WO1993010220A1 - Molecules solubles du complexe majeur d'histocompatibilite et leurs utilisations - Google Patents

Molecules solubles du complexe majeur d'histocompatibilite et leurs utilisations Download PDF

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Publication number
WO1993010220A1
WO1993010220A1 PCT/US1992/010030 US9210030W WO9310220A1 WO 1993010220 A1 WO1993010220 A1 WO 1993010220A1 US 9210030 W US9210030 W US 9210030W WO 9310220 A1 WO9310220 A1 WO 9310220A1
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Prior art keywords
mhc
component
constant region
immunoglobulin constant
composition
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PCT/US1992/010030
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English (en)
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H. E. Selick
Randall J. Armstrong
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Anergen, Inc.
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Publication of WO1993010220A1 publication Critical patent/WO1993010220A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present invention relates generally to chimeric proteins and, in particular, to chimeric proteins comprising an MHC component and an immunoglobulin constant region component.
  • the chimeric molecules specifically bind T cell receptors through the MHC component while retaining desired functions of the constant region component.
  • MHC major histocompatibility complex
  • T cells unlike B C €-lls, do not directly recognize antigens. Instead, an accessory cell must first process the antigen and present it in association with an MHC molecule in order to elicit an immunological response.
  • MHC glycoproteins appears to be the binding and presentation of processed antigen in the form of short (10-20 amino acids in length) antigenic peptides. in addition to binding 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-dependenz diabetes mellitus (IDDM) , etc.
  • MG myasthenia gravis
  • MS multiple sclerosis
  • SLE systemic lupus erythematosus
  • RA rheumatoid arthritis
  • IDDM insulin-dependenz diabetes mellitus
  • autoimmune disease and related conditions consists primarily of treating the symptoms, but not intervening in the etiology of the disease.
  • Broad spectrum chemotherapeutic agents are typically employed, which agents are often associated with numerous undesirable side effects.
  • Compounds capable of selectively suppressing autoimmune responses by blocking MHC binding provide a safer, more effective treatment.
  • selective immunosuppressive compounds are useful in the treatment of non- autoimmune diseases, such as graft versus host disease (GVHD) or various allergic responses. For instance, chronic GVHD patients frequently present conditions and symptoms similar to certain autoimmune diseases.
  • a desirable approach to treating autoimmune diseases and other pathological conditions mediated by MHC is to use antagonists to block binding to the T cell receptor.
  • MHC molecules themselves 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 noresponsiveness, in targeted T cells.
  • Production of soluble MHC molecules for this purpose would be particularly desirable.
  • the use of soluble MHC as a therapeutic is promising, although a number of improvements in this approach can be made. For instance, improvement of the relatively short serum half-life of soluble MHC molecules is desirable.
  • the therapeutic effectiveness of these molecules can be increased if they can be modified to be able to cross the placental or other biological barriers.
  • the ability to eliminate target cells with out the use of toxins would be advantageous.
  • the prior art thus lacks a soluble agent capable of blocking MHC restricted T cell activation, which agent inter alia has an extended serum half-life and provides a mechanism for eliminating targeted T cells without the necessity of using toxic conjugates.
  • the present invention provides soluble chimeric proteins comprising an MHC component linked to an immunoglobulin constant region component.
  • the chimeric protein is capable of binding a T cell receptor through the MHC component, while the immunoglobulin component retains normal effector functions and provides the chimeric proteins with an extended serum half-life and other advantages.
  • the MHC component preferably consists of the extracellular region of a full length MHC glycoprotein. Typically, MHC class II glycoproteins are used. If the chimeric protein is used to treat autoimmune disease, the MHC component will include an autoantigenic peptide associated with the autoimmune disease.
  • the immunoglobulin constant region component is preferably of the human IgGl isotype.
  • the immunoglobulin component preferably retains certain effector functions such as the ability to fix complement or to mediate antibody dependent cell cytotoxicity.
  • the chimeric proteins of the present invention may contain a protease recognition site between the MHC component and the immunoglobulin component.
  • the protease recognition site is preferably one that is not present in the MHC component.
  • suitable proteases include. Factor Xa and collagenase. Methods for purifying the soluble MHC component using the protease recognition site are also provided.
  • the present invention further provides pharmaceutical compositions and methods suitable for treating a patient with autoimmune disease.
  • the compositions can also be used for a variety of diagnostic purposes, as well.
  • the invention provides recombinant expression cassettes which are incorporated in expression vectors and used to transform a variety of host cells. Methods are disclosed for transforming appropriate host cells and recovering the expressed chimeric proteins from the cell culture.
  • FIG. 1 is a schematic representation of an MHC molecule showing the extracellular, the transmembrane, and cytoplasmic regions.
  • FIG. 2 is a schematic of a a MHC class 11/immunoglobulin chimeric molecule of the present invention.
  • FIG. 3 is a schematic of a ⁇ homodimeric molecule of the present invention comprising two chains, each comprising an MHC II ⁇ chain attached to the immunoglobulin constant region.
  • FIG. 4 is a schematic of a + ⁇ heavy chain homodimeric molecule of the present invention comprising an MHC II ⁇ chain attached to the immunoglobulin constant region and a MHC II ⁇ chain linked to the ⁇ chain.
  • FIGS. 5a and 5b show two expression vectors encoding chimeric proteins of the present invention.
  • FIG. 5a shows an expression cassette comprising a cDNA segment encoding the immunoglobulin component.
  • FIG. 5b shows an expression cassette comprising a genomic DNA segment encoding the immunoglobulin component.
  • the chimeric proteins of the present invention comprise a major histocompatibility complex (MHC) component linked to an immunoglobulin constant region component.
