccg cgcggtgacc 300 gagctggggc ggccagaccc cgagtactac aataagcagt acctggagca aacgcgggcc 360 gagctggaca cggtgtgcag acacaactac gagggggtgg agacccacac ctccctgcgg 420 cggcttgaac agcccaatgt cgtcatctcc ctgtccagga cagaggccct caaccaccac 480 aacactctgg tctgctcagt gacagatttc tacccagcca agatcaaagt gcgctggttc 540 cggaatggcc aggaggagac ggtgggggtc tcatccacac agcttattag gaatggggac 600 tggaccttcc aggtcctggt catgctggag atgacccctc ggcggggaga ggtctacacc 660 tgccacgtgg agcatccgag cctgaagagc cccatcactg tggagtggac tagtggtggc 720 ggtggcagcg gcggtggtgg ttccggtggc ggcggttctg gcggtggcgg ttcctcgagt 780 gaagacgaca ttgaggccga ccacgtaggc gtctatggta caactgtata tcagtctcct 840 ggagacattg gccagtacac acatgaattt gatggtgatg agtggttcta tgtggacttg 900 gataagaagg agactatctg gatgcttcct gagtttggcc aattgacaag ctttgacccc 960 caaggtggac tgcaaaacat agctacagga aaatacacct tgggaatctt gactaagagg 1020 tcaaattcca ccccagctac caatgaggct cctcaagcga ctgtgttccc caagtcccct 1080 gtgctgctgg gtcagcccaa caccctcatc tgctttgtgg acaacatctt ccctcctgtg 1140 atcaacatca catggctcag aaatagtaag tcagtcacag acggcgttta tgagaccagc 1200 ttccttgtca accgtgacca ttccttccac aagctgtctt atctcacctt catcccttct 1260 gacgatgata tttatgactg caaggtggag cactggggcc tggaggagcc ggttctgaaa 1320 cactgggcta gcggaggggg cggaagcggc ggagggggag ccaaaacgac acccccatct 1380 gtctatccac tggcccctgg atctgctgcc caaactaact ccatggtgac cctgggatgc 1440 ctggtcaagg gctatttccc tgagccagtg acagtgacct ggaactctgg atccctgtcc 1500 agcggtgtgc acaccttccc agctgtcctg cagtctgacc tctacactct gagcagctca 1560 gtgactgtcc cctccagcac ctggcccagc gagaccgtca cctgcaacgt tgcccacccg 1620 gccagcagca ccaaggtgga caagaaaatt gtgcccaggg attgtggttg taagccttgc 1680 atatgtacag tctaaggtac c 1701
<210> IAS MBP 90-101 CH1.H.CH2
<211> 2053
<212> DNA <213> murine gcggccgcca ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca 60 ggttccactg gtgacttcaa gaacattgtg acacctcgaa caccacctcc agctagcgga 120 gggggcggaa gcggcggagg gggagactcc gaaaggcatt tcgtgttcca gttcaagggc 180 gagtgctact tcaccaacgg gacgcagcgc atacgatctg tggacagata catctacaac 240 cgggaggagt acctgcgctt cgacagcgac gtgggcgagt accgcgcggt gaccgagctg 300 gggcggccag accccgagta ctacaataag cagtacctgg agcaaacgcg ggccgagctg 360 gacacggtgt gcagacacaa ctacgagggg gtggagaccc acacctccct gcggcggctt 420 gaacagccca atgtcgtcat ctccctgtcc aggacagagg ccctcaacca ccacaacact 480 ctggtctgct cagtgacaga tttctaccca gccaagatca aagtgcgctg gttccggaat' 540 ggccaggagg agacggtggg ggtctcatcc acacagctta ttaggaatgg ggactggacc 600 ttccaggtcc tggtcatgct ggagatgacc cctcggcggg gagaggtcta cacctgccac 660 gtggagcatc cgagcctgaa gagccccatc actgtggagt ggactagtgg tggcggtggc 720 agcggcggtg gtggttccgg tggcggcggt tctggcggtg gcggttcctc gagtgaagac 780 gacattgagg ccgaccacgt aggcgtctat ggtacaactg tatatcagtc tcctggagac 840 attggccagt acacacatga atttgatggt gatgagtggt tctatgtgga cttggataag 900 aaggagacta tctggatgct tcctgagttt ggccaattga caagctttga cccccaaggt 960 ggactgcaaa acatagctac aggaaaatac accttgggaa tcttgactaa gaggtcaaat 1020 tccaccccag ctaccaatga ggctcctcaa gcgactgtgt tccccaagtc ccctgtgctg 1080 ctgggtcagc ccaacaccct catctgcttt gtggacaaca. tcttccctcc tgtgatcaac 1140 atcacatggc tcagaaatag taagtcagtc acagacggcg tttatgagac cagcttcctt 1200 gtcaaccgtg accattcctt ccacaagctg tcttatctca ccttcatccc ttctgacgat 1260 gatatttatg actgcaaggt ggagcactgg ggcctggagg agccggttct gaaacactgg 1320 gctagcggag ggggcggaag cggcggaggg ggagccaaaa caacaccccc atcagtctat 1380 ccactggccc ctgggtgtgg agatacaact ggttcctccg tgactctggg atgcctggtc 1440 aagggctact tccctgagtc agtgactgtg acttggaact ctggctccct gtccagcagt 1500 gtgcacacct tcccagctct cctgcagtct ggactctaca ctatgagcag ctcagtgact 1560 gtcccctcca gcacctggcc aagtcagacc gtcacctgca gcgttgctca cccagccagc 1620 agcaccacgg tggacaaaaa acttgagccc agcgggccca tttcaacaat caacccctgt 1680 cctccatgca aggagtgtca caaatgccca gctcctaacc tggagggtgg accatccgtc 1740 ttcatcttcc ctccaaatat caaggatgta ctcatgatct ccctgacacc caaggtcacg 1800 tgtgtggtgg tggatgtgag cgaggatgac ccagacgtcc agatcagctg gtttgtgaac 1860 aacgtggaag tacacacagc tcagacacaa acccatagag aggattacaa cagtactatc 1920 cgggtggtca gcaccctccc catccagcac caggactgga tgagtggcaa ggagttcaaa 1980 tgcaaggtca acaacaaaga cctcccatca cccatcgaga gaaccatctc aaaaattaaa 2040 tagggtaccc cga 2053
<210> IAS MBP 90-101 CH1.H <211> 1707 <212> DNA <213> murine gcggccgcca ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca 60 ggttccactg gtgacttcaa gaacattgtg acacctcgaa caccacctcc agctagcgct 120 agcggagggg gcggaagcgg cggaggggga gactccgaaa ggcatttcgt gttccagttc 180 aagggcgagt gctacttcac caacgggacg cagcgcatac gatctgtgga cagatacatc 240 tacaaccggg aggagtacct gcgcttcgac agcgacgtgg gcgagtaccg cgcggtgacc 300 gagctggggc ggccagaccc cgagtactac aataagcagt acctggagca aacgcgggcc 360 gagctggaca cggtgtgcag acacaactac gagggggtgg agacccacac ctccctgcgg 420 cggcttgaac agcccaatgt cgtcatctcc ctgtccagga cagaggccct caaccaccac 480 aacactctgg tctgctcagt gacagatttc tacccagcca agatcaaagt gcgctggttc 540 cggaatggcc aggaggagac ggtgggggtc tcatccacac agcttattag gaatggggac 600 tggaccttcc aggtcctggt catgctggag atgacccctc ggcggggaga ggtctacacc 660 tgccacgtgg agcatccgag cctgaagagc cccatcactg tggagtggac tagtggtggc 720 ggtggcagcg gcggtggtgg ttccggtggc ggcggttctg gcggtggcgg ttcctcgagt 780 gaagacgaca ttgaggccga ccacgtaggc gtctatggta caactgtata tcagtctcct 840 ggagacattg gccagtacac acatgaattt gatggtgatg agtggttcta tgtggacttg 900 gataagaagg agactatctg gatgcttcct gagtttggcc aattgacaag cttaagcttt 960 gacccccaag gtggactgca aaacatagct acaggaaaat acaccttggg aatcttgact 1020 aagaggtcaa attccacccc agctaccaat gaggctcctc aagcgactgt gttccccaag 1080 tcccctgtgc tgctgggtca gcccaacacc ctcatctgct ttgtggacaa catcttccct 1140 cctgtgatca acatcacatg gctcagaaat agtaagtcag tcacagacgg cgtttatgag 1200 accagcttcc ttgtcaaccg tgaccattcc ttccacaagc tgtcttatct caccttcatc 1260 ccttctgacg atgatattta tgactgcaag gtggagcact ggggcctgga ggagccggtt 1320 ctgaaacact gggctagcgg agggggcgga agcggcggag ggggagccaa aacgacaccc 1380 ccatctgtct atccactggc ccctggatct gctgcccaaa ctaactccat ggtgaccctg 1440 ggatgcctgg tcaagggcta tttccctgag ccagtgacag tgacctggaa ctctggatcc 1500 ctgtccagcg gtgtgcacac cttcccagct gtcctgcagt ctgacctcta cactctgagc 1560 agctcagtga ctgtcccctc cagcacctgg cccagcgaga ccgtcacctg caacgttgcc 1620 cacccggcca gcagcaccaa ggtggacaag aaaattgtgc ccagggattg tggttgtaag 1680 ccttgcatat gtacagtcta aggtacc 1707
<210> MBP 1-14 CK <211> 1686 <212> DNA <213> murine gcggccgcca ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca 60 ggttccactg gtgacatggc gtcacagaag agaccctccc agaggcacgg atccaaggct 120 agcggagggg gcggaagcgg cggaggggga gactccgaaa ggcatttcgt gttccagttc 180 aagggcgagt gctacttcac caacgggacg cagcgcatac gatctgtgga cagatacatc 240 tacaaccggg aggagtacct gcgcttcgac agcgacgtgg gcgagtaccg cgcggtgacc 300 gagctggggc ggccagaccc cgagtactac aataagcagt acctggagca aacgcgggcc 360 gagctggaca cggtgtgcag acacaactac gagggggtgg agacccacac ctccctgcgg 420 cggcttgaac agcccaatgt cgtcatctcc ctgtccagga cagaggccct caaccaccac 480 aacactctgg tctgctcagt gacagatttc tacccagcca agatcaaagt gcgctggttc 540 cggaatggcc aggaggagac ggtgggggtc tcatccacac agcttattag gaatggggac 600 tggaccttcc aggtcctggt catgctggag atgacccctc ggcggggaga ggtctacacc 660 tgccacgtgg agcatccgag cctgaagagc cccatcactg tggagtggac tagtggtggc 720 ggtggcagcg gcggtggtgg ttccggtggc ggcggttctg gcggtggcgg ttcctcgagt 780 gaagacgaca ttgaggccga ccacgtaggc gtctatggta caactgtata tcagtctcct 840 ggagacattg gccagtacac acatgaattt gatggtgatg agtggttcta tgtggacttg 900 gataagaagg agactatctg gatgcttcct gagtttggcc aattgacaag ctttgacccc 960 caaggtggac tgcaaaacat agctacagga aaatacacct tgggaatctt gactaagagg 1020 tcaaattcca ccccagctac caatgaggct cctcaagcga ctgtgttccc caagtcccct 1080 gtgctgctgg gtcagcccaa caccctcatc tgctttgtgg acaacatctt ccctcctgtg 1140 atcaacatca catggctcag aaatagtaag tcagtcacag acggcgttta tgagaccagc 1200 ttccttgtca accgtgacca ttccttccac aagctgtctt atctcacctt catcccttct 1260 gacgatgata tttatgactg caaggtggag cactggggcc tggaggagcc ggttctgaaa 1320 cactgggcta gcggaggggg cggaagcggc ggagggggag ctgatgctgc accaactgta 1380 tccatcttcc caccatccag tgagcagtta acatctggag gtgcctcagt cgtgtgcttc 1440 ttgaacaact tctaccccaa agacatcaat gtcaagtgga agattgatgg cagtgaacga 1500 caaaatggcg' tcctgaacag ttggactgat caggacagca aagacagcac ctacagcatg 1560 agcagcaccc tcacgttgac caaggacgag tatgaacgac ataacagcta tacctgtgag 1620 gccactcaca agacatcaac ttcacccatt gtcaagagct' tcaacaggaa tgagtgttag 1680 ggtacc 1686
<210> MBP 1-14 CH1.H.CH2
<211> 2059 <212> DNA
