EP1054984A1 - Proteines hybrides monovalentes, multivalentes et multimeres caracterisees par un domaine de liaison cmh (complexe majeur d'histocompatibilite), conjugues de ces proteines, et utilisations correspondantes - Google Patents

Proteines hybrides monovalentes, multivalentes et multimeres caracterisees par un domaine de liaison cmh (complexe majeur d'histocompatibilite), conjugues de ces proteines, et utilisations correspondantes

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Publication number
EP1054984A1
EP1054984A1 EP99908272A EP99908272A EP1054984A1 EP 1054984 A1 EP1054984 A1 EP 1054984A1 EP 99908272 A EP99908272 A EP 99908272A EP 99908272 A EP99908272 A EP 99908272A EP 1054984 A1 EP1054984 A1 EP 1054984A1
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EP
European Patent Office
Prior art keywords
mhc
class
major histocompatibility
histocompatibility complex
fusion protein
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EP99908272A
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German (de)
English (en)
Inventor
Kai W. Wucherpfennig
Jack L. Strominger
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Harvard College
Dana Farber Cancer Institute Inc
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Harvard College
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Publication of EP1054984A1 publication Critical patent/EP1054984A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/02—Peptides being immobilised on, or in, an organic carrier
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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

Definitions

  • the present invention is directed to the field of immunology.
  • the present invention is directed to the design, production, and use of Major Histocompatibility Complex binding domain fusion proteins and conjugates.
  • MHC molecules are highly polymorphic dimeric proteins which determine the specificity of T cell mediated immune responses by binding peptides from foreign antigens in an intracellular processing compartment, and by presenting these peptides on the surface of antigen presenting cells, where they may be recognized by specialized T cell receptors (TCRs) (reviewed in Strominger and Wiley, 1995).
  • TCRs T cell receptors
  • MHC Class II DR ⁇ chain gene with 137 known DRB-1 alleles (Marsh and Bodmer, 1995), is the most polymorphic human gene that has been identified.
  • the polymorphic residues of these proteins are clustered in peptide binding domains which define the large repertoire of peptides that may be presented to T cells (Bjorkman et al, 1987; Stern et al, 1994).
  • T cells should not normally react to self peptides presented in syngeneic MHC molecules, some alleles of the MHC genes are believed to confer susceptibility to autoimmune diseases through the presentation of pathogenic self-peptides.
  • the MHC Class II HLA-DR2 subtypes confer an increased risk for multiple sclerosis (MS), while subtypes of HLA-DR4 confer susceptibility to rheumatoid arthritis (reviewed in Todd et al, 1988; Wucherpfennig and Strominger, 1995b).
  • soluble, "empty” MHC Class II molecules i.e., molecules which do not have peptides bound within the MHC Class II peptide binding domains
  • Such soluble, MHC/peptide complexes have several important investigational and therapeutic uses.
  • soluble MHC Class II molecules are required for crystallographic studies of single MHC/peptide complexes, and for studying the biochemical interaction of particular MHC/peptide complexes with their cognate TCRs.
  • soluble MHC/peptide complexes are useful for the treatment of autoimmune diseases.
  • EAE murine experimental autoimmune encephalomyelitis
  • Such complexes are expected to be useful in the treatment of several human autoimmune diseases, including multiple sclerosis (MS) and rheumatoid arthritis (RA).
  • MHC Class II molecules can be purified from mammalian cells by affinity chromatography following detergent solubilization of B cell membranes (Gorga et al., 1987). MHC molecules purified from B cell lines, however, have already passed through the intracellular MHC Class II peptide loading compartment and, therefore, are already loaded with a diverse set of peptides (Chicz et al, 1992). Furthermore, removal of these peptides from B cell derived MHC complexes (e.g., by low pH treatment) is very difficult and typically results in MHC protein denaturation.
  • HLA-DR1 and HLA-DR4 molecules have been expressed in the baculovirus/insect cell system using cDNA constructs for the DR ⁇ and DR ⁇ extracellular domains without the hydrophobic transmembrane domains (Stern and Wiley, 1992). These molecules were assembled and secreted but had a tendency to aggregate unless they were loaded with a high affinity peptide. Moreover, this approach has not been successful with HLA-DR2 molecules. For example, the product of the DRA, DRB5*0101 genes showed a strong tendency to aggregate even when high affinity peptides were added (Vranovsky and Strominger, unpublished observations).
  • dimerization domains of known, stable dimeric proteins may be genetically engineered into fusion proteins to promote the formation of stable dimeric fusion proteins.
  • synthetic peptides of the isolated Fos and Jun leucine zipper dimerization domains, with added N-terminal cysteine residues and (Gly) 2 linkers were shown to assemble as soluble heterodimers with interchain disulfide bridges (O'Shea et al., 1989).
  • Fusion proteins including artificial leucine zipper dimerization domains were also employed to express ⁇ heterodimers of the TCR extracellular domains with interchain disulfide bridges (Chang et al., 1994).
  • TCR chimeras were bound by antibodies to native TCRs, they were not shown to retain MHC/peptide complex specificity.
  • Gregiore et al. (1991) produced soluble ⁇ heterodimers of TCR extracellular domains by co-expressing proteins in which the variable and constant (first exon only) domains of ⁇ and ⁇ TCR chains were each fused to the same constant domain of an immunoglobulin K light chain.
  • the present invention is directed to monovalent and multivalent fusion proteins, and multimeric protein conjugates, comprising human Major Histocompatibility Complex binding domains, with or without bound MHC binding peptides, which are useful in diagnostic and therapeutic methods, as well as laboratory assays.
  • the present invention provides MHC binding domain fusion proteins of MHC Class II ⁇ and ⁇ chain proteins in which substantially all of the C-terminal transmembrane and cytoplasmic domains have been replaced by dimerization domains and, optionally, interposing linker sequences.
  • a Class II MHC binding domain fusion protein is provided comprising a fusion of, toward the N-terminus, at least an MHC Class II binding domain of an MHC Class II ⁇ chain and, toward the C-terminus, a dimerization domain.
  • the MHC Class II binding domain comprises an extracellular domain of an MHC Class II ⁇ chain, preferably at least residues 5-180 of an MHC Class II ⁇ chain, more preferably residues 5-190, and most preferably residues 5-200.
  • the MHC Class II ⁇ chains from which the fusion proteins of the invention may be derived include HLA-DRl, HLA-DR2, HLA-DR4, HLA-DQ 1, HLA-DQ2 and HLA-DQ8 ⁇ chains, and particularly ⁇ chains encoded by DRA*0101, DRA*0102, DQA1*0301 or DQA1 *0501 alleles.
  • a Class II MHC binding domain fusion protein comprising a fusion of, toward the N-terminus, at least an MHC Class II binding domain of an MHC Class II ⁇ chain and, toward the C-terminus, a dimerization domain.
  • the MHC Class II binding domain comprises an extracellular domain of an MHC Class II ⁇ chain, preferably at least residues 5-185 of an MHC Class II ⁇ chain, more preferably residues 5-195, and most preferably residues 5-205.
  • the MHC Class II ⁇ chains from which the fusion proteins of the invention may be derived include HLA-DRl , HLA-DR2, HLA-DR4, HLA-DQ 1 , HLA-DQ2 and HLA-DQ8 ⁇ chains, and particularly ⁇ chains encoded by DRB1*01, DRB1*15, DRB1*16, DRB5*01, DQB1 *03 and DQB1 *02 alleles.
  • the dimerization domains of the Class II MHC binding domain fusion proteins comprise coiled-coil dimerization domains, such as leucine zipper domains.
  • the leucine zipper domains include at least four leucine heptads.
  • the leucine zipper domain is a Fos or Jun leucine zipper domain.
  • the dimerization domain is an immunoglobulin Fab constant domain, such as an immunoglobulin heavy chain C H 1 constant region or an immunoglobulin light chain constant region.
  • a flexible molecular linker optionally may be interposed between, and covalently join, the MHC Class II binding domain and the dimerization domain.
  • the flexible molecular linker comprises a peptide sequence of 1-15 amino acid residues, more preferably 5-7 amino acid residues.
  • polypeptide linkers it is preferred that a majority of the amino acid residues in the linker are alanine, glycine, serine, leucine, isoleucine, or valine residues.
  • an MHC Class II binding peptide optionally may be covalently joined to the N-terminus of the MHC Class II ⁇ or ⁇ chain binding domain, such that the binding peptide is capable of selectively binding to an MHC Class II binding domain formed by the ⁇ or ⁇ chain and another ( ⁇ or ⁇ , respectively) MHC Class II chain.
  • the MHC binding peptide and the MHC Class II binding domain form an MHC/peptide complex.
  • the MHC binding peptide is joined to the N-terminus of the ⁇ chain.
  • any MHC binding peptides may be joined to the N-termini of MHC Class II chains with which they selectively bind in nature.
  • the MHC Class II binding domain is an HLA-DR2 binding domain and the binding peptide is selected from residues 85-99, 84-102 and 148-162 of human myelin basic protein.
  • the MHC Class II binding domain is an HLA-DR4 or HLA-DQ 1 binding domain and said binding peptide is selected from residues 78-93, 97-111, 190-204, 206-220, 251-265, 512-526 and 762-786 of the human desmoglein 3 protein.
  • a flexible molecular linker optionally may be interposed between, and covalently join, the MHC Class II chain and the MHC binding peptide.
  • the linker is a polypeptide sequence of 10-20 amino acid residues, more preferably 12-18 amino acid residues.
  • a polypeptide linker it is preferred that a majority of the amino acid residues are alanine, glycine, serine, leucine, isoleucine, and valine residues.
  • the present invention provides Class II MHC binding domain fusion proteins comprising a heterodimer of a first polypeptide chain and a second polypeptide chain, in which the first polypeptide chain comprises a fusion of, toward the N-terminus, at least an MHC binding domain of an MHC Class II ⁇ chain and, toward the C-terminus, a first dimerization domain, and the second polypeptide chain comprises a fusion of, toward the N-terminus, at least an MHC binding domain of an MHC Class II ⁇ chain and, toward the C-terminus, a second dimerization domain.
  • the first dimerization domain and the second dimerization domain associate in solution at physiological conditions to form a heterodimer capable of selectively binding an MHC binding peptide.
  • the dimerization domains as described above, may be coiled-coil dimerization domains and, preferably, leucine zipper domains.
  • Flexible molecular linkers as described above, may be interposed between and covalently join the MHC chains and dimerization domains, and MHC binding peptides may be covalently joined to one of the MHC chains.
  • a Class II MHC binding domain fusion protein comprising a heterodimer of a first polypeptide chain and a second polypeptide chain, in which the first polypeptide chain comprises a fusion of, toward the N-terminus, at least an MHC binding domain of an MHC Class II ⁇ chain and, toward the C-terminus, an immunoglobulin heavy chain C H I constant region, and the second polypeptide chain comprises a fusion of, toward the N-terminus, at least an MHC binding domain of an MHC Class II ⁇ chain and, toward the C-terminus, an immunoglobulin light chain constant region.
  • the immunoglobulin heavy chain C H 1 constant region and the immunoglobulin light chain constant region dimerize in solution at physiological conditions to form a heterodimer capable of selectively binding an MHC binding peptide.
  • a Class II MHC binding domain fusion protein comprising a heterodimer of a first polypeptide chain and a second polypeptide chain, in which the first polypeptide chain comprises a fusion of, toward the N-terminus, at least an extracellular domain of an MHC Class II ⁇ chain and, toward the C-terminus, an immunoglobulin light chain constant region, and the second polypeptide chain comprises a fusion of, toward the N-terminus, at least an extracellular domain of an MHC Class II ⁇ chain and, toward the C-terminus, an immunoglobulin heavy chain C H I constant region.
  • the immunoglobulin heavy chain C H I constant region and the immunoglobulin light chain constant region dimerize in solution at physiological conditions to form a heterodimer capable of selectively binding an MHC binding peptide.
  • the Class II MHC fusion protein may further comprise an immunoglobulin Fc region covalently joined to the immunoglobulin heavy chain C H I constant region.
  • Such Fc regions may be IgE or IgM Fc regions, and a flexible molecular linker may optionally be interposed between, and covalently join, the immunoglobulin heavy chain C H I constant region and immunoglobulin Fc region.
  • the Fc regions may be IgA, IgD or IgG Fc regions.
  • a flexible molecular linker may be optionally interposed between, and covalently join, the immunoglobulin heavy chain C H 1 constant region and immunoglobulin Fc region and, in these embodiments, may be immunoglobulin hinge regions.
  • a multivalent Class II MHC binding domain fusion protein comprising two Class II MHC binding domain fusion proteins as described above, in which the Fc regions are covalently joined by at least one disulfide bond.
  • a multivalent Class II MHC binding domain fusion protein is provided comprising five pairs of Class II MHC binding domain fusion proteins in which the Fc regions are IgM regions, each pair is covalently joined by at least one disulfide bond between Fc regions of each pair, and the five pairs are covalently joined by disulfide bridges to form a ring structure such that each adjacent pair in the ring is joined by at least one disulfide bond.
  • the Class II MHC binding domain fusion proteins may further comprise an N-terminal secretory signal sequence covalently joined to the N-terminus of the fusion protein.
  • the secretory signal sequence comprises a yeast ⁇ -mating factor secretion signal or a human MHC Class II protein secretion signal.
  • the present invention provides for multimeric MHC binding domain conjugates comprising a carrier conjugated to a multiplicity of MHC binding domains, with or without peptide bound thereto.
  • the multimeric MHC binding domain conjugates comprise about 5 to about 500 MHC binding domains per carrier, preferably about 10 to about 200 MHC binding domains per carrier, and most preferably about 20 to about 100 MHC binding domains per carrier.
  • the multimeric MHC binding domain conjugates are characterized by the presence of MHC binding domains at an average density of about 4 x 10 "3 to 20 MHC binding domains/nm 2 on the surface of the carrier, preferably about 4 x 10 "2 to 20 MHC binding domains/nm 2 , and most preferably about 0.4 to 20 MHC binding domains/nm 2 on said surface.
  • the multimeric MHC binding domain conjugates comprise a carrier having a maximum diameter of about 5 to about 1000 nm, preferably about 5 to about 500 nm, and most preferably about 5 to about 100 nm. In some embodiments, the multimeric MHC binding domain conjugates define a minimal surface area of less than approximately 3.1 x 10 6 nm 2 , preferably less than 7.9 x 10 5 nm 2 , and more preferably less than 3.1 x 10 4 nm 2 . In most preferred embodiments, MHC binding domain conjugates define minimal surface areas of approxiamtely 78.5 to 5.0 x 10 3 nm 2 .
  • the multimeric MHC binding domain conjugates define a minimal volume of less than approximately 5.2 x 10 8 nm 3 , preerably less than 6.5 x 10 7 nm 3 , and more preferably less than 5.2 x 10 5 nm 3 . In most preferred embodiments, MHC binding domain conjugates define minimal volumes of 65.4 to 3.4 x 10 nm 3 .
  • the multimeric MHC binding domain conjugates comprise a carrier weighing about 100 kDa to about 10,000 kDa, preferably about 100 kDa to about 5,000 kDa, more preferably about 100 kDa to about 1,000 kDa, and most preferably about 100 kDa to about 500 kDa.
  • the multimeric MHC binding domain conjugates weigh about 400 kDa to about 10,000 kDa, preferably about 400 kDa to about 5,000 kDa, more preferably about 400 kDa to about 1,000 kDa, and most preferably about 400 kDa to about 500 kDa.
  • the multimeric MHC binding domain conjugate is particulate, the carrier is biodegradable, the carrier is non-immunogenic, the carrier has a net neutral or negative charge, and/or the carrier is fluorescently labeled.
  • the carrier may be covalently or non-covalently bound to the MHC binding domains.
  • the multimeric MHC binding domain conjugate comprises a carrier which is a substantially spherical bead or a porous bead.
  • the carrier is a bead
  • the bead preferably comprises a material selected from the group consisting of glass, silica, polyesters of hydroxy carboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxy carboxylic acids and dicarboxylic acids.
  • the multimeric MHC binding domain conjugate comprises a carrier which is a branched polymer, such as a dendrimer.
  • the dendrimer comprises a material selected from the group consisting of a polyamidoamine, a polyamidoalcohol, a polyalkyleneimine, a polyalkylene, a polyether, a polythioether, a polyphosphonium, a polysiloxane, a polyamide, and a polyaryl polymer.