  • MHC major histocompatibility complex
  • the chimeric proteins may also comprise a protease recognition site between the two components. Treatment of the chimeric protein with an appropriate protease allows purification of the MHC component, if, for instance, the chimeric protein is bound to an affinity chromatography column through the immunoglobulin component.
  • the MHC component of the chimeric proteins determines the specificity of the proteins by binding selected T cell receptors.
  • the glycoproteins encoded by the MHC have been extensively studied in both the human and murine systems. They are classified according to the kinds of cells on which they are expressed and the T cells which recognize them.
  • Class I MHC molecules e.g., HLA-A, -B and -C molecules in the human system
  • Class II MHC molecules are expressed primarily on antigen presenting cells such as B lymphocytes, macrophages, etc.
  • MHC glycoproteins of both classes have been iso; * ,ed and characterized (see. Fundamental Immunology. 2d Ed., W.E. Paul ed.. Ravens Press, N.Y., (1989), and Roitt, et al., Immunology. 2d Ed., Gower Medical Publishing, London, (1989) which are both incorporated herein by reference) .
  • the resulting antigenic peptide first forms a complex with the antigen binding pocket of the MHC molecule through various noncovalent associations. This complex then fits into a single recognition site in a T cell receptor on a cytotoxic or helper T cell, depending upon the class of MHC molecule.
  • a general discussion of the function of MHC molecules see Grey, H.M. , et al.. Scientific American pp 56-64 (November, 1989) which is incorporated herein by reference and Paul, supra. Chapter 18.
  • FIG. 1 presents a schematic representation of a Class II MHC molecule.
  • Class II MHC antigens are heterodimeric transmembrane glycoproteins consisting of an chain (MW 25-33 kD) and a ⁇ chain (MW 24-29 kD) , which are noncovalently associated.
  • Figure 1 shows that each chain consists of two globular domains, a linker peptide, a transmembrane region, and a cytoplasmic tail. Both globular domains of the ⁇ chain are stabilized by intrachain disulfide bonds, whereas the chain contains only one such disulfide bond (see, Paul, supra. Chapters 16 and 17) .
  • the human Class I proteins have also been studied.
  • the Class I MHC of humans on chromosome 6 has three loci, HLA-, HLA-B, and HLA-C, the first two of which have a large number of alleles.
  • Class I molecules consist of a 44 kd subunit noncovalently associated with a 12 kd / 3 2 -microglobulin subunit. ,3 2 -microglobulin is not encoded by a locus in the MHC but is common to all Class I molecules. Although ⁇ -microglobulin does not form part of the antigen binding pocket of the molecule, it is necessary for processing and expression of Class I MHC molecules.
  • Soluble HLA-A2 can be purified after papain digestion of plasma membranes from the homozygous human lymphoblastoid cell line J-Y as described by Turner, M.J. et al., J. Biol. Chem. 252:7555-7567 (1977), which is incorporated herein by reference.
  • Papain cleaves the 44 kd chain close to the transmembrane region yielding a molecule comprised of ⁇ ,, ⁇ 2 , c_ 3 , and 3 2 -microglobulin.
  • MHC component refers to a purified a MHC glycoprotein or portion thereof which is in other than its native state, that is, not associated with the cell membrane of a cell that normally expresses MHC.
  • the "extracellular region" of the molecule is a water soluble portion of the molecule consisting of the sequences extending from the N-terminus to the transmembrane region and comprises the antigen binding pocket as well as other sequences necessary for recognition by the appropriate T cell receptor.
  • the extracellular region may comprise sequences from the transmembrane region (up to about ten amino acids) , so long as solubility is not significantly affected.
  • the second major component of the chimeric proteins of the present invention is the immunoglobulin constant region component.
  • Immunoglobulins ? e a group of glycoproteins present in the serum and tissue fluids of all mammals.
  • the basic immunoglobulin structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25kD) and one "heavy" chain (about 50-70kD) .
  • the N-terminal region of the chains defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the C-terminal region of the chains defines a constant region primarily responsible for effector function.
  • Light chains are classified as either kappa or lambda.
  • Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. (see. Paul and Roitt et al., supra) .
  • V H interacts with the N-terminal domain of the light chain, V L , to produce the binding region of the antibody.
  • the C H 1 domain is associated with the constant region of the light chain, C L .
  • the remaining domains on gamma, alpha and delta heavy chains are designated C H 2 and C H 3, respectively.
  • the mu and epsilon heavy chains contain an additional domain, C H 4.
  • Most heavy chains have a hincre region, consisting of a small number of amino acid residues, between the C H 1 and C H 2 domains.
  • the hinge is flexible and allows the binding region to move freely relative to the rest of the molecule.
  • At the hinge region are the disulfide bridges which hold the two dimers together, creating the tetramer structural unit.
  • the hinge region is the point on the molecule most susceptible to the action of protease.
  • Treatment with the protease papain splits the molecule into three fragments, two of which are designated F afa fragments, and the other, the F c fragment.
  • the F ab fragments each consist of an antigen binding domain and a C H 1 domain.
  • the F c fragment which consists of the C H 2 and C H 3 domains, is the portion of the immunoglobulin molecule that mediates effector functions. Depending upon the heavy chain in the immunoglobulin, a variety of effector functions are possible. These include complement fix ⁇ ition, mediation of antibody dependent cell toxicity, stimulation of B cells, and transport across the placenta. (See. Roitt et al.
  • the IgGl isotype is of particular interest for use in the chimeric proteins of the present invention because of its demonstrated effectiveness in complement lysis and antibody- dependent cell mediated cytotoxicity (Reichmann et al.. Nature. 332:323-327 (1988), which is incorporated herein by reference).
  • the chimeric protein can be conjugated to a cytotoxic agent to create an immunotoxin.