<213> murine gcggccgcca ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca 60 ggttccactg gtgacatggc gtcacagaag agaccctccc agaggcacgg atccaaggct 120 agcggagggg gcggaagcgg cggaggggga gactccgaaa ggcatttcgt gttccagttc 180 aagggcgagt gctacttcac caacgggacg cagcgcatac gatctgtgga cagatacatc 240 tacaaccggg aggagtacct gcgcttcgac agcgacgtgg gcgagtaccg cgcggtgacc 300 gagctggggc ggccagaccc cgagtactac aataagcagt acctggagca aacgcgggcc 360 gagctggaca cggtgtgcag acacaactac gagggggtgg agacccacac ctccctgcgg 420 cggcttgaac agcccaatgt cgtcatctcc ctgtccagga cagaggccct caaccaccac 480 aacactctgg tctgctcagt gacagatttc tacccagcca agatcaaagt gcgctggttc 540 cggaatggcc aggaggagac ggtgggggtc tcatccacac agcttattag gaatggggac 600 tggaccttcc aggtcctggt catgctggag atgacccctc ggcggggaga ggtctacacc 660 tgccacgtgg agcatccgag cctgaagagc cccatcactg tggagtggac tagtggtggc 720 ggtggcagcg gcggtggtgg ttccggtggc ggcggttctg gcggtggcgg ttcctcgagt 780 gaagacgaca ttgaggccga ccacgtaggc gtctatggta caactgtata tcagtctcct 840 ggagacattg gccagtacac acatgaattt gatggtgatg agtggttcta tgtggacttg 900 gataagaagg agactatctg gatgcttcct gagtttggcc aattgacaag ctttgacccc 960 caaggtggac tgcaaaacat agctacagga aaatacacct tgggaatctt gactaagagg 1020 tcaaattcca ccccagctac caatgaggct cctcaagcga ctgtgttccc caagtcccct 1080 gtgctgctgg gtcagcccaa caccctcatc tgctttgtgg acaacatctt ccctcctgtg 1140 atcaacatca catggctcag aaatagtaag tcagtcacag acggcgttta tgagaccagc 1200 ttccttgtca accgtgacca ttccttccac aagctgtctt atctcacctt catcccttct 1260 gacgatgata tttatgactg caaggtggag cactggggcc tggaggagcc ggttctgaaa 1320 cactgggcta gcggaggggg cggaagcggc ggagggggag ccaaaacaac acccccatca 1380 gtctatccac tggcccctgg gtgtggagat acaactggtt cctccgtgac tctgggatgc 1440 ctggtcaagg gctacttccc tgagtcagtg actgtgactt ggaactctgg ctccctgtcc 1500 agcagtgtgc acaccttccc agctctcctg cagtctggac tctacactat gagcagctca 1560 gtgactgtcc cctccagcac ctggccaagt cagaccgtca cctgcagcgt tgctcaccca 1620 gccagcagca ccacggtgga caaaaaactt gagcccagcg ggcccatttc aacaatcaac 1680 ccctgtcctc catgcaagga gtgtcacaaa tgcccagctc ctaacctgga gggtggacca 1740 tccgtcttca tcttccctcc aaatatcaag gatgtactca tgatctccct gacacccaag 1800 gtcacgtgtg tggtggtgga tgtgagcgag gatgacccag acgtccagat cagctggttt 1860 gtgaacaacg tggaagtaca cacagctcag acacaaaccc atagagagga ttacaacagt 1920 actatccggg tggtcagcac cctccccatc cagcaccagg actggatgag tggcaaggag 1980 ttcaaatgca aggtcaacaa caaagacctc ccatcaccca tcgagagaac catctcaaaa 2040 attaaatagg gtaccccga 2059
<210> MBP 90-101 CHI . H . CH2. CH3
<211> 2343
<212> DNA--
<213> murine gcggccgcca ccatggagac agacacactc ctgctatggg tactgctgct ctgggttcca 60 ggttccactg gtgacttcaa gaacattgtg acacctcgaa caccacctcc agctagcgga 120 gggggcggaa gcggcggagg gggagactcc gaaaggcatt tcgtgttcca gttcaagggc 180 gagtgctact tcaccaacgg gacgcagcgc atacgatctg tggacagata catctacaac 240 cgggaggagt acctgcgctt cgacagcgac gtgggcgagt accgcgcggt gaccgagctg 300 gggcggccag accccgagta ctacaataag cagtacctgg agcaaacgcg ggccgagctg 360 gacacgacgt gcagacacaa ctacgagggg gtggagaccc acacctccct gcggcggctt 420 gaacagccca atgtcgtcat ctccctgtcc aggacagagg ccctcaacca ccacaacact 480 ctggtctgct cagtgacaga tttctaccca gccaagatca aagtgcgctg gttccggaat 540 ggccaggagg agacggtggg ggtctcatcc acacagctta ttaggaatgg ggactggacc 600 ttccaggtcc tggtcatgct ggagatgacc cctcggcggg gagaggtcta cacctgccac 660 gtggagcatc cgagcctgaa gagccccatc actgtggagt ggactagtgg tggcggtggc 720 agcggcggtg gtggttccgg tggcggcggt tctggcggtg gcggttcctc gagtgaagac 780 gacattgagg ccgaccacgt aggcgtctat ggtacaactg tatatcagtc tcctggagac 840 attggccagt acacacatga atttgatggt gatgagtggt tctatgtgga cttggataag 900 aaggagacta tctggatgct tcctgagttt ggccaattga caagctttga cccccaaggt 960 ggactgcaaa acatagctac aggaaaatac accttgggaa tcttgactaa gaggtcaaat 1020 tccaccccag ctaccaatga ggctcctcaa gcgactgtgt tccccaagtc ccctgtgctg 1080 ctgggtcagc ccaacaccct catctgcttt gtggacaaca tcttccctcc tgtgatcaac 1140 atcacatggc tcagaaatag taagtcagtc acagacggcg tttatgagac cagcttcctt 1200 gtcaaccgtg accattcctt ccacaagctg tcttatctca ccttcatccc ttctgacgat 1260 gatatttatg actgcaaggt ggagcactgg ggcctggagg agccggttct gaaacactgg 1320 gctagcggag ggggcggaag cggcggagga agcttagcca aaacgacacc cccatctgtc 1380 tatccactgg cccctggatc tgctgcccaa actaactcca tggtgaccct gggatgcctg 1440 gtcaagggct atttccctga gccagtgaca gtgacctgga actctggatc cctgtccagc 1500 ggtgtgcaca ccttcccagc tgtcctgcag tctgacctct acactctgag cagctcagtg 1560 actgtcccct ccagcacctg gcccagcgag accgtcacct gcaacgttgc ccacccggcc 1620 agcagcacca aggtggacaa gaaaattgtg cccagggatt gtggttgtaa gccttgcata 1680 tgtacagtcc cagaagtatc atctgtcttc atcttccccc caaagcccaa ggatgtgctc 1740 accattactc tgactcctaa ggtcacgtgt gttgtggtag acatcagcaa ggatgatccc 1800 gaggtccagt tcagctggtt tgtagatgat gtggaggtgc acacagctca gacgcaaccc 1860 cgggaggagc agttcaacag cactttccgc tcagtcagtg aacttcccat catgcaccag 1920 gactggctca atggcaagga gttcaaatgc agggtcaaca gtgcagcttt ccctgccccc 1980 atcgagaaaa ccatctccaa aaccaaaggc agaccgaagg ctccacaggt gtacaccatt 2040 ccacctccca aggagcagat ggccaaggat aaagtcagtc tgacctgcat gataacagac 2100 ttcttccctg aagacattac tgtggagtgg cagtggaatg ggcagccagc ggagaactac 2160 aagaacactc agcccatcat ggacacagat ggctcttact tcgtctacag caagctcaat 2220 gtgcagaaga gcaactggga ggcaggaaat actttcacct gctctgtgtt acatgagggc 2280 ctgcacaacc accatactga gaagagcctc tcccactctc ctggtaaatg atctggtacc 2340 tgc 2343
<210> IASMBP 1-14 CHI . CH2. CH3 <211> 679 <212> PRT-- <213> murine
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
5 10 15
Gly Ser Thr Gly Asp Met Ala Ser Gin Lys Arg Pro Ser Gin Arg His 20 25 30
Gly Ser Lys Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser 35 40 45
Glu Arg His Phe Val Phe Gin Phe Lys Gly Glu Cys Tyr Phe Thr Asn 50 55 60 Gly Thr Gin Arg He Arg Ser Val Asp Arg Tyr He Tyr Asn Arg Glu 65 70 75 80
Glu Tyr Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr 85 90 95
Glu Leu Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys Gin Tyr Leu Glu 100 105 110 Gin Thr Arg Ala Glu Leu Asp Thr Val Cys Arg His Asn Tyr Glu Gly 115 120 125
Val Glu Thr His Thr Ser Leu Arg Arg Leu Glu Gin Pro Asn Val Val 130 135 140
He Ser Leu Ser Arg Thr Glu Ala Leu Asn His His Asn Thr Leu Val 145 150 155 160
Cys Ser Val Thr Asp Phe Tyr Pro Ala Lys He Lys Val Arg Trp Phe 165 170 175
Arg Asn Gly Gin Glu Glu Thr Val Gly Val Ser Ser Thr Gin Leu He 180 185 190 Arg Asn Gly Asp Trp Thr Phe Gin Val Leu Val Met Leu Glu Met Thr ' 195 200 205
Pro Arg Arg Gly Glu Val Tyr Thr Cys His Val Glu His Pro Ser Leu 210 215 220
Lys Ser Pro He Thr Val Glu Trp Thr Ser Gly Gly Gly Gly Ser Gly 225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser 245 250 255
Glu Asp Asp He Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr Val 260 265 270 Tyr Gin Ser Pro Gly Asp He Gly Gin Tyr Thr His Glu Phe Asp Gly 275 280 285
Asp Glu Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr He Trp Met 290 295 300
Leu Pro Glu Phe Gly Gin Leu Thr Ser Phe Asp Pro Gin Gly Gly Leu 305 310 315 320
Gin Asn He Ala Thr Gly Lys Tyr Thr Leu Gly He Leu Thr Lys Arg 325 330 335
Ser Asn Ser Thr Pro Ala Thr Asn Glu Ala Pro Gin Ala Thr Val Phe 340 345 350 Pro Lys Ser Pro Val Leu Leu Gly Gin Pro Asn Thr Leu He Cys Phe 355 360 365
Val Asp Asn He Phe Pro Pro Val He Asn He Thr Trp Leu Arg Asn 370 375 380 Ser Lys Ser Val Thr Asp Gly Val Tyr Glu Thr Ser Phe Leu Val Asn 385 390 395 400
Arg Asp His Ser Phe His Lys Leu Ser Tyr Leu Thr Phe He Pro Ser 405 410 415
Asp Asp Asp He Tyr Asp Cys Lys Val Glu His Trp Gly Leu Glu Glu 420 425 ' 430
Pro Val Leu Lys His Trp Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly 435 440 445
Gly Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly. Cys 450 455 460
Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly 465 470 475 480 Tyr Phe Pro Glu Ser Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser
485 490 495
Ser Ser Val His Thr Phe Pro Ala Leu Leu Gin Ser Gly Leu Tyr Thr 500 505 510
Met Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gin Thr 515 520 525
Val Thr Cys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val Asp Lys 530 535 540
Lys Leu Glu Pro Ser Gly Pro He Ser Thr He Asn Pro Cys Pro Pro
545 550 555 560 Cys Lys Glu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro
565 570 575
Ser Val Phe He Phe Pro Pro Asn He Lys Asp Val Leu Met He Ser 580 585 590
Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp 595 600 605
Pro Asp Val Gin He Ser Trp Phe Val Asn Asn Val Glu Val His Thr 610 615 620
Ala Gin Thr Gin Thr His Arg Glu Asp Tyr Asn Ser Thr He Arg Val 625 630 635 640 Val Ser Thr Leu Pro He Gin His Gin Asp Trp Met Ser Gly Lys Glu
645 650 655
Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro He Glu Arg 660 665 670
Thr He Ser Lys He Lys 675 <210> IAS MBP 1-14 CH1.H <211> 677 <212> PRT-- <213> murine
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
5 10 15
Gly Ser Thr Gly Asp Phe Lys Asn He Val Thr Pro Arg Thr Pro Pro 20 25 30
Pro Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser Glu Arg 35 40 45 His Phe Val Phe Gin Phe Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr 50 55 60
Gin Arg He Arg Ser Val Asp Arg Tyr He Tyr Asn Arg Glu Glu Tyr 65 70 75 80
Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu 85 90 95
Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys Gin Tyr Leu Glu Gin Thr 100 105 110
Arg Ala Glu Leu Asp Thr Val Cys Arg His Asn Tyr Glu Gly Val Glu 115 120 125 Thr His Thr Ser Leu Arg Arg Leu Glu Gin Pro Asn Val Val He Ser 130 135 140
Leu Ser Arg Thr Glu Ala Leu Asn His His Asn Thr Leu Val Cys Ser 145 150 155 160
Val Thr Asp Phe Tyr Pro Ala Lys He Lys Val Arg Trp Phe Arg Asn 165 170 175
Gly Gin Glu Glu Thr Val Gly Val Ser Ser Thr Gin Leu He Arg Asn 180 185 190
Gly Asp Trp Thr Phe Gin Val Leu Val Met Leu Glu Met Thr Pro Arg 195 200 205 Arg Gly Glu Val Tyr Thr Cys His Val Glu His Pro Ser Leu Lys Ser 210 215 220
Pro He Thr Val Glu Trp Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser Glu Asp 245 250 255
Asp He Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr Val Tyr Gin 260 265 270
Ser Pro Gly Asp He Gly Gin Tyr Thr His Glu Phe Asp Gly Asp Glu 275 280 285 Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr He Trp Met Leu Pro 290 295 300
Glu Phe Gly Gin Leu Thr Ser Phe Asp Pro Gin Gly Gly Leu Gin Asn 305 310 315 320
He Ala Thr Gly Lys Tyr Thr Leu Gly He Leu Thr Lys Arg Ser Asn 325 330 335 Ser Thr Pro Ala Thr Asn Glu Ala Pro Gin Ala Thr Val Phe Pro Lys 340 345 350
Ser Pro Val Leu Leu Gly Gin Pro Asn Thr Leu He Cys Phe Val Asp 355 360 365