  • the multimeric MHC binding domain conjugate comprises a carrier which is a liposome.
  • the liposome preferably comprises a material selected from the group consisting of phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidic acid, dicetyl phosphate, . monosialoganglioside, polyethylene glycol, stearyl amine, ovolecithin and cholesterol.
  • the multimeric MHC binding domain conjugates may further comprise a multiplicity of MHC binding peptides bound to the MHC binding domains, either covalently or non-covalently.
  • each MHC binding domain preferably comprises a heterodimer of at least the peptide binding domain of an MHC Class I ⁇ chain and an MHC Class I ⁇ chain, or a heterodimer of at least the peptide binding domain of an MHC Class II ⁇ chain and an MHC Class II ⁇ chain.
  • the MHC binding domains may comprise a part of a monovalent or multivalent MHC binding domain fusion protein of the invention.
  • the present invention provides a method for detecting T cells having a defined MHC/peptide complex specificity comprising providing a monovalent, multivalent or multimeric MHC binding domain fusion protein or conjugate, as described above and comprising the defined MHC/peptide complex, contacting a population of T cells with the fusion protein or conjugate, and detecting the presence or absence of binding of the fusion protein or conjugate and T cells in the population. Also provided is a method further comprising isolating T cells reactive with the defined MHC/peptide complex from the population of T cells by, for example, means of fluorescence activated cell sorting.
  • the present invention provides a method of conferring to a subject adoptive immunity to a defined MHC/peptide complex comprising providing a monovalent, multivalent or multimeric MHC binding domain fusion protein or conjugate, as described above and comprising the defined MHC/peptide complex, contacting a population of T cells with the fusion protein or conjugate, isolating T cells reactive with the defined MHC/peptide complex from the population of T cells, and administering the isolated T cells to the subject to provide adoptive immunity.
  • the present invention provides a method for stimulating or activating T cells reactive to a defined MHC/peptide complex comprising providing a monovalent, multivalent ' or multimeric MHC binding domain fusion protein or conjugate, as described above and comprising the defined MHC/peptide-complex, and contacting a population of T cells with an immunogenic amount of the fusion protein or conjugate.
  • the fusion protein or conjugate is contacted with the population of T cells in vivo in a human subject, and the MHC fusion protein or conjugate comprises an MHC binding domain which is syngeneic to the subject.
  • the present invention provides a method for selectively killing T cells reactive to a defined MHC/peptide complex comprising providing a monovalent, multivalent or multimeric MHC binding domain fusion protein or conjugate, as described above and comprising the defined MHC/peptide-complex, and contacting a population of T cells with the fusion protein or conjugate, in which the fusion protein or conjugate comprises a domain of an immunoglobulin effective to activate the complement system and cause the complement system to kill the T cells.
  • the present invention provides a method for selectively killing T cells reactive to a defined MHC/peptide complex comprising providing a monovalent, multivalent or multimeric MHC binding domain fusion protein or conjugate, as described above and comprising the defined MHC/peptide-complex, and contacting a population of T cells with the fusion protein or conjugate, in which the fusion protein or conjugate comprises a cytotoxic substance associated with the protein or conjugate and capable of killing T cells to which the fusion protein or conjugate selectively binds.
  • the present invention provides a method for tolerizing a human subject to a defined MHC/peptide complex comprising providing a monovalent, multivalent or multimeric MHC binding domain fusion protein or conjugate, as described above and comprising the defined MHC/peptide-complex, and administering to the subject an amount of the fusion protein or conjugate effective to induce tolerization to said MHC/peptide complex.
  • the MHC fusion protein or conjugate comprises an MHC binding domain which is syngeneic to the subject. In other preferred embodiments, however, the MHC fusion protein or conjugate comprises an MHC binding domain which is allogeneic to the subject.
  • the present invention provides nucleic acid sequences encoding the above-described MHC binding domain fusion proteins.
  • FIG. 1 is a schematic representation of one embodiment of a monovalent MHC binding domain fusion protein of the invention.
  • an extracellular or peptide binding domain of an MHC Class II ⁇ chain 10 is joined to a first dimerization domain 30
  • an extracellular or peptide binding domain of an MHC Class II ⁇ chain 20 is joined to a second dimerization domain 40
  • these two fusion constructs form a heterodimeric molecule which binds an MHC binding peptide 110 in the cleft formed by the MHC Class II binding domains of the ⁇ and ⁇ chains, 10 and 20.
  • flexible molecular linkers are interposed between the MHC domains (10, 20) and the dimerization domains (30, 40).
  • FIG. 1 is a schematic representation of one embodiment of a divalent MHC binding domain fusion protein construct of the invention.
  • an extracellular or peptide binding domain of an MHC Class II ⁇ chain 10 is joined to either a first coiled-coil or dimerization domain or an immunoglobulin heavy chain C H 1 constant region 30, and an extracellular or peptide binding domain of an MHC Class II ⁇ chain 20 is joined to a second coiled-coil dimerization domain or an immunoglobulin light chain constant region 40.
  • the domain 30 fused to the MHC ⁇ chain domain 10 is further fused to a hinge region 50 (optional) and Fc region 60 of an immunoglobulin chain.
  • the MHC ⁇ and ⁇ chain domains 10 and 20 may be switched such that the MHC ⁇ chain domain is fused to the immunoglobulin heavy chain domains 50 and 60.
  • the dimerization domains 30 and 40 promote the assembly of these two fusion constructs to form a heterodimeric structure which binds an MHC binding peptide 110 in the cleft formed by the MHC Class II binding domains of the ⁇ and ⁇ chains, 10 and 20.
  • flexible molecular linkers are interposed between the MHC domains (10, 20) and the dimerization domains (30, 40), and/or between the dimerization domain 30 and the immunoglobulin hinge 50 or Fc region 60.
  • the Fc regions 60 and 60' of two of these heterodimeric MHC-immunoglobulin fusion proteins associate in the manner of an antibody to form a divalent MHC binding domain fusion protein construct.
  • Horizontal lines between the Fc regions 60 and 60* represent disulfide bridges between the immunoglobulin heavy chain domains.
  • FIG. 3 This figure is a schematic representation of one embodiment of a decavalent MHC binding domain fusion protein construct of the invention.
  • an extracellular or peptide binding domain of an MHC Class II ⁇ chain 10 is joined to either a first coiled-coil dimerization domain or an IgM immunoglobulin heavy chain CHI (C ⁇ l) constant domain
  • an extracellular or peptide binding domain of an MHC Class II ⁇ chain 20 is joined to either a second coiled-coil dimerization domain or an IgM immunoglobulin light chain constant region 40, and these two fusion constructs assemble to form a heterodimeric molecule which binds an MHC binding peptide 110 in the cleft formed by the MHC Class II binding domains of the ⁇ and ⁇ chains, 10 and 20.
  • the domain 30 fused to the MHC ⁇ chain domain 10 is further fused to an IgM Fc domain (C H 2, C H 3, C H 4) 60.
  • the MHC ⁇ and ⁇ chain domains 10 and 20 may be switched such that the MHC ⁇ chain domains are fused to the immunoglobulin heavy chain domains 60.
  • the Fc regions 60 and 60' of two heterodimeric MHC-immunoglobulin fusion proteins associate in the manner of a single IgM subunit to form a divalent MHC-IgM fusion structure joined by a disulfide bond.
  • MHC-IgM fusion subunits Five of these divalent MHC-IgM fusion subunits assemble to form a characteristic IgM pentamer, joined by disulfide bonds 90 between IgM subunits and including a J-chain peptide 100, and resulting in a decavalent MHC-IgM fusion structure.
  • flexible molecular linkers are interposed between the MHC domains (10, 20) and the coiled-coil or IgM dimerization domains (30, 40), and/or between the dimerization domains (30) and the IgM Fc domains (60).
  • FIG. 4 This figure is a schematic representation of one embodiment of a tetravalent MHC binding domain fusion protein construct of the invention.
  • an extracellular or peptide binding domain of an MHC Class II ⁇ chain 10 is joined to a first dimerization domain 30
  • an extracellular or peptide binding domain of an MHC Class II ⁇ chain 20 is joined to a second dimerization domain 40, and these two fusion constructs assemble to form a heterodimeric molecule which binds an MHC binding peptide 110 in the cleft formed by the MHC Class II binding domains of the ⁇ and ⁇ chains, 10 and 20.
  • the domain 30 fused to the MHC ⁇ chain domain 10 is further fused to a ligand tag 70 which binds to anti-ligand 80.
  • the MHC ⁇ and ⁇ chain domains 10 and 20 may be switched such that the MHC ⁇ chain domain is fused to the ligand tag 70.
  • each anti-ligand binds four ligand moieties, and the MHC binding domain fusion protein complex is tetravalent.
  • flexible molecular linkers are interposed between the MHC domains (10, 20) and the dimerization domains (30, 40), and/or between the dimerization domain 30 and the ligand tag 70.
  • Figure 5 is a schematic representation of one embodiment of a multimeric
  • an extracellular or peptide binding domain of a first MHC chain ( ⁇ or ⁇ ) 10 and an extracellular or peptide binding domain of a second MHC chain ( ⁇ or ⁇ ) 20 assemble to form a heterodimeric molecule which binds an MHC binding peptide 110 in the cleft formed by the MHC binding domains of the ⁇ and ⁇ chains, 10 and 20.
  • a conjugating moiety 200 conjugates, covalently or non-covalently, one of the MHC chains 10 to a carrier 300.
  • FIG. 6 This figure is a schematic representation of one embodiment of a multimeric MHC binding domain conjugate of the invention.
  • an extracellular or peptide binding domain of an MHC ⁇ chain 10 is joined to a first dimerization domain 30
  • an extracellular or peptide binding domain of an MHC ⁇ chain 20 is joined to a second dimerization domain 40, and these two fusion constructs assemble to form a heterodimeric molecule which binds an MHC binding peptide 110 in the cleft formed by the MHC binding domains of the ⁇ and ⁇ chains, 10 and 20.
  • the domain 30 fused to the MHC ⁇ chain domain 10 is bound, covalently or non-covalently, to a conjugating moiety 200 which is bound, covalently or non-covalently, to a carrier 300.
  • the carrier 300 is shown as a dendrimer.
  • the MHC ⁇ and ⁇ chain domains 10 and 20 may be switched such that the MHC ⁇ chain domain is bound to the conjugating moiety 200.
  • flexible molecular linkers are interposed between the MHC domains (10, 20) and the dimerization domains (30, 40), and/or between the dimerization domain 30 and the conjugating moiety 200, and/or between the conjugating moiety 200 and the carrier 300.
  • Figure 7. This figure is a schematic representation of one embodiment of a multimeric
  • an extracellular or peptide binding domain of an MHC ⁇ chain 10 is joined to a first dimerization domain 30, an extracellular or peptide binding domain of an MHC ⁇ chain 20 is joined to a second dimerization domain 40, and these two fusion constructs assemble to form a heterodimeric molecule which binds an MHC binding peptide 110 in the cleft formed by the MHC binding domains of the ⁇ and ⁇ chains, 10 and 20.
  • the domain 30 fused to the MHC ⁇ chain domain 10 is further fused to a ligand tag 70 which binds to anti-ligand 80, which is bound to the surface of a carrier 300.
  • the MHC ⁇ and ⁇ chain domains 10 and 20 may be switched such that the MHC ⁇ chain domain is fused to the ligand tag 70.
  • flexible molecular linkers are interposed between the MHC domains (10, 20) and the dimerization domains (30, 40), and/or between the dimerization domain 30 and the ligand tag 70, and/or between the anti-ligand 80 and the carrier 300.
  • FIG. 8 This figure graphically presents the results of experiments demonstrating the assembly and secretion of recombinant HLA-DR2 fusion proteins by Pichia pastoris.
  • Expression of DR2 fusion proteins (DR ⁇ -Fos and DR ⁇ -Jun) were examined by sandwich ELISA of cell culture supernatants (top graph) or cell culture lysates (bottom graph) using a mAb (L243) specific for the DR2 ⁇ heterodimer for capture, and a polyclonal DR antiserum for detection. Binding of the secondary antibody was quantitated with a peroxidase conjugated anti-rabbit IgG antiserum, with ABTS as the peroxidase substrate and detection at 405 nm. Results are from cells transfected with: DR ⁇ -Fos only, open squares; DR ⁇ -Jun only, solid circles; and both DR ⁇ - Fos and DR ⁇ -Jun, open circles.
  • FIG. 9 This figure presents the results of experiments demonstrating the specificity of peptide binding to recombinant HLA-DR2 (rDR2) fusion proteins.
  • Peptide binding was examined using a biotinylated MBP(85-99) peptide ("MBP") that was previously shown to bind with high affinity to detergent soluble DR2.
  • MBP biotinylated MBP(85-99) peptide
  • rDR2-MBP complexes were captured on an ELISA plate using a DR specific mAb (L243) and DR bound biotinylated MBP was quantitated using peroxidase labeled streptavidin, with ABTS as the peroxidase substrate and detection at 405 nm.
  • the top graph shows the effect of rDR2 concentration on peptide binding with: 2 ⁇ M biotinylated MBP peptide, open circles; 2 ⁇ M biotinylated MBP peptide with 100 ⁇ M unbiotinylated MBP as a competitor, solid triangles; and no peptide, solid squares.
  • the same ELISA assay was used with 200 nM rDR2 and 2 ⁇ M biotinylated MBP to demonstrate binding specificity.
  • the bottom graph shows the effect of varying concentrations of competitor peptides on biotinylated MBP peptide binding to rDR2 fusion proteins: unbiotinylated MBP competitor, open squares; Val89— »Asp MBP competitor, closed circles.
  • Figure 10 shows the effect of rDR2 concentration on peptide binding with: 2 ⁇ M biotinylated MBP peptide, open circles; 2 ⁇ M biotinylated MBP peptide with 100 ⁇ M unbio
  • This figure presents the results of experiments demonstrating the kinetics of peptide binding to recombinant HLA-DR2 fusion proteins (rDR2).
  • the kinetics of peptide binding were compared for rDR2 and for detergent soluble DR2 purified from an EB V transformed B cell line.
  • the DR2 proteins 200 nM were incubated with biotinylated MBP peptide (2 ⁇ M) at 37°C for different periods of time; the amount of DR bound peptide was examined by ELISA using a DR specific antibody for capture and streptavidin-peroxidase for quantification of bound peptide, with ABTS as the peroxidase substrate and detection at 405 nm.
  • the graph shows biotinylated MBP peptide binding over time for: recombinant DR2 fusions, open squares; detergent solubilized B cell DR2 molecules, closed triangles.
  • MHC Major Histocompatibility Complex
  • TCRs T cell receptors
  • the human MHC region also referred to as HLA, is found on chromosome six and includes the Class I region (including the Class I ⁇ genes HLA-A, HLA-B and HLA-C) and the Class II region (including the subregions for Class II ⁇ and ⁇ genes DP, DQ and DR).
  • MHC Class I refers to the human Major Histocompatibility Complex Class I proteins, binding peptides, or genes. Within the MHC Class I region are found the HLA-A, HLA-B and HLA-C subregions.
  • MHC Class I molecule means a covalently or non-covalently joined complex of an MHC Class I ⁇ chain and a ⁇ 2 -microglobulin chain.
  • MHC Class II refers to the human Major Histocompatibility Complex Class II proteins, binding peptides, or genes. Within the MHC Class II region are found the DP, DQ and DR subregions for Class II ⁇ chain and ⁇ chain genes (i.e., DP ⁇ , DP ⁇ , DQ ⁇ , DQ ⁇ , DR ⁇ , and DR ⁇ ). As used herein, the term “MHC Class II molecule” means a covalently or non-covalently joined complex of an MHC Class II ⁇ chain and an MHC Class II ⁇ chain.
  • MHC Class I ⁇ chain means a naturally occurring polypeptide, or one encoded by an artificially mutated ⁇ gene, essentially corresponding to at least the ⁇ i and ⁇ 2 domains of one of the gene products of an MHC Class I ⁇ gene (e.g. HLA-A HLA-B or HLA-C gene).
  • MHC Class I ⁇ chain is intended to include variants with and without the usual glycosylation of the ⁇ domain.
  • MHC Class I ⁇ chain may also be referred to herein as an "MHC Class I heavy chain.”
  • Class I ⁇ chain or " ⁇ -microglobulin” means a naturally occurring polypeptide, or one encoded by an artificially mutated ⁇ 2 -microglobulin gene, essentially corresponding to the gene product of a ⁇ 2 -micro globulin gene.