  • Methods for the production of various immunotoxins is well known in the art. See, generally. "Monoclonal Antibody-Toxin Conjugates: Aiming the Magic Bullet," Thorpe, et al., Monoclonal Antibodies in Clinical Medicine. Academic Press, pp. 168-190 (1982) , which is incorporated herein by reference.
  • the chimeric proteins can also be used to induce anergy in targeted T cells.
  • anergy or nonresponsiver.ess can be induced in autoreactive lymphocytes (see. Schwartz, Cell (1989) 1073-1081, which is incorporated herein by reference) .
  • In vitro experiments suggest that antigen presentation by MHC Class II molecules in the absence of an unknown co-stimulatory signal induces a state of proliferative non-responsiveness in syngeneic T cells (Quill et al., J. Immunol. (1987) 138:3704- 3712, which is incorporated herein by reference).
  • the immunoglobulin constant region component of the present invention typically comprises the hinge region and the C H 1, C H 2, and C H 3 domains. If mu or epsilon heavy chains are used, a C H 4 may be included, as well. As defined here, the constant region component may also comprise a portion of the variable region of the particular immunoglobulin chain, usually less than about 10 amino acids. In certain embodiments, the immunoglobulin component may lack the C H 1 domain. An immunoglobulin constant region lacking a C H 1 domain is particularly preferred in combination with homodimeric constructs, discussed below. The C H 1 domain is believed to be responsible for the phenomenon of "heavy chain toxicity" which is observed when heavy chains are expressed in the absence of their corresponding light chains.
  • the chimeric molecules of the present invention can exist as either heterodimers or homodimers. Homodimers exist in two classes or forms, ⁇ /3 homodimers and + ⁇ homodimers.
  • One example of a chimeric molecule of the present invention is illustrated in Figure 2, and comprises constant domains from both the heavy and light chains of the immunoglobulin. Either an MHC II ⁇ or ⁇ chain is linked to each of the immunoglobulin chains.
  • an MHC II ⁇ chain is linked to the heavy chain and the ⁇ chain is linked to the light chain.
  • the linkage can, of course, be reversed (i.e.. a chain to light chain and ⁇ chain to heavy chain) .
  • chimeric molecules of the invention comprise a single chain MHC molecule capable of binding the appropriate antigenic peptide and T cell receptor (MHC II ⁇ or ⁇ chain or MHC I heavy chain) linked to an immunoglobulin heavy chain.
  • MHC II ⁇ or ⁇ chain or MHC I heavy chain the appropriate antigenic peptide and T cell receptor
  • two a chains are linked to an immunoglobulin heavy chain homodimer.
  • This figure also illustrates a construct lacking C H 1 domains, which, as discussed above, are correlated with heavy chain toxicity.
  • the efficacy of single chain MHC components is disclosed and claimed in copending application U.S.S.N. (Attorney Docket No. 14048-16) which is incorporated herein by reference.
  • each immunoglobulin chain comprises ⁇ and ⁇ chains, or portions thereof, linked together.
  • the ⁇ and ⁇ chains are attached with a peptide linker as described in Huston et al., Proc. Nat. Acad. Sci. USA 85:5879- 5883 (1988) and Chaudary et al.. Nature 339:394-397 (1989), both of which are incorporated herein by reference.
  • This technique involves construction of recombinant expression vector encoding the appropriate portions of the ⁇ and ⁇ chains. Preparation and expression of a recombinant DNA constructs is discussed more fully, below.
  • the expression vectors suitable for expressing ct+ ⁇ homodimers comprise a sequence encoding the peptide linker placed between the two MHC sequences.
  • the linker preferably exhibits little or no ordered secondary structure and does not substantially interfere with proper folding of the MHC chains.
  • the appropriate linker length is determined by measuring the distance between the C-terminu ⁇ of one chain and the N-terminus of the other chain. Calculation of the appropriate number of amino acids in the linker is based on a typical peptide unit length of about 0.38 nm.
  • the chimeric proteins of the present invention may also comprise a protease recognition site between the MHC component and the immunoglobulin component.
  • a protease recognition site allows cleavage of the two components and recovery of the purified MHC component.
  • the sequence of the protease recognition site preferably does not occur in the MHC component.
  • Proteases suitable for the present invention include Factor Xa and collagenase. Treatment of the chimeric protein bound to an affinity chromatography column through the immunoglobulin component can be used to isolate the soluble MHC component in pure form. Suitable columns for this purpose include protean A/G sepharose columns and the like.
  • purified chimeric protein is treated in solution and the MHC component is purified by anti-MHC affinity chromatography.
  • the MHC and immunoglobulin components of the chimeric proteins may be conjugated by a number of methods.
  • the linkage may be by way of heterobifunctional cross-linkers, such as SPDP, carbodiimide, glutaraldehyde or the like. Methods for linking protein molecules are well known in the art.
  • the present invention is directed to recombinant DNA constructs comprising expression cassettes encoding the MHC components and the immunoglobulin components.
  • the nomenclature used hereafter and the laboratory procedures in recombinant DNA technology described below are those well known and commonly employed in the art. Standard techniques are used for DNA and RNA isolation, amplification. and cloning. Generally enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases and the like are performed according to the manufacturer's specifications. These techniques and various other techniques are generally performed according to Sambrook et al. , Molecular Cloning - A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989, which is incorporated herein by reference. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader.
  • the DNA constructs will typically include an expression control DNA sequence, including naturally-associated or heterologous promoter regions, operably linked to protein coding sequences.
  • the term "operably linked” as used herein refers to linkage of a promoter upstream from one or more DNA sequences such that the promoter mediates transcription of the DNA sequences.
