WO2010037397A1 - Multimères de mhc dans la surveillance immunitaire contre le cmv - Google Patents

Multimères de mhc dans la surveillance immunitaire contre le cmv Download PDF

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Publication number
WO2010037397A1
WO2010037397A1 PCT/DK2009/050257 DK2009050257W WO2010037397A1 WO 2010037397 A1 WO2010037397 A1 WO 2010037397A1 DK 2009050257 W DK2009050257 W DK 2009050257W WO 2010037397 A1 WO2010037397 A1 WO 2010037397A1
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mhc
multimer according
mhc multimer
peptide
group
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PCT/DK2009/050257
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English (en)
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Liselotte Brix
Jørgen SCHØLLER
Henrik Pedersen
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Dako Denmark A/S
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Publication of WO2010037397A1 publication Critical patent/WO2010037397A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/69—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 conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6901—Conjugates being cells, cell fragments, viruses, ghosts, red blood cells or viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/73—Fusion polypeptide containing domain for protein-protein interaction containing coiled-coiled motif (leucine zippers)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases

Definitions

  • PCT/DK2009/050185, PCT/DK2008/0001 18, and PCT/DK2008/050167 are hereby also incorporated herein by reference in their entirety. All patent and non-patent references cited in PCT/DK2009/050185, PCT/DK2008/0001 18, and PCT/DK2008/050167 are hereby also incorporated by reference in their entirety.
  • the present invention relates to MHC-peptide complexes and uses thereof in the treatment of a disease in an individual.
  • MHC Major Histocompatibility Complex
  • TCR T-cell receptors
  • the immune response is divided into two parts termed the innate immune response and the adaptive immune response. Both responses work together to eliminate pathogens (antigens). Innate immunity is present at all times and is the first line of defense against invading pathogens.
  • the immediate response by means of pre- existing elements, i.e. various proteins and phagocytic cells that recognize conserved features on the pathogens, is important in clearing and control of spreading of pathogens. If a pathogen is persistent in the body and thus only partially cleared by the actions of the innate immune system, the adaptive immune system initiate a response against the pathogen.
  • the adaptive immune system is capable of eliciting a response against virtually any type of pathogen and is unlike the innate immune system capable of establishing immunological memory.
  • the adaptive response is highly specific to the particular pathogen that activated it but it is not so quickly launched as the innate when first encountering a pathogen.
  • the adaptive response is carried out by two distinct sets of lymphocytes, the B cells producing antibodies leading to the humoral or antibody mediated immune response, and the T cells leading to the cell mediated immune response.
  • T cells express a clonotypic T cell receptor (TCR) on the surface.
  • TCR clonotypic T cell receptor
  • MHC major histocompatibility complex
  • HLA human leukocyte antigens
  • MHC class I or MHC class II MHC class II
  • TCR recognition of MHC-peptide complexes result in T cell activation, clonal expansion and differentiation of the T cells into effector, memory and regulatory T cells.
  • B cells express a membrane bound form of immunoglobulin (Ig) called the B cell receptor (BCR).
  • BCR immunoglobulin
  • the BCR recognizes an epitope that is part of an intact three dimensional antigenic molecule.
  • the BCR:antigen complex is internalized and fragments from the internalized antigen is presented in the context of MHC class Il on the surface of the B cell to CD4+ helper T-cells (Th).
  • Th helper T-cells
  • a very important feature of the adaptive immune system is its ability to distinguish between self and non-self antigens, and preferably respond against non-self. If the immune system fails to discriminate between the two, specific immune responses against self-antigens are generated. These autoimmune reactions can lead to damage of self-tissue.
  • the adaptive immune response is initiated when antigens are taken up by professional antigen presenting cells such as dendritic cells, Macrophages, Langerhans cells and B- cells. These cells present peptide fragments, resulting from the degradation of proteins, in the context of MHC class Il proteins (Major Histocompatibility Complex) to helper T cells.
  • the T helper cells then mediate help to B-cells and antigen-specific cytotoxic T cells, both of which have received primary activation signals via their BCR respective TCR.
  • the help from the Th-cell is mediated by means of soluble mediators e.g. cytokines.
  • the interactions between the various cells of the cellular immune response is governed by receptor-ligand interactions directly between the cells and by production of various soluble reporter substances e.g. cytokines by activated cells.
  • MHC-peptide complexes function as antigenic peptide receptors, collecting peptides inside the cell and transporting them to the cell surface, where the MHC-peptide complex can be recognized by T-lymphocytes.
  • MHC class I and II Two classes of classical MHC complexes exist, MHC class I and II. The most important difference between these two molecules lies in the protein source from which they obtain their associated peptides.
  • MHC class I molecules present peptides derived from endogenous antigens degraded in the cytosol and are thus able to display fragments of viral proteins and unique proteins derived from cancerous cells. Almost all nucleated cells express MHC class I on their surface even though the expression level varies among different cell types.
  • MHC class Il molecules bind peptides derived from exogenous antigens. Exogenous proteins enter the cells by endocytosis or phagocytosis, and these proteins are degraded by proteases in acidified intracellular vesicles before presentation by MHC class Il molecules. MHC class Il molecules are only expressed on professional antigen presenting cells like B cells and macrophages.
  • MHC class I molecules consist of two polypeptide chains, a heavy chain, ⁇ , spanning the membrane and a light chain, ⁇ 2-microglobulin ( ⁇ 2m).
  • the heavy chain is encoded in the gene complex termed the major histocompatibility complex (MHC), and its extracellular portion comprises three domains, ⁇ 1 , ⁇ 2 and ⁇ 3.