  • the term is particularly intended to embrace all allelic variants of ⁇ -microglobulin, as well as any equivalents, including those which may be produced synthetically of recombinantly by, for example, site-directed mutagenesis of a naturally occurring variant.
  • MHC ⁇ chain is used without specifying whether the chain is Class I or Class II, the term is intended to include ⁇ 2 - microglobulin as well as MHC Class II ⁇ chains.
  • a ⁇ 2 -microglobulin or MHC Class I ⁇ chain may also be referred to herein as an "MHC Class I light chain.”
  • MHC Class II ⁇ chain means a naturally occurring polypeptide, or one encoded by an artificially mutated ⁇ gene, essentially corresponding to at least the ⁇ i and ⁇ 2 extracellular domains of one of the gene products of an MHC Class II ⁇ gene (e.g., a DP, DQ or DR ⁇ gene).
  • an MHC Class II ⁇ gene e.g., a DP, DQ or DR ⁇ gene.
  • the C-terminal transmembrane and cytoplasmic portions of the ⁇ chain are not necessary for antigenic peptide binding in the present invention, they may be omitted while retaining biological activity.
  • MHC Class II ⁇ chain is intended to include variants with and without the usual glycosylation of the ⁇ i and ⁇ 2 domains. The term is particularly intended to embrace all allelic variants of the Class II ⁇ genes, as well as any equivalents which may be produced synthetically or recombinantly by, for example, site-directed mutagenesis of
  • MHC Class II ⁇ chain means a naturally occurring polypeptide, or one encoded by an artificially mutated ⁇ gene, essentially corresponding to at least the ⁇ i and ⁇ 2 extracellular domain of one of the gene products of an MHC Class II ⁇ gene (e.g., DP, DQ or DR ⁇ gene).
  • MHC Class II ⁇ chain is intended to include variants with and without the usual glycosylation of the ⁇ i domain. The term is particularly intended to embrace all allelic variants of the Class II ⁇ genes, as well as any equivalents which may be produced synthetically or recombinantly by, for example, site-directed mutagenesis of a naturally occurring variant.
  • MHC binding domain means an MHC Class I binding domain and/or an MHC Class II binding domain.
  • MHC Class I binding domain refers to the region of an MHC Class I molecule which is necessary for binding an antigenic peptide.
  • An MHC Class I binding domain is formed primarily by the ⁇ i and ⁇ domains of the MHC Class I ⁇ chain. Although the ⁇ 3 domain of the ⁇ chain and ⁇ 2 -microglobulin are not essential parts of the binding domain, they are believed to be important in stabilizing the over-all structure of the MHC Class I molecule and, therefore, an MHC Class I binding domain of the present invention preferably includes these regions.
  • An MHC Class I binding domain may also be essentially defined as the extracellular domain of an MHC Class I molecule, distinguishing it from the transmembrane and cytoplasmic domains, although it is likely that some portion of the extracellular domain may be omitted while retaining biological activity.
  • MHC Class II binding domain refers to the region of an MHC Class II molecule which is necessary for binding an antigenic peptide.
  • An MHC Class II binding domain is formed primarily by the ⁇ i and ⁇ i domains of the MHC Class II ⁇ and ⁇ chains and, therefore, an MHC Class II binding domain minimally includes these regions.
  • the ⁇ and ⁇ 2 domains of these proteins are also believed to be important to stabilizing the over-all structure of the MHC binding cleft and, therefore, an MHC Class II binding domain of the present invention preferably includes these regions.
  • An MHC Class II binding domain may also be essentially defined as the extracellular domain of an MHC Class II molecule, distinguishing it from the transmembrane and cytoplasmic domains, although it is likely that some portion of the extracellular domain may be omitted while retaining biological activity.
  • MHC binding peptide or “binding peptide” means an MHC Class I binding peptide and/or an MHC Class II binding peptide.
  • MHC Class I binding peptide means a polypeptide which is capable of selectively binding within the binding cleft formed by a specified MHC Class I molecule to form a Class I MHC/peptide complex.
  • An MHC Class I binding peptide may be a processed self or non-self peptide or may be a synthetic peptide.
  • the binding peptides are typically 8-10 amino acid residues in length, although longer and shorter ones may be effective.
  • MHC Class II binding peptide means a polypeptide which is capable of selectively binding within the binding cleft formed by the ⁇ and ⁇ chains of a specified MHC Class II molecule to form a Class II MHC/peptide complex.
  • An MHC Class II binding peptide may be a processed self or non-self peptide or may be a synthetic peptide.
  • the binding peptides are typically 10-25 amino acids in length, and more typically 13-18 residues in length, although longer and shorter ones may be effective.
  • MHC/peptide complex means a covalently or non-covalently joined ternary complex of either (a) the binding domain of an MHC Class I molecule and an MHC Class I binding peptide which binds to that MHC Class I binding domain or (b) the binding domain of an MHC Class II molecule and an MHC Class II binding peptide which binds to that MHC Class II binding domain.
  • multimeric Major Histocompatibility Complex binding domain conjugate or “multimeric MHC binding domain conjugate” means a conjugate of a multiplicity of MHC binding domains directly or indirectly joined, bound (covalently or noncovalently), attached, adsorbed, or otherwise conjugated to a carrier.
  • the MHC binding domains may be, but need not be, part of the monovalent or multivalent MHC fusion proteins of the invention.
  • carrier means a molecule, particle, composition, or other microscopic object to which may be conjugated, directly or indirectly, a multiplicity of MHC binding domains, so as to form a multimeric MHC binding domain conjugate.
  • the MHC binding domains may be, but need not be, part of the monovalent or multivalent MHC fusion proteins of the invention.
  • the term “dendrimer” refers to a branched polymer in which a multiplicity of core polymer branches extend outwards from a core or initiator molecule, each branch forming additional sub-branches as it extends further outward, thereby forming a structure in which the number of terminal branches exceeds the number of core polymer branches by at least a factor of two.
  • liposome refers to an aqueous compartment enclosed by at least one bilayer of amphipathic molecules (e.g., phospholipids).
  • amphipathic molecules e.g., phospholipids.
  • liposome is intended to embrace unilamellar and multilamellar liposomes.
  • the term “porous” means, with respect to a carrier, that there are a multiplicity of openings in the surface of the carrier which are in fluid communication with each other, and which define passages within said carrier of sufficient diameter to permit diffusion of low molecular weight compounds (e.g., less than 5 kDa) therethrough, but are of insufficient diameter to permit unimpeded movement of higher molecular weight compounds therethrough.
  • the term “flexible molecular linker” or “linker” means a chemical moiety having a length equal to or greater than that of three carbon to carbon bonds and including at least one freely rotating bond along said length.
  • a flexible molecular linker is comprised of one or more amino acid residues but this need not be the case.
  • the flexible molecular linkers of the invention comprise at least three and, more preferably, at least seven amino acid residues.
  • conjugating moiety refers to a chemical moiety which directly or indirectly joins, binds (covalently or noncovalently), attaches, adsorbs, or otherwise conjugates an MHC binding domain, or a fusion protein comprising an MHC binding domain, and a carrier.
  • selective binding means capable of binding in the electro- and stereospecific manner of an antibody to antigen or ligand to receptor.
  • selective binding entails the non-covalent binding of specific side chains of the peptide within the binding pockets present in the MHC binding domain in order to form an MHC/peptide complex (see, e.g., Brown et al., 1993; Stern et al., 1994).
  • substantially pure means, with respect to the MHC binding peptides and various MHC binding domain fusion proteins of the invention, that these peptides or proteins are essentially free of other substances to an extent practical and appropriate for their intended use.
  • the peptides and proteins are sufficiently pure and are sufficiently free from other biological constituents of their hosts cells so as to be useful in, for example, generating antibodies or producing pharmaceutical preparations.
  • a substantially pure preparation of the peptides or proteins of the invention need not be absolutely free of all other proteins or cell components and, for purposes of administration, may be relatively dilute.
  • One of ordinary skill in the art may produce such substantially pure preparations by application or serial application of well-known methods including, but not limited to, HPLC, affinity chromatography or electrophoretic separation.
  • the substantially pure preparations of the invention may also comprise other active ingredients and, therefore, the percentage by weight of the MHC binding peptides and/or various MHC binding domain fusion proteins of the invention may be reduced in such a preparation.
  • articulate describes a structure which extends in three dimensions and defines a minimal surface area and a minimal volume, and which includes at least one surface capable of being conjugated to a multiplicity of MHC binding domains in a substantially two dimensional array.
  • the term “particulate” is intended to embrace carriers which are generally spherical, ellipsoidal, rod-shaped, globular, or polyhedral.
  • minimal surface means, with respect to a carrier, the surface area of the smallest continuous surface which defines a volume which may contain the carrier.
  • minimal volume means the volume contained within a minimal surface.
  • the present invention depends, in part, upon the discovery that fusion proteins, comprising MHC Class II binding domains and coiled-coil and/or immunoglobulin constant domains, may be recombinantly produced, and that these fusion proteins may form heterodimers which include biologically functional MHC Class II binding domains in monovalent or multivalent fusion proteins.
  • heterodimeric MHC Class II binding domains including those of certain MHC Class II molecules which previously could not be produced as empty, soluble, stable heterodimers, may be produced using fusion proteins incorporating dimerization domains, and (2) heterodimeric MHC Class II binding domains, with or without dimerization domains, may be produced in the form of multivalent fusion protein constructs by incorporating them as fusions in multivalent immunoglobulin or ligand/anti-ligand structures.
  • MHC Class II binding domains of these fusion proteins retain their biological activity despite the functional requirement for highly specific tertiary and quaternary structural interactions within and between the ⁇ and ⁇ chains of the MHC molecule, and despite the substitution of relatively large, relatively hydrophilic fusion domains for the natural, hydrophobic transmembrane domains of the MHC Class II proteins.
  • the present invention provides for the production of fusion proteins of MHC Class II ⁇ and ⁇ chain proteins in which substantially all of the C-terminal transmembrane and cytoplasmic domains have been replaced by coiled-coil dimerization domains and, optionally, interposing linker sequences.
  • Figure 1 schematically illustrates such a monovalent MHC Class II binding domain fusion protein. At least the peptide binding domain, and preferably the entire extracellular domain, of an MHC Class II ⁇ chain 10 may be fused to a first dimerization domain 30 (e.g., a leucine zipper domain or an immunoglobulin Fab constant domain).
  • a first dimerization domain 30 e.g., a leucine zipper domain or an immunoglobulin Fab constant domain.
  • an MHC Class II ⁇ chain 20 may be fused to a second dimerization domain 40 (e.g., a leucine zipper domain or an immunoglobulin Fab constant domain).
  • a second dimerization domain 40 e.g., a leucine zipper domain or an immunoglobulin Fab constant domain.
  • the dimerization domains (30 and 40) associate in solution to promote formation of a heterodimeric fusion protein in which the MHC Class II ⁇ and ⁇ chain components (10 and 20) stably associate to form a biologically active MHC Class II binding domain which is capable of binding, or being "loaded” with, an MHC binding peptide 110 so as to form a stable MHC/peptide complex which can selectively bind to cognate T cell receptors and/or selectively activate T cell clones bearing cognate TCRs.
  • flexible molecular linkers may be interposed between the MHC components (10 and 20) and dimerization domains (30 and 40) so as to better approximate the normal distance between the MHC components and their natural MHC transmembrane domains, and/or to provide for free rotation between the MHC components and the dimerization domains such that the geometry of the association between dimerization domains does not constrain or interfere with the geometry of association of the MHC binding domains.
  • the present invention provides for the production of divalent fusion proteins of MHC Class II ⁇ and ⁇ chain proteins in which substantially all of the C-terminal transmembrane and cytoplasmic domains have been replaced by immunoglobulin constant chain domains and, optionally, interposing linker sequences and/or coiled-coil dimerization domains.
  • the immunoglobulin constant domains are chosen so as to promote the formation of divalent antibody-like molecules bearing two MHC Class II binding domains and, optionally, to promote certain effector functions (e.g., complement activation, cell binding).
  • Figure 2 schematically illustrates such a divalent MHC Class II binding domain fusion protein.
  • At least the peptide binding domain, and preferably the entire extracellular domain, of an MHC Class II ⁇ chain 10 may be fused to a first dimerization domain 30 (e.g., a leucine zipper domain or an immunoglobulin Fab constant domain).
  • a first dimerization domain 30 e.g., a leucine zipper domain or an immunoglobulin Fab constant domain
  • a second dimerization domain 40 e.g., a leucine zipper domain or an immunoglobulin Fab constant domain.
  • the dimerization domains (30 and 40) associate in solution to promote formation of a heterodimeric fusion protein in which the MHC Class II ⁇ and ⁇ components (10 and 20) stably associate to form a biologically active MHC Class II binding domain which is capable of binding, or being "loaded” with, an MHC binding peptide 110 so as to form a stable MHC/peptide complex which can selectively bind to cognate T cell receptors and/or selectively activate T cell clones bearing cognate TCRs.
  • some MHC Class II molecules e.g., HLA-DRl, HLA-DR4
  • HLA-DRl HLA-DR4
  • one of the two MHC fusion proteins further comprises an immunoglobulin Fc region 60, with or without an interposing immunoglobulin hinge region 50 appropriate to the Fc region (IgA, IgD and IgG molecules include hinge regions; IgE and IgM molecules do not).
  • the MHC Class II ⁇ chain fusion protein which is fused to the immunoglobulin heavy chain Fc region because the MHC Class II ⁇ chains are less variable than the ⁇ chains and, therefore, such an ⁇ chain fusion protein can be used with a number of different MHC Class II ⁇ chain fusion proteins to form a variety of different divalent molecules with different HLA specificity. It should, however, be noted that there is no reason that the ⁇ chain construct can not include the immunoglobulin Fc regions.
  • flexible molecular linkers may be optionally interposed between the MHC components (10 and 20), dimerization domains (30 and 40), and/or immunoglobulin components (50 and/or 60) so as to better approximate the normal distance between the MHC components and their natural MHC transmembrane domains, and/or to provide for free rotation between the MHC components, the dimerization domains, and/or the immunoglobulin domains such that the geometry of the association between any pair of dimerizing components does not constrain or interfere with the geometry of association or dimerization of the others.
  • the immunoglobulin heavy chain Fc regions 60 and 60' of two such MHC Class II fusion proteins associate and form a divalent structure, with one or more disulfide linkages between chains, analogous to the structure of natural antibodies.
  • the present invention provides for the production of decavalent fusion proteins of MHC Class II ⁇ and ⁇ chain proteins in which substantially all of the C-terminal transmembrane and cytoplasmic domains have been replaced by IgM immunoglobulin constant chain domains and, optionally, interposing linker sequences and/or coiled-coil dimerization domains.
  • immunoglobulin constant domains are specifically chosen to be IgM chains, which form divalent subunits which are then assembled into decavalent pentamers, and (2) that the cells producing these MHC-IgM fusions are cotransfected with a J-chain gene in order to facilitate the assembly and secretion of IgM molecules (Matsuuchi et al., 1986).
  • Figure 3 schematically illustrates such a decavalent MHC Class II fusion protein.
  • the peptide binding domains, and preferably the entire extracellular domains, of MHC Class II ⁇ 10 and ⁇ 20 chains may be fused to dimerization domains 30 and 40 (e.g., a leucine zipper domain or an immunoglobulin Fab constant domain).
  • the dimerization domains (30 and 40) associate in solution to promote formation of a heterodimeric fusion protein in which the MHC Class II ⁇ and ⁇ components (10 and 20) stably associate to form a biologically active MHC Class II binding domain which is capable of binding, or being "loaded” with, an MHC binding peptide 110.
  • MHC Class II molecules e.g., HLA-DRl, HLA-DR4
  • HLA-DRl HLA-DR4
  • coiled-coil dimerization domains may be omitted entirely or may be replaced by Fab constant domains (i.e., heavy chain C H 1 domains or light chain C L domains).
  • Fab constant domains i.e., heavy chain C H 1 domains or light chain C L domains.
  • either the ⁇ or ⁇ chain construct further comprises an immunoglobulin Fc region 60 which, in these embodiments, is an IgM Fc region (C H 2, C H 3, C H 4).
  • flexible molecular linkers may be optionally interposed between the MHC components (10 and 20), dimerization domains (30 and 40), and/or IgM Fc components (60) so as to better approximate the normal distance between the MHC components and their natural MHC transmembrane domains, and/or to provide for free rotation between the MHC components, the dimerization domains, and/or the immunoglobulin domains.