  • the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and the collection and purification of the chimeric proteins.
  • Human MHC and immunoglobulin constant region DNA sequences can be isolated in accordance with well known procedures from a variety of human cells.
  • immortalized B cells are suitable for isolating immunoglobulin sequences, e.g.. cDNA or genomic DNA.
  • MHC cDNA and genomic clones from a variety of cells have been extensively characterized (see, e.g. , Paul, Chapter 17, and Kabat et al. , Seguences of Proteins of Immunological Interest. (U.S. Dept. of Health and Human Services, NIH, 1987) , which is incorporated herein by reference) .
  • traditional screening of a cDNA library prepared from RNA isolated from appropriate cells is used.
  • PCR amplification of the desired sequences can also be used (See, PCR Protocols, Innis et al. , eds. Academic Press, 1990) .
  • Suitable source cells for the DNA sequences and host cells for expression and secretion can be obtained from a number of sources, such as the American Type Culture Collection ("Catalogue of Cell Lines and Hybridomas," 6th edition (1988) Rockville, Maryland, U.S.A. and NIGMS Human Genetic Mutant Cell Repository 1990/1991 Catalogue of Cell Lines. 15th edition, NIH Publication No. 91-2011, which are incorporated herein by reference) .
  • the nucleotide sequences used to transfect the host cells can be modified according to-standard techniques to yield chimeric molecules with a variety of desired properties.
  • the molecules of the present invention can be readily designed and manufactured utilizing various recombinant DNA techniques well known to those skilled in the art and described in detail, below.
  • the chains can vary from the naturally- occurring sequence at the primary structure level by amino acid 7 insertions, substitutions, deletions, and the like. These modifications can be used in a number of combinations to produce the final modified protein chain.
  • the amino acid sequence variants can be prepared with various objectives in mind, including increasing the affinity of the molecule for target T cells, or for facilitating purification and preparation of the chimeric molecule.
  • the modified molecules are also useful for modifying plasma half life, improving therapeutic efficacy, and lessening the severity or occurrence of side effects during therapeutic use.
  • the amino acid sequence variants are usually predetermined variants not found in nature.
  • the variants typically exhibit the same biological activity as naturally occurring MHC molecule.
  • the variants and derivatives that are not capable of binding are useful nonetheless (a) as a reagent in diagnostic assays for particular MHC allelles, (b) as agents for purifying anti-MHC antibodies from antisera or hybridoma culture supernatants when insolubilized in accord with known methods, and (c) as immunogens for raising antibodies to MHC alleles so long as at least one MHC epitope remains active.
  • Polypeptide fragments comprising only a portion (usually at least about 60-80%, typically 90-95%) of the primary structure may be produced.
  • the immunoglobulin and MHC genes contain separate functional regions, each having one or more distinct biological activities.
  • the immunoglobulin component may be modified so as to retain certain functions (e.g. , complement fixation activity) , while exhibiting lower immunogenicity.
  • modifications of the genes encoding the chimeric molecule may be readily accomplished by a variety of well-known techniques, such as site-directed mutagenesis (see. Gillman and Smith, Gene 8:81-97 (1979) and Roberts, S. et al.. Nature 328:731-734 (1987), both of which are incorporated herein by reference) .
  • site-directed mutagenesis see. Gillman and Smith, Gene 8:81-97 (1979) and Roberts, S. et al.. Nature 328:731-734 (1987), both of which are incorporated herein by reference
  • a change in the immunological character of the chimeric molecule can be detected by an appropriate competitive binding assay.
  • the effect of a modification on the ability of the chimeric molecule to bind target T cell receptors can be tested using .in vitro cellular assays as described below. Modifications of other properties such as redox or thermal stability, hydrophobicity, susceptibility to proteolysis, or the tendency to aggregate are all assayed according to standard techniques.
  • Insertional variants of the present invention are those in which one or more amino acid residues are introduced into a predetermined site in the protein and which displace the preexisting residues.
  • cleavable sequences may be fused to the protein (e.g.. sequences form viral proteins) which allow ready affinity chromatographic purification of the fusion protein. Once isolated, the cleavable sequences are removed by treatment with an appropriate protease and the desired GMP-140 molecule is recovered.
  • Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place.
  • Non-natural amino acid i.e.. amino acids not normally found in native proteins
  • isosteric analogs amino acid or otherwise
  • Substantial changes in function or immunological identity are made by selecting substituting residues that differ in their effect on the structure of the polypeptide backbone (e.g.. as a sheet or helical conformation) , the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chain.
  • substitutions which in general are expected to produce the greatest changes in function will be those in which (a) a hydrophilic residue, e.g., serine or threonine, is substituted for (or by) a hydrophobic residue, e.g.
  • electropositive side chain e.g., lysine, arginine, or histidine
  • an electronegative residue e.g., glutamine or aspartine
  • a residue having a bulky side chain e.g., phenylalanine
  • Substitutional variants of the subunits also include variants in which functionally homologous (having at least about 70% homology) domains of other proteins are substituted by routine methods for one or more of the MHC domains.
  • Particularly preferred proteins for this purpose are other members of the immunoglobulin superfamily.
  • deletional variants are characterized by the removal of one or more amino acid residues from the MHC sequence. Typically, the transmembrane and cytoplasmic domains are deleted. Deletions of cysteine or other labile residues also may be desirable, for example in increasing the oxidative stability of the protein. Deletion or substitutions of potential proteolysis sites, e.g., Arg Arg, is accomplished by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues. Typically, the transmembrane domain is inactivated by deletion of all the transmembrane domain residues. Inactivation of the membrane binding function is also accomplished by deletion of sufficient residues (not necessarily all the residues) to produce a substantially hydrophilic hydropathy profile at this site or by substituting with heterologous residues which accomplish the same result.