  • MHC major histocompatibility complex
  • the ⁇ 2m chain is not encoded in the MHC gene and consists of a single domain, which together with the ⁇ 3 domain of the heavy chain make up a folded structure that closely resembles that of the immunoglobulin.
  • mice The ⁇ 1 and ⁇ 2 domains pair to form the peptide binding cleft, consisting of two segmented ⁇ helices lying on a sheet of eight ⁇ -strands.
  • MHC class I molecule In humans as well as in mice three different types of MHC class I molecule exist. HLA- A, B, C are found in humans while MHC class I molecules in mice are designated H- 2K, H-2D and H-2L
  • the MHC class Il molecule is composed of two membrane spanning polypeptide chains, ⁇ and ⁇ , of similar size (about 30000 Da). Genes located in the major histocompatibility complex encode both chains. Each chain consists of two domains, where ⁇ 1 and ⁇ 1 forms a 9-pocket peptide-binding cleft, where pocket 1 , 4, 6 and 9 are considered as major peptide binding pockets.
  • the ⁇ 2 and ⁇ 2, like the ⁇ 2 and ⁇ 2m in the MHC class I molecules, have amino acid sequence and structural similarities to immunoglobulin constant domains. In contrast to MHC class I complexes, where the ends of the antigenic peptide is buried, peptide-ends in MHC class Il complexes are not.
  • HLA-DR, DQ and DP are the human class Il molecules
  • H-2A, M and E are those of the mice.
  • MHC genes A remarkable feature of MHC genes is their polymorphism accomplished by multiple alleles at each gene.
  • the polygenic and polymorphic nature of MHC genes is reflected in the peptide-binding cleft so that different MHC complexes bind different sets of peptides.
  • the variable amino acids in the peptide binding cleft form pockets where the amino acid side chains of the bound peptide can be buried. This permits a specific variant of MHC to bind some peptides better than others.
  • MHC multimers have been developed. These are complexes that include multiple copies of MHC-peptide complexes, providing these complexes with an increased affinity and half-life of interaction, compared to that of the monomer MHC-peptide complex. The multiple copies of MHC-peptide complexes are attached, covalently or non-covalently, to a multimerization domain.
  • MHC multimers include the following:
  • MHC-dimers Each MHC dimer contains two copies of MHC-peptide. IgG is used as multimerization domain, and one of the domains of the MHC protein is covalently linked to IgG.
  • MHC-tetramers Each MHC-tetramer contains four copies of MHC-peptide, each of which is biotinylated. The MHC complexes are held together in a complex by the streptavidin tetramer protein, providing a non-covalent linkage between a streptavidin monomer and the MHC protein. Tetramers are described in US patent 5,635,363.
  • MHC pentamers Five copies of MHC-peptide complexes are multimerised by a self-assembling coiled-coil domain, to form a MHC pentamer. MHC pentamers are described in the US patent 2004209295
  • MHC dextramers A large number of MHC-peptide complexes, typically more than ten, are attached to a dextran polymer. MHC-dextramers are described in the patent application WO 02/072631 A2.
  • MHC streptamers 8-12 MHC-peptide complexes attached to Streptactin. MHC streptamers are described in Knabel M et al. Reversibel MHC multimer staining for functional isolation of T-cell populations and effective adoptive transfer. Nature medicine 6. 631 -637 (2002).
  • the concentration of antigen-specific T-cells in samples from e.g. peripheral blood can be very low.
  • Flow cytometry and related methods offer the ability to analyze a large number of cells and simultaneously identify the few of interest. MHC multimers have turned out to be very valuable reagents for detection and characterization of antigen- specific T-cells in flow cytometer experiments.
  • the relative amount of antigen-specific T cells in a sample can be determined and also the affinity of the binding of MHC multimer to the T-cell receptor can be determined.
  • the basic function of a flow cytometer is its ability to analyse and identify fluorochrome labelled entities in a liquid sample, by means of its excitation, using a light source such as a laser beam and the light emission from the bound fluorochrome.
  • MHC multimers is used as detections molecule for identification of antigen-specific T- cells in flow cytometry, by labelling the MHC multimer with a specific fluorochrome, which is detectable, by the flow cytometer used.
  • the cells can be sub- categorized using antibodies or other fluorochrome labelled detections molecules directed against surface markers other than the TCR on the specific T-cells population.
  • Antibodies or other fluorochrome labelled detections molecules can also be used to identify cells known not to be antigen-specific T-cells. Both kinds of detections molecules are in the following referred to as gating reagents.
  • Gating reagents helps identify the "true" antigen-specific T cells bound by MHC multimers by identifying specific subpopulations in a sample, e.g. T cells and by excluding cells that for some reason bind MHC mulimers without being antigen-specific T-cells.
  • Other cytometry methods e.g. fluorescence microscopy and IHC can like flow cytometry be employed in identification of antigen-specific T cells in a cell sample using MHC multimers.
  • T cells are pivotal for mounting an adaptive immune response. It is therefore of importance to be able to measure the number of specific T cells when performing a monitoring of a given immune response, for example in connection with vaccine development, infectious diseases e.g. tuberculosis, toxicity studies etc.
  • the present invention further provides powerful tools in the fields of vaccines, therapy and diagnosis.
  • One objective is to isolate antigen-specific T-cells and culture these in the presence of co-stimulatory molecules. Ex vivo priming and expansion of T-cell populations allows the T-cells to be used in immunotherapy of various types of infectious diseases.
  • a second objective of the present invention is to identify and label specific subsets of cells with relevance for the development or treatment of diseases.