Asn He Phe Pro Pro Val He Asn He Thr Trp Leu Arg Asn Ser Lys 370 375 380
Ser Val Thr Asp Gly Val Tyr Glu Thr Ser Phe Leu Val Asn Arg Asp 385 390 395 400
His Ser Phe His Lys Leu Ser Tyr Leu Thr Phe He Pro Ser Asp Asp 405 410 415 Asp He Tyr Asp Cys Lys Val Glu His Trp Gly Leu Glu Glu Pro Val 420 425 430
Leu Lys His Trp Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ala 435 440 445
Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Cys Gly Asp 450 455 460
Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe 465 470 475 480
Pro Glu Ser Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Ser
485 490 495 Val His Thr Phe Pro Ala Leu Leu Gin Ser Gly Leu Tyr Thr Met Ser 500 505 510
Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Gin Thr Val Thr
515 520 525
Cys Ser Val Ala His Pro Ala Ser Ser Thr Thr Val Asp Lys Lys Leu 530 535 540
Glu Pro Ser Gly Pro He Ser Thr He Asn Pro Cys Pro Pro Cys Lys 545 550 555 560
Glu Cys His Lys Cys Pro Ala Pro Asn Leu Glu Gly Gly Pro Ser Val 565 570 575 Phe He Phe Pro Pro Asn He Lys Asp Val Leu Met He Ser Leu Thr 580 585 590
Pro Lys Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp 595 600 605 Val Gin He Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gin 610 615 620 Thr Gin Thr His Arg Glu Asp Tyr Asn Ser Thr He Arg Val Val Ser 625 630 635 640
Thr Leu Pro He Gin His Gin Asp Trp Met Ser Gly Lys Glu Phe Lys 645 650 655
Cys Lys Val Asn Asn Lys Asp Leu Pro Ser Pro He Glu Arg Thr He 660 665 670
Ser Lys He Lys 675
<210> IAS MBP 90-101 CH1.H.CH2 <211> 563 <212> PRT-- <213> murine
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
5 10 15
Gly Ser Thr Gly Asp Phe Lys Asn He Val Thr Pro Arg Thr Pro Pro 20 25 30
Pro Ala Ser Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser 35 40 45
Glu Arg His Phe Val Phe Gin Phe Lys Gly Glu Cys Tyr Phe Thr Asn 50 55 60 Gly Thr Gin Arg He Arg Ser Val Asp Arg Tyr He Tyr Asn Arg Glu 65 70 75 80
Glu Tyr Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr 85 90 95
Glu Leu Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys Gin Tyr Leu Glu 100 105 110
Gin Thr Arg Ala Glu Leu Asp Thr Val Cys Arg His Asn Tyr Glu Gly 115 120 125
Val Glu Thr His Thr Ser Leu Arg Arg Leu Glu Gin Pro Asn Val Val 130 135 140 He Ser Leu Ser Arg Thr Glu Ala Leu Asn His His Asn Thr Leu Val 145 150 155 160
Cys Ser Val Thr Asp Phe Tyr Pro Ala Lys He Lys Val Arg Trp Phe 165 170 175
Arg Asn Gly Gin Glu Glu Thr Val Gly Val Ser Ser Thr Gin Leu He 180 185 190
Arg Asn Gly Asp Trp Thr Phe Gin Val Leu Val Met Leu Glu Met Thr 195 200 205
Pro Arg Arg Gly Glu Val Tyr Thr Cys His Val Glu His Pro Ser Leu 210 215 220
Lys Ser Pro He Thr Val Glu Trp Thr Ser Gly Gly Gly Gly Ser Gly 225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser 245 250 255
Glu Asp Asp He Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr Val 260 265 270 Tyr Gin Ser Pro Gly Asp He Gly Gin Tyr Thr His Glu Phe Asp Gly 275 280 285
Asp Glu Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr He Trp Met 290 295 300
Leu Pro Glu Phe Gly Gin Leu Thr Ser Leu Ser Phe Asp Pro Gin Gly 305 310 315 320
Gly Leu Gin Asn He Ala Thr Gly Lys Tyr Thr Leu Gly He Leu Thr 325 330 335
Lys Arg Ser Asn Ser Thr Pro Ala Thr Asn Glu Ala Pro Gin Ala Thr 340 345 350 Val Phe Pro Lys Ser Pro Val Leu Leu Gly Gin Pro Asn Thr Leu He 355 360 365
Cys Phe Val Asp Asn He Phe Pro Pro Val He Asn He Thr Trp Leu 370 375 380
Arg Asn Ser Lys Ser Val Thr Asp Gly Val Tyr Glu Thr Ser Phe Leu 385 390 395 400
Val Asn Arg Asp His Ser Phe His Lys Leu Ser Tyr Leu Thr Phe He 405 410 415
Pro Ser Asp Asp Asp He Tyr Asp Cys Lys Val Glu His Trp Gly Leu 420 425 430 Glu Glu Pro Val Leu Lys His Trp Ala Ser Gly Gly Gly Gly Ser Gly 435 440 445
Gly Gly Gly Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro 450 455 460
Gly Ser Ala Ala Gin Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val 465 470 475 480
Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser 485 490 495
Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Asp Leu
500 505 510 Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser 515 520 525
Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val 530 535 540
Asp Lys Lys He Val Pro Arg Asp Cys Gly Cys Lys Pro Cys He Cys 545 550 555 560 Thr Val
<210> IAS MBP 90-101 CH1.H <211> 556 <212> PRT <213> murine
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 5 10 15
Gly Ser Thr Gly Asp Met Ala Ser Gin Lys Arg Pro Ser Gin Arg His 20 25 30 Gly Ser Lys Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser 35 40 45
Glu Arg His Phe Val Phe Gin Phe Lys Gly Glu Cys Tyr Phe Thr Asn 50 55 60
Gly Thr Gin Arg He Arg Ser Val Asp Arg Tyr He Tyr Asn Arg Glu 65 70 75 80
Glu Tyr Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr 85 90 95
Glu Leu Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys Gin Tyr Leu Glu
100 105 110 Gin Thr Arg Ala Glu Leu Asp Thr Val Cys Arg His Asn Tyr Glu Gly
115 120 125
Val Glu Thr His Thr Ser Leu Arg Arg Leu Glu Gin Pro Asn Val Val 130 135 140
He Ser Leu Ser Arg Thr Glu Ala Leu Asn His His Asn Thr Leu Val 145 150 155 160
Cys Ser Val Thr Asp Phe Tyr Pro Ala Lys He Lys Val Arg Trp Phe 165 170 175
Arg Asn Gly Gin Glu Glu Thr Val Gly Val Ser Ser Thr Gin Leu He 180 185 190 Arg Asn Gly Asp Trp Thr Phe Gin Val Leu Val Met Leu Glu Met Thr 195 200 205
Pro Arg Arg Gly Glu Val Tyr Thr Cys His Val Glu His Pro Ser Leu 210 215 220 Lys Ser Pro He Thr Val Glu Trp Thr Ser Gly Gly Gly Gly Ser Gly 225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser 245 250 255
Glu Asp Asp He Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr Val 260 265 270
Tyr Gin Ser Pro Gly Asp He Gly Gin Tyr Thr His Glu Phe Asp Gly 275 280 285
Asp Glu Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr He Trp Met 290 295 300
Leu Pro Glu Phe Gly Gin Leu Thr Ser Phe Asp Pro Gin Gly Gly Leu
305 310 315 320 Gin Asn He Ala Thr Gly Lys Tyr Thr Leu Gly He Leu Thr Lys Arg
325 330 335
Ser Asn Ser Thr Pro Ala Thr Asn Glu Ala Pro Gin Ala Thr Val Phe 340 345 350
Pro Lys Ser Pro Val Leu Leu Gly Gin Pro Asn Thr Leu He Cys Phe 355 360 365
Val Asp Asn He Phe Pro Pro Val He Asn He Thr Trp Leu Arg Asn 370 375 38.0
Ser Lys Ser Val Thr Asp Gly Val Tyr Glu Thr Ser Phe Leu Val Asn 385 390 395 400 Arg Asp His Ser Phe His Lys Leu Ser Tyr Leu Thr Phe He Pro Ser
405 410 415
Asp Asp Asp He Tyr Asp Cys Lys Val Glu His Trp Gly Leu Glu Glu 420 425 430
Pro Val Leu Lys His Trp Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly 435 440 445
Gly Ala Asp Ala Ala Pro Thr Val Ser He Phe Pro Pro Ser Ser Glu 450 455 460
Gin Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 465 470 475 480 Tyr Pro Lys Asp He Asn Val Lys Trp Lys He Asp Gly Ser Glu Arg
485 490 495
Gin Asn Gly Val Leu Asn Ser Trp Thr Asp Gin Asp Ser Lys Asp Ser 500 505 510
Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 515 520 525
Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 530 535 540
Pro He Val Lys Ser Phe Asn Arg Asn Glu Cys 545 550 555
<210> IAS MBP 1-14 CK
<211> 775
<212> PRT <213> murine
Thr Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val
5 10 15 Pro Gly Ser Thr Gly Asp Met Ala Ser Gin Lys Arg Pro Ser Gin Arg
20 25 30
His Gly Ser Lys Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp 35 40 45
Ser Glu Arg His Phe Val Phe Gin Phe Lys Gly Glu Cys Tyr Phe Thr 50 55 60
Asn Gly Thr Gin Arg He Arg Ser Val Asp Arg Tyr He Tyr Asn Arg 65 70 75 80
Glu Glu Tyr Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val 85 90 95 Thr Glu Leu Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys Gin Tyr Leu 100 105 110
Glu Gin Thr Arg Ala Glu Leu Asp Thr Val Cys Arg His Asn Tyr Glu 115 120 125
Gly Val Glu Thr His Thr Ser Leu Arg Arg Leu Glu Gin Pro Asn Val 130 135 140
Val He Ser Leu Ser Arg Thr Glu Ala Leu Asn His His Asn Thr Leu 145 150 155 160
Val Cys Ser Val Thr Asp Phe Tyr Pro Ala Lys He Lys Val Arg Trp 165 170 175 Phe Arg Asn Gly Gin Glu Glu Thr Val Gly Val Ser Ser Thr Gin Leu 180 185 190
He Arg Asn Gly Asp Trp Thr Phe Gin Val Leu Val Met Leu Glu Met 195 200 205
Thr Pro Arg Arg Gly Glu Val Tyr Thr Cys His Val Glu His Pro Ser 210 215 ' 220
Leu Lys Ser Pro He Thr Val Glu Trp Thr Ser Gly Gly Gly Gly Ser 225 230 235 240
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser 245 250 255 Ser Glu Asp Asp He Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr 260 265 270
Val Tyr Gin Ser Pro Gly Asp He Gly Gin Tyr Thr His Glu Phe Asp 275 280 285
Gly Asp Glu Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr He Trp 290 295 300 Met Leu Pro Glu Phe Gly Gin Leu Thr Ser Phe Asp Pro Gin Gly Gly 305 310 315 320
Leu Gin Asn He Ala Thr Gly Lys Tyr Thr Leu Gly He Leu Thr Lys 325 330 335
Arg Ser Asn Ser Thr Pro Ala Thr Asn Glu Ala Pro Gin Ala Thr Val 340 345 350
Phe Pro Lys Ser Pro Val Leu Leu Gly Gin Pro Asn Thr Leu He Cys 355 360 365
Phe Val Asp Asn He Phe Pro Pro Val He Asn He Thr Trp Leu Arg 370 375 380 Asn Ser Lys Ser Val Thr Asp Gly Val Tyr Glu Thr Ser Phe Leu Val 385 390 395 400
Asn Arg Asp His Ser Phe His Lys Leu Ser Tyr Leu Thr Phe He Pro 405 410 415
Ser Asp Asp Asp He Tyr Asp Cys Lys Val Glu His Trp Gly Leu Glu 420 425 430
Glu Pro Val Leu Lys His Trp Ala Ser Gly Gly Gly Gly Ser Gly Gly 435 440 445
Gly Gly Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly 450 ■ 455 460
Ser Ala Ala Gin Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys 465 470 475 480
Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu 485 490 495
Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Asp Leu Tyr 500 505 510
Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu 515 520 525
Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp 530 535 540 Lys Lys He Val Pro Arg Asp Cys Gly Cys Lys Pro Cys He Cys Thr 545 550 555 560
Val Pro Glu Val Ser Ser Val Phe He Phe Pro Pro Lys Pro Lys Asp 565 570 575 Val Leu Thr He Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp 580 585 590
He Ser Lys Asp Asp Pro Glu Val Gin Phe Ser Trp Phe Val Asp Asp
595 600 605
Val Glu Val His Thr Ala Gin Thr Gin Pro Arg Glu Glu Gin Phe Asn 610 615 620
Ser Thr Phe Arg Ser Val Ser Glu Leu Pro He Met His Gin Asp Trp 625 630 635 640
Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro 645 650 655
Ala Pro He Glu Lys Thr He Ser Lys Thr Lys Gly Arg Pro Lys Ala 660 665 670 Pro Gin Val Tyr Thr He Pro Pro Pro Lys Glu Gin Met Ala Lys Asp 675 680 685
Lys Val Ser Leu Thr Cys Met He Thr Asp Phe Phe Pro Glu Asp He 690 695 700
Thr Val Glu Trp Gin Trp Asn Gly Gin Pro Ala Glu Asn Tyr Lys Asn 705 710 715 720
Thr Gin Pro He Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys 725 730 735
Leu Asn Val Gin Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys 740 745 750 Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu 755 760 765
Ser His Ser Pro Gly Lys 770
<210> IAS MBP 1-14 CH1.H.CH2 <211> 561 <212> PRT <213> murine
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
5 10 15 Gly Ser Thr Gly Asp Met Ala Ser Gin Lys Arg Pro Ser Gin Arg His
20 25 30
Gly Ser Lys Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser 35 40 45
Glu Arg His Phe Val Phe Gin Phe Lys Gly Glu Cys Tyr Phe Thr Asn 50 55 60
Gly Thr Gin Arg He Arg Ser Val Asp Arg Tyr He Tyr Asn Arg Glu 65 70 75 80
Glu Tyr Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr 85 90 95
Glu Leu Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys Gin Tyr Leu Glu 100 105 110
Gin Thr Arg Ala Glu Leu Asp Thr Val Cys Arg His Asn Tyr Glu Gly 115 120 125
Val Glu Thr His Thr Ser Leu Arg Arg Leu Glu Gin Pro Asn Val Val 130 135 140 He Ser Leu Ser Arg Thr Glu Ala Leu Asn His His Asn Thr Leu Val 145 150 155 160
Cys Ser Val Thr Asp Phe Tyr Pro Ala Lys He Lys Val Arg Trp Phe
165 170 175
Arg Asn Gly Gin Glu Glu Thr Val Gly Val Ser Ser Thr Gin Leu He 180 185 190
Arg Asn Gly Asp Trp Thr Phe Gin Val Leu Val Met Leu Glu Met Thr 195 200 205
Pro Arg Arg Gly Glu Val Tyr Thr Cys His Val Glu His Pro Ser Leu 210 215 220 Lys Ser Pro He Thr Val Glu Trp Thr Ser Gly Gly Gly Gly Ser Gly 225 230 235 240
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser 245 250 255
Glu Asp Asp He Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr Val 260 265 270
Tyr Gin Ser Pro Gly Asp He Gly Gin Tyr Thr His Glu Phe Asp Gly 275 280 285
Asp Glu Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr He Trp Met 290 295 300 Leu Pro Glu Phe Gly Gin Leu Thr Ser Phe Asp Pro Gin Gly Gly Leu 305 310 315 320
Gin Asn He Ala Thr Gly Lys Tyr Thr Leu Gly He Leu Thr Lys Arg 325 330 335
Ser Asn Ser Thr Pro Ala Thr Asn Glu Ala Pro Gin Ala Thr Val Phe 340 345 350
Pro Lys Ser Pro Val Leu Leu Gly Gin Pro Asn Thr Leu He Cys Phe 355 360 365
Val Asp Asn He Phe Pro Pro Val He Asn He Thr Trp Leu Arg Asn
370 375 380 Ser Lys Ser Val Thr Asp Gly Val Tyr Glu Thr Ser Phe Leu Val Asn 385 390 395 400
Arg Asp His Ser Phe His Lys Leu Ser Tyr Leu Thr Phe He Pro Ser 405 410 415
Asp Asp Asp He Tyr Asp Cys Lys Val Glu His Trp Gly Leu Glu Glu 420 425 430 Pro Val Leu Lys His Trp Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly 435 • 440 445
Gly Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser 450 455 460
Ala Ala Gin Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly 465 470 475 480
Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser 485 490 495
Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Asp Leu Tyr Thr 500 505 510 Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr 515 520 525
Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys 530 535 540
Lys He Val Pro Arg Asp Cys Gly Cys Lys Pro Cys He Cys Thr Val 545 550 555 560
<210> IAS MBP 90-101 CHI . H. CH2. CH3 <211> 773 <212> PRT <213> murine Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
5 10 15
Gly Ser Thr Gly Asp Phe Lys Asn He Val Thr Pro Arg Thr Pro Pro 20 25 30
Pro Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser Glu Arg 35 40 45
His Phe Val Phe Gin Phe Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr 50 55 60
Gin Arg He Arg Ser Val Asp Arg Tyr He Tyr Asn Arg Glu Glu Tyr 65 70 75 80 Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu
85 90 95
Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys Gin Tyr Leu Glu Gin Thr 100 105 110 • Arg Ala Glu Leu Asp Thr Thr Cys Arg His Asn Tyr Glu Gly Val Glu 115 120 125 Thr His Thr Ser Leu Arg Arg Leu Glu Gin Pro Asn Val Val He Ser 130 135 140
Leu Ser Arg Thr Glu Ala Leu Asn His His Asn Thr Leu Val Cys Ser 145 150 155 160
Val Thr Asp Phe Tyr Pro Ala Lys He Lys Val Arg Trp Phe Arg Asn 165 170 175
Gly Gin Glu Glu Thr Val Gly Val Ser Ser Thr Gin Leu He Arg Asn 180 185 190
Gly Asp Trp Thr Phe Gin Val Leu Val Met Leu Glu Met Thr Pro Arg 195 200 205 Arg Gly Glu Val Tyr Thr Cys His Val Glu His Pro Ser Leu Lys Ser 210 215 220
Pro He Thr Val Glu Trp Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser Glu Asp 245 250 255
Asp He Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr Val Tyr Gin 260 265 270
Ser Pro Gly Asp He Gly Gin Tyr Thr His Glu Phe Asp Gly Asp Glu 275 280 285 Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr He Trp Met Leu Pro 290 295 300
Glu Phe Gly Gin Leu Thr Ser Phe Asp Pro Gin Gly Gly Leu Gin Asn 305 310 315 320
He Ala Thr Gly Lys Tyr Thr Leu Gly He Leu Thr Lys Arg Ser Asn
325 330 335
Ser Thr Pro Ala Thr Asn Glu Ala Pro Gin Ala Thr Val Phe Pro Lys 340 345 350
Ser Pro Val Leu Leu Gly Gin Pro Asn Thr Leu He Cys Phe Val Asp 355 360 365 Asn He Phe Pro Pro Val He Asn He Thr Trp Leu Arg Asn Ser Lys 370 375 380
Ser Val Thr Asp Gly Val Tyr Glu Thr Ser Phe Leu Val Asn Arg Asp
385 390 395 400
His Ser Phe His Lys Leu Ser Tyr Leu Thr Phe He Pro Ser Asp Asp
405 410 415
Asp He Tyr Asp Cys Lys Val Glu His Trp Gly Leu Glu Glu Pro Val 420 425 430
Leu Lys His Trp Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Leu 435 440 445
Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala 450 455 460
Ala Gin Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr 465 470 475 480
Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser 485 490 495 Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Asp Leu Tyr Thr Leu 500 505 510
Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val 515 520 525
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys 530 535 540
He Val Pro Arg Asp Cys Gly Cys Lys Pro Cys He Cys Thr Val Pro 545 550 555 560
Glu Val Ser Ser Val Phe He Phe Pro Pro Lys Pro Lys Asp Val Leu
565 570 575 Thr He Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp He Ser 580 585 590
Lys Asp Asp Pro Glu Val Gin Phe Ser Trp Phe Val Asp Asp Val Glu 595 600 605
Val His Thr Ala Gin Thr Gin Pro Arg Glu Glu Gin Phe Asn Ser Thr 610 615 620
Phe Arg Ser Val Ser Glu Leu Pro He Met His Gin Asp Trp Leu Asn 625 630 635 640
Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro 645 650 655 He Glu Lys Thr He Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gin 660 665 670
Val Tyr Thr He Pro Pro Pro Lys Glu Gin Met Ala Lys Asp Lys Val 675 680 685
Ser Leu Thr Cys Met He Thr Asp Phe Phe Pro Glu Asp He Thr Val 690 695 700
Glu Trp Gin Trp Asn Gly Gin Pro Ala Glu Asn Tyr Lys Asn Thr Gin 705 710 715 720
Pro He Met Asp Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn 725 730 735 Val Gin Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val 740 745 750
Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His 755 760 765
Ser Pro Gly Lys 770 <210> pCRC203
<211> 609
<212> DNA
<213> Murine <400> 1 catatgttca agaacattgt gacacctcga acaccacctc caggaggagg atccggagac 60 tcggaaaggc atttcgtgtt ccagttcaag ggcgagtgct acttcaccaa cgggacgcag 120 cgcatacgat ctgtggacag atacatctac aaccgggagg agtacctgcg cttcgacagc 180 gacgtgggcg agtaccgcgc ggtgaccgag ctggggcggc cagaccccga gtactacaat 240 aagcagtacc tggagcaaac gcgggccgag ctggacacgg tgtgcagaca caactacgag 300 ggggtggaga cccacacctc cctgcggcgg cttggaggtg aagacgacat tgaggccgac 360 cacgtaggcg tctatggtac aactgtatat cagtctcctg gagacattgg ccagtacaca 420 catgaatttg atggtgatga gtggttctat gtggacttgg ataagaagga gactatctgg 480 atgcttcctg agtttggcca attgacaagc tttgaccccc aaggtggact gcaaaacata 540 gctacaggaa aatacacctt gggaatcttg actaagaggt caaattccac cccagctacc 600 aatctcgag 609
<210> pCRC201 <211> 614 <212> DNA
<213> Murine
<400> 2 catatgttca agaacattgt gacacctcga acaccacctc caggaggagg atccggagac 60 tcggaaaggc atttcgtgtt ccagttcaag ggcgagtgct acttcaccaa cgggacgcag 120 cgcatacgat ctgtggacag atacatctac aaccgggagg agtacctgcg cttcgacagc 180 gacgtgggcg agtaccgcgc ggtgaccgag ctggggcggc cagaccccga gtactacaat 240 aagcagtacc tggagcaaac gcgggccgag ctggacacgg tgtgcagaca caactacgag 300 ggggtggaga cccacacctc cctgcggcgg cttggaggtg aagacgacat tgaggccgac 360 cacgtaggcg tctatggtac aactgtatat cagtctcctg gagacattgg ccagtacaca 420 catgaatttg atggtgatga gtggttctat gtggacttgg ataagaagga gactatctgg 480 atgcttcctg agtttggcca attgacaagc tttgaccccc aaggtggact gcaaaacata 540 gctacaggaa aatacacctt gggaatcttg actaagaggt caaattccac cccagctacc 600 aattaagcgg ccgc 614
<210> pCRC199 <211> 642 <212> DNA <213> Murine