  • Glycosylation variants are included within the scope of this invention. They include variants completely lacking in glycosylation (unglycosylated) and.variants having at least one less glycosylated site than the native form (deglycosylated) as well as variants in which the glycosylation has been changed. Included are deglycosylated and unglycosylated amino acid sequence variants, deglycosylated and unglycosylated subunits having the native, unmodified amino acid sequence. For example, substitutional or deletional mutagenesis is employed to eliminate the N- or 0-linked glycosylation sites of the protein, e.g., the asparagine residue is deleted or substituted for by another basic residue such as lysine or histidine.
  • flanking residues making up the glycosylation site are substituted or deleted, even though the asparagine residues remain unchanged, in order to prevent glycosylation by eliminating the glycosylation recognition site.
  • unglycosylated subunits which have the amino acid sequence of the native subunits are produced in recombinant prokaryotic cell culture because prokaryotes are incapable of introducing glycosylation into polypeptides.
  • Glycosylation variants are conveniently produced by selecting appropriate host cells or by in vitro methods.
  • Yeast for example, introduce glycosylation which varies significantly from that of mammalian systems.
  • mammalian cells from a different species e.g., hamster, urine, insect, porcine, bovine or ovine
  • tissue than the GMP-140 source are routinely screened for the ability to introduce variant glycosylation as characterized for example by elevated levels of mannose or variant ratios of mannose, fucose, sialic acid, and other sugars typically found in mammalian glycoproteins.
  • In vitro processing of the subunit typically is accomplished by enzymatic hydrolysis, e.g., neuraminidase digestion.
  • the nucleotide sequences encoding the MHC and immunoglobulin components will be expressed in hosts after the sequences have been operably linked to an expression control sequence (i.e., positioned to ensure the translation of the structural gene) along with other sequences (e.g.. enhancers, polyadenylation sites, etc.) necessary for efficient transcription and translation of the desired sequences.
  • an expression control sequence i.e., positioned to ensure the translation of the structural gene
  • other sequences e.g. enhancers, polyadenylation sites, etc.
  • expression cassette which is typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA.
  • expression vectors comprising the expression cassette will contain selection markers, e.g..
  • prokaryotes are used for cloning the chimeric protein nucleotide sequences.
  • E. coli is particularly useful for cloning the nucleotide sequences of the present invention.
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilus. and other enterobacteriaceae, such as Salmonella. Serratia. and various Pseudomonas species.
  • expression vectors which will typically contain expression control sequences compatible with the host cell (e.g.. an origin of replication) .
  • expression control sequences compatible with the host cell (e.g.. an origin of replication)
  • any of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences and the like, for initiating and completing transcription and translation.
  • Other microbes, such as yeast may also be used for expression.
  • Saccharomyces is a preferred host, with suitable vectors having expression control sequences, an origin of replication, termination sequences and the like as desired.
  • Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes.
  • Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase 2, isocytochrome C, and enzymes responsible for maltose and galactose utilization.
  • the plas id YRp7 can be used (Stinchcomb, et al.. Nature, 282: 39 (1979) , which is incorporated by reference) .
  • This plasmid contains the trpl gene which is a selectable marker for a mutant strain which is unable to grow on media lacking tryptophan. The presence of the trpl gene allows transformed mutant cells to grow on selective media and to be identified.
  • Mammalian tissue cell culture will typically be used to produce the polypeptides of the present invention (see. Winnacker, "From Genes to Clones,” VCH Publishers, N.Y., N.Y. (1987) , which is incorporated herein by reference) .
  • Eukaryotic cells are actually preferred, because a number of suitable host cell lines capable of secreting intact immunoglobulins or MHC molecules have been developed in the art, and include the CHO cell lines, various COS cell lines, HeLa cells, myeloma cell lines such as SP2/0 or lymphoma lines such as BW5147, etc.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer (Queen, C.
  • Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, hCMV early enhancer/promoter (Boshart et al.. Cell 41:521-530 (1985)), SR ⁇ (Takebe et al., Mol. and Cell Biol.. 8:486-472 (1988)) and the like.
  • Enhancers are cis-acting sequences of between 10 to 300bp that increase transcription by a promoter. Enhancers can effectively increase transcription when either 5* or 3' to the transcription unit. They are also effective if located within an intron or within the coding sequence itself.
  • viral enhancers are used, including SV40 enhancers, cytomegalovirus enhancers, polyoma enhancers, and adenovirus enhancers. Enhancer sequences from mammalian systems such as the mouse immunoglobulin heavy chain enhancer are also commonly used. Mammalian expression vector systems will also typically include a selectable marker gene.
  • markers include, the hypoxanthine-guanine phosp ⁇ cribosyl transferase gen (HGPT) , the thymidine kinase gene t"K) , or various prokaryotic genes conferring drug resistance.
  • Amplifiable marker genes such as dihydrofolate reductase gene (DHFR) , may also be used (see, generally. Kriegler, supra..
  • prokaryotic drug resistance genes useful as markers include genes conferring resistance to neomycin, G418, and hygromycin.
  • the chimeric proteins may also comprise protease recognition sites between the MHC and immunoglobulin components.
  • nucleotides encoding the appropriate protease recognition site will be included.
  • the recognition site is Ile(Glu/Asp)GlyArg.
  • the recognition site is ProLeuGlyPro(D-Arg) .
  • the vectors containing the nucleotide segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, cationic liposomes, or electroporation may be used for other cellular hosts. Other methods used to transform mammalian cells include the use of Polybrene, protoplast fusion, microprojectiles and icroinjection. See, generally, Sambrook et al., supra.