  • MHC multimers of the present invention can be used in immune monitoring following transplantation but may also be used for immune monitoring in other immuno suppressive conditions e.g. in HIV infected patients or patients receiving chemotherapy SUMMARY OF INVENTION.
  • MHC multimers are crucial reagents in monitoring of antigen-specific T cells.
  • the present invention describes novel methods to generate MHC multimers and methods to improve existing and new MHC multimers.
  • the invention also describes improved methods for the use of MHC multimers in analysis of T cells in samples including diagnostic and prognostic methods.
  • MHC multimers in therapy are described, e.g. anti- tumour and anti-virus therapy, including isolation of antigen-specific T cells capable of inactivation or elimination of undesirable target cells or isolation of specific T cells capable of regulation of other immune cells.
  • the present invention also relates to MHC multimers comprising one or more CMV derived peptides.
  • MHC multimers are used in monitoring immune status following e.g. transplantation, during immunosuppresive treatment, during anticancer treatment, in HIV infected individuals or other conditions where the immune system is supressed.
  • the present invention relates to a MHC multimer such as a MHC dextramer such as a chemically biotinylated MHC dextramer.
  • MHC multimers prefereably comprise one or more peptides derived from one or more CMV antigens such as one or more peptides derived from pp28, pp50, pp65, pp150, pp71 , gH, gB, IE-1 , IE-2, US2, US3, US6, US1 1 , and UL18.
  • the measurement of antigen-specific T cells according to the present invention is based on measurement of the number of T cells and not on measurement of activation of T cells.
  • the measurement of antigen-specific T cells according to the present invention is based on a binding assay and not on a functional assay.
  • the method for measurement of T cells is not based on and/or does not involve priming of Antigen presenting cells.
  • the method for measurement of T cells can in one preferred embodiment be performed at experimental condition that will only allow limited or completely prevent priming of Antigen presenting cells. These experimental conditions can involve one or more factors that do not allow priming of Antigen presenting cells such as an incubation time that is too short to allow priming or a pH or salt concentration that will prevent or inhibit priming of Antigen presenting cells.
  • the present invention relates to a CMV vaccine.
  • a CMV vaccine the peptides bound in the peptide binding cleft of MHC are derived from CMV proteins.
  • adjuvants are drugs that have few or no pharmacological effects by themselves, but can increase the efficacy or potency of other drugs when given at the same time.
  • an adjuvant is an agent which, while not having any specific antigenic effect in itself, can stimulate the immune system, increasing the response to a vaccine.
  • Agonist as used herein is a substance that binds to a specific receptor and triggers a response in the cell. It mimics the action of an endogenous ligand that binds to the same receptor.
  • Antagonist as used herein is a substance that binds to a specific receptor and blocks the response in the cell. It blocks the action of an endogenous ligand that binds to the same receptor.
  • an antibody means an isolated or recombinant binding agent that comprises the necessary variable region sequences to specifically bind an antigenic epitope. Therefore, an antibody is any form of antibody or fragment thereof that exhibits the desired biological activity, e.g., binding the specific target antigen.
  • Antibodies can derive from multiple species. For example, antibodies include rodent (such as mouse and rat), rabbit, sheep, camel, and human antibodies. Antibodies can also include chimeric antibodies, which join variable regions from one species to constant regions from another species.
  • antibodies can be humanized, that is constructed by recombinant DNA technology to produce immunoglobulins which have human framework regions from one species combined with complementarity determining regions (CDR's) from a another species' immunoglobulin.
  • the antibody can be monoclonal or polyclonal.
  • Antibodies can be divided into isotypes (IgA, IgG, IgM, IgD, IgE, IgGI , lgG2, lgG3, lgG4, IgAI , lgA2, IgMI , lgM2)
  • antibody refers to an intact antibody, or a fragment of an antibody that competes with the intact antibody for antigen binding.
  • antibody fragments are produced by recombinant DNA techniques.
  • antibody fragments are produced by enzymatic or chemical cleavage of intact antibodies.
  • Exemplary antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, and scFv.
  • Exemplary antibody fragments also include, but are not limited to, domain antibodies, nanobodies, minibodies ((scFv- C.sub.H3).sub.2), maxibodies ((scFv-C.sub.H2-C. sub. H3).sub.2), diabodies (noncovalent dimer of scFv).
  • Antigen presenting cell An antigen-presenting cell (APC) as used herein is a cell that displays foreign antigen complexed with MHC on its surface.
  • Antigenic peptide Used interchangeably with binding peptide. Any peptide molecule that is bound or able to bind into the binding groove of either MHC class 1 or MHC class 2.
  • Aptamer the term aptamer as used herein is defined as oligonucleic acid or peptide molecules that bind a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist. Aptamers can be divided into DNA aptamers, RNA aptamers and peptide aptamers.
  • Avidin as used herein is a glycoprotein found in the egg white and tissues of birds, reptiles and amphibians. It contains four identical subunits having a combined mass of 67,000-68,000 daltons. Each subunit consists of 128 amino acids and binds one molecule of biotin.
  • a biologically active molecule is a molecule having itself a biological activity/effect or is able to induce a biological activity/effect when administered to a biological system.
  • Biologically active molecules include adjuvants, immune targets (e.g. antigens), enzymes, regulators of receptor activity, receptor ligands, immune potentiators, drugs, toxins, cytotoxic molecules, co-receptors, proteins and peptides in general, sugar moieties, lipid groups, nucleic acids including siRNA, nanoparticles, and small molecules.
  • Bioluminescent is the production and emission of light by a living organism as the result of a chemical reaction during which chemical energy is converted to light energy.