<400> 3 catatgttca agaacattgt gacacctcga acaccacctc cagctagcgg agggggcgga 60 agcggcggag ggggagactc cgaaaggcat ttcgtgttcc agtttaaagg cgagtgctac 120 ttcaccaacg ggacgcagcg catacgatct gtggacagat acatctacaa ccgggaggag 180 tacctgcgct tcgacagcga cgtgggcgag taccgcgcgg tgaccgagct ggggcggcca 240 gaccccgagt actacaataa gcagtacctg gagcaaacgc gggccgagct ggacacggtg 300 tgcagacaca actacgaggg ggtggagacc cacacctccc tgcggcggct tactagtggt 360 ggcggtggca gcgaagacga cattgaggcc gaccacgtag gcgtctatgg tacaactgta 420 tatcagtctc ctggagacat tggccagtac acacatgaat ttgatggtga tgagtggttc 480 tatgtggact tggataagaa ggagactatc tggatgcttc ctgagtttgg ccaattgaca 540 agctttgacc cccaaggtgg actgcaaaac atagctacag gaaaatacac cttgggaatc 600 ttgactaaga ggtcaaattc caccccagct accaatctcg ag 642 <210> pCRC197 <211> 647 <212> DNA <213> Murine <400> 4 catatgttca agaacattgt gacacctcga acaccacctc cagctagcgg agggggcgga 60 agcggcggag ggggagactc cgaaaggcat ttcgtgttcc agtttaaagg cgagtgctac 120 ttcaccaacg ggacgcagcg catacgatct gtggacagat acatctacaa ccgggaggag 180 tacctgcgct tcgacagcga cgtgggcgag taccgcgcgg tgaccgagct ggggcggcca 240 gaccccgagt actacaataa gcagtacctg gagcaaacgc gggccgagct ggacacggtg 300 tgcagacaca actacgaggg ggtggagacc cacacctccc tgcggcggct tactagtggt 360 ggcggtggca gcgaagacga cattgaggcc gaccacgtag gcgtctatgg tacaactgta 420 tatcagtctc ctggagacat tggccagtac acacatgaat ttgatggtga tgagtggttc 480 tatgtggact tggataagaa ggagactatc tggatgcttc ctgagtttgg ccaattgaca 540 agctttgacc cccaaggtgg actgcaaaac atagctacag gaaaatacac cttgggaatc 600 ttgactaaga ggtcaaattc caccccagct accaattaag cggccgc 647
<210> pCRC188 <211> 1698 <212> DNA
<213> Murine
<400> 5 gcggccgcca ccatggctct gcagatcccc agcctcctcc tctcggctgc tgtggtggtg 60 ctgatggtgc tgagcagccc agggactgag ggcttcaaga acattgtgac acctcgaaca 120 ccacctccag ctagcggagg gggcggaagc ggcggagggg gagactccga aaggcatttc 180 gtgttccagt tcaagggcga gtgctacttc accaacggga cgcagcgcat acgatctgtg 240 gacagataca tctacaaccg ggaggagtac ctgcgcttcg acagcgacgt gggcgagtac 300 cgcgcggtga ccgagctggg gcggccagac cccgagtact acaataagca gtacctggag 360 caaacgcggg ccgagctgga cacggtgtgc agacacaact acgagggggt ggagacccac 420 acctccctgc ggcggcttga acagcccaat gtcgtcatct ccctgtccag gacagaggcc 480 ctcaaccacc acaacactct ggtctgctca gtgacagatt tctacccagc caagatcaaa 540 gtgcgctggt tccggaatgg ccaggaggag acggtggggg tctcatccac acagcttatt 600 aggaatgggg actggacctt ccaggtcctg gtcatgctgg agatgacccc tcggcgggga 660 gaggtctaca cctgccacgt ggagcatccg agcctgaaga gccccatcac tgtggagtgg 720 actagtggtg gcggtggcag cggcggtggt ggttccggtg gcggcggttc tggcggtggc 780 ggttcctcga gtgaagacga cattgaggcc gaccacgtag gcgtctatgg tacaactgta 840 tatcagtctc ctggagacat tggccagtac acacatgaat ttgatggtga tgagtggttc 900 tatgtggact tggataagaa ggagactatc tggatgcttc ctgagtttgg ccaattgaca 960 agctttgacc cccaaggtgg actgcaaaac atagctacag gaaaatacac cttgggaatc 1020 ttgactaaga ggtcaaattc caccccagct accaatgagg ctcctcaagc gactgtgttc 1080 cccaagtccc ctgtgctgct gggtcagccc aacaccctca tctgctttgt ggacaacatc 1140 ttccctcctg tgatcaacat cacatggctc agaaatagta agtcagtcac agacggcgtt 1200 tatgagacca gcttccttgt caaccgtgac cattccttcc acaagctgtc ttatctcacc 1260 ttcatccctt ctgacgatga tatttatgac tgcaaggtgg agcactgggg cctggaggag 1320 ccggttctga aacactgggc tagcggaggg ggcggaagcg gcggaggggg agctgatgct 1380 gcaccaactg tatccatctt cccaccatcc agtgagcagt taacatctgg aggtgcctca 1440 gtcgtgtgct tcttgaacaa cttctacccc aaagacatca atgtcaagtg gaagattgat 1500 ggcagtgaac gacaaaatgg cgtcctgaac agttggactg atcaggacag caaagacagc 1560 acctacagca tgagcagcac cctca.cgttg accaaggacg agtatgaacg acataacagc 1620 tatacctgtg aggccactca caagacatca acttcaccca ttgtcaagag cttcaacagg 1680 aatgagtgtt agggtacc 1698
<210> pCRC187 <211> 1662 <212> DNA <213> Murine <400> 6 gcggccgcca ccatggctct gcagatcccc agcctcctcc tctcggctgc tgtggtggtg 60 ctgatggtgc tgagcagccc agggactgag ggcgctagcg gagggggcgg aagcggcgga 120 gggggagact ccgaaaggca tttcgtgttc cagttcaagg gcgagtgcta cttcaccaac 180 gggacgcagc gcatacgatc tgtggacaga tacatctaca accgggagga gtacctgcgc 240 ttcgacagcg acgtgggcga gtaccgcgcg gtgaccgagc tggggcggcc agaccccgag 300 tactacaata agcagtacct ggagcaaacg cgggccgagc tggacacggt gtgcagacac 360 aactacgagg gggtggagac ccacacctcc ctgcggcggc ttgaacagcc caatgtcgtc 420 atctccctgt ccaggacaga ggccctcaac caccacaaca ctctggtctg ctcagtgaca 480 gatttctacc cagccaagat caaagtgcgc tggttccgga atggccagga ggagacggtg 540 ggggtctcat ccacacagct tattaggaat ggggactgga ccttccaggt cctggtcatg 600 ctggagatga cccctcggcg gggagaggtc tacacctgcc acgtggagca tccgagcctg 660 aagagcccca tcactgtgga gtggactagt ggtggcggtg gcagcggcgg tggtggttcc 720 ggtggcggcg gttctggcgg tggcggttcc tcgagtgaag acgacattga ggccgaccac 780 gtaggcgtct atggtacaac tgtatatcag tctcctggag acattggcca gtacacacat 840 gaatttgatg gtgatgagtg gttctatgtg gacttggata agaaggagac tatctggatg 900 cttcctgagt ttggccaatt gacaagcttt gacccccaag gtggactgca aaacatagct 960 acaggaaaat acaccttggg aatcttgact aagaggtcaa attccacccc agctaccaat 1020 gaggctcctc aagcgactgt gttccccaag tcccctgtgc tgctgggtca gcccaacacc 1080 ctcatctgct ttgtggacaa catcttccct cctgtgatca acatcacatg gctcagaaat 1140 agtaagtcag tcacagacgg cgtttatgag accagcttcc ttgtcaaccg tgaccattcc 1200 ttccacaagc tgtcttatct caccttcatc ccttctgacg atgatattta tgactgcaag 1260 gtggagcact ggggcctgga ggagccggtt ctgaaacact gggctagcgg agggggcgga 1320 agcggcggag ggggagctga tgctgcacca actgtatcca tcttcccacc atccagtgag 1380 cagttaacat ctggaggtgc ctcagtcgtg tgcttcttga acaacttcta ccccaaagac 1440 atcaatgtca agtggaagat tgatggcagt gaacgacaaa atggcgtcct gaacagttgg 1500 actgatcagg acagcaaaga cagcacctac agcatgagca gcaccctcac gttgaccaag 1560 gacgagtatg aacgacataa cagctatacc tgtgaggcca ctcacaagac atcaacttca 1620 cccattgtca agagcttcaa caggaatgag tgttagggta cc 1662 <210> pCB229
<211> 1085
<212> DNA <213> Murine <400> 7 gaattcggta ccaccatggc tctgcagatc cccagcctcc tcctctcggc tgctgtggtg 60 gtgctgatgg tgctgagcag cccagggact gagggct ca agaacattgt gacacctcga 120 acaccacctc cagctagcgg agggggcgga agcggcggag ggggagactc cgaaaggcat 180 ttcgtgttcc agttcaaggg cgagtgctac ttcaccaacg ggacgcagcg catacgatct 240 gtggacagat acatctacaa ccgggaggag tacctgcgct tcgacagcga cgtgggcgag 300 taccgcgcgg tgaccgagct ggggcggcca gaccccgagt actacaataa gcagtacctg 360 gagcaaacgc gggccgagct ggacacggtg tgcagacaca actacgaggg ggtggagacc 420 cacacctccc tgcggcggct tgaacagccc aatgtcgtca tctccctgtc caggacagag 480 gccctcaacc accacaacac tctggtctgc tcagtgacag atttctaccc agccaagatc 540 aaagtgcgct ggttccggaa tggccaggag gagacggtgg gggtctcatc cacacagctt 600 attaggaatg gggactggac cttccaggtc ctggtcatgc tggagatgac ccctcggcgg 660 ggagaggtct acacctgcca cgtggagcat ccgagcctga agagccccat cactgtggag 720 tggagggcac agtctgagtc tgcccggagc ggatccgctg atgctgcacc aactgtatcc 780 atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 840 aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 900 aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 960 agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 1020 actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgttaggcg 1080 gccgc 1085 <210> pCB223 <211> 1676 <212> DNA <213> Murine
<400> 8 gaattcggta ccatggctct gcagatcccc agcctcctcc tctcggctgc tgtggtggtg 60 ctgatggtgc tgagcagccc agggactgag ggcgaagacg acattgaggc cgaccacgta 120 ggcgtctatg gtacaactgt atatcagtct cctggagaca ttggccagta cacacatgaa 180 tttgatggtg atgagtggtt ctatgtggac ttggataaga aggagactat ctggatgctt 240 cctgagtttg gccaattgac aagctttgac ccccaaggtg gactgcaaaa catagctaca 300 ggaaaataca ccttgggaat cttgactaag aggtcaaatt ccaccccagc taccaatgag 360 gctcctcaag cgactgtgtt ccccaagtcc cctgtgctgc tgggtcagcc caacaccctc 420 atctgctttg tggacaacat cttccctcct gtgatcaaca tcacatggct cagaaatagt 480 aagtcagtca cagacggcgt ttatgagacc agcttccttg tcaaccgtga ccattccttc 540 cacaagctgt cttatctcac cttcatccct tctgacgatg atatttatga ctgcaaggtg 600 gagcactggg gcctggagga gccggttctg aaacactggg aacctgagat tccagccccc 660 atgtcagaag gatctgccaa aacaacagcc ccatcggtct atccactggc ccctgtgtgt 720 ggagatacaa ctggctcctc ggtgactcta ggatgcctgg tcaagggtta tttccctgag 780 ccagtgacct tgacctggaa ctctggatcc ctgtccagtg gtgtgcacac cttcccagct 840 gtcctgcagt ctgacctcta caccctcagc agctcagtga ctgtaacctc gagcacctgg 900 cccagccagt ccatcacctg caatgtggcc cacccggcaa gcagcaccaa ggtggacaag 960 aaaattgagc ccagagggcc cacaatcaag ccctgtcctc catgcaaatg cccagcacct 1020 aacctcttgg gtggaccatc cgtcttcatc ttccctccaa agatcaagga tgtactcatg 1080 atctccctga gccccatagt cacatgtgtg gtggtggatg tgagcgagga tgacccagat 1140 gtccagatca gctggtttgt gaacaacgtg gaagtacaca cagctcagac acaaacccat 1200 agagaggatt acaacagtac tctccgggtg gtcagtgccc tccccatcca gcaccaggac 1260 tggatgagtg gcaaggagtt caaatgcaag gtcaacaaca aagacctccc agcgcccatc 1320 gagagaacca tctcaaaacc caaagggtca gtaagagctc cacaggtata tgtcttgcct 1380 ccaccagaag aagagatgac taagaaacag gtcactctga cctgcatggt cacagacttc 1440 atgcctgaag acatttacgt ggagtggacc aacaacggga aaacagagct aaactacaag 1500 aacactgaac cagtcctgga ctctgatggt tcttacttca tgtacagcaa gctgagagtg 1560 gaaaagaaga actgggtgga aagaaatagc tactcctgtt cagtggtcca cgagggtctg 1620 cacaatcacc acacgactaa gagcttctcc cggactccgg gtaaatgagc ggccgc 1676
<210> pCB212 <211> 773 <212> DNA <213> Murine