  • the immunoglobulin heavy chain Fc regions 60 and 60' of two such MHC- IgM fusion proteins are associated to form a divalent structure with one or more disulfide linkages between chains.
  • IgM subunits will further associate to form a multimer with one or more disulfide bonds 90 between divalent subunits.
  • J- chain protein 100 IgM subunits are assembled into decavalent pentamers as shown in Figure 3, analogous to naturally occurring IgM pentamers.
  • the present invention provides for the production of tetravalent fusion proteins of MHC Class II ⁇ and ⁇ chain binding domains in which substantially all of the C-terminal transmembrane and cytoplasmic domains have been replaced by dimerization domains and, optionally, interposing linker sequences, and in which a C-terminal ligand "tag" sequence allows a multiplicity of MHC -tag fusions to bind to an anti-ligand and form a multivalent MHC binding domain fusion protein complex.
  • the ligand tag sequence may be any sequence for which an anti-ligand is available, or any sequence which facilitates the addition of a ligand to the tag.
  • the tag sequence may be a poly-His sequence, which may serve as a ligand for a Ni + -bearing anti-ligand.
  • the tag sequence may be the epitope of an antibody, and the anti-ligand may be that antibody.
  • the tag is a recognition sequence which may be biotinylated by biotin ligase, and the anti-ligand may be avidin or streptavidin.
  • Figure 4 schematically illustrates a tetravalent MHC binding domain fusion protein complex in which a biotinylated tag serves as the ligand, and avidin or streptavidin serves as the anti-ligand.
  • At least the peptide binding domain, and preferably the entire extracellular domain, of MHC Class II ⁇ chains 10 and ⁇ chains 20 may be fused to dimerization domains (30 and 40) (e.g., a leucine zipper domain or an immunoglobulin Fab constant domain).
  • the dimerization domains (30 and 40) associate in solution to promote formation of a heterodimeric fusion protein in which the MHC Class II ⁇ and ⁇ components (10 and 20) stably associate to form a biologically active MHC Class II binding domain which is capable of binding, or being "loaded” with, an MHC binding peptide 110.
  • a biotin ligase recognition sequence or "tag” is fused to the C-terminus of at least one of the MHC binding domain fusion chains.
  • This sequence tag may be biotinylated by enzymes within the cells which produce these MHC binding domain fusion proteins, or may be subsequently biotinylated in vitro.
  • the biotinylated tag 70 can be used to cause the monovalent MHC binding domain fusion proteins to bind to avidin (or streptavidin) 80.
  • an MHC-biotin/(strept)avidin fusion protein complex can be produced which is tetravalent (with lower valencies at lower molar ratios of biotin:(strept)avidin).
  • flexible molecular linkers may optionally be interposed between the MHC components (10 and 20), the dimerization domains (30 and 40) and/or the biotin sequence tag so as to better approximate the normal distance between the MHC components and their natural MHC transmembrane domains, and/or to provide for free rotation between the MHC components, the dimerization domains, and/or biotinylated tag.
  • the MHC binding peptide 110 may be covalently joined to either the MHC Class II ⁇ or ⁇ components (10 and 20) with a flexible molecular linker (not shown in the Figures).
  • a flexible molecular linker are polypeptide sequences of 10-20 amino acid residues, more preferably 12-18 amino acid residues.
  • the MHC binding peptide, linker and MHC Class II ⁇ or ⁇ components may all be expressed as a single fusion protein, further comprising dimerization domains toward the C-terminus.
  • the present invention provides (a) isolated nucleic acid sequences encoding such fusion proteins; (b) vectors for transiently or stably transfecting host cells with these nucleic acids; (c) host cells transformed with these sequences or vectors; (d) methods for producing the fusion proteins employing these sequences, vectors and host cells; and (e) the substantially purified fusion proteins themselves.
  • the present invention provides for a number of utilities for these products and processes including, but not limited to, the treatment of allergic and autoimmune diseases, the detection and/or isolation of T cells with defined MHC/peptide specificities, and the selective activation, anergization, or killing of T cells with defined MHC/peptide specificities.
  • MHC Class II proteins are preferably human HLA Class II proteins.
  • the present invention may be practiced with either of the known HLA-DRA alleles (DRA*0101 and DRA*0102), any of the approximately 160 known HLA-DRB alleles (including at least 137 known HLA-DRBl alleles), any of the approximately 15 known HLA-DQ Al alleles, any of the approximately 25 known HLA-DQB1 alleles, any of the approximately 8 known HLA-DPA1 alleles, or any of the approximately 65 known HLA-DPBl alleles.
  • the known HLA-DRA alleles DRA*0101 and DRA*0102
  • any of the approximately 160 known HLA-DRB alleles including at least 137 known HLA-DRBl alleles
  • any of the approximately 15 known HLA-DQ Al alleles any of the approximately 25 known HLA-DQB1 alleles
  • any of the approximately 8 known HLA-DPA1 alleles any of the approximately 65 known HLA-DPBl alleles.
  • Embodiments employing coiled-coil dimerization domains are particularly preferred for use with those MHC Class II binding domains which, without their transmembrane and cytoplasmic domains, do not form stable heterodimers in solution.
  • the coiled- coil domains add stability to the heterodimer while allowing for the production of soluble, non- aggregated proteins.
  • HLA-DR2 serotypes e.g., those encoded by DRA and DRB1 *15 or DRB1 *16 alleles
  • HLA-DQ8 encoded by, for example, the DQA1*0301 and DQB1 *0302 alleles
  • HLA-DQ2 encoded by, for example, DQA1*0501 and DQB 1 *0201 alleles
  • coiled-coil dimerization domains may be employed with any of the human MHC Class II binding domains, including those which have previously been successfully expressed as stable, soluble heterodimers without their transmembrane domains (e.g., DR1 and DR4).
  • splice points for the MHC components of the MHC Class II binding domain fusion proteins must be chosen so as to include sufficient N- terminal sequence for proper formation of an MHC binding domain while excluding most if not all of the C-terminal transmembrane and cytoplasmic domains of the MHC chains.
  • the MHC Class II ⁇ and ⁇ chains are each characterized by two N-terminal, globular, extracellular domains ( ⁇ l and ⁇ 2, or ⁇ l and ⁇ 2), followed by a short loop or connecting peptide, a hydrophobic transmembrane domain, and a C-terminal hydrophilic cytoplasmic domain.
  • the binding cleft of the MHC Class II molecules is formed primarily by the interaction of the ⁇ l and ⁇ 1 domains in the heterodimer and, therefore, these domains must minimally be included in the fusion proteins of the present invention.
  • the ⁇ 2 and ⁇ 2 domains are also preferably included because they may aid in stabilizing the MHC binding domain, are believed to be involved in the formation of dimers of the MHC chains, and are believed to be involved in CD4 receptor binding.
  • the splice points for the MHC Class II fusion peptides are chosen to be in the regions approximately between the ends of the ⁇ 2 or ⁇ 2 domains and the beginnings of the transmembrane domains.
  • the transmembrane domains essentially begin after the Glu residue at position 191 or the Asn residue at position 192.
  • the transmembrane domains essentially begin after the Lys residue at position 198 or the Met residue at position 199.
  • the HLA-DQ Al transmembrane domains essentially begin after the Glu residue at position 195 or the Thr residue at position 196
  • the HLA-DQBl transmembrane domains essentially begin after the Lys residue at position 200 or the Met residue at position 201.
  • allelic variants there may be amino acid insertions or deletions prior to these sites which alter the residue numbering.
  • the connecting peptide and transmembrane regions of the MHC Class II ⁇ and ⁇ chains are not, however, highly polymorphic and, indeed, appear invariant for all known DRA and DRB alleles (see Marsh and Bodmer, 1995). Therefore, working with any given MHC Class II ⁇ and ⁇ chains, one of ordinary skill in the art can easily identify the transmembrane domains both by homology to the above-described alleles, and by their essential hydrophobic nature.
  • the fusion proteins of the present invention include several residues from the transmembrane domain or that several residues of the ⁇ 2 or ⁇ 2 domains be omitted.
  • the inclusion of 1-5 residues of the transmembrane domain may be included in the present invention and still yield a soluble fusion protein, but this is not preferred.
  • the omission of, for example, 1-5 residues from the ⁇ 2 or ⁇ 2 domains may not result in structural alterations which disrupt MHC peptide binding, heterodimer formation, or T cell interactions.
  • ⁇ 2 and ⁇ 2 structural domains may be omitted in accordance with the present invention (e.g., replacing portions of the Class II ⁇ or ⁇ 2 domains with equivalent portions of the Class I ⁇ 3 or ⁇ -microglobulin proteins).
  • the splice points are chosen to be within the loop or connecting peptide sequence near the N-terminal end of the transmembrane domain.
  • the entire extracellular domains of the MHC Class II ⁇ and ⁇ chains are included in the MHC fusion proteins of the invention. 3. MHC Class II Binding Peptide Fusions
  • the MHC binding peptide is joined to the N-terminus of the ⁇ chain because, when the ⁇ and ⁇ chains associate to form a heterodimeric MHC molecule, the N-terminus of the ⁇ chain is more accessible than the N- terminus of the ⁇ chain.
  • the ⁇ chains of MHC Class II molecules are more polymorphic than the ⁇ chains and, therefore, the specificity of an MHC binding domain is more dependent upon which ⁇ chain is included in the molecule.
  • the MHC binding peptide is preferably linked to the MHC binding domain using a flexible molecular linker, as described below.
  • the flexible molecular linker is a polypeptide sequence of approximately 10-20 amino acid residues, more preferably 12-18 amino acid residues, which joins the binding peptide and MHC binding domains by standard polypeptide linkages to form a larger fusion protein which may be encoded by a single nucleic acid construct and expressed as a single fusion protein.
  • amino acids be chosen from the relatively small residues (e.g., alanine, glycine, serine, leucine, isoleucine, valine) in order to minimize the potential for steric hindrance.
  • the MHC binding peptide is chosen such that it is capable of selectively binding to the MHC molecule to which it is attached.
  • Thousands of combinations of MHC binding peptides and MHC molecules are known in the art and can be identified by standard methods (see, e.g., Chicz et al., 1993). Of particular interest are those pairs of MHC binding peptides and MHC molecules which are implicated in diseases, including infections and autoimmune diseases.
  • MHC binding peptides derived from the human myelin basic protein e.g., residues 85-99, 84-102 and 148-162
  • particular MHC alleles e.g., HLA-DR2 or DRA DRB 1*1501
  • HLA-DR2 or DRA DRB 1*1501 have been implicated in the development of multiple sclerosis.
  • monovalent or multivalent MHC Class II binding domain fusion proteins are produced in which an immunogenic myelin basic protein (MBP) peptide is covalently joined by a polypeptide linker sequence to the N-terminus of the peptide binding domain of an HLA-DRB1*1501 protein, and this fusion is covalently joined, with or without an interposing flexible molecular linker, to a dimerization domain.
  • MBP myelin basic protein
  • Such a fusion protein may then be dimerized with a corresponding HLA-DRA ⁇ chain fusion protein such that the MBP peptide binds in the cleft formed by association of the MHC Class II ⁇ and ⁇ chain binding domains.
  • residues of the human desmoglein 3 protein e.g., residues 78-93, 97-111, 190-204, 206-220, 251-265, 512-526 and 762-786
  • certain MHC alleles e.g., HLA- DR4 or DRA DRB 1 *0402, and HLA-DQ 1 or DQA/DQB 1 *05032
  • HLA- DR4 or DRA DRB 1 *0402 have been implicated in the development of pemphigus vulgaris (see, e.g., WO 96/27387).
  • immunodominant self peptides may be identified which selectively bind to particular MHC Class II molecules.
  • monovalent or multivalent MHC Class II binding domain fusion proteins may be produced, having the autoimmunogenic MHC binding peptides covalently joined to the MHC binding domains, and these may be used, as further described below, in identifying, sorting, selecting or targeting autoreactive T cells, or in tolerizing or anergizing the immune response to the autoantigens.
  • MHC binding domain fusion proteins may be produced which optionally include flexible molecular linkers which covalently join, as described above, (1) MHC binding domains to dimerization domains; (2) dimerization domains to immunoglobulin Fc domains or ligand tag domains; or (3) MHC binding peptides to MHC binding domains.
  • Appropriate linkers include, but are not limited to, short polypeptide chains which can be encoded with the MHC domains, dimerization domains, immunoglobulin domains, and/or tag domains in recombinant DNA constructs.
  • appropriate linkers include any relatively small (e.g., ⁇ 2 kDa, preferably ⁇ 1 kDa) organic chemical moieties which are flexible because they include at least one single bond located between their termini and about which there is free rotation.
  • bifunctional molecules e.g., an ⁇ , ⁇ - dicarboxylic acid or an ⁇ , ⁇ -diamine
  • a lower alkyl chain may be employed, and such flexible molecular linkers may be reacted with the C-termini of the MHC components and the N-termini of the coiled-coil, immunoglobulin or ligand tag components (or with reactive groups of the amino acid side chains of any of these).
  • Many other cross-linking agents are well known in the art and may be employed as substantial equivalents.
  • the flexible molecular linkers of the present invention comprise a series of amino acid residues which can be encoded in a fusion gene construct.
  • a linker of 1-15 generally small amino acid residues e.g., alanine, glycine, serine, leucine, isoleucine, valine
  • one or more hydrophilic residues e.g., aspartate, glutamate, lysine
  • the length of the linker may be chosen so as to maintain, approximately, the spacing naturally found between the MHC binding domains and the transmembrane domains of the MHC proteins and, therefore, the length of the linker may depend upon whether some or all of the naturally occurring loop or connecting residues between the binding domains and transmembrane domains have been included or omitted.
  • linker sequences may be particularly chosen so as to introduce specific proteinase cleavage sites in the fusion protein or, for ease of recombinant DNA manipulations, to introduce specific restriction endonuclease sites into the recombinant construct.
  • the included portion of the loop or connecting peptide may be varied, the linker length may be varied, or the loop peptide and/or linker may be omitted entirely.
  • site-directed mutagenesis, or restriction and ligation of recombinant constructs with a variety of different endonucleases one of ordinary skill in the art can easily produce many variations on the fusion protein constructs and, as described below, rapidly test them for cognate TCR binding and/or T cell activation.
  • some presently preferred embodiments employ the entire extracellular domains of the MHC molecules joined by particular linkers to dimerization, immunoglobulin or ligand tag domains, the invention is not limited to such embodiments. 5.
  • Coiled-coils are common structural features of dimeric proteins in which two ⁇ -helical polypeptides ("coils") are twisted (“coiled") about each other to form a larger quaternary structure or "coiled-coil” (see, e.g., Hu et al., 1990; Oas and Endow, 1994). Indeed, the transmembrane regions of HLA-DR ⁇ and ⁇ chains are thought to be ⁇ helices that assemble as a coiled-coil within the hydrophobic environment of the cell membrane (Cosson and Bonifacino, 1992). Other coiled-coils, however, are hydrophilic and may be found in secreted, cytosolic and nuclear proteins.
  • leucine zippers are coiled-coil domains which are present in a large number of DNA binding proteins and which may mediate either homodimer or heterodimer formation (see, e.g., Ferre-D'Amare et al., 1993; O'Shea et al, 1989; O'Shea et al., 1991).
  • artificial coiled-coil domains including pairs of basic and acidic amphipathic helices and artificial leucine zippers, which have been expressed and assembled in recombinant homodimeric and heterodimeric proteins (see, e.g., Pack and Pluckthun, 1992; Chang et al, 1994).
  • the dimerization domains are leucine zipper domains.
  • These leucine zippers are characterized by at least 4 and, preferably, at least 5-7 leucine residues that are spaced periodically at approximately every seventh residue (heptad repeat), with each heptad repeat contributing two turns of the ⁇ -helix (3.5 residues/turn).
  • the leucine residues appear to have a special function in coiled-coil dimerization, and form part of the hydrophobic interface between the two ⁇ -helices in the coiled-coil.
  • the 40 amino acid leucine zipper domains of the proteins Fos and Jun are preferred examples of leucine zipper dimerization domains.
  • MHC Class II molecules e.g., HLA-DRl, HLA-DR4
  • HLA-DRl HLA-DR4
  • a coiled-coil dimerization domain may not be necessary.
  • such domains may be omitted entirely or, alternatively, other domains which promote dimerization may be substituted.