  • Biotin as used herein, is also known as vitamin H or B 7 .
  • Niotin has the chemical formula Ci 0 H 16 N 2 O 3 S.
  • bispecific antibodies are defined as monoclonal, preferably but not limited to human or humanized, antibodies that have binding specificities for at least two different antigens.
  • the antibody can also be trispecific or multispecific.
  • a carrier as used herin can be any type of molecule that is directly or indirectly associated with the MHC peptide complex.
  • a carrier will typically refer to a functionalized polymer (e.g. dextran) that is capable of reacting with MHC-peptide complexes, thus covalently attaching the MHC-peptide complex to the carrier, or that is capable of reacting with scaffold molecules (e.g. streptavidin), thus covalently attaching streptavidin to the carrier; the streptavidin then may bind MHC-peptide complexes.
  • scaffold molecules e.g. streptavidin
  • Chelating chemical compound is the process of reversible bindingof a ligand to a metal ion, forming a metal complex.
  • Chemiluminescent is the emission of light (luminescence) without emission of heat as the result of a chemical reaction.
  • Chromophore A chromophore, as used herein, is the part of a visibly coloured molecule responsible for light absorption over a range of wavelengths thus giving rise to the colour. By extension the term can be applied to uv or ir absorbing parts of molecules.
  • CMV Cytomegalovirus
  • Coiled-coil polypeptide Used interchangeably with coiled-coil peptide and coiled-coil structure.
  • the term coiled-coil polypeptide as used herein is a structural motif in proteins, in which 2-7 alpha-helices are coiled together like the strands of a rope
  • Covalent binding is used herein to describe a form of chemical bonding that is characterized by the sharing of pairs of electrons between atoms. Attraction-to-repulsion stability that forms between atoms when they share electrons is known as covalent bonding.
  • Crosslinking is the process of chemically joining two or more molecules by a covalent bond.
  • Crosslinking reagents contain reactive ends to specific functional groups (primary amines, sulfhydryls, etc.) on proteins or other molecules.
  • Diagnosis The act or process of identifying or determining the nature and cause of a disease or injury through evaluation
  • Diabodies refers to small antibody fragments with two antigen- binding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL).
  • VH heavy-chain variable domain
  • VL light-chain variable domain
  • Dendritic cell The term dendritic cell as used herein is a type of immune cells. Their main function is to process antigen material and present it on the surface to other cells of the immune system, thus functioning as antigen-presenting cells.
  • Detection In this invention detection means any method capable of measuringen one molecule bound to anoher molecule.
  • the molecules are typically proteins but can be any type of molecule
  • Dextran the term dextran as used herein is is a complex, branched polysaccharide made of many glucose molecules joined into chains of varying lengths.
  • the straight chain consists of ⁇ 1 ->6 glycosidic linkages between glucose molecules, while branches begin from ⁇ 1 ->3 linkages (and in some cases, ⁇ 1 ->2 and ⁇ 1 ->4 linkages as well).
  • Direct detection of T cells is used herein interchangeably with direct detection of TCR and direct detection of T cell receptor.
  • direct detection of T cells is detection directly of the binding interaction between a specific T cell receptor and a MHC multimer.
  • DNA duplex As used herein is a polymer of simple units called nucleotides, with a backbone made of sugars and phosphate atoms joined by ester bonds. Attached to each sugar is one of four types of molecules called bases.
  • DNA duplex In living organisms, DNA does not usually exist as a single molecule, but instead as a tightly-associated pair of molecules. These two long strands entwine like vines, in the shape of a double helix.
  • Electrophilic is a reagent attracted to electrons that participates in a chemical reaction by accepting an electron pair in order to bond to a nucleophile.
  • Enzyme label involves a detection method comprising a reaction catalysed by an enzyme.
  • Epitope-focused antibody Antibodies also include epitope-focused antibodies, which have at least one minimal essential binding specificity determinant from a heavy chain or light chain CDR3 from a reference antibody, methods for making such epitope- focused antibodies are described in U.S. patent application Ser. No. 1 1/040,159, which is incorporated herein by reference in its entirety.
  • Flow cytomerty The analysis of single cells using a flow cytometer.
  • Flow cytometer Instrument that measures cell size, granularity and flourescence due to bound fluorescent marker molecules as single cells pass in a stream past photodectors. A flow cytomter carry out the measurements and/or sorting of individual cells.
  • Fluorescent the term fluorescent as used herein is to have the ability to emit light of a certain wavelength when activated by light of another wavelength.
  • Fluorochromes is any fluorescent compound used as a dye to mark e.g. protein with a fluorescent label.
  • Fluorophore A fluorophore, as used herein, is a component of a molecule which causes a molecule to be fluorescent.
  • folding means in vitro or in vivo folding of proteins in a tertiery structure.
  • Fusion antibody refers to a molecule in which an antibody is fused to a non-antibody polypeptide at the N- or C-terminus of the antibody polypeptide.
  • Glycosylation is the process or result of addition of saccharides to proteins and lipids.
  • Hapten A residue on a molecule for which there is a specific molecule that can bind, e.g. an antibody.
  • Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells.
  • IgG IgG as used herein is a monomeric immunoglobulin, built of two heavy chains and two light chains. Each molecule has two antigen binding sites.
  • Isolated antibody The term "isolated” antibody as used herein is an antibody which has been identified and separated and/or recovered from a component of its natural environment.