  • the whole chimeric proteins, or individual chains of the present invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity and fraction column chromatography, gel electrophoresis and the like, (See. generally. Scopes, R., Protein Purification. Springer-Verlag, N.Y. (1982), which is incorporated herein by reference.)
  • the polypeptides may then be used therapeutically or in developing and performing assay procedures, immunofluorescent stainings, and the like. (See, generally. Immunological Methods, Vols. I and II, Eds. Lefkovits and Pernis, Academic Press, New York, N.Y.
  • Therapeutic uses of the chimeric molecules of the present invention require identification of the MHC haplotypes and antigens useful in treating a particular disease.
  • the present invention is particularly suitable for treatment of autoimmune disease. Based on knowledge of the pathogenesis of human autoimmune disease and the results of studies in relevant animal models, one skilled in the art can readily identify and isolate the MHC haplotype and autoantigen associated with a variety of autoimmune diseases.
  • the MHC antigen encoded by the allele is also identifiable.
  • the chimerics of the present invention used for treatment or diagnosis of an individual with rheumatoid arthritis would include a MHC II component encoded by the DR, DQ or DP gene of the DR4 haplotype which contains the Dw4 or Dwl4 DR allelic determinant.
  • Contemporary knowledge of the autoantigens associated with particular autoimmune diseases is extensive.
  • Identified autoantigens include acetylcholine receptor in myasthenia gravis, myelin basic protein in multiple sclerosis, mitochondrial dihydrolipoamide acy1transferase in primary biliary cirrhosis, type II collagen in rheumatoid arthritis, thyroglobulin in autoimmune thyroiditis, S antigen in autoimmune uveitis, and desmoplakin I in paraneoplastic pemphigus.
  • small autoantigenic peptide fragments (epitopes) of the macromolecular autoantigen have been shown to be recognized by a defined subset of helper T cells (Livingstone et al., Ann. Rev. Immunol. 5:477-501 (1987), which is incorporated herein by reference) .
  • the assay methods typically use antigenic fragments generated by enzymatic digestion of the whole autoantigen or by cloning and expression of fragments of the gene encoding the autoantigen. When the amino acid sequence of the autoantigenic peptide fragment is known, sets of overlapping peptides are then synthesized. These assays identify epitopic sequences by the ability of the fragments or synthetic peptides to stimulate disease associated T cell clones or hybrido as in syngeneic antigen-presenting systems (see, e.g.. Watts et al., Ann. Rev. Immunol.
  • the chimeric molecule with the appropriate autoantigenic peptide, identified by the methods described above. This is typically done by incubating the purified chimeric with excess peptide at a low or high pH so as to open the antigen binding pocket (see. Harding et al., Proc. Nat. Acad. Sci. USA 88:2740-2744 (1991), which is incorporated herein by reference) .
  • the peptide is thus noncovalently linked with the antigen binding pocket of the MHC component.
  • the antigenic peptide can also be covalently bound using standard procedures such as photo- affinity labelling, (see e.g., Hall et al..
  • the chimeric molecules of the invention can be assayed using an in vitro system or using an in vivo model.
  • the in vitro system the molecule is incubated with peripheral blood T cells from subjects immunized with, or showing immunity to, the protein or antigen responsible for the condition associated with the peptide of the complex.
  • the successful molecules will eliminate (or induce anergy in) syngeneic T cells as measured in the assays described above.
  • T cells that proliferate in response to the isolated epitope or to the full length antigen in the presence of antigen presenting cells are cloned.
  • the clones are injected into histocompatible animals which have not been immunized in order to induce the autoimmune disease. Symptoms related to the relevant complex should ameliorate or eliminate the symptoms of the disease.
  • Chimeric proteins of the present invention can find a wide variety of in vitro and in vivo utilities. By way of example, they can be used to prepare purified MHC compositions.
  • a protein A/G sepharose column can be used to bind the immunoglobulin component of a chimeric protein of the present invention. Treatment of the bound chimeric protein with the appropriate protease will, release the soluble MHC component in a pure form.
  • the chimeric protein can be protease treated in solution and the MHC component can be isolated using the general methods described above for purifying chimeric proteins.
  • an anti-MHC antibody column can be used to obtain a pure MHC component preparation.
  • the chimeric proteins can also be used in a variety of in vivo applications, such as treating or monitoring autoimmune diseases. In vitro uses include, diagnostic applications, T cell typing, isolating or labeling specific cells, and the like. For any of these purposes, the chimeric proteins may be labeled or unlabeled.
  • Unlabeled chimeric proteins can be used in combination with labeled antibodies that are reactive with the immunoglobulin portion of the molecule.
  • Antibodies specific for human immunoglobulin constant regions are well known in the art. See, generally. Harlow and Lane, Antibodies: A Laboratory Manual (1988) .
  • Labeled protein A or protein G may also be used for this purpose. These proteins are normal constituents of the cell walls of streptococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species. See, generally Kronval, et al., J. Immunol.. 111:1401-1406 (1973), and Akerstro , et al., J. Immunol.. 135:2589-2542 (1985), all of which are incorporated herein by reference.
  • the chimeric protein can be directly labeled.
  • labels may be employed, such as radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chemiluminescent compounds, bioluminescent compounds, etc.
  • Those of ordinary skill in the art will know of other suitable labels for binding to the chimeric protein, or will be able to ascertain such using routine experimentation. The binding of these labels can be done using standard techniques common to those of ordinary skill in the art.
  • the detectably labeled chimeric protein is given in a dose which is diagnostically effective.
  • diagnostically effective means that the amount of detectably labeled protein is administered in sufficient quantity to enable detection of cells having the receptor for which the MHC component is specific.