  • the constant domains of the Fab fragments of immunoglobulins i.e., the C H I and C L domains
  • the constant domains of the Fab fragments of immunoglobulins i.e., the C H I and C L domains
  • the light chain and the portion of the heavy chain associated with it, corresponding to elements 10, 20, 30 and 40, are referred to as the Fab fragment.
  • the portions of the two heavy chains which are closely associated with each other, 60 and 60', are referred to as the Fc fragment.
  • immunoglobulins i.e., IgA IgD, and IgG
  • the immunoglobulin light chains include a variable domain V L , corresponding essentially to element 20 of Figure 2 (but not to scale), and a constant domain C L , corresponding essentially to element 40 (but not to scale).
  • each heavy chain includes an N-terminal variable domain VH, corresponding essentially to element 10 (but not to scale), and three or four constant domains C H I through C H 4, corresponding essentially to elements 30, 50 and 60 (but not to scale).
  • VH N-terminal variable domain
  • C H I through C H 4 constant domains
  • C H I through C H 4 corresponding essentially to elements 30, 50 and 60 (but not to scale).
  • the immunoglobulin constant domains are relatively invariant in the human population.
  • light chains are broadly classified as either K or ⁇ and, in humans, ⁇ chains are further divided into four subtypes.
  • immunoglobulin heavy chains are the basis of the division of these molecules into five broad classes ( ⁇ , ⁇ , ⁇ , ⁇ and ⁇ chains in IgA, IgD, IgE, IgG and IgM, respectively). Based on minor differences in amino acid sequences in humans, the ⁇ chains have been further subdivided into four subclasses, and the ⁇ chains into two. The amino acid sequences of these various immunoglobulin light and heavy chain constant domains have long been known in the art (see, e.g., Kabat et al., 1979) and will not be reproduced here.
  • the MHC-immunoglobulin fusion proteins of the present invention include the Fc regions of either IgG (subtypes 1, 2 or 3) or IgM because these Fc domains are capable of activating the classical complement pathway and, therefore, are more useful in some of the therapeutic methods described below.
  • IgG subtypes 1, 2 or 3
  • IgM immunoglobulin isotypes
  • the IgM isotypes are preferred in some embodiments because they can form pentamers of divalent MHC-IgM fusion proteins. 7.
  • MHC binding domain fusion proteins of the present invention may be expressed in any standard protein expression system which allows for proper folding and secretion of the desired molecules, or which allows for their recovery as properly folded molecules from inclusion bodies.
  • eukaryotic expression systems are preferred because they are most likely to produce a high yield of properly folded, glycosylated and disulfide-linked molecules.
  • Mammalian cell lines especially those which are well characterized for protein expression (e.g., CHO cells, COS cells) or those which are known to secrete properly folded, glycosylated and disulfide linked immunoglobulins (e.g., any mAb producing cell line), may be preferred for some uses.
  • the expression vector will include a strong promoter to drive expression of the recombinant constructs and, optionally, a number of marker genes which will simplify the identification and/or selection of transformants.
  • the present invention depends, in part, upon the discovery that multimeric MHC binding domain conjugates comprising a multiplicity of MHC binding domains conjugated to a carrier may be produced, and that these multimeric conjugates have far greater avidity for their cognate TCRs, and far greater biological activity, than monovalent MHC binding domains, or even divalent or tetravalent MHC binding domain constructs.
  • a great increase in avidity of T cell binding and/or activation may be achieved by providing a multiplicity of MHC binding domains on a single carrier such that a substantially two-dimensional array of MHC binding domains may make contact with an area of a T cell membrane bearing a multiplicity of T cell receptors.
  • the present invention provides for the production of multimeric MHC binding domain conjugates in which about 5-500 MHC binding domains, preferably about 10-200 MHC binding domains, and more preferably about 20-100 MHC binding domains, are conjugated to a single carrier.
  • the carrier can be characterized as defining a minimal surface area and, preferably, the average density of the MHC binding domains on that surface is between about 4 x 10 '3 to 20 MHC binding domains/nm 2 ; more preferably about 4 x 10 "2 to 20 MHC binding domains/nm 2 , and most preferably about 0.4 to 20 MHC binding domains/nm 2 .
  • the size and weight of the multimeric MHC binding conjugates are limited to aid in maintaining solubility, and to avoid possible complications caused by aggregation in vivo.
  • the largest cross-sectional diameters of the MHC binding domain conjugates of the invention are less than about 1,000 nm, preferably less than about 500 nm, and more preferably less than about 100 nm.
  • such conjugates would define a minimal surface area of less than approximately 3.1 x 10 6 nm 2 , 7.9 x 10 5 nm 2 , and 3.1 x 10 4 nm 2 , respectively, and would define a minimal volume of 5.2 x 10 8 nm 3 , 6.5 x 10 7 nm 3 , and 5.2 x 10 5 nm 3 .
  • the MHC binding domain conjugates have maximum diameters of about 5-40 nm.
  • the overall weights of the MHC binding domain conjugates are less than about 10,000 kDa, preferably less than about 5,000 kDa, and more preferably less than about 1,000 kDa. In the most preferred embodiments, as described below, the MHC binding domain conjugates have weights of about 200-500 kDa.
  • Figure 5 schematically illustrates one embodiment of a multimeric MHC binding domain conjugate comprising a multiplicity of MHC binding domains conjugated to a carrier.
  • the conjugate comprises at least the binding domains, and preferably the entire extracellular domains, of a multiplicity of MHC ⁇ chains 10 which are stably associated with at least the binding domains, and preferably the entire extracellular domains, of MHC ⁇ chains 20 to form biologically active MHC binding domains which are capable of binding, or being "loaded” with, MHC binding peptides 110.
  • the MHC ⁇ chain 10 is shown as being conjugated to a carrier 300 by a conjugating moiety 200.
  • the MHC ⁇ chain 20 may be conjugated to the carrier 300 by a conjugating moiety 200.
  • the conjugating moiety 200 may be omitted and one of the MHC chains (10 or 20) may be directly conjugated to the carrier 300.
  • the MHC binding domains are conjugated to the carrier in an orientation which allows interaction of the MHC/peptide complexes with the TCRs on cognate T cells.
  • the carrier 300 is depicted as a substantially spherical particle, but this need not be the case.
  • Figure 6 schematically illustrates another embodiment of a multimeric MHC binding domain conjugate of the invention.
  • the conjugate comprises a multiplicity of MHC binding domain fusion proteins, such as those described above.
  • At least the peptide binding domains of a multiplicity of MHC ⁇ chains 10 have been joined to first dimerization domains 30, at least the peptide binding domains of a multiplicity of MHC ⁇ chains 20 have been joined to second dimerization domains 40, and these fusion proteins have assembled to form heterodimeric MHC binding domains which may bind MHC binding peptides 110.
  • Flexible molecular linkers optionally may be interposed between the MHC domains (10, 20) and the dimerization domains (30, 40).
  • the first dimerization domains 30 are shown as being conjugated to a carrier 300 by a conjugating moiety 200.
  • the second dimerization domains 40 may be conjugated to the carrier 300 by a conjugating moiety 200.
  • the conjugating moiety 200 may be omitted and one of the dimerization domains (30 or 40) may be directly conjugated to the carrier 300.
  • the MHC binding domain fusion proteins are conjugated to the carrier in an orientation which allows interaction of the MHC/peptide complexes with the TCRs on cognate T cells.
  • the carrier 300 is depicted as a substantially spherical branched polymer or dendrimer, but this need not be the case.
  • Figure 7 schematically illustrates another embodiment of a multimeric MHC binding domain conjugate of the invention.
  • the conjugate comprises a multiplicity of MHC binding domain fusion proteins, such as those described above, and the numbered elements 10, 20, 30, 40 and 110 are as described in Figure 6.
  • the conjugating moiety 200 of Figure 6 has been replaced by two elements, 70 and 80.
  • a first conjugating moiety 70 is bound, covalently or non-covalently, to the second dimerization domain 40
  • the second conjugating moiety 80 is bound, covalently or non-covalently, to the carrier 300.
  • the first conjugating moiety 70 may be a biotin-tag, as described above, and the second conjugating moiety 80 may be avidin or streptavidin.
  • the conjugating moieties 70 and 80 may be any pair of molecules which are capable of binding to each other, covalently or non-covalently, so as to conjugate the MHC binding domains to the carrier.
  • the various elements depicted in Figures 1-7 may be interchanged or mixed.
  • the monovalent MHC binding domain fusion protein of Figure 1 may be conjugated by conjugating moieties as in Figures 5 or 6, or by first and second conjugating moieties as in Figure 7, to a particulate carrier as in Figures 5 or 7, or to a branched polymer or dendrimer carrier as in Figure 6.
  • conjugating moieties as in Figures 5 or 6 may be conjugated by conjugating moieties as in Figures 5 or 6, or by first and second conjugating moieties as in Figure 7, to a particulate carrier as in Figures 5 or 7, or to a branched polymer or dendrimer carrier as in Figure 6.
  • other embodiments not depicted in the figures may be employed.
  • the multimeric MHC binding domain conjugates of the present invention may employ either Class I or Class II MHC binding domains.
  • Class I and MHC Class II proteins are preferably human MHC proteins.
  • any of the MHC binding domains which were described above for use in MHC binding domain fusion proteins may also be used in the production of multimeric MHC binding domain conjugates.
  • the same MHC splice points, described above, may be employed to obtain the complete extracellular portion of MHC Class II ⁇ and ⁇ chains, or just the minimal portions of those chains which include the MHC binding domain.
  • any of the above-described monovalent or multivalent MHC binding domain fusion proteins may be employed as the MHC component in a multimeric MHC binding domain conjugate.
  • divalent MHC binding domain fusion proteins comprising at least the MHC binding domain of an MHC Class II molecule joined to immunoglobulin domains (with or without intervening coiled-coil dimerization domains or interposing flexible molecular linkers) may be conjugated to a carrier to produce a multimeric MHC binding domain conjugate of the invention.
  • the tetravalent and decavalent MHC binding domain fusion protein constructs described above may be conjugated to a carrier to produce a multimeric MHC binding domain conjugate.
  • the monovalent MHC binding domain fusion proteins, employing coiled-coil or other dimerization domains as described above may be conjugated to a carrier to produce a multimeric MHC binding domain conjugate.
  • multimeric MHC binding domain conjugates may be produced using MHC Class II binding domains which are free of exogenous coiled-coil or dimerization domains.
  • the extracellular or peptide binding domains of those MHC Class II molecules which are stable under physiological conditions without exogenous dimerization domains such as those which may be produced by the methods of Stern and Wiley (1992)
  • the MHC binding domains of the Class II HLA-DRl and HLA-DR4 molecules may, for example, be produced in truncated form, and may be conjugated to a carrier without dimerization domains to aid in the stabilization of the heterodimer.
  • the considerations in the choice of splice points for such truncated MHC Class II proteins are the same as those described above for the MHC binding domain fusion proteins and will not be repeated here.
  • multimeric MHC binding domain conjugates may be produced in which the MHC binding domains are derived from MHC Class I as well as MHC Class II proteins.
  • Class I MHC binding domains do not require stabilization by dimerization domains because the light chains of these molecules (i.e., ⁇ 2 -microglobulin) lack a transmembrane domain in nature, but are nonetheless able to stably associate with Class I ⁇ chains under physiological conditions. Therefore, MHC binding domain conjugates may be produced employing ⁇ 2- microglobulin in association with at least the peptide binding domain of an MHC Class I ⁇ chain conjugated to a carrier.
  • the choice of splice points for MHC Class I ⁇ chains is within the ability of one of ordinary skill in the art. Specifically, however, the splice point is preferably chosen between residues at the C-terminus of the ⁇ 3 domain and residues at the N-terminus of the transmembrane domain (e.g., between about residues 273-283 of the mature HLA-A2 protein, preferably after the Pro residue at position 283 or the He residue at position 284). Preferably, the entire extracellular domain of an MHC Class I ⁇ chain is employed.
  • the MHC binding domain conjugates of the present invention may be produced with any of a large variety of carriers including, but not limited to, particles, beads, branched polymers, dendrimers, or liposomes.
  • the carriers must be capable of being conjugated, either directly or indirectly, to a multiplicity of MHC binding domains and, therefore, preferably comprise a multiplicity of reactive groups near the surface which can be used in conjugation reactions.
  • the carrier may have a surface to which conjugating moieties may be adsorbed without chemical bond formation.
  • the carrier is particulate, and generally spherical, ellipsoidal, rod-shaped, globular, or polyhedral in shape. Alternatively, however, the carrier may be of an irregular or branched shape.
  • the carrier is composed of material which is biodegradable and non-immunogenic. It is further preferred that the carrier have a net neutral or negative charge, in order to reduce non-specific binding to cell surfaces which, in general, bear a net negative charge.
  • the overall size and weight of the carriers are important considerations.
  • the carriers are microscopic or nanoscopic in size, both to enhance solubility, and to avoid possible complications caused by aggregation in vivo.
  • the largest cross-sectional diameters of the carriers of the invention are less than about 1,000 nm, preferably less than about 500 nm, and more preferably less than about 100 nm.
  • carriers have maximum diameters of about 5-40 nm.
  • the overall weights of the carriers are less than about 10,000 kDa, preferably less than about 5,000 kDa, and more preferably less than about 1,000 kDa.
  • the carriers have weights of about 200-500 kDa.
  • the present invention provides for the production of multimeric MHC binding domain conjugates in which a multiplicity of MHC binding domains are conjugated to a substantially spherical microbead or nanobead.
  • the beads may be solid, hollow, or porous.
  • a marker e.g., a fluorescent agent
  • therapeutic agent e.g., a cytotoxin or lymphokine
  • Carrier beads can be formed from a wide range of materials.
  • beads may be composed of glass, silica, polyesters of hydroxy carboxylic acids, polyanhydrides of dicarboxylic acids, or copolymers of hydroxy carboxylic acids and dicarboxylic acids.
  • the carrier beads may be composed of polyesters of straight chain or branched, substituted or unsubstituted, saturated or unsaturated, linear or cross-linked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy hydroxy acids, or polyanhydrides of straight chain or branched, substituted or unsubstituted, saturated or unsaturated, linear or cross-linked, alkanyl, haloalkyl, thioalkyl, aminoalkyl, aryl, aralkyl, alkenyl, aralkenyl, heteroaryl, or alkoxy dicarboxylic acids.
  • Carrier beads including mixtures of ester and anhydride bonds may also be employed.
  • carrier beads may comprise materials including polyglycolic acid polymers (PGA), polylactic acid polymers (PLA), polysebacic acid polymers (PSA), poly(lactic-co-glycolic) acid copolymers (PLGA), poly(lactic-co-sebacic) acid copolymers (PLSA), poly(glycolic-co-sebacic) acid copolymers (PGSA), etc.
  • biocompatible, biodegradable polymers useful in the present invention include polymers or copolymers of caprolactones, carbonates, amides, amino acids, orthoesters, acetals, cyanoacrylates and degradable urethanes, as well as copolymers of these with straight chain or branched, substituted or unsubstituted, alkanyl, haloalkyl, thioalkyl, aminoalkyl, alkenyl, or aromatic hydroxy- or di-carboxylic acids.
  • the biologically important amino acids with reactive side chain groups such as lysine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine and cysteine, or their enantiomers, may be included in copolymers with any of the aforementioned materials to provide reactive groups for conjugating to MHC binding domains or conjugating moieties.
  • preferred biodegradable materials include PLA, PG and PLGA polymers. See, generally, U.S. Pat. Nos. 1,995,970; 2,703,316; 2,758,987; 2,951,828; 2,676,945; 2,683,136 and 3,531,561.
  • Biocompatible but non-biodegradable materials may also be used in the carrier beads of the invention.
  • non-biodegradable polymers of acrylates, ethylene- vinyl acetates, acyl substituted cellulose acetates, non-degradable urethanes, styrenes, vinyl chlorides, vinyl fluorides, vinyl imidazoles, chlorosulphonated olefins, ethylene oxide, vinyl alcohols, TEFLON® (DuPont, Wilmington, DE), and nylons may be employed. See, generally, U.S. Pat. Nos. 2,609,347; 2,653,917; 2,659,935; 2,664,366; 2,664,367; and 2,846,407.
  • the beads are composed of polystyrene, silica, PGA, PLA, PSA, PLGA PLSA or PGSA.