  • Immunoconjugates comprising an antibody or a MHC-peptide complex conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
  • Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
  • diphtheria A chain nonbinding active fragments of diphtheria toxin
  • exotoxin A chain from Pseudomonas aeruginosa
  • ricin A chain abrin A chain
  • modeccin A chain alpha-
  • Conjugates of the antibody or MHC-peptide complex and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as N-succinimidyl-3-(2-pyridyldithiol) propionate (
  • Immune monitoring of the present invention refers to testing of immune status in the diagnosis and therapy of diseases like but not limited to cancer, immunoproliferative and immunodeficiency disorders, autoimmune abnormalities, and infectious diseases. It also refers to testing of immune status before, during and after vaccination and transplantation procedures.
  • Immune monitoring process a series of one or more immune monitoring analysis lmmuno profiling: lmmuno profiling as used herein defines the profiling of an individual's antigen-specific T-cell repertoire
  • Indirect detection of T cells Indirect detection of T cells is used interchangeably herein with Indirect detection of TCR and indirect detection of T cell receptor.
  • indirect detection of T cells is detection of the binding interaction between a specific T cell receptor and a MHC multimer by measurement of the effect of the binding interaction.
  • ionophore is a lipid-soluble molecule usually synthesized by microorganisms capable of transporting ions.
  • Label herein is used interchangeable with labeling molecule. Label as described herein is an identifiable substance that is detectable in an assay and that can be attached to a molecule creating a labeled molecule. The behavior of the labeled molecule can then be studied.
  • Labelling herein means attachment of a label to a molecule.
  • Lanthanide as used herein, series comprises the 15 elements with atomic numbers 57 through 71 , from lanthanum to lutetium.
  • Linker molecule Linker molecule and linker is used interchangeable herein.
  • a linker molecule is a molecule that covalently or non-covalently connects two or more molecules, thereby creating a larger complex consisting of all molecules including the linker molecule.
  • Liposomes The term liposomes as used herein is defined as a spherical vesicle with a membrane composed of a phospholipid and cholesterol bilayer. Liposomes, usually but not by definition, contain a core of aqueous solution; lipid spheres that contain no aqueous material are called micelles.
  • Immunoliposomes The antibodies or MHC-peptide complexes disclosed herein can also be formulated as immunoliposomes.
  • Liposomes comprising the antibody or MHC- peptide complexes are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.
  • Particularly useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG-PE).
  • Marker is used interchangeably with marker molecule herein.
  • a marker is molecule that specifically associates covalently or non-covalently with a molecule belonging to or associated with an entity.
  • MHC Denotes the major histocompatibility complex.
  • MHC Denotes the major histocompatibility complex.
  • MHC I is used interchangeably herein with MHC class I and denotes the major histocompatibility complex class I.
  • MHC Il is used interchangeably herein with MHC class Il and denotes the major histocompatibility complex class I.
  • MHC molecule a MHC molecule as used everywhere herein is defined as any MHC class I molecule or MHC class Il molecule as defined herein.
  • MHC Class I molecule as used everywhere herein is used interchangeably with MHC I molecule and is defined as a molecule which comprises 1 -3 subunits, including a MHC I heavy chain, a MHC I heavy chain combined with a MHC I beta2microglobulin chain, a MHC I heavy chain combined with MHC I beta2microglobulin chain through a flexible linker, a MHC I heavy chain combined with an antigenic peptide, a MHC I heavy chain combined with an antigenic peptide through a linker, a MHC I heavy chain/ MHC I beta2microglobulin dimer combined with an antigenic peptide, and a MHC I heavy chain/ MHC I beta2microglobulin dimer combined with an antigenic peptide through a flexible linker to the heavy chain or beta2microglobulin.
  • MHC complex is herein used interchangeably with MHC-peptide complex, and defines any MHC I and/or MHC Il molecule combined with antigenic peptide unless it is specified that the MHC complex is empty, i.e. is not complexed with antigenic peptide
  • MHC Class I like molecules include CD1d, HLA E, HLA G, HLA F, HLA H, MIC A, MIC B, ULBP-1 , ULBP-2, and ULBP-3.
  • MHC Class Il molecule as used everywhere herein is used interchangeably with MHC Il molecule and is defined as a molecule which comprises 2-3 subunits including a MHC Il alpha-chain and a MHC Il beta-chain (i.e. a MHC Il alpha/beta-dimer), an MHC Il alpha/beta dimer with an antigenic peptide, and an MHC Il alpha/beta dimer combined with an antigenic peptide through a flexible linker to the MHC Il alpha or MHC Il beta chain, a MHC Il alpha/beta dimer combined through an interaction by affinity tags e.g.
  • MHC Il alpha/beta dimer combined through an interaction by affinity tags e.g. jun-fos and further combined with an antigenic peptide through a flexible linker to the MHC Il alpha or MHC Il beta chain.
  • the MHC Il molecule chains can be changed by substitution of single or by cohorts of native amino acids, or by inserts, or deletions to enhance or impair the functions attributed to said molecule.
  • the "MHC Class Il molecule" can comprise only 1 subunit or 2 subunits if antigenic peptide also. Included.
  • MHC Class Il like molecules include HLA DM, HLA DO, I-A beta2, and I-E beta2.
  • a "peptide free MHC Class I molecule” is used interchangeably herein with "peptide free MHC I molecule” and as used everywhere herein is meant to be a MHC Class I molecule as defined above with no peptide.
  • a "peptide free MHC Class Il molecule” is used interchangeably herein with "peptide free MHC Il molecule” and as used everywhere herein is meant to be a MHC Class Il molecule as defined above with no peptide.
  • Such peptide free MHC Class I and Il molecules are also called "empty" MHC Class I and Il molecules.
  • the MHC molecule may suitably be a vertebrate MHC molecule such as a human, a mouse, a rat, a porcine, a bovine or an avian MHC molecule.