  • concentration of detectably labeled protein which is administered should be sufficient such that the binding to those cells having the receptor is detectable compared to the background signal. Further, it is desirable that the detectably labeled protein be rapidly cleared from the circulatory system in order to give the best target-to- background signal ratio.
  • the dosage of detectably labeled protein for in vivo diagnosis will vary depending on such factors as age, sex and extent of disease of the individual.
  • radioisotopes are typically used.
  • the type of detection instrument available is a major factor in selecting the radioisotope used.
  • the radioisotope chosen must have a type of decay which is detectable for a given type of instrument.
  • Still another important factor in selecting a radioisotope for in vivo diagnosis is that the half-life of the radioisotope be long enough so that it is still detectable at the time of maximum uptake by the target, but short enough so that deleterious radiation with respect to the host is minimized.
  • a radioisotope used for m vivo imaging will lack a particle emission, but produce a large number of photons in the 140-250 keV range, which may be readily detected by conventional gamma cameras.
  • radioisotopes may be bound to the protein either directly or indirectly by using an intermediate functional group.
  • Intermediate functional groups which often are used to bind radioisotopes which exist as metallic ions to proteins are the bi-functional chelating agents such as diethylenetriaminepentacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA) and similar molecules.
  • DTPA diethylenetriaminepentacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • the proteins of the invention can also be labeled with a paramagnetic isotope for purposes of in vivo diagnosis, as in magnetic resonance imaging (MRI) or electron spin resonance (ESR) .
  • MRI magnetic resonance imaging
  • ESR electron spin resonance
  • any conventional method for visualizing diagnostic imaging can be utilized.
  • gamma and positron emitting radioisotopes are used for camera imaging and paramagnetic isotopes for MRI.
  • the proteins of the present invention can be used to monitor the course of amelioration of an immune response mediated disorder (such as autoimmunity) in an individual.
  • an immune response mediated disorder such as autoimmunity
  • compositions comprising the claimed proteins can be prepared.
  • Pharmaceutical compositions comprising the proteins are useful for, e.g.. parenteral administration, i.e.. subcutaneously, intramuscularly or intravenously.
  • parenteral administration i.e.. subcutaneously, intramuscularly or intravenously.
  • new drug delivery approaches are being developed.
  • the pharmaceutical compositions of the present invention are suitable for administration using these new methods, as well. See, Langer, Science 249:1527-1533 (1990), which is incorporated herein by reference.
  • compositions for parenteral administration will commonly comprise a solution of the antibody or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used. e.g.. water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter.
  • These compositions may be sterilized by conventional, well known sterilization techniques.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc.
  • the concentration of the chimeric protein in these formulations can vary widely, i.e. , from less than about 1 ⁇ g/ml, usually at least about 0.1 mg/ml to as much as 10 - 100 mg/ml by weight and will be selected primarily based on fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • a typical pharmaceutical composition for intramuscular injection could be made up to contain 1 ml sterile buffered water, and 0.1 mg of chimeric protein.
  • a typical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 10 mg of chimeric protein.
  • Actual methods for preparing parenterally ad inistrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example. Remington's Pharmaceutical Science. 17th Ed. , Mack Publishing Company, Easton, Pennsylvania (1985) , which is incorporated herein by reference.
  • the chimeric proteins of this invention can be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and commonly used lyophilization and reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilization and reconstitution can lead to varying degrees of activity loss and that use levels may have to be adjusted to compensate.
  • compositions containing the present chimeric proteins or a cocktail thereof can be administered for the prophylactic and/or therapeutic treatments.
  • compositions are administered to a patient already affected by the particular disease, in an amount sufficient to cure or at least partially arrest the disease process and its complications.
  • An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's own immune system, but generally range from about 0.01 to about 1000 mg of chimeric protein per dose, with dosages of from about 10 to about 100 mg per patient being more commonly used.
  • compositions containing the chimeric proteins or a cocktail thereof are administered to a patient not already in a disease state to enhance the patient's resistance.
  • Such an amount is defined to be a
  • prophylactically effective dose In this use, the precise amounts again depend upon the patient's state of health and general level of immunity, but generally range from 0.01 to 1000 mg per dose, especially about 10 to about 100 mg per patient.
  • compositions can be carried out with dose levels and pattern being selected by the treating physician.
  • pharmaceutical formulations should provide a quantity of the chimeric proteins of this invention sufficient to effectively treat the patient.
  • Kits can also be supplied for therapeutic or diagnostic uses.
  • the subject composition of the present invention may be provided, usually in a lyophilized form in a container.
  • the proteins which may be conjugated to a label or toxin, or unconjugated, are included in the kits with buffers, such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g. serum albumin, or the like, and a set of instructions for use.
  • buffers such as Tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g. serum albumin, or the like
  • these materials will be present in less than about 5% wt. based on the amount of active chimeric protein and usually present in total amount of at least about 0.001% wt. based again on the protein concentration.
  • an inert extender or excipient to dilute the active ingredients, where the excipient may be present in from about 1 to 99% wt. of the total composition.
  • a second antibody capable of binding to the chimeric protein is employed in an assay, this will usually be present in a separate vial.
  • the second antibody is typically conjugated to a label and formulated in an analogous manner with the antibody formulations described above.
  • the following example is offered by way of illustration, not by way of limitation.
  • EXAMPLE I This example shows the construction of an expression vector capable of directing expression of a chimeric protein in an appropriate mammalian cell.
  • Nucleotide sequences from the Class II MHC gene, HLA DR4 (DW4) are obtained using standard procedures from GMD6821a cells, which can be obtained from NIGMS Human Genetic Mutant Cell Repository, supra.
  • a unique restriction site Xbal followed by a Kozak consensus ribosomal binding site is added to the 5' end of an isolated Class II MHC gene immediately upstream of the translational start codon, ATG, of the leader peptide.