  • Suitable beads which are currently available commercially include polystyrene beads such as FluoSpheresTM (Molecular Probes, Eugene, OR), and silica beads such as SpherisorbTM (Phase Separation, North Wales, UK).
  • carrier beads are employed having an average diameter of about 10-400 nm, more preferably 20-100 nm, and most preferably about 40 nm.
  • the present invention provides for the production of conjugates wherein a multiplicity of MHC binding domains are conjugated to a branched polymer.
  • Branched polymers are preferable to linear polymers because they have numerous chain-ends or termini which can be functionalized and, therefore, can be conjugated to a multiplicity of MHC binding domains, either directly or indirectly through conjugating moieties.
  • the branched polymer carriers of the invention are dendrimers. Dendrimers, also known as arborols, cascade molecules, dendritic polymers, or fractal polymers, are highly branched macromolecules in which the branches emanate from a central core.
  • dendrimers are synthesized outward from a core molecule by sequential addition of layers of monomers.
  • the first round of dendrimer synthesis adds a single layer or "generation" of monomers to the core, with each monomer having at least one free, reactive terminus.
  • Each subsequent round of polymerization results in the expansion of the dendrimer by one layer and increases the number of free, reactive termini. This process can be repeated numerous times to produce dendrimers of desired diameter or mass.
  • the outermost branches arrange themselves in the form of a sphere surrounding a lower density core. See, for example, U.S. Pat. No. 5,338,532.
  • dendrimers may be produced in rod-shaped, disk-like, and comb-like forms.
  • the resulting dendrimers may possess an arbitrarily large number of free, reactive termini, to which a multiplicity of MHC binding domains may be conjugated, either directly or indirectly.
  • Figure 6 provides a schematic depiction of a multimeric MHC binding domain conjugate comprising a dendrimer carrier 300.
  • the dendrimer comprises a polyamidoamine; a polyamidoalcohol; a polyalkyleneimine such as polypropyleneimine or polyethyleneimine; a polyalkylene such as polystyrene or polyethylene; a polyether; a polythioether; a polyphosphonium; a polysiloxane; a polyamide; or a polyaryl polymer.
  • Dendrimers have also been prepared from amino acids (e.g., polylysine). Suitable dendrimers which are currently available commercially include polyamidoamine dendrimers such as StarburstTM dendrimers (Dendritech, Midland, MI).
  • the StarburstTM dendrimers terminate in either amine groups or carboxymethyl groups which may be used, with or without further modification, and with or without interposing conjugating moieties, to conjugate MHC binding domains to the surface of these carriers.
  • dendrimers are employed which terminate in carboxyl or other negatively charged reactive groups.
  • Generation 1 polyamidoamine StarburstTM dendrimers have a molecular weight of- 1.0 kDa, a diameter of- 1.6 nm, and 6 terminal groups;
  • Generation 2 have a molecular weight of- 2.4 kDa, a diameter of- 2.2 nm, and 12 terminal groups;
  • Generation 3 have a molecular weight of - 5.1 kDa, a diameter of - 3.1 nm, and 24 terminal groups;
  • Generation 4 have a molecular weight of- 10.6 kDa, a diameter of- 4.0 nm, and 48 terminal groups;
  • Generation 5 have a molecular weight of- 21.6 kDa, a diameter of- 5.3 nm, and 96 terminal groups;
  • Generation 6 have a molecular weight of- 43.5 kDa, a diameter of- 6.7 nm, and 192 terminal groups;
  • Generation 7 have a molecular weight of- 43.5 kDa, a
  • Non-dendrimer branched polymers may also be employed in the invention, and may be produced from the same general classes of materials as dendrimers. The synthesis of such branched polymers is also well known in the art.
  • a "branched polymer” means a polymer having at least 5 termini, preferably at least 10 termini, and more preferably 20-500 termini, formed by branching of a carbon and/or heteroatom backbone, (c) Liposome Carriers
  • the present invention provides for the production of multimeric MHC binding domain conjugates in which a multiplicity of MHC binding domains are conjugated to the outer surface of a liposome.
  • Liposomes also called lipid vesicles, are aqueous compartments enclosed by lipid membranes, and are typically formed by suspending a suitable lipid in an aqueous medium, and shaking, extruding, or sonicating the mixture to yield a dispersion of vesicles.
  • lipid membranes typically formed by suspending a suitable lipid in an aqueous medium, and shaking, extruding, or sonicating the mixture to yield a dispersion of vesicles.
  • Various forms of liposomes including unilamellar vesicles and multilamellar vesicles, may be used in the present invention.
  • Liposomes may be prepared from a variety of lipid materials including, but not limited to, lipids of phosphatidyl choline, phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidic acid, dicetyl phosphate, monosialoganglioside, polyethylene glycol, stearyl amine, ovolecithin and cholesterol, as well as mixtures of these in varying stoichiometries.
  • lipids of phosphatidyl choline phosphatidyl serine, phosphatidyl inositol, phosphatidyl glycerol, phosphatidyl ethanolamine, phosphatidic acid, dicetyl phosphate, monosialoganglioside, polyethylene glycol, stearyl amine, ovolecithin and cholesterol, as well as mixtures
  • Liposomes may also be formed from non-lipid amphipathic molecules, such as block copolymers of poly(oxyethylene- >-isoprene-Z>-oxyethylene) and the like.
  • the liposomes are prepared from lipids that will form negatively charged liposomes, such as those produced from phosphatidyl serine, dicetyl phosphate, and dimyristoyl phosphatidic acid.
  • liposomes may also be modified to reduce immunogenicity or to provide convenient reactive groups for conjugation.
  • sialic acid or other carbohydrates, or polyethylene glycol or other alkyl or alkenyl polymers may be attached to the surface of a liposome to reduce immunogenicity.
  • liposomes may be produced bearing a conjugating moiety such as biotin by inclusion of a small molar percentage of, for example, biotin- X-dipalmitoylphosphatidylethanolamine (Molecular Probes, Eugene, OR) in the liposome. 3.
  • Means of Conjugating MHC Binding Domains to a Carrier may be used to conjugate MHC binding domains to carriers to produce the MHC binding domain conjugates of the invention. These methods include any standard chemistries which do not destroy or severely limit the biological activity of the MHC binding domains, and which allow for a sufficient number of MHC binding domains to be conjugated to the carrier an orientation which allows for interaction of the MHC binding domain with a cognate T cell receptor. Generally, methods are preferred which conjugate the C-terminal regions of an MHC binding domain, or the C-terminal regions of an MHC binding domain fusion protein, to the carrier.
  • the exact chemistries will, of course, depend upon the nature of the carrier material, the presence or absence of C-terminal fusions to the MHC binding domain, and/or the presence or absence of conjugating moieties.
  • the MHC binding domains are bound to the carrier via a covalent chemical bond.
  • a reactive group or moiety near the C-terminus of the MHC ⁇ or ⁇ chain may be conjugated directly to a reactive group or moiety on the surface of the carrier (e.g., a hydroxyl or carboxyl group of a PLA or PGA polymer, a terminal amine or carboxyl group of a dendrimer, or a hydroxyl, carboxyl or phosphate group of a phospholipid) by direct chemical reaction.
  • the carrier e.g., a hydroxyl or carboxyl group of a PLA or PGA polymer, a terminal amine or carboxyl group of a dendrimer, or a hydroxyl, carboxyl or phosphate group of a phospholipid
  • reactive carboxyl groups on the surface of a carrier may be joined to free amines (e.g., from Lys residues) on MHC binding domains, or MHC binding domain fusion proteins, by reacting them with, for example, l-ethyl-3-[3,9-dimethyl aminopropyl] carbodiimide hydrochloride (EDC) or N-hydroxysuccinimide ester (NHS).
  • EDC l-ethyl-3-[3,9-dimethyl aminopropyl] carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide ester
  • the same chemistry may be used to conjugate free amines on the surface of a carrier with free carboxyls (e.g., from the C-terminus, or from Asp or Glu residues) on MHC binding domains, or MHC binding domain fusion proteins.
  • free amine groups on the surface of a carrier may be covalently bound to MHC binding domains, or MHC binding domain fusion proteins, using sulfo-SIAB chemistry, essentially as described by Arano et al. (1991).
  • a non-covalent bond between a ligand bound to the MHC binding domain and an anti-ligand attached to the carrier may conjugate the MHC binding domains to the carrier.
  • a biotin ligase recognition sequence tag may be joined to the C-terminus of either (or both) of the MHC ⁇ or ⁇ chain binding domains, or to the C-terminus of an MHC binding domain fusion protein, and this tag may be biotinylated by biotin ligase.
  • the biotin may then serve as a ligand to non-covalently conjugate the MHC binding domain to avidin or streptavidin which is adsorbed or otherwise bound to the surface of the carrier as an anti-ligand.
  • the Fc domain may act as a ligand and protein A, either covalently or non-covalently bound to the surface of the carrier, may serve as the anti-ligand to non-covalently conjugate the MHC binding domain to the carrier.
  • Other means are well known in the art which may be employed to non-covalently conjugate MHC binding domains to carriers, including metal ion chelation techniques (e.g., using a poly-His tag at the C-terminus of the MHC binding domain or MHC binding domain fusion proteins, and a Ni + - coated carrier), and these methods may be substituted for those described here.
  • MHC binding domain fusion proteins and conjugates of the present invention may also be associated with, or bound to, various accessory molecules or moieties which are suitable to particular utilities.
  • the fusion proteins or conjugates may be associated with, or bound to, molecules or moieties including cytotoxins (e.g., genistein, ricin, diphtheria toxins, Pseudomonas toxins, the Fas ligand, and radioactive isotopes) for killing T cells, or to T cell- modulating molecules (such as the B7-1, B7-2, LFA-3, CD40 or I-CAM proteins) for activating or anergizing T cells.
  • cytotoxins e.g., genistein, ricin, diphtheria toxins, Pseudomonas toxins, the Fas ligand, and radioactive isotopes
  • T cell- modulating molecules such as the B7-1, B7-2, LFA-3, CD40 or I-CAM proteins
  • the MHC binding domain fusion proteins and conjugates may be associated with, or bound to, various molecules or moieties which are useful for detecting the presence of the fusion proteins or conjugates, such as radioactive or fluorescent labels.
  • such accessory molecules or moieties are preferably attached to the C-terminal region of the fusion protein or at some other point which is not expected to interfere with its ability to bind its cognate TCR (e.g., along an Fc domain, dimerization domain or flexible molecular linker).
  • the accessory molecules may be similar attached to the MHC binding domain or a fusion protein component (e.g., dimerization domains), to flexible molecular linkers or conjugating moieties, or to the carrier.
  • an accessory molecule or moiety may be included within the interior or pores of the carrier. Inclusion within the interior of a carrier is particularly preferred for cytotoxic agents which may exert their effect after the MHC binding domain conjugate is endocytosed within a T cell.
  • the accessory molecule is preferably bound to the exterior of a carrier such as a bead, dendrimer, or liposome.
  • Accessory molecules may be bound to MHC binding domain conjugates by standard chemical techniques known in the art, including those described above for binding MHC binding domains, or MHC binding domain fusion proteins, to carriers. Accessory molecules may be associated within porous carriers, or included within hollow carriers, by standard techniques which are known in the art.
  • the present invention provides a method for detecting and/or isolating T cells of a defined MHC/peptide complex specificity comprising contacting a population of T cells with monovalent, multivalent or multimeric MHC binding domain fusion proteins or conjugates of the invention, as described above, which are loaded with a particular MHC binding peptide and which, therefore, define a particular MHC/peptide complex.
  • the activation or proliferation of the T cells may then be determined and used, with an appropriate control, as an indication of whether the T cell population includes T cells specific for the defined MHC/peptide complex.
  • the monovalent, multivalent or multimeric MHC binding domain fusion proteins and conjugates of the invention having a defined specificity, may be immobilized on a substrate and a population of T cells may be contacted with the immobilized MHC binding domains. After allowing a period of time for the binding, if any, of T cells specific for the defined MHC/peptide complex, unbound cells may be washed away and the presence or absence of bound T cells may be used as an indication of whether the T cell population includes T cells specific for the defined MHC/peptide complex.
  • the monovalent, multivalent or multimeric MHC binding domain fusion proteins and conjugates of the invention may be contacted with a T cell population and, after allowing a period of time for the binding, if any, of the MHC binding domains to T cells specific for the defined MHC/peptide complex, unbound fusion proteins or conjugates may be washed away and the presence or absence of bound fusion proteins or conjugates may be used as an indication of whether the T cell population includes T cells specific for the defined MHC/peptide complex.
  • the labeling of the T cells, fusion proteins or conjugates, or complexes of the MHC/peptide complex with a reactive T cell receptor with fluorescent, radioactive or other markers is preferred to simplify detection.
  • fluorescent labels may be used in conjunction with FACS (fluorescence-activated cell sorting) techniques to isolate a desired subpopulation of T cells with a defined MHC/peptide specificity.
  • FACS fluorescence-activated cell sorting
  • MHC/peptide complex are detected and isolated as described above, and are then used, preferably after proliferation in vitro, for adoptive immunotherapy.
  • a population of T cells may be obtained from a host (either the subject to be treated or a syngeneic donor).
  • T cells which are reactive for a particular MHC/peptide complex are then detected and isolated using the methods described above.
  • the cells preferably after several rounds of proliferation to increase their numbers, are then administered (e.g., intravenously, intraperitoneally) to the subject to confer adoptive immunity.
  • Such a procedure may be of particular utility in stimulating adoptive immunity against weak antigens such as tumor-associated antigens.
  • the present invention provides methods for stimulating or activating T cells, in vivo or in vitro.
  • the present invention provides for the production of soluble Class II MHC fusion proteins for which no soluble counterparts had previously existed, and for the production of multivalent and multimeric Class I and Class II MHC binding domains at higher valencies than previously obtained.
  • these monovalent, multivalent and multimeric MHC binding domain fusion proteins and conjugates, loaded with appropriate MHC binding peptides and defining a specific MHC/peptide complex may now be contacted with T cells in solution, in vivo or in vitro, to specifically stimulate or activate T cells which are reactive to the defined MHC/peptide complex.
  • the multivalent and multimeric MHC binding domain fusion proteins and conjugates of the invention are expected to be particularly potent for these purposes.
  • this serves as a method of vaccination against the MHC binding peptide when presented in the defined
  • MHC/peptide complex When used for vaccination purposes against pathogens including the MHC binding peptide, of course, the MHC binding domain components of the fusion proteins or conjugates are chosen to syngeneic to the subject being vaccinated.
  • the monovalent, multivalent or multimeric MHC binding domain fusion proteins and conjugates of the present invention may be used to kill or anergize T cells reactive to a defined MHC/peptide complex, or to tolerize an individual to a particular MHC/peptide complex.
  • the MHC binding domain fusion proteins may include Fc regions which activate the complement system and, thereby, cause the destruction of T cells to which they bind.
  • the fusion proteins may be designed to include a cytotoxic substance attached to, for example, the C-terminus, or at some other point which does not interfere with the binding of the MHC/peptide complex to cognate T cell receptors (e.g., to a dimerization domain, Fc domain, ligand tag domain, or flexible molecular linker).
  • the MHC binding domain conjugates may be designed to include a cytotoxic substance attached to the MHC binding domains, to fusion protein components (e.g., a dimerization domain, Fc domain, ligand tag domain), to flexible molecular linkers or conjugating moieties, or to the carrier.
  • a cytotoxic substance may be included within the interior or pores of the carrier.
  • useful cytotoxic substances include, for example, genistein, ricin, diphtheria toxins, Pseudomonas toxins, and radioactive isotopes (e.g., 125 I). It is also known in the art that high doses of many antigens have a T cell tolerizing or anergizing effect rather than a T cell stimulating effect.
  • administering can cause tolerization to the MHC/peptide complex, even when lower doses would cause sensitization (i.e., vaccination or immunization).
  • sensitization i.e., vaccination or immunization
  • the MHC binding domain components of the fusion protein or conjugate are chosen so as to be syngeneic with the subject. Such cases would include tolerization to the antigens which cause allergic reactions, as well as autoantigens which are implicated in autoimmune disease.
  • the MHC components may be specifically allogeneic so as to tolerize the subject to an MHC/peptide complex which is foreign.
  • Such cases would include tolerization to foreign tissue before or after organ or tissue transplantation in which the donor and recipient are not identical with respect to one or more MHC alleles.