<400> 9 ccatgggtaa gaaacagacc gctgttgcat tcgctctggc gctcctggct ctttctatga 60 ccccggcgta cgctttcaag aacattgtga cacctcgaac accacctcca gctagcggag 120 ggggcggaag cggcggaggg ggagactccg aaaggcattt cgtgttccag tttaaaggcg 180 agtgctactt caccaacggg acgcagcgca tacgatctgt ggacagatac atctacaacc 240 gggaggagta cctgcgcttc gacagcgacg tgggcgagta ccgcgcggtg accgagctgg 300 ggcggccaga ccccgagtac tacaataagc agtacctgga gcaaacgcgg gccgagctgg 360 acacggtgtg cagacacaac tacgaggggg tggagaccca cacctccctg cggcggcttg 420 gtggcggtgg cagcggcggt ggtggttccg gtggcggcgg ttctggcggt ggcggttccg 480 gtggcggtgg cagcgaagac gacattgagg ccgaccacgt aggcgtctat ggtacaactg 540 tatatcagtc tcctggagac attggccagt acacacatga atttgatggt gatgagtggt 600 tctatgtgga cttggataag aaggagacta tctggatgct tcctgagttt ggccaattga 660 caagctttga cccccaaggt ggactgcaaa acatagctac aggaaaatac accttgggaa 720 tcttgactaa gaggtcaaat tccaccccag ctaccaatta aggtaccgga tec 773
<210> pCB214 <211> 702 <212_> DNA <213> Murine
<400> 10 atgttcaaga acattgtgac acctcgaaca ccacctccag ctagcggagg gggcggaagc 60 ggcggagggg gagactccga aaggcatttc gtgttccagt ttaaaggcga gtgctacttc 120 accaacggga cgcagcgcat acgatctgtg gacagataca tctacaaccg ggaggagtac 180 ctgcgcttcg acagcgacgt gggcgagtac cgcgcggtga ccgagctggg gcggccagac 240 cccgagtact acaataagca gtacctggag caaacgcggg ccgagctgga cacggtgtgc 300 agacacaact acgagggggt ggagacccac acctccctgc ggcggcttgg tggcggtggc 360 agcggcggtg gtggttccgg tggcggcggt tctggcggtg gcggttccgg tggcggtggc 420 agcgaagacg acattgaggc cgaccacgta ggcgtctatg gtacaactgt atatcagtct 480 cctggagaca ttggccagta cacacatgaa tttgatggtg atgagtggtt ctatgtggac 540 ttggataaga aggagactat ctggatgctt cctgagtttg gccaattgac aagctttgac 600 ccccaaggtg gactgcaaaa catagctaca ggaaaataca ccttgggaat cttgactaag 660 aggtcaaatt ccaccccagc taccaattaa ggtaccggat cc 702
<210> pCB220 <211> 1013 <212> DNA <213> Murine
<400> 11 gaattcggta ccatggctct gcagatcccc agcctcctcc tctcggctgc tgtggtggtg 60 ctgatggtgc tgagcagccc agggactgag ggcgaagacg acattgaggc cgaccacgta 120 ggcgtctatg gtacaactgt atatcagtct cctggagaca ttggccagta cacacatgaa 180 tttgatggtg atgagtggtt ctatgtggac ttggataaga aggagactat ctggatgctt 240 cctgagtttg gccaattgac aagctttgac ccccaaggtg gactgcaaaa catagctaca 300 ggaaaataca ccttgggaat cttgactaag aggtcaaatt ccaccccagc taccaatgag 360 gctcctcaag cgactgtgtt ccccaagtcc cctgtgctgc tgggtcagcc caacaccctc 420 atctgctttg tggacaacat cttccctcct gtgatcaaca tcacatggct cagaaatagt 480 aagtcagtca cagacggcgt ttatgagacc agcttccttg tcaaccgtga ccattccttc 540 cacaagctgt cttatctcac cttcatccct tctgacgatg atatttatga ctgcaaggtg 600 gagcactggg gcctggagga gccggttctg aaacactggg aacctgagat tccagccccc 660 atgtcagaag gatccgccaa aacaacagcc ccatcggtct atccactggc ccctgtgtgt 720 ggagatacaa ctggctcctc ggtgactcta ggatgcctgg tcaagggtta tttccctgag 780 ccagtgacct tgacctggaa ctctggatct ctgtccagtg gtgtgcacac cttcccagct 840 gtcctgcagt ctgacctcta caccctcagc agctcagtga ctgtaacctc gagcacctgg 900 cccagccagt ccatcacctg caatgtggcc cacccggcaa gcagcaccaa ggtggacaag 960 aaaattgagc ccagagggcc cacaatcaag ccctgtgctg cataggcggc cgc 1013
<210> pCRC203 <211> 207 <212> PRT <213> Murine
<400> 12
Met Phe Lys Asn He Val Thr Pro Arg Thr Pro Pro Pro Gly Gly Gly
5 10 15 Ser Gly Asp Ser Glu Arg His Phe Val Phe Gin Phe Lys Gly Glu Cys
20 25 30
Tyr Phe Thr Asn Gly Thr Gin Arg He Arg Ser Val Asp Arg Tyr He 35 40 45
Tyr Asn Arg Glu Glu Tyr Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr 50 55 60
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys 65 70 75 80
Gin Tyr Leu Glu Gin Thr Arg Ala Glu Leu Asp Thr Val Cys Arg His 85 90 95
Asn Tyr Glu Gly Val Glu Thr His Thr Ser Leu Arg Arg Leu Gly Gly 100 105 110
Glu Asp Asp He Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr Val 115 120 125
Tyr Gin Ser Pro Gly Asp He Gly Gin Tyr Thr His Glu Phe Asp Gly 130 135 140 Asp Glu Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr He Trp Met 145 150 155 160
Leu Pro Glu Phe Gly Gin Leu Thr Ser Phe Asp Pro Gin Gly Gly Leu 165 170 175
Gin Asn He Ala Thr Gly Lys Tyr Thr Leu Gly He Leu Thr Lys Arg 180 185 190
Ser Asn Ser Thr Pro Ala Thr Asn His His His His His His 195 200 205
<210> pCRC201 <211> 201 <212> PRT
<213> Murine
<400> 13
Met Phe Lys Asn He Val Thr Pro Arg Thr Pro Pro Pro Gly Gly Gly 5 10 15
Ser Gly Asp Ser Glu Arg His Phe Val Phe Gin Phe Lys Gly Glu Cys 20 25 30 Tyr Phe Thr Asn Gly Thr Gin Arg He Arg Ser Val Asp Arg Tyr He 35 40 45
Tyr Asn Arg Glu Glu Tyr Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr 50 55 60
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys 65 70 75 80
Gin Tyr Leu Glu Gin Thr Arg Ala Glu Leu Asp Thr Val Cys Arg His 85 90 95
Asn Tyr Glu Gly Val Glu Thr His Thr Ser Leu Arg Arg Leu Gly Gly 100 105 110 Glu Asp Asp He Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr Val 115 120 125
Tyr Gin Ser Pro Gly Asp He Gly Gin Tyr Thr His Glu Phe Asp Gly 130 135 140 ' Asp Glu Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr He Trp Met 145 150 155 160 Leu Pro Glu Phe Gly Gin Leu Thr Ser Phe Asp Pro Gin Gly Gly Leu
165 170 175
Gin Asn He Ala Thr Gly Lys Tyr Thr Leu Gly He Leu Thr Lys Arg 180 185 190
Ser Asn Ser Thr Pro Ala Thr Asn 195 200
<210> pCRC199 <211> 218 <212> PRT <213> Murine <400> 14
Met Phe Lys Asn He Val Thr Pro Arg Thr Pro Pro Pro Ala Ser Gly
5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser Glu Arg His Phe Val Phe 20 25 30
Gin Phe Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr Gin Arg He Arg 35 40 45 Ser Val Asp Arg Tyr He Tyr Asn Arg Glu Glu Tyr Leu Arg Phe Asp 50 55 60
Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro Asp 65 70 75 80
Pro Glu Tyr Tyr Asn Lys Gin Tyr Leu Glu Gin Thr Arg Ala Glu Leu 85 90 95
Asp Thr Val Cys Arg His Asn Tyr Glu Gly Val Glu Thr His Thr Ser 100 105 110
Leu Arg Arg Leu Thr Ser Gly Gly Gly Gly Ser Glu Asp Asp He Glu 115 120 125 Ala Asp His Val Gly Val Tyr Gly Thr Thr Val Tyr Gin Ser Pro Gly 130 135 140
Asp He Gly Gin Tyr Thr His Glu Phe Asp Gly Asp Glu Trp Phe Tyr
145 150 155 160
Val Asp Leu Asp Lys Lys Glu Thr He Trp Met Leu Pro Glu Phe Gly
165 170 175
Gin Leu Thr Ser Phe Asp Pro Gin Gly Gly Leu Gin Asn He Ala Thr 180 185 190
Gly Lys Tyr Thr Leu Gly He Leu Thr Lys Arg Ser Asn Ser Thr Pro 195 200 205 Ala Thr Asn His His His His His His 210 215
<210> pCRC197 <211> 212 <212> PRT <213> Murine <400> 15
Met Phe Lys Asn He Val Thr Pro Arg Thr Pro Pro Pro Ala Ser Gly
5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser Glu Arg His Phe Val Phe 20 25 30
Gin Phe Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr Gin Arg He Arg 35 40 45 Ser Val Asp Arg Tyr He Tyr Asn Arg Glu Glu Tyr Leu Arg Phe Asp 50 55 60
Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro Asp 65 70 75 80
Pro Glu Tyr Tyr Asn Lys Gin Tyr Leu Glu Gin Thr Arg Ala Glu Leu 85 90 95
Asp Thr Val Cys Arg His Asn Tyr Glu Gly Val Glu Thr His Thr Ser 100 105 110
Leu Arg Arg Leu Thr Ser Gly Gly Gly Gly Ser Glu Asp Asp He Glu 115 120 125 Ala Asp His Val Gly Val Tyr Gly Thr Thr Val Tyr Gin Ser Pro Gly 130 135 140
Asp He Gly Gin Tyr Thr His Glu Phe Asp Gly Asp Glu Trp Phe Tyr 145 150 155 160
Val Asp Leu Asp Lys Lys Glu Thr He Trp Met Leu Pro Glu Phe Gly 165 170 175
Gin Leu Thr Ser Phe Asp Pro Gin Gly Gly Leu Gin Asn He Ala Thr 180 185 190
Gly Lys Tyr Thr Leu Gly He Leu Thr Lys Arg Ser Asn Ser Thr Pro 195 200 205 Ala Thr Asn 210
<210> pCRC188 <211> 548 <212> PRT <213> Murine
<400> 16 Met Ala Leu Gin He Pro Ser Leύ Leu Leu Ser Ala Ala Val Val Val
5 10 15
Leu Met Val Leu Ser Ser Pro Gly Thr Glu Gly Ala Ser Gly Gly Gly 20 25 30
Gly Ser Gly Gly Gly Gly Asp Ser Glu Arg His Phe Val Phe Gin Phe 35 40 45 Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr Gin Arg He Arg Ser Val 50 55 60
Asp Arg Tyr He Tyr Asn Arg Glu Glu Tyr Leu Arg Phe Asp Ser Asp 65 70 75 80
Val Gly Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Pro Glu 85 90 95
Tyr Tyr Asn Lys Gin Tyr Leu Glu Gin Thr Arg Ala Glu Leu Asp Thr 100 105 110
Val Cys Arg His Asn Tyr Glu Gly Val Glu Thr His Thr Ser Leu Arg 115 120 125 Arg Leu Glu Gin Pro Asn Val Val He Ser Leu Ser Arg Thr Glu Ala 130 135 140
Leu Asn His His Asn Thr Leu Val Cys Ser Val Thr Asp Phe Tyr Pro 145 150 155 160
Ala Lys He Lys Val Arg Trp Phe Arg Asn Gly Gin Glu Glu Thr Val 165 170 175
Gly Val Ser Ser Thr Gin Leu He Arg Asn Gly Asp Trp Thr Phe Gin 180 185 190
Val Leu Val Met Leu Glu Met Thr Pro Arg Arg Gly Glu Val Tyr Thr 195 200 205 Cys His Val Glu His Pro Ser Leu Lys Ser Pro He Thr Val Glu Trp 210 215 220
Thr Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 225 230 235 240
Ser Gly Gly Gly Gly Ser Ser Ser Glu Asp Asp He Glu Ala Asp His 245 250 255
Val Gly Val Tyr Gly Thr Thr Val Tyr Gin Ser Pro Gly Asp He Gly 260 265 270
Gin Tyr Thr His Glu Phe Asp Gly Asp Glu Trp Phe Tyr Val Asp Leu 275 280 285 Asp Lys Lys Glu Thr He Trp Met Leu Pro Glu Phe Gly Gin Leu Thr 290 295 300
Ser Phe Asp Pro Gin Gly Gly Leu Gin Asn He Ala Thr Gly Lys Tyr 305 310 315 320 Thr Leu Gly He Leu Thr Lys Arg Ser Asn Ser Thr Pro Ala Thr Asn 325 330 335
Glu Ala Pro Gin Ala Thr Val Phe Pro Lys Ser Pro Val Leu Leu Gly 340 345 350
Gin Pro Asn Thr Leu He Cys Phe Val Asp Asn He Phe Pro Pro Val 355 360 365
He Asn He Thr Trp Leu Arg Asn Ser Lys Ser Val Thr Asp Gly Val 370 375 380
Tyr Glu Thr Ser Phe Leu Val Asn Arg Asp His Ser Phe His Lys Leu 385 390 395 400
Ser Tyr Leu Thr Phe He Pro Ser Asp Asp Asp He Tyr Asp Cys Lys 405 410 415 Val Glu His Trp Gly Leu Glu Glu Pro Val Leu Lys His Trp Ala Ser 420 425 430
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ala Asp Ala Ala Pro Thr Val 435 440 445
Ser He Phe Pro Pro Ser Ser Glu Gin Leu Thr Ser Gly Gly Ala Ser 450 455 460
Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp He Asn Val Lys 465 470 475 480