  • a vertebrate MHC molecule such as a human, a mouse, a rat, a porcine, a bovine or an avian MHC molecule.
  • Such MHC complexes from different species have different names. E.g. in humans, MHC complexes are denoted HLA. The person skilled in the art will readily know the name of the MHC complexes from various species.
  • MHC molecule is intended to include all alleles.
  • HLA A, HLA B, HLA C, HLA D, HLA E, HLA F, HLA G, HLA H, HLA DR, HLA DQ and HLA DP alleles are of interestshall be included, and in the mouse system, H-2 alleles are of interestshall be included.
  • RT1 -alleles in the porcine system SLA-alleles, in the bovine system BoLA, in the avian system e.g. chicken-B alleles, are of interestshall be included.
  • MHC complexes and “MHC constructs” are used interchangeably herein.
  • MHC complexes and “MHC multimers” as used herein are meant such complexes and multimers thereof, which are capable of performing at least one of the functions attributed to said complex or multimer.
  • the terms include both classical and non-classical MHC complexes.
  • the meaning of “classical” and “non-classical” in connection with MHC complexes is well known to the person skilled in the art.
  • Non- classical MHC complexes are subgroups of MHC-like complexes.
  • MHC complex includes MHC Class I molecules, MHC Class Il molecules, as well as MHC- like molecules (both Class I and Class II), including the subgroup non-classical MHC Class I and Class Il molecules.
  • MHC multimer The terms MHC multimer, MHC-multimer, MHCmer and MHC'mer herein are used interchangeably, to denote a complex comprising more than one MHC- peptide complexes, held together by covalent or non-covalent bonds.
  • Monoclonal antibodies are antibodies that are identical because they were produced by one type of immune cell and are all clones of a single parent cell.
  • Monovalent antibodies The antibodies in the present invention can be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly, Fab fragments, can be accomplished using routine techniques known in the art.
  • a multimerization domain is a molecule, a complex of molecules, or a solid support, to which one or more MHC or MHC-peptide complexes can be attached.
  • a multimerization domain consist of one or more carriers and/or one or more scaffolds and may also contain one or more linkers connecting carrier to scaffold, carrier to carrier, scaffold to scaffold.
  • the multimerization domain may also contain one or more linkers that can be used for attachment of MHC complexes and/or other molecules to the multimerization domain.
  • Multimerization domains thus include IgG, streptavidin, streptactin, micelles, cells, polymers, beads and other types of solid support, and small organic molecules carrying reactive groups or carrying chemical motifs that can bind MHC complexes and other molecules.
  • Nanobodies as used herein is a type of antibodies derived from camels, and are much smaller than traditional antibodies.
  • Neutralizing antibodies as used herein is an antibody which, on mixture with the homologous infectious agent, reduces the infectious titer.
  • NMR Nuclear magnetic resonance
  • Non-covalent The term noncovalent bond as used herein is a type of chemical bond, that does not involve the sharing of pairs of electrons, but rather involves more dispersed variations of electromagnetic interactions.
  • Nucleic acid duplex A nucleic acid is a complex, high-molecular-weight biochemical macromolecule composed of nucleotide chains that convey genetic information. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • nucleophilic a nucleophile, as used herein, is a reagent that forms a chemical bond to its reaction partner (the electrophile) by donating both bonding electrons.
  • One or more as used everywhere herein is intended to include one and a plurality.
  • a "peptide free MHC Class I molecule" as used everywhere herein is meant to be a MHC Class I molecule as defined above with no peptide.
  • a "peptide free MHC Class Il molecule" as used everywhere herein is meant to be a MHC Class Il molecule as defined above with no peptide.
  • Such peptide free MHC Class I and Il molecules are also called "empty" MHC Class I and Il molecules.
  • Pegylated pegylated, as used herein, is conjugation of Polyethylene glycol (PEG) to proteins.
  • Pentamer, MHC pentamer and pentamer MHC multimer is used interchangeable herein and refers to a MHC multimer comprising 5 MHC molecules and optionally one or more labelling compunds.
  • Peptide or protein Any molecule composed of at least two amino acids. Peptide normally refers to smaller molecules of up to around 30 amino acids and protein to larger molecules containing more amino acids. Phosphorylated; phosphorylated, as used herein, is is the addition of a phosphate (PO 4 ) group to a protein molecule or a small molecule. "A plurality" as used everywhere herein should be interpreted as two or more.
  • PNA PNA (Peptide nucleic acid) as used herein is a chemical similar to DNA or RNA. PNA is not known to occur naturally in existing life on Earth but is artificially synthesized and used in some biological research and medical treatments.
  • DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA's backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds.
  • the various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds.
  • PNAs are depicted like peptides, with the N-terminus at the first (left) position and the C-terminus at the right.
  • a plurality as used everywhere herein should be interpreted as two or more. This applies i.a. to the MHC peptide complex and the binding entity.
  • the number of MHC peptide complexes need only be limited by the capacity of the multimerization domain.
  • Polyclonal antibodies a polyclonal antibody as used herein is an antibody that is derived from different B-cell lines. They are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognising a different epitope.
  • Polymer the tern polymer as used herein is defined as a compound composed of repeating structural units, or monomers, connected by covalent chemical bonds.
  • Polypeptide Peptides are the family of short molecules formed from the linking, in a defined order, of various ⁇ -amino acids. The link between one amino acid residue and the next is an amide bond and is sometimes referred to as a peptide bond. Longer peptides are reffered to as proteins or polypeptide.