  • an effective amount with respect to tolerizing an individual to an MHC/peptide complex, is meant an amount sufficient to render T cells, otherwise specific for the MHC/peptide complex, unresponsive to the MHC/peptide complex. T cells which are unresponsive fail to activate or proliferate when presented with the complex for which they are specific.
  • an effective amount with respect to immunizing an individual to an antigen, is meant an amount sufficient to induce an immune response which results in activation or proliferation of T cells specific for the antigen in an MHC/peptide complex. Typical ranges of dosages are from 1 nanogram/kilogram to 100 milligrams/kilogram or even 500 milligrams/kilogram of body weight. Effective amounts will vary according to such factors as age, sex and sensitivity to the antigen.
  • MHC multiple sclerosis
  • RA rheumatoid arthritis
  • PV pemphigus vulgaris
  • SLE systemic lupus erythematosus
  • MHC binding domain fusion proteins and conjugates of the invention may be used to detect T cells having any defined specificity by constructing an MHC binding domain fusion protein or conjugate loaded with the appropriate MHC binding peptide (covalently or non-covalently joined) and detecting the binding and/or activation of T cells contacted with the MHC binding domains.
  • MHC binding domain fusion proteins and conjugates of the invention may be used to detect T cells having any defined specificity by constructing an MHC binding domain fusion protein or conjugate loaded with the appropriate MHC binding peptide (covalently or non-covalently joined) and detecting the binding and/or activation of T cells contacted with the MHC binding domains.
  • MHC class II haplotypes (DR4, DR6) have been associated with susceptibility to MS in particular populations (Italians, Jordanian Arabs); however, these associations are not as strong as the association with DR2 (Marrosu et al., 1988; Kurdi et al., 1977).
  • HLA-DR2 (encoded by the DRA, DRB1 *1501 genes) has been shown to present at least two peptides of human myelin basic protein (residues 85-99 and 148-162) to T cells.
  • the MBP(85-99) peptide binds with high affinity to purified DR2, and the affinity of the MBP(148- 162) peptide is lower but significant.
  • DR2 transfectants (DRA DRB 1*1501) were found to present these MBP peptides to T cell clones that had been generated from blood lymphocytes of MS patients (Chou et al., 1989; Pette et al., 1990, Martin et al., 1990; Ota et al., 1990; Wucherpfennig et al, 1990; Valli et al., 1993, Wucherpfennig et al., 1994). These studies support the hypothesis that T cells specific for MBP and other myelin antigens are involved in the inflammatory response in MS.
  • T cells in MS lesions are, however, required to prove this hypothesis and this will require soluble, stable MHC complexes with single peptides, such as those provided by the present invention.
  • therapeutic intervention whether by tolerization or killing of T cells, will require soluble, stable MHC complexes with a high avidity for binding T cells specific for particular MHC/peptide complexes, such as the multivalent and multimeric MHC binding domain fusion proteins and conjugates provided by the present invention.
  • a principal difficulty with using soluble MHC/peptide complexes as probes and therapeutics is that the affinity for the TCR is relatively low.
  • T cells compensate for the relatively low affinity of TCRs for MHC/peptide complexes by the interaction of multiple TCR molecules with MHC/peptide complexes on the surface of antigen presenting cells. Indeed, such dimerization of MHC Class II molecules may be important in T cell activation since HLA-DRl is found as a dimer when crystallized (Brown et al., 1993).
  • the present invention by providing multivalent and multimeric MHC fusion proteins and conjugates addresses this problem.
  • IgG molecules and F(ab) fragments bind monovalent antigen in solution with equal affinity; however, the binding to multivalent antigens (i.e. cell surface antigens) is greatly strengthened by the bivalent nature of the IgG molecule (Crothers and Metzger, 1972; Dower et al., 1984; Hornick and Karush, 1972).
  • the multivalent and multimeric MHC binding domain fusion proteins and conjugates of the present invention are expected to have far greater avidity for their cognate TCRs than standard, solubilized MHC proteins.
  • the extracellular domains of DR ⁇ and DR ⁇ as well as the Fos and Jun dimerization domains were generated by PCR with primers designed to include a seven amino acid linker (VDGGGGG, residues 199-205 of SEQ LD NO: 2) with a Sail restriction site at the C-terminus of the MHC extracellular domains and at the N-terminus of the Fos or Jun leucine zipper domains.
  • the MHC segments were then joined with the Fos or Jun segments through the Sail restriction site.
  • This linker was included between the DR and leucine zipper segments both to facilitate cloning (through the Sail site) and to allow for greater rotational freedom of the chains (through the poly- Gly sequence).
  • constructs were reamplified by PCR to permit cloning into the XhoI-EcoRI sites of pPIC9 as in frame fusions with the ⁇ -mating factor secretion signal.
  • the in-frame cloning into this vector preserved the Lys-Arg-Glu recognition sequence (cleavage C-terminal to Arg) required for cleavage of the ⁇ -mating secretion signal by the KEX2 gene product (Brake, 1990).
  • DR ⁇ forward primer 5' GTA TCT CTC GAG AAA AGA GAG ATC AAA GAA GAA CAT GTG ATC 3', Xhol site underlined SEQ LD NO: 5
  • DR ⁇ reverse primer 5' GTC ATA GAA TTC TCA ATG GGC GGC CAG GAT GAA CTC CAG 3', EcoRI site underlined (encodes 3' end of Fos segment, stop codon and EcoRI restriction site) SEQ LD NO: 6
  • DR ⁇ forward primer 5' GTA TCT CTC GAG AAA AGA GAG GGG GAC ACC CGA CCA CGT TTC 3', Xhol site underlined SEQ LD NO: 7
  • DR ⁇ reverse primer 5' GTC ATA GAA TTC TCA ATG GTT CAT GAC TTT CTG TTT AAG 3' EcoRI site underlined encodes 3' end of Jun segment, stop codon and EcoRI restriction site
  • the resulting PCR products are disclosed as SEQ LD NO: 1 and SEQ LD NO: 3. These PCR products were cloned into the XhoI-EcoRI sites of pPIC9 and were verified by restriction mapping and dideoxy-sequencing.
  • DR ⁇ and ⁇ chains were cloned into Pichia pastoris expression vector pPIC9 as in frame fusions with the ⁇ -mating factor secretion signal (Brake, 1990).
  • the ⁇ -mating factor secretion signal is cleaved by the KEX2 gene product at the sequence Leu-Glu-Lys-Arg-Glu (residues 3-7 of SEQ LD NO: 2 and SEQ LD NO: 4), with the cleavage C-terminal to the Arg residue.
  • the expression cassette of pPIC9 can be excised as a Bglll fragment; the cassette carries 5' and 3' sequences of the AOX1 gene to allow for integration into the AOX1 locus as well as the HIS4 gene that allows for selection of transformants in histidine deficient media.
  • Genes integrate into the AOX1 locus by homologous recombination; integration into the AOX1 gene disrupts the gene and leads to slow growth if methanol is the only carbon source (methanol utilization deficient phenotype, Mut s ) (Cregg et al., 1987).
  • pPIC9 plasmid DNA was purified on CsCl gradients and digested with Bglll to release the expression cassette (5 1 end of AOX1 gene-DR ⁇ or DR ⁇ chain construct- polyadenylation signal-HIS4 gene-3' end of the AOX1 gene). Transformations were done by spheroplasting of the GS115 strain (following the procedure provided by Invitrogen, San Diego, CA). Briefly, GS 115 cells were grown to mid-log phase in YPD media and spheroplasts were prepared by limited digestion of the yeast cell wall with zymolase (approximately 70% of spheroplasting) (Cregg et al., 1987).
  • Cells were transfected with 5 mg of DR ⁇ and DR ⁇ plasmid DNA and transfectants that expressed the FQS4 gene (present in the pPIC9 expression cassette) were selected on HIS" plates. Integration of plasmids into the AOX1 locus was confirmed by replica plating of colonies on minimal media plates with methanol or dextrose as the sole carbon source. Transformants that had integrated the plasmid DNA into the AOX1 locus showed little or no growth on methanol plates due to disruption of the alcohol oxidase gene.
  • Pichia pastoris system A major advantage of the Pichia pastoris system is that transformants can be readily identified: Integration into the AOX1 locus confers a methanol utilization deficient (Mut s ) phenotype that can be determined by comparing the growth of duplicate colonies on plates with methanol or dextrose as the sole carbon source. Mut s colonies obtained after cotransformation of plasmids carrying the DR ⁇ and DR ⁇ chain constructs were tested by PCR analysis of genomic DNA for the integration of DR ⁇ and ⁇ chain genes. 27 of 28 colonies with a Mut s phenotype carried DR ⁇ and/or DR ⁇ chain genes; four of these colonies (14.2%) had integrated both genes.
  • Mot s methanol utilization deficient
  • DR ⁇ and DR ⁇ chain constructs were examined by PCR analysis of genomic DNA isolated from individual Mut s colonies.
  • Replica colonies were transferred into 200 ml of lysis buffer (2.5 M LiCl, 50 mM Tris, pH 8.0, 4% triton X-100, 62 mM EDTA) using a sterile toothpick. Acid washed glass beads and an equal volume of phenol/chloroform (1 : 1) were added and samples were vigorously vortexed. Following centrifugation, the upper phase was transferred to a clean tube and genomic DNA was precipitated by addition of 2.5 vol of cold EtOH.
  • lysis buffer 2.5 M LiCl, 50 mM Tris, pH 8.0, 4% triton X-100, 62 mM EDTA
  • DR ⁇ and DR ⁇ chains were amplified by PCR for 35 cycles (94°C 1 min, 55°C 2 min, 72°C 2 min) using the oligonucleotides that had been used to generate the DNA constructs; PCR products were resolved on 1% agarose gels stained with ethidium bromide.
  • Affinity purification yielded approximately 300-400 mg of HLA-DR2 fusion protein per liter of culture.
  • SDS-PAGE revealed two bands, the identity of these bands (upper band DR ⁇ , lower band DR ⁇ ) and appropriate cleavage of the ⁇ -mating factor signal peptide were confirmed by N-terminal sequence analysis following separation of DR ⁇ and ⁇ chains by SDS-PAGE and transfer to a PVDF membrane.
  • Induction of high density cultures was carried out using a Inceltech LH series fermenter equipped with monitors and controls for pH, dissolved 02, agitation, temperature, and air flow.
  • a 100 ml YNB-glycerol overnight culture was used to inoculate the fermenter which contained 10 liters of fermentation basal salts medium (0.93 g/L calcium sulfate 2 H2O, 18.2 g/L potassium sulfate, 14.9 g/L magnesium sulfate 7 H2O, and 6.5 g/L potassium hydroxide) containing 4% glycerol (w/v) plus 43.5 ml of PTMi trace salts (24 mM CuSO4, 0.53 mM Nal, 19.87 mM MnSO4, 0.83 mM Na2MoO4, 0.32 mM boric acid, 2.1 mM C0CI2, 0.15 mM ZnCl2, 0.23 mM FeSO4, and 0.82 mM biotin) at 30
  • Dissolved O2 was maintained above 20% by adjusting aeration and agitation, and pH was maintained at 6.0 by the addition of 28% (v/v) ammonium hydroxide. Growth was continued until the glycerol was exhausted (20 hours).
  • a glycerol fed- batch phase was initiated by the limited addition of 50% (w/v) glycerol and 12 ml PTMi salts per liter of glycerol at 18.15 ml/hr/L initial fermentation volume until the culture reached a wet cell weight (wcw) of 200 g/L (22 hours).
  • the culture was induced by replacing the glycerol feed with a methanol-batch feed (100% methanol containing 12 ml
  • PTMi trace salts per liter of methanol at 1 ml/hr/L.
  • the methanol feed was gradually increased in 10% increments every 30 minutes to a rate of 3 ml/hr/L and the fermentation continued for a duration of 96 hours.
  • Supernatants were concentrated by ultrafiltration on a YM30 membrane (Amicon) and passed over an anti-DR (mAb L243) affinity column at a flow rate of approximately 10 ml hour. Following extensive washing with PBS, heterodimers were eluted with 50 mM glycine, pH 11.5. Eluates were immediately neutralized by addition of 2 M Tris, pH 8.0, dialyzed against PBS and concentrated by ultrafiltration. Protein concentrations were determined by Coomassie Plus Protein Assay (Pierce, Rockford, LL) using bovine serum albumin as a standard.
  • Binding was shown to be specific because it could be blocked by an excess of non- biotinylated MBP(85-99) peptide, but not by an analog peptide in which the PI anchor residue (Val 89) of MBP(85-99) had been substituted by aspartic acid (Figure 9).
  • the DR2 fusion protein 400 nM was incubated with biotinylated peptide (2 ⁇ M) in a 50 ml volume in PBS, 1 mM EDTA 1 mM PMSF, pH 7.2 for 24 hours at 37°C.
  • DR2 -peptide complexes were precipitated with streptavidin-agarose beads. Beads were first blocked with 3% bovine serum albumin in PBS, 0.1% NP40 for 1 hour at 4°C; beads were then pelleted and the DR2/peptide samples added. Following a 1 hour incubation, beads were washed three times with blocking buffer.
  • DR2-peptide complexes were eluted from streptavidin beads by heating in lxSDS-PAGE buffer at 94°C for 3 minutes. Samples were resolved on a 12.5% SDS-PAGE and transferred to immobilon membrane (Millipore). Blots were blocked overnight with 5% non-fat dry milk in 50 mM Tris, pH 8.0, 150 mM NaCl, 0.2% Tween 20 (TBST buffer). Precipitated DR ⁇ and ⁇ chain fusions were detected with a polyclonal DR antiserum (CHAMP, 1 :50,000 in blocking buffer for 90 min).
  • CHMP polyclonal DR antiserum
  • Blots were washed in TBST buffer and incubated for 30 min with a peroxidase conjugated anti-rabbit IgG antibody (1 : 10,000 in blocking buffer). Following extensive washing in TBST, bands were detected by enhanced chemiluminescence (Amersham, Arlington Heights, LL).
  • peptide binding to recombinant DR2 fusion proteins was quantitated by capturing DR2 fusions to ELISA plates with an immobilized DR antibody.
  • Standard binding conditions were: 37°C for 24 hours in PBS, pH 7.2, 1 mM EDTA, 1 mM PMSF.
  • bound peptide was quantitated by ELISA. Plates were coated with 200 ng/well of the purified L234 mAb in 0.1 M bicarbonate, pH 9.6 overnight at 4°C. Nonspecific binding sites were blocked with 3% BSA in PBS, 0.05% Tween 20 for 2 hours. Samples were diluted in blocking buffer and added to the wells (1 hour). HLA-DR2 bound biotinylated peptide was quantitated with streptavidin-peroxidase using ABTS as a peroxidase substrate; absorbance was read at 405 nm.
  • HLA-DQ MHC Binding Domain Fusion Proteins The leucine zipper dimerization domains of Fos and Jun were also used to express soluble HLA-DQ MHC binding domain fusion proteins for DQ1 and DQ8 alleles, which are associated with susceptibility to pemphigus vulgaris and insulin dependent diabetes, respectively. The same design was used as described above for recombinant DR2 (including splice points). Stable transfectants were generated using Drosophila Schneider cells and soluble DQ molecules were affinity purified. Peptide binding studies using peptides that were previously shown to bind to DQ1 or DQ8 demonstrated that the molecules were functional.
  • Divalent HLA- DR2 MHC binding domain fusion proteins were expressed by fusing the Fc part of IgG2a to the 3' end of the DR ⁇ -Fos cDNA construct described above.
  • the DR ⁇ -Fc chain corresponds to an antibody heavy chain and the DR ⁇ -Jun construct to an antibody light chain.
  • the DR2-IgG design was chosen both to increase the affinity for the T cell receptor by increasing valency, and to attach an effector domain, the Fc region of IgG2a. Complement fixation may result in the lysis of target T cells following binding of DR2-IgG molecules to the T cell receptor.
  • the DR2-IgG molecules may therefore be useful for the selective depletion of autoaggressive T cells.
  • the nucleic acid sequence encoding the DR2-IgG construct is disclosed as SEQ LD NO: 11 and the encoded fusion protein is disclosed as SEQ ID NO: 12.
  • the Fc part of IgG2a was amplified by RT-PCR from a mouse hybridoma (L243) that secretes an IgG2a mAb.
  • the PCR product was fused in frame with the DR ⁇ -Fos construct by overlapping PCR with a primer for the Fc part that overlapped by 20 bp with the 3' end of the DR ⁇ -Fos construct.