Trp Lys He Asp Gly Ser Glu Arg Gin Asn Gly Val Leu Asn Ser Trp 485 490 495 Thr Asp Gin Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu 500 505 510
Thr Leu 'Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu 515 520 525
Ala Thr His Lys Thr Ser Thr Ser Pro He Val Lys Ser Phe Asn Arg 530 535 540
Asn Glu Cys 545
<210> pCRC187 <211> 253 <212> PRT
<213> Murine
<400> 17
Met Gly Lys Lys Gin Thr Ala Val Ala Phe Ala Leu Ala Leu Leu Ala 5 10 15
Leu Ser Met Thr Pro Ala Tyr Ala Phe Lys Asn He Val Thr Pro Arg 20 25 30 Thr Pro Pro Pro Ala Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Asp 35 40 45
Ser Glu Arg His Phe Val Phe Gin Phe Lys Gly Glu Cys Tyr Phe Thr 50 55 60
Asn Gly Thr Gin Arg He Arg Ser Val Asp Arg Tyr He Tyr Asn Arg 65 70 75 80 Glu Glu Tyr Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr Arg Ala Val
85 90 95
Thr Glu Leu Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys Gin Tyr Leu 100 105 110
Glu Gin Thr Arg Ala Glu Leu Asp Thr Val Cys Arg His Asn Tyr Glu 115 120 125
Gly Val Glu Thr His Thr Ser Leu Arg Arg Leu Gly Gly Gly Gly Ser 130 135 140
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 145 150 155 160 Gly Gly Gly Ser Glu Asp Asp He Glu Ala Asp His Val Gly Val Tyr
165 170 175
Gly Thr Thr Val Tyr Gin Ser Pro Gly Asp He Gly Gin Tyr Thr His 180 185 190
Glu Phe Asp Gly Asp Glu Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu 195 200 205
Thr He Trp Met Leu Pro Glu Phe Gly Gin Leu Thr Ser Phe Asp Pro 210 215 220
Gin Gly Gly Leu Gin Asn He Ala Thr Gly Lys Tyr Thr Leu Gly He 225 230 235 240 Leu Thr Lys Arg Ser Asn Ser Thr Pro Ala Thr Asn
245 250
<210> pCB229 <211> 354 <212> PRT <213> Murine
<400> 18 Met Ala Leu Gin He Pro Ser Leu Leu Leu Ser Ala Ala Val Val Val
5 10 15
Leu Met Val Leu Ser Ser Pro Gly Thr Glu Gly Phe Lys Asn He Val 20 25 30
Thr Pro Arg Thr Pro Pro Pro Ala Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45
Gly Gly Asp Ser Glu Arg His Phe Val Phe Gin Phe Lys Gly Glu Cys 50 55 60
Tyr Phe Thr Asn Gly Thr Gin Arg He Arg Ser Val Asp Arg Tyr He 65 70 75 80
Tyr Asn Arg Glu Glu Tyr Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr 85 90 95
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys 100 105 110
Gin Tyr Leu Glu Gin Thr Arg Ala Glu Leu Asp Thr Val Cys Arg His 115 120 125 Asn Tyr Glu Gly Val Glu Thr His Thr Ser Leu Arg Arg Leu Glu Gin 130 135 140
Pro Asn Val Val He Ser Leu Ser Arg Thr Glu Ala Leu Asn His His 145 150 155 160
Asn Thr Leu Val Cys Ser Val Thr Asp Phe Tyr Pro Ala Lys He Lys 165 170 175
Val Arg Trp Phe Arg Asn Gly Gin Glu Glu Thr Val Gly Val Ser Ser 180 185 190
Thr Gin Leu He Arg Asn Gly Asp Trp Thr Phe Gin Val Leu Val Met 195 200 205 Leu Glu Met Thr Pro Arg Arg Gly Glu Val Tyr Thr Cys His Val Glu 210 215 220
His Pro Ser Leu Lys Ser Pro He Thr Val Glu Trp Arg Ala Gin Ser 225 230 235 240
Glu Ser Ala Arg Ser Gly Ser Ala Asp Ala Ala Pro Thr Val Ser He 245 250 255
Phe Pro Pro Ser Ser Glu Gin Leu Thr Ser Gly Gly Ala Ser Val Val 260 265 270
*
Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp He Asn Val Lys Trp Lys 275 * 280 285 He Asp Gly Ser Glu Arg Gin Asn Gly Val Leu Asn Ser Trp Thr Asp 290 295 300
Gin Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu
305 310 315 320
Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr
325 330 335
His Lys Thr Ser Thr Ser Pro He Val Lys Ser Phe Asn Arg Asn Glu 340 345 350
Cys <210> p/cb223 <211> 552 <212> PRT <213> Murine
<400> 19
Met Ala Leu Gin He Pro Ser Leu Leu Leu Ser Ala Ala Val Val Val
5 10 15
Leu Met Val Leu Ser Ser Pro Gly Thr Glu Gly Glu Asp Asp He Glu 20 25 30
Ala Asp His Val Gly Val Tyr Gly Thr Thr Val Tyr Gin Ser Pro Gly 35 40 45
Asp He Gly Gin Tyr Thr His Glu Phe Asp Gly Asp Glu Trp Phe Tyr
50 55 60 Val Asp Leu Asp Lys Lys Glu Thr He Trp Met Leu Pro Glu Phe Gly 65 70 75 80
Gin Leu Thr Ser Phe Asp Pro Gin Gly Gly Leu Gin Asn He Ala Thr 85 90 95
Gly Lys Tyr Thr Leu Gly He Leu Thr Lys Arg Ser Asn Ser Thr Pro 100 105 110
Ala Thr Asn Glu Ala Pro Gin Ala Thr Val Phe Pro Lys Ser Pro Val 115 120 125
Leu Leu Gly Gin Pro Asn Thr Leu He Cys Phe Val Asp Asn He Phe 130 135 140 Pro Pro Val He Asn He Thr Trp Leu Arg Asn Ser Lys Ser Val Thr 145 150 155 160
Asp Gly Val Tyr Glu Thr Ser Phe Leu Val Asn Arg Asp His Ser Phe 165 170 175
His Lys Leu Ser Tyr Leu Thr Phe He Pro Ser Asp Asp Asp He Tyr 180 185 190
Asp Cys Lys Val Glu His Trp Gly Leu Glu Glu Pro Val Leu Lys His 195 200 205
Trp Glu Pro Glu He Pro Ala Pro Met Ser Glu Gly Ser Ala Lys Thr 210 215 220 Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr 225 230 235 240
Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu 245 250. 255
Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His 260 265 270
Thr Phe Pro Ala Val Leu Gin Ser Asp Leu Tyr Thr Leu Ser Ser Ser 275 280 285
Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gin Ser He Thr Cys Asn 290 295 300
Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys He Glu Pro 305 310 315 320
Arg Gly Pro Thr He Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro 325 330 335
Asn Leu Leu Gly Gly Pro Ser Val Phe He Phe Pro Pro Lys He Lys 340 345 350 Asp Val Leu Met He Ser Leu Ser Pro He Val Thr Cys Val Val Val 355 360 365
Asp Val Ser Glu Asp Asp Pro Asp Val Gin He Ser Trp Phe Val Asn 370 375 380
Asn Val Glu Val His Thr Ala Gin Thr Gin Thr His Arg Glu Asp Tyr 385 390 395 400
Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro He Gin His Gin Asp 405 410 415
Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu 420 425 430 Pro Ala Pro He Glu Arg Thr He Ser Lys Pro Lys Gly Ser Val Arg 435 440 445
Ala Pro Gin Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys 450 455 460
Lys Gin Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp 465 470 475 480
He Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr- Lys 485 490 495
Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser 500 505 510 Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser 515 520 525
Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser 530 535 540
Phe Ser Arg Thr Pro Gly Lys 545 550
<210> pCB212
<211> 331
<212> PRT
<213> Murine <400> 20
Met Ala Leu Gin He Pro Ser Leu Leu Leu Ser Ala Ala Val Val Val
5 10 15
Leu Met Val Leu Ser Ser Pro Gly Thr Glu Gly Glu Asp Asp He Glu 20 25 30
Ala Asp His Val Gly Val Tyr Gly Thr Thr Val Tyr Gin Ser Pro Gly 35 40 45
Asp He Gly Gin Tyr Thr His Glu Phe Asp Gly Asp Glu Trp Phe Tyr 50 55 60
Val Asp Leu Asp Lys Lys Glu Thr He Trp Met Leu Pro Glu Phe Gly 65 70 75 80
Gin Leu Thr Ser Phe Asp Pro Gin Gly Gly Leu Gin Asn He Ala Thr 85 90 95 Gly Lys Tyr Thr Leu Gly He Leu Thr Lys Arg Ser Asn Ser Thr Pro 100 105 110
Ala Thr Asn Glu Ala Pro Gin Ala Thr Val Phe Pro Lys Ser Pro Val 115 120 125
Leu Leu Gly Gin Pro Asn Thr Leu He Cys Phe Val Asp Asn He Phe 130 135 140
Pro Pro Val He Asn He Thr Trp Leu Arg Asn Ser Lys Ser Val Thr 145 150 155 160
Asp Gly Val Tyr Glu Thr Ser Phe Leu Val Asn Arg Asp His Ser Phe 165 170 175 His Lys Leu Ser Tyr Leu Thr Phe He Pro Ser Asp Asp Asp He Tyr 180 185 190
Asp Cys Lys Val Glu His Trp Gly Leu Glu Glu Pro Val Leu Lys His 195 200 205
Trp Glu Pro Glu He Pro Ala Pro Met Ser Glu Gly Ser Ala Lys Thr 210 215 220
Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr 225 230 235 240
Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu
245 250 255 Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His 260 265 270
Thr Phe Pro Ala Val Leu Gin Ser Asp Leu Tyr Thr Leu Ser Ser Ser 275 280 285
Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gin Ser He Thr Cys Asn 290 295 300
Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys He Glu Pro ' 305 310 315 320
Arg Gly Pro Thr He Lys Pro Cys Ala Ala 325 330
<210> pCB214 <211> 234 <212> PRT <213> Murine
<400> 21
Met Phe Lys Asn He Val Thr Pro Arg Thr Pro Pro Pro Ala Ser Gly
5 10 15
Gly Gly Gly Ser Gly Gly Gly Gly Asp Ser Glu Arg His Phe Val Phe 20 25 30
Gin Phe Lys Gly Glu Cys Tyr Phe Thr Asn Gly Thr Gin Arg He Arg 35 40 45
Ser Val Asp Arg Tyr He Tyr Asn Arg Glu Glu Tyr Leu Arg Phe Asp 50 55 60 Ser Asp Val Gly Glu Tyr Arg Ala Val Thr Glu Leu Gly Arg Pro Asp 65 70 75 80
Pro Glu Tyr Tyr Asn Lys Gin Tyr Leu Glu Gin Thr Arg Ala Glu Leu
85 90 95
Asp Thr Val Cys Arg His Asn Tyr Glu Gly Val Glu Thr His Thr Ser 100 105 110
Leu Arg Arg Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Asp Asp 130' 135 140 He Glu Ala Asp His Val Gly Val Tyr Gly Thr Thr Val Tyr Gin Ser 145 150 155 160
Pro Gly Asp He Gly Gin Tyr Thr His Glu Phe Asp Gly Asp Glu Trp
165 170 175
Phe Tyr Val Asp Leu Asp Lys Lys Glu Thr He Trp Met Leu Pro Glu 180 185 190
Phe Gly Gin Leu Thr Ser Phe Asp Pro Gin Gly Gly Leu Gin Asn He 195 200 205
Ala Thr Gly Lys Tyr Thr Leu Gly He Leu Thr Lys Arg Ser Asn Ser 210 215 220 Thr Pro Ala Thr Asn Gly Thr Gly Ser 225
<210> pCB220 <211> 560 <212> PRT <213> Murine <400> 22
Met Ala Leu Gin He Pro Ser Leu Leu Leu Ser Ala Ala Val Val Val
5 10 15
Leu Met Val Leu Ser Ser Pro Gly Thr Glu Gly Phe Lys Asn He Val 20 25 30
Thr Pro Arg Thr Pro Pro Pro Ala Ser Gly Gly Gly Gly Ser Gly Gly 35 40 45 Gly Gly Asp Ser Glu Arg His Phe Val Phe Gin Phe Lys Gly Glu Cys 50 55 60
Tyr Phe Thr Asn Gly Thr Gin Arg He Arg Ser Val Asp Arg Tyr He 65 70 75 80
Tyr Asn Arg Glu Glu Tyr Leu Arg Phe Asp Ser Asp Val Gly Glu Tyr 85 90 95
Arg Ala Val Thr Glu Leu Gly Arg Pro Asp Pro Glu Tyr Tyr Asn Lys 100 105 110
Gin Tyr Leu Glu Gin Thr Arg Ala Glu Leu Asp Thr Val Cys Arg His 115 120 125 Asn Tyr Glu Gly Val Glu Thr His Thr Ser Leu Arg Arg Leu Glu Gin 130 135 140