  • Polysaccharide The term polysaccharide as used herein is defined as polymers made up of many monosaccharides joined together by glycosidic linkages.
  • Radicals radicals, as used herein, are atomic or molecular species with unpaired electrons on an otherwise open shell configuration. These unpaired electrons are usually highly reactive, so radicals are likely to take part in chemical reactions.
  • Radioactivity Radioactive decay is the process in which an unstable atomic nucleus loses energy by emitting radiation in the form of particles or electromagnetic waves.
  • RNA Ribonucleic acid
  • RNA Ribonucleic acid
  • a scaffold is typically an organic molecule carrying reactive groups, capable of reacting with reactive groups on a MHC-peptide complex.
  • Particularly small organic molecules of cyclic structure e.g. functionalized cycloalkanes or functionalized aromatic ring structures
  • Scaffold and carrier are used interchangeably herein where scaffold typically refers to smaller molecules of a multimerization domain and carrier typically refers to larger molecule and/or cell like structures.
  • staining means specific or unspecific labelling of cells by binding labeled molecules to defined proteins or other structures on the surface of cells or inside cells.
  • the cells are either in suspension or part of a tissue.
  • the labeled molecules can be MHC multimers, antibodies or similar molecules capable of binding specific structures on the surface of cells.
  • Streptavidin as used herein is a tetrameric protein purified from the bacterium Streptomyces avidinii. Streptavidin is widely use in molecular biology through its extraordinarily strong affinity for biotin.
  • Sugars as used herein include monosaccharides, disaccharides, trisaccharides and the oligosaccharides - comprising 1 , 2, 3, and 4 or more monosaccharide units respectively.
  • a vaccine is an antigenic preparation used to establish immunity to a disease or illness and thereby protects or cure the body from a specific disease or illness.
  • Vaccines are either prophylactic and prevent disease or therapeutic and treat disease.
  • Vaccines may contain more than one type of antigen and is then called a combined vaccine.
  • Vaccination The introduction of vaccine into the body of human or animals for the purpose of inducing immunity.
  • the present invention in one aspect refers to a MHC monomer comprising a-b-P, or a MHC multimer comprising (a-b-P) n , wherein n > 1 ,
  • (a-b-P) is the MHC-peptide complex formed when the peptide P binds to the functional MHC protein
  • each MHC peptide complex of a MHC multimer is associated with one or more multimerization domains.
  • the peptide is in one embodiment a CMV peptide such as e.g. a peptide derived from the CMV internal matrix protein pp65.
  • MHC monomers and MHC multimers comprising one or more MHC peptide complexes of class 1 or class 2 MHC are covered by the present invention.
  • the peptide P can have a length of e.g. 8, 9 ,10, 1 1 , 12, 13, 14, 15, 16, 16-20, or 20-30 amino acid residues.
  • the peptide P can be selected from the group consisting of sequences disclosed in the electronically enclosed "Sequence Listing" and annotated consecutively (using integers) starting with SEQ ID NO 1 and ending with SEQ ID NO 9697.
  • the present invention is directed to a composition
  • a composition comprising a plurality of MHC monomers and/or MHC multimers according to the present invention, wherein the MHC multimers are identical or different, and a carrier.
  • kits comprising a MHC monomer or a MHC multimer according to the present invention, or a composition according to the present invention, and at least one additional component, such as a positive control and/or instructions for use.
  • a method for immune monitoring one or more diseases comprising monitoring of antigen-specific T cells, said method comprising the steps of
  • the present invention makes it possible to pursue different immune monitoring methods using the MHC monomers and MHC multimers according to the present invention.
  • the immune monitoring methods include e.g. flow cytometry, ELISPOT, LDA, Quantaferon and Quantaferon-like methods.
  • the MHC monomers and/or the MHC multimers can be provided as a MHC peptide complex, or the peptide and the MHC monomer and/or multimer can be provided separately.
  • recognition of TCR's can be achieved by direct or indirect detection, e.g. by using one or more of the following methods:
  • ELISPOT technique using indirect detection, e.g. by adding the antigenic peptide optionally associated with a MHC monomer or MHC multimer, followed by measurement of INF-gamma secretion from a population of cells or from individual cells.
  • Another technique involves a Quantaferon-like detection assays, e.g. by using indirect detection, e.g. by adding the antigenic peptide optionally associated with a MHC monomer or MHC multimer, followed by measurement of INF-gamma secretion from a population of cells or from individual cells.
  • indirect detection e.g. by adding the antigenic peptide optionally associated with a MHC monomer or MHC multimer, followed by measurement of INF-gamma secretion from a population of cells or from individual cells.
  • Flow cytometry offers another alternative for performing detection assays, e.g. by using direct detection (e.g. of MHC tetramers), e.g. by adding the antigenic peptide optionally associated with a MHC monomer or MHC multimer, followed by detection of a fluorescein label, thereby measuring the number of TCRs on specific T-cells.
  • direct detection e.g. of MHC tetramers
  • MHC monomer or MHC multimer e.g. by adding the antigenic peptide optionally associated with a MHC monomer or MHC multimer, followed by detection of a fluorescein label, thereby measuring the number of TCRs on specific T-cells.
  • Flow cytometry can also be used for indirect detection, e.g. by adding the antigenic peptide optionally associated with a MHC monomer or MHC multimer, followed by addition of a "cell-permeabilizing factor", and subsequent measurement of an intracellular component (e.g. INF-gamma mRNA), from individual cells or populations of cells.
  • an intracellular component e.g. INF-gamma mRNA
  • infectious diseases caused e.g. by mycobacetrium, Gram positive bacteria, Gram negative bacteria, Spirochetes, intracellular bacterium, extracellular bacterium, Borrelia, TB, CMV, HPV, Hepatitis, BK, fungal organisms and microorganisms.