  • DR ⁇ -Fos and Fc were amplified separately, gel purified, mixed and amplified using oligos representing the 5' end of DR ⁇ and the 3' end of IgG2a.
  • the construct was cloned into the EcoRI-BamHI sites of the pRmHa-3 expression vector under the control of the metalothionein promoter. The insert was checked by restriction mapping and dideoxy- sequencing.
  • DR2-IgG fusion proteins were expressed in the Drosophila Schneider cell system.
  • the Drosophila Schneider cell system was chosen for the expression of the DR2-IgG fusion protein for the following reasons: (1) recombinant antibodies have previously been expressed in insect cells, (2) in the pRmHa-3 expression vector, genes are under the control of the strongly inducible metalothionein promoter, (3) Schneider cells can be grown to a high cell density in serum free media, and (4) large scale production of protein is more straightforward than in another insect cell system (the Baculovirus system) since stable transfectants are generated.
  • Stable transfectants were generated by the contransfecting Schneider cells with the DR ⁇ - IgG and DR ⁇ chains vectors as well as with plasmid pH8CO. This vector confers resistance to selection by methotrexate. Transfectants were selected with 0.1 ⁇ M methotrexate in Schneider media, 10% fetal calf serum. Transfectants were cloned by limiting dilution, and the secretion of DR2-IgG fusion proteins was examined by ELISA using an antibody specific for the Fc segment of IgG, as well as an antibody specific for the DR ⁇ heterodimer.
  • Transfectants were grow to a density of -lOxloVml and expression was induced by adding CuSO 4 to a final concentration of lmM. Supernatants were harvested five days following induction and concentrated by ultrafiltration. DR2-IgG fusion proteins were purified by affinity chromatography using the L243 mAb. Purity was examined by SDS-PAGE; for comparison, purified mouse IgG was also run on the gel. Western blot analysis with a polyclonal antiserum confirmed the identity of the two bands. Peptide binding experiments demonstrated that DR2- IgG fusion proteins were properly folded and functional. C.
  • DR2-IgM fusion proteins molecules comprise ten MHC binding domains (five IgM monomers per IgM pentamer; two MHC binding domains per IgM monomer). Since DR2-IgG fusion proteins have only two MHC binding domains, the functional affinity of DR2-IgM fusion proteins for cognate T cell receptors is expected to be much higher. A significant increase in affinity would improve the sensitivity for immunohisto chemical staining as well as the therapeutic effectiveness of these molecules.
  • DR2-IgM fusion proteins may be particularly useful for immunotherapy for the following reasons: (1) higher avidity for the T cell receptors on cognate T cells, (2) complement fixation by the Fc segment of IgM, and (3) longer serum half life.
  • the Fc segment of IgM is fused in frame to the 3' end of the DR ⁇ -Fos segment, as previously described for the DR ⁇ -IgG construct.
  • DR2-IgM construct is disclosed as SEQ ID NO: 13 and the encoded fusion protein is disclosed as SEQ LD NO: 13.
  • the DR ⁇ -IgM construct is cloned into, for example, the EcoRI-BamHI sites of the pRmHa-3 expression vector, under the control of the inducible metalothionein promoter (Bunch et al, 1988).
  • the DR ⁇ -IgM and DR ⁇ chain fusion constructs are cotransfected with a gene encoding the J-chain.
  • the J-chain facilitates assembly and secretion of IgM molecules by mammalian cells (Matsuuchi et al., 1986).
  • the J-chain may be cloned into, for example, expression vector pUC-hygMT which confers resistance to hygromycin. Stable transfectants may then be selected using hygromycin at 100 ⁇ g/ml in Schneider cell media (Sigma) supplemented with 10% insect cell tested fetal calf serum. Transfectants are cloned by limiting dilution and tested for expression of DR2-IgM fusion proteins following induction with CuSO 4 .
  • DR2-IgM fusion proteins may be assessed by immunoprecipitation with mAb L243, followed by Western blot analysis with antibodies specific for the Fc segment of the IgM.
  • transfectants can be adapted to serum free media (ExCell 400, JRH Biosciences). These constructs also can be transfected into CHO cells or into a murine B cell line
  • CHO cells were previously shown to secrete recombinant IgM antibodies at high levels and have been used for the expression of a CD2-IgM fusion protein (Wood et al., 1990; Arulanandam et al., 1993).
  • the DR ⁇ -IgM and DR ⁇ chain constructs are cloned into eukaryotic expression vectors.
  • the DR ⁇ -IgM construct can be cloned into, for example, pcDNA3, which carries the neomycin resistance gene, and the DR ⁇ -Jun construct can be cloned in the pcDNAI vector (Invitrogen, San Diego, CA).
  • Cells can be transfected by electroporation and stable transfectants can be selected with G418. Secretion of DR2-IgM fusion proteins can be assessed by immunoprecipitation with mAb L243 and by Western blot analysis.
  • DR2-IgM fusion proteins were not secreted by Drosophila Schneider cells and, therefore, expression in COS cells was performed. DR2-IgM fusion proteins were secreted when COS cells were transfected with the cDNA constructs.
  • Biotin ligase specifically biotinylates a lysine residue within a 14-amino acid recognition sequence (LGGIFEAMKMELRD, SEQ ID NO: 9) (Shatz, 1993) and, therefore, a DNA sequence encoding this sequence was added to the DR ⁇ -Fos construct.
  • This "DR ⁇ -Fos-tag" construct was cloned into the EcoRI and Sail sites of Drosophila expression vector pRmHa-3 under the control of the inducible metalothionein promoter.
  • Drosophila Schneider cells stably co-transfected with the DR ⁇ -Fos-tag and DR ⁇ -Jun constructs were generated as described above for the DR2-IgG fusion proteins.
  • the resulting "DR2-tag" fusion molecules differ from the DR2-Fos/Jun fusion proteins only by the addition of the biotinylation sequence tag to the C-terminus of the DR ⁇ -Fos construct.
  • the DR2-tag fusion proteins were affinity purified from supernatants using the L243 mAb as described above.
  • DR2-tag-biotin tetramers Site specific biotinylation of these DR2-tag molecules allows assembly of DR2-tag-biotin tetramers on avidin or streptavidin because avidin and streptavidin have four biotin binding sites. Thus, tetramers are made by mixing the DR2-tag-biotin molecules and streptavidin at a 4:1 molar ratio.
  • Biotinylation of MHC Binding Domain-Biotin-Tag Fusion Proteins A biotin ligase cDNA (provided by S. Lesley, Promega Corporation) was cloned as a Ndell-Xhol fragment into the prokaryotic expression vector pET22b under the control of the T7 promoter. This construct was transfected into E. coli strain BL21/DE3 which is lysogenic for the T7 RNA polymerase gene under the control of the lacZ promoter. Protein expression was induced by addition of LPTG to ImM for 4 hours. Cells were then harvested by centrifugation, resuspended in 20 mM Tris, pH 8.0, 100 mM NaCl.
  • Biotinylation was performed at 37°C in a 100 ⁇ l volume with 0.1 to lO ⁇ l of enzyme, 1 mM of ATP and 1 or 10 ⁇ M of biotin.
  • recombinant DR2-tag-biotin molecules were captured on a 96-well plate coated with the L243 mAb and the degree of biotinylation was quantitated using peroxidase conjugated streptavidin.
  • a Western blot was sequentially probed with a polyclonal DR antiserum and with streptavidin peroxidase. This experiment demonstrated specific biotinylation of the DR ⁇ chain (which carried the 14-amino acid biotin ligase recognition sequence) by biotin ligase.
  • Fluorescein-labeled streptavidin was used to examine the formation of DR2-tag-biotin tetramers. Fluorescein absorbs at 492 nm, allowing detection during HLPC gel filtration chromatography (Bio-Gel SEC 300 mm x 7.8 mm; flow rate 1 ml/min, PBS pH 6.8). Streptavidin (MW 60 kDa) eluted as a single peak at 8.3 minutes on the HPLC gel filtration column. The streptavidin-DR2-tag-biotin complex eluted at 5.8 minutes. Intermediates with one, two or three DR2 fusion molecules bound to streptavidin were observed when smaller amounts of DR2-tag- biotin were used for complex formation. MW standards confirmed the predicted molecular weight of streptavidin and the streptavidin-DR complex.
  • Binding was examined by capturing biotinylated DR2-tag molecules on a streptavidin coated plate. Non-specific binding sites were blocked with 0.1% BSA in PBS. T cells were labeled with BCEFC-AM, a fluorescent membrane probe, for 30 minutes at 37°C, washed and added to the plate for 20 minutes at 37°C. Following three washes, the fraction of T cells that bound to DR2/peptide complexes or to the anti-CD3 mAb was determined in a fluorescent plate reader.
  • BCEFC-AM a fluorescent membrane probe
  • DR2-biotin tag molecules were used to generate highly multimeric MHC binding domain conjugates for the specific staining of antigen specific T cells.
  • DR2/peptide complexes were bound to highly fluorescent microbeads, purchased from Molecular Probes (Eugene, OR), to which streptavidin had been conjugated.
  • Polystyrene beads similar in size to viral particles (40 nm) were selected based on their ability to remain soluble; these beads pellet in an ultracentrifuge but not under the low G-forces used to wash cells. Staining of antigen specific T cells was examined by FACS.
  • biotinylated mAbs specific for CD4 positive control
  • a murine MHC class II (10-2.16) negative control
  • Approximately 10 6 T cells were used for each assay. T cells were pelleted and resuspended in cold PBS, 0.1% sodium azide. Staining was observed with both DR2/MBP(85-99) specific T cell clones and multivalent DR2 MBP(85-99) peptide complexes; the staining intensity was similar to that observed with the CD4 mAb. Binding was highly specific because a single amino acid substitution in the MBP peptide at a TCR contact residue greatly reduce the staining intensity.
  • control T cell clones specific for other MHC class II/peptide combinations. These control clones were specific for MBP(85-99) bound to HLA-DQ 1 (clone HY.1B11), a desmoglein 3 peptide (190-204) bound to HLA-DR4 (clone Go.P3) and a tetanus toxoid peptide (830-843) bound to HLA-DR2a (clone Kw-TTl).
  • DR2-Ig fusion proteins are generated to allow multivalent binding to TCRs on target T cells (2 DR2/peptide arms in the DR2-IgG fusion protein, 10 DR2/peptide arms in the DR2-IgM fusion protein).
  • DR2 molecules were expressed with a covalently linked MBP peptide.
  • the MBP(85-99) sequence was attached to the N-terminus of the mature DR ⁇ chain through a 16- amino acid linker (linker sequence: SGGGSLVPRGSGGGGS, SEQ LD NO: 10).
  • This cDNA construct was used to express DR2 molecules and DR2-IgG molecules with a linked MBP peptide in Drosophila Schneider cells.
  • DR2-Ig Fusion proteins may be useful for the selective depletion of T cells that recognize DR2 bound self- peptides. Binding of DR2-Ig fusion proteins by the T cell receptor may lead to complement fixation and lysis of target T cells. Multivalent DR2 molecules could also be conjugated to genistein, a tyrosine kinase inhibitor that induces apoptosis following uptake by target cells.
  • Affinity of Multivalent DR2/Pe ⁇ tide Complexes for the T Cell Receptor The binding of multivalent DR2/peptide complexes to the TCR will be examined using human DR2 restricted T cell clones. DR2 molecules will be loaded with the MBP(85-99) peptide and labeled with [ 25 I] using immobilized chloramine T (lodobeads, Pierce). In the binding assay, a fixed number of T cells (lxlO 6 cells, 1 ml) will be incubated with 6-10 different concentrations of radiolabeled DR2/peptide complexes in PBS, 1.0% BSA, 0.02% NaN 3 .
  • Radiolabeled molecules will be used at concentrations at which only a small fraction (less than 10%) of TCRs on target cells will be occupied.
  • Cell bound radioactivity will be quantitated in a ⁇ -counter and data will be analyzed on Scatchard plots to determine K (dissociation constant) and n (number of TCR molecules on target cells).
  • a mAb specific for human CD3 (OKT3, IgG2a) that fixes complement will be used as a positive control. Specificity of lysis will be assessed using control T cell clones as well as DR2 molecules loaded with control peptides.
  • Multivalent DR2 molecules of all three designs, DR2-IgG, DR2-IgM and DR2 -tetramers, will be conjugated to toxin moieties as another means of mediating selective T cell death.
  • Genistein a tyrosine kinase inhibitor, may be particularly effective for this purpose.
  • genistein coupled to CD 19 mAb was found to be highly effective in eradicating a human B cell leukemia from SCLD mice (Uckun et al., 1995).
  • a single dose of 25 ⁇ g of a genistein-mAb conjugate provided complete protection from a lethal challenge with the B cell leukemia.
  • CD 19 is a B lineage specific surface molecule; the antibody conjugate was shown to induce apoptosis following internalization by receptor mediated endocytosis.
  • T cell receptors are endocytosed following recognition of DR2/peptide complexes (Valitutti et al., 1995); it is therefore likely that multivalent DR2/peptide complexes will be taken up target T cells following binding to the T cell receptor.
  • Genistein will be conjugated in multivalent DR2/peptide complexes by photoaffinity crosslinking using a photosensitive 18.2 A long non-cleavable hetero-bifunctional crosslinking agent (Sulfo-SANPAH) as described by Uckun et al. (1995).
  • the DR2-toxin conjugates will be tested using the human DR2 restricted T cell clones. T cells will be incubated with the DR2-toxin conjugates and the induction of apoptosis will be assessed by agarose gel electrophoresis of genomic DNA. Nucleosomal fragmentation of DNA will be examined by ethidium bromide staining.
  • DR2 molecules loaded with control peptides as well as control T cell clones will be used to demonstrate the specificity of apoptosis induction.
  • Apoptosis induction by DR2-toxin conjugates will be quantitated by flow cytometry following end labeling of fragmented DNA ends (TUNEL procedure).
  • TUNEL procedure end labeling of fragmented DNA ends
  • the free ends of nuclear DNA fragments will be labeled with dioxygenin-conjugated nucleotides, using the enzyme terminal deoxynucleotidyl transferase (TdT).
  • TdT enzyme terminal deoxynucleotidyl transferase
  • the 3'-OH ends of nuclear DNA fragments will be labeled with dioxygenin- dUTP, dioxygenin-dATP and TdT, followed by detection of labeled DNA ends with a fluorescein labeled anti-dioxygenin antibody (ApopTag, in situ apoptosis detection kit, Oncor). FACS analysis will be used to determine the fraction of cells that have undergone apoptosis. Cells grown for 12 hours at a low serum concentration (1% serum) will be used as a positive control. Specificity of apoptosis induction will be demonstrated by using control T cells clones and DR2 molecules loaded with control peptides.
  • T Cell Binding to Immobilized DR2/Peptide Complexes Previous studies had demonstrated that recombinant, soluble DR2 molecules specifically bind peptides. To examine if recombinant DR2/peptide complexes are recognized by T cell receptors, T cell adhesion assays were performed using biotinylated DR2/peptide complexes that were captured on streptavidin coated microtiter plates. MBP(85-99) specific T cell clones and control T cell clones were labeled with BCEFC-AM, a fluorescent membrane probe, washed and incubated for 30 minutes at 37°C with immobilized DR2/peptide complexes. Following washing, the fraction of bound T cells was determined in a fluorometer.

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Abstract

L'invention concerne le secteur de l'immunologie, et en particulier la conception, la production et l'utilisation de protéines hybrides monovalentes, multivalentes et multimères caractérisées par un domaine de liaison CMH (complexe majeur d'histocompatibilité), et de conjugués de ces protéines.
EP99908272A 1998-02-19 1999-02-19 Proteines hybrides monovalentes, multivalentes et multimeres caracterisees par un domaine de liaison cmh (complexe majeur d'histocompatibilite), conjugues de ces proteines, et utilisations correspondantes Ceased EP1054984A1 (fr)

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US7535198P 1998-02-19 1998-02-19
US75351P 1998-02-19
PCT/US1999/003603 WO1999042597A1 (fr) 1998-02-19 1999-02-19 Proteines hybrides monovalentes, multivalentes et multimeres caracterisees par un domaine de liaison cmh (complexe majeur d'histocompatibilite), conjugues de ces proteines, et utilisations correspondantes

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AU765378B2 (en) 2003-09-18
JP2002504342A (ja) 2002-02-12
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WO1999042597A1 (fr) 1999-08-26
NZ506071A (en) 2003-05-30

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