Pro Asn Val Val He Ser Leu Ser Arg Thr Glu Ala Leu Asn His His
145 150 155 160
Asn Thr Leu Val Cys Ser Val Thr Asp Phe Tyr Pro Ala Lys He Lys 165 170 175
Val Arg Trp Phe Arg Asn Gly Gin Glu Glu Thr Val Gly Val Ser Ser 180 185 190
Thr Gin Leu He Arg Asn Gly Asp Trp Thr Phe Gin Val Leu Val Met 195 200 205 Leu Glu Met Thr Pro Arg Arg Gly Glu Val Tyr Thr Cys His Val Glu 210 215 220
His Pro Ser Leu Lys Ser Pro He Thr Val Glu Trp Thr Ser Gly Gly 225 230 235 240
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 245 250 255
Gly Ser Ser Ser Glu Asp Asp He Glu Ala Asp His Val Gly Val Tyr 260 265 270
Gly Thr Thr Val Tyr Gin Ser Pro Gly Asp He Gly Gin Tyr Thr His 275 280 285 Glu Phe Asp Gly Asp Glu Trp Phe Tyr Val Asp Leu Asp Lys Lys Glu 290 295 300
Thr He Trp Met Leu Pro Glu Phe Gly Gin Leu Thr Ser Phe Asp Pro 305 310 315 320
Gin Gly Gly Leu Gin Asn He Ala Thr Gly Lys Tyr Thr Leu Gly He 325 330 335 Leu Thr Lys Arg Ser Asn Ser Thr Pro Ala Thr Asn Glu Ala Pro Gin 340 345 350
Ala Thr Val Phe Pro Lys Ser Pro Val Leu Leu Gly Gin Pro Asn Thr 355 360 365
Leu He Cys Phe Val Asp Asn He Phe Pro Pro Val He Asn He Thr 370 375 380
Trp Leu Arg Asn Ser Lys Ser Val Thr Asp Gly Val Tyr Glu Thr Ser 385 390 395 400
Phe Leu Val Asn Arg Asp His Ser Phe His Lys Leu Ser Tyr Leu Thr 405 410 415 Phe He Pro Ser Asp Asp Asp He Tyr Asp Cys Lys Val Glu His Trp 420 425 430
Gly Leu Glu Glu Pro Val Leu Lys His Trp Ala Ser Gly Gly Gly Gly 435 440 445
Ser Gly Gly Gly Gly Ala Asp Ala Ala Pro Thr Val Ser He Phe Pro 450 455 460
Pro Ser Ser Glu Gin Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe 465 470 475 480
Leu Asn Asn Phe Tyr Pro Lys Asp He Asn Val Lys Trp Lys He Asp 485 490 495 Gly Ser Glu Arg Gin Asn Gly Val Leu Asn Ser Trp Thr Asp Gin Asp 500 505 510
Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys 515 520 525
Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys 530 535 540
Thr Ser Thr Ser Pro He Val Lys Ser Phe Asn Arg Asn Glu Cys 545 550 555

Claims

WHAT IS CLAIMED IS:
1. A multimeric complex comprising a first recombinant single chain MHC class II molecule and a second recombinant single chain MHC class II molecule, wherein the first and the second single chain MHC class II molecule each comprise an αl domain and a βl domain linked via an amino acid linker and a multimerization domain, and wherein the first and the second single chain MHC class II molecule are linked via the multimerization domain to form a multimeric complex.
2. The multimeric complex of claim 1, wherein the first and the second single chain MHC class II molecules are each linked to an antigenic peptide via an amino acid linker.
3. The multimeric complex of claim 2, wherein the peptides are the same.
4. The multimeric complex of claim 2, wherein the peptides are from the same antigen.
5. The multimeric complex of claim 2, wherein the peptides are from different antigens.
6. The multimeric complex of claim 2, wherein the peptides are selected from the group consisting of MBP83-102Y83, PLP 40-60, PLP 89-106, PLP 95- 117, and PLP 185-206.
7. The multimeric complex of claim 1, wherein the first and the second MHC class II molecules are from the same MHC class II allele.
8. The multimeric complex of claim 1, wherein the first and the second MHC class II molecules are from different MHC class II alleles.
9. The multimeric complex of claim 7 or 8, wherein the alleles are selected from the group consisting of DRB1*1501 and DRB5*0101.
10. The multimeric complex of claim 7 or 8, wherein the first and the second single chain MHC class II molecules are each linked to an antigenic peptide via an amino acid linker.
11. The multimeric complex of claim 10, wherein the peptides are the same.
12. The multimeric complex of claim 10, wherein the peptides are from the same antigen.
13. The multimeric complex of claim 10, wherein the peptides are from different antigens.
14. The multimeric complex of claim 10, wherein the peptides are selected from the group consisting of MBP83-102Y83, PLP 40-60, PLP 89-106, PLP 95- 117, and PLP 185-206.
15. The multimeric complex of claim 1 , wherein the first and second MHC class II molecules are from a human.
16. The multimeric complex of claim 1, wherein the multimerization domains are covalently linked.
17. The multimeric complex of claim 1, wherein the multimerization domains are non-covalently linked.
18. The multimeric complex of claim 1 , wherein the multimerization domain is a leucine zipper domain.
19. The multimeric complex of claim 1, wherein the multimerization domain is an immunoglobulin domain.
20. The multimeric complex of claim 19, wherein the immunoglobulin domain is a light chain constant domain or a heavy chain constant domain.
21. The multimeric complex of claim 1 , wherein the first or the second MHC class II molecule is selected from the group consisting of I-As MBP.βl β2αlα2.Cκ, I-As MBP.βl β2αlα2.CHl.H, I-As MBP.βlβ2αlα2.CHl.H.CH2, and I-As MBP.βlβ2αlα2.CHl.H.CH2.CH3.
22. The multimeric complex of claim 1, further comprising a third and a fourth MHC class II molecule, wherein the third and the fourth MHC class II molecule each comprise an αl domain and a βl domain linked via an amino acid linker and a multimerization domain, and wherein the first, second, third and fourth single chain MHC class II molecules are linked via the multimerization domain to form a multimeric complex.
23. The multimeric complex of claim 1 , wherein the first and the second MHC class II molecules comprise βl β2 domains and αl α2 domains linked via an amino acid linker.
24. The multimeric complex of claim 1 or 2, wherein the linker is selected from the group consisting of GG, GGGG, GGGGS, GGGSG, ASGGGSGGG, TSGGGGSGGGGSSS, GSPGGGGSGGGPGS, GSPPGGPPGS, GSPGGGGPGS, TSGGGGS, SGGSGGS, FDAPSPLP, and NYPENTN.
25. The multimeric complex of claim 1 or 2, wherein the linker is from a CD4 molecule.
26. The multimeric complex of claim 25, wherein the linker is selected from the group consisting of RΝGQEEKAGNNSTGLI ,RΝGQETKAGNNSTGLI, YΝQQEEKAGGVSTGLI, FRΝGQEEKAGVNSTGLI, FRΝGQETKAGNVSTGLI, FYΝQQEEKAGGNSTGLI, and LΝGQEEKAGMNSTGLI.
27. The multimeric complex of claim 1, wherein the first or the second MHC class II molecule is selected from the group consisting of CO523, CO543, CO563, CO567, CO580, CO581, CO582, CO583, CO584, CO585, CO586, CO587, CO588, CO589, CO590, CO591, CO592, CO593, CO594, CO595, CO596, CO597, and CO608.
28. The multimeric complex of claim 1, wherein the first and the second MHC class II molecule are independently selected from the group consisting of CO523, CO543, CO563, CO567, CO580, CO581, CO582, CO583, CO584, CO585, CO586, CO587, CO588, CO589, CO590, CO591, CO592, CO593, CO594, CO595, CO596, CO597, and CO608.
29. The multimeric complex of claim 1, wherein the first MHC class II or the second MHC class II molecule is encoded by a nucleic acid that codons optimized for prokaryotic expression.
30. The multimeric complex of claim 29, wherein the prokaryote is E. coli.
31. The multimeric complex of claim 29, wherein the nucleic acid encoding the first or the second MHC class II molecule is selected from the group consisting of CO528-AC and CO608-AC.
32. A pharmaceutical composition comprising the multimeric complex of claim 2.
33. The pharmaceutical composition of claim 32, further comprising an adjuvant.
34. A method of treating autoimmune disease in a subject, the method comprising administering an immunogenically effective amount of a pharmaceutical composition comprising the multimeric complex of claim 2.
35. The method of claim 34, wherein the subject is a human.
36. A recombinant nucleic acid encoding a single chain MHC class II molecule comprising an αl domain and a βl domain linked via an amino acid linker, wherein the linker is selected from the group consisting of GG, GGGG, GGGGS, GGGSG, ASGGGSGGG, TSGGGGSGGGGSSS, GSPGGGGSGGGPGS, GSPPGGPPGS, GSPGGGGPGS, TSGGGGS, SGGSGGS, FDAPSPLP, and NYPΕNTV.
37. The nucleic acid of claim 36, wherein the MHC class II molecule is selected from the group consisting of CO523, CO543, CO563, CO567, CO580, CO581, CO582, CO583, CO584, CO585, CO586, CO587, CO588, CO589, CO590, CO591, CO592, CO593, CO594, CO595, CO596, CO597, and CO608.
38. A recombinant nucleic acid encoding a single chain MHC class II molecule comprising an αl domain and a βl domain linked via an amino acid linker, wherein the linker is wherein the linker is from a CD4 molecule.
39. The nucleic acid of claim 38, wherein the linker is selected from the group consisting of RNGQEEKAGNVSTGLI ,RΝGQETKAGNVSTGLI, YΝQQEEKAGGVSTGLI, FRΝGQEEKAGVNSTGLI, FRΝGQETKAGNVSTGLI, FYΝQQEEKAGGVSTGLI, and LΝGQEEKAGMVSTGLI.
40. A recombinant nucleic acid encoding a single chain MHC class II molecule selected from the group consisting of CO528-AC and CO608-AC.
41. A recombinant nucleic acid encoding a single chain MHC class II molecule selected from the group consisting I-As MBP.βl β2αlα2.Cκ, I-As MBP.βlβ2αlα2.CHl.H, I-As MBP.βl β2αlα2.CHl.H.CH2, and I-As MBP.βlβ2αlα2.CHl.H.CH2.CH3.
42. A recombinant nucleic acid encoding a single chain MHC class II molecule selected from the group consisting CO523, CO543, CO563, CO567, CO580, CO581, CO582, CO583, CO584, CO585, CO586, CO587, CO588, CO589, CO590, CO591, CO592, CO593, CO594, CO595, CO596, CO597, and CO608.
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