  • nucleotides encoding the appropriate protease recognition site flanked by nucleotides encoding are inserted immediately upstream of the hydrophobic transmembrane domain.
  • the amino acid sequence is lle(Glu/Asp)GlyArg, for collagenase, ProLeuGlyPro(D-Arg) .
  • the splice donor sequence C/AAGGTA/GAGT and the Xbal restriction site are placed immediately downstream of the [ (Gly 4 )protease recognition(Gly 4 ) ] site.
  • the splice donor is placed in such a manner that the translational reading frame of the MHC II/IgG hybrid mRNA is not altered.
  • the splice donor will be replaced with an Xbal or another restriction site that has the minimum effect on the amino acid sequence of the chimeric protein.
  • the final genes are excised with Xbal (or a combination of Xbal and a second restriction enzyme) and inserted into expression vectors.
  • the expression vectors are depicted schematically in Figure 5.
  • An expression vector will comprise sequences encoding either the ⁇ or the ⁇ MHC chain and the immunoglobulin constant region gene.
  • Figure 5a shows a vector comprising immunoglobulin cDNA
  • Figure 5b shows the construct with genomic immunoglobulin DNA.
  • the important components of the vectors include the following.
  • An ampicillin resistance gene and bacterial origin of replication derived from the pUC series of plasmids is included to allow cloning in a bacterial host.
  • a drug resistance gene mycophenolic acid
  • Transcription of this gene is driven by an SV40 early/late promoter followed by a SV40 early/late poly-adenylation signal.
  • a strong enhancer/promoter drives expression of the inserted chimeric protein gene.
  • a splice acceptor sequence T/CT/CT/CT/CT/CT/CT/CT/CT/CT/CT/CT/CT/CT/CNC/TAGG/A, is used, followed by the sequence encoding either the rearranged genomic heavy or light chain IgGl constant region (Hieter et al.. Cell. 22:197- 207 (1980); Ellison et al., Nuc. Acids Res.. 10:4071-4079
  • the splice acceptor will be placed in such a manner that the translational reading frame of the hybrid mRNA will be unaffected.
  • the mature cDNA sequences of the heavy or light chain IgG constant regions are linked directly to the appropriate MHC sequence. Following the constant region coding sequences will be either the endogenous polyadenylation signal (genomic) or a heterologous signal (cDNA) .

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Abstract

La présente invention se rapporte à des nouvelles protéines chimèriques comprenant un composant majeur du complexe d'histocompatibilité (CHM) lié à un composant de région constante d'immunoglobuline dans lequel la protéine chimèrique est capable de se fixer sélectivement à un récepteur des lympocytes T par l'intermédiaire du composant CHM. Les molécules chimèriques de l'invention peuvent également comprendre un site de reconnaissance de protéases situé entre les deux composants de façon à faciliter la purification de l'un des composants. Les protéines chimèriques sont utiles pour le traitement des affections associées au CHM, telles que les maladies auto-immunes.
PCT/US1992/010030 1991-11-19 1992-11-18 Molecules solubles du complexe majeur d'histocompatibilite et leurs utilisations WO1993010220A1 (fr)

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WO1996020215A2 (fr) * 1994-12-23 1996-07-04 Laboratoires Om S.A. Utilisation de molecules se fixant a cnh-ii et/ou simulant cnh-ii dans la prevention et/ou le traitement des etats inflammatoires
EP1437366A1 (fr) * 1995-02-28 2004-07-14 The Board Of Trustees Of The Leland Stanford Junior University Complexes antigènes/MHC pour détecter et purifier les lymphocytes T spécifiques aux antigènes
EP0812331A4 (fr) * 1995-02-28 2000-06-07 Trustees Of Board Of Complexes antigenes/mhc pour detecter et purifier les lymphocytes t specifiques aux antigenes
US7141656B2 (en) 1996-01-31 2006-11-28 Altor Bioscience Corporation MHC complexes and uses thereof
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US6140113A (en) * 1996-03-28 2000-10-31 The Johns Hopkins University Polynucleotides encoding molecular complexes which modify immune responses
AU729406B2 (en) * 1996-03-28 2001-02-01 Johns Hopkins University, The Soluble divalent and multivalent heterodimeric analogs of proteins
WO1997035991A1 (fr) * 1996-03-28 1997-10-02 The Johns Hopkins University Analogues solubles heterodimeres divalents et multivalents de proteines
US6211342B1 (en) * 1996-07-18 2001-04-03 Children's Hospital Medical Center Multivalent MHC complex peptide fusion protein complex for stimulating specific T cell function
WO1998006749A3 (fr) * 1996-08-16 1998-06-11 Harvard College Proteines de fusion de classe ii du cmh, solubles, monovalentes ou polyvalentes, et utilisations associees
WO1998006749A2 (fr) * 1996-08-16 1998-02-19 President And Fellows Of Harvard College Proteines de fusion de classe ii du cmh, solubles, monovalentes ou polyvalentes, et utilisations associees
US6737057B1 (en) 1997-01-07 2004-05-18 The University Of Tennessee Research Corporation Compounds, compositions and methods for the endocytic presentation of immunosuppressive factors
US6734013B2 (en) 1997-03-28 2004-05-11 The Johns Hopkins University Use of multivalent chimeric peptide-loaded, MHC/Ig molecules to detect, activate or suppress antigen-specific T cell-dependent immune responses
WO1999009064A1 (fr) 1997-08-19 1999-02-25 Mount Sinai School Of Medicine Of New York University ; Element de la classe ii du complexe majeur d'histocompatibilite portant un epitope/molecules chimeres d'immunoglobuline
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