  • infectious diseases caused e.g. by mycobacetrium, Gram positive bacteria, Gram negative bacteria, Spirochetes, intracellular bacterium, extracellular bacterium, Borrelia, TB, CMV, HPV, Hepatitis, BK, fungal organisms and microorganisms.
  • the diagnosis and/or monitoring of a particular disease can greatly aid in
  • T-cells Introducing said isolated or obtained T-cells into an individual to be subjected to a therapeutic treatment, wherein the individual can be the same individual or a different individual from the source individual.
  • the present invention provides:
  • a method for performing a control experiment comprising the step of counting of particles comprising the MHC multimer according to the present invention.
  • a method for performing a control experiment comprising the step of sorting of particles comprising the MHC multimer according to the present invention.
  • a method for performing a control experiment comprising the step of performing flow cytometry analysis of particles comprising the MHC multimer according to the present invention.
  • a method for performing a control experiment comprising the step of performing a immunohistochemistry analysis comprising the MHC multimer according to the present invention.
  • a method for performing a control experiment comprising the step of performing a immunocytochemistry analysis comprising the MHC multimer according to the present invention.
  • a method for performing a control experiment comprising the step of performing an ELISA analysis comprising the MHC multimer according to the present invention.
  • the method can also be performed by initially providing one or more antigenic peptide(s) P and one or more functional MHC proteins to generate a MHC-peptide complex (a-b-P); subsequently providing one or more multimerisation domain(s); and reacting the one or more MHC-peptide complexes and the one or more multimerization domain(s) to generate a MHC multimer according to the present invention.
  • the present invention is directed to novel MHC complexes optionally comprising a multimerization domain preferably comprising a carrier molecule and/or a scaffold.
  • MHC multimer comprising 2 or more MHC-peptide complexes and a multimerization domain to which the 2 or more MHC-peptide complexes are associated.
  • the MHC multimer can generally be formed by association of the 2 or more MHC-peptide complexes with the multimerization domain to which the 2 or more MHC-peptide complexes are capable of associating.
  • the multimerization domain can be a scaffold associated with one or more MHC- peptide complexes, or a carrier associated with one or more, preferably more than one, MHC-peptide complex(es), or a carrier associated with a plurality of scaffolds each associated with one or more MHC-peptide complexes, such as 2 MHC-peptide complexes, 3 MHC-peptide complexes, 4 MHC-peptide complexes, 5 MHC-peptide complexes or more than 5 MHC-peptide complexes. Accordingly, multimerization domain collectively refers to each and every of the above. It will be clear from the detailed description of the invention provided herein below when the multimerization domain refers to a scaffold or a carrier or a carrier comprising one or more scaffolds.
  • the MHC complexes can be associated with this domain either directly or via one or more binding entities.
  • the association can be covalent or non-covalent.
  • a MHC complex comprising one or more entities (a-b-P) n , wherein a and b together form a functional MHC protein capable of binding a peptide P, and wherein (a-b-P) is the MHC-peptide complex formed when the peptide P binds to the functional MHC protein, said MHC complex optionally further comprising a multimerization domain comprising a carrier molecule and/or a scaffold.
  • MHC complex refers to any MHC complex, including MHC monomers in the form of a single MHC-peptide complex and MHC multimers comprising a multimerization domain to which more than one MHC peptide complex is associated.
  • MHC multimer i.e. a plurality of MHC peptide complexes of the general composition (a-b-P) n associated with a multimerization domain
  • n is by definition more than 1 , i.e. at least 2 or more.
  • MHC multimer is used herein specifically to indicate that more than one MHC-peptide complex is associated with a multimerization domain, such as a scaffold or carrier or carrier comprising one or more scaffolds.
  • a single MHC-peptide complex can be associated with a scaffold or a carrier or a carrier comprising a scaffold and a MHC-multimer comprising 2 or more MHC-peptide complexes can be formed by association of the individual MHC-peptide complexes with a scaffold or a carrier or a carrier comprising one or more scaffolds each associated with one or more MHC-peptide complexes.
  • the association can be a covalent linkage so that each or at least some of the n MHC-peptide complexes is covalently linked to the multimerization domain, or the association can be a non-covalent association so that each or at least some of the n MHC-peptide complexes are non-covalently associated with the multimerization domain.
  • the MHC complexes of the invention may be provided in non-soluble or soluble form, depending on the intended application.
  • MHC complexes of the present invention overcome low intrinsic affinities of monomer ligands and counter receptors.
  • the MHC complexes have a large variety of applications that include targeting of high affinity receptors (e.g. hormone peptide receptors for insulin) on target cells. Taken together poly-ligand binding to target cells has numerous practical, clinical and scientifically uses.
  • the present invention provides MHC complexes which present mono-valent or multi-valent binding sites for MHC recognising cells, such as MHC complexes optionally comprising a multimerization domain, such as a scaffold or a carrier molecule, which multimerization domain have attached thereto, directly or indirectly via one or more linkers, covalently or non-covalently, one or more MHC peptide complexes.
  • MHC complexes optionally comprising a multimerization domain, such as a scaffold or a carrier molecule, which multimerization domain have attached thereto, directly or indirectly via one or more linkers, covalently or non-covalently, one or more MHC peptide complexes.
  • a multimerization domain such as a scaffold or a carrier molecule
  • linkers covalently or non-covalently, one or more MHC peptide complexes.
  • the product of the present invention is a MHC monomer or a MHC multimer as described above.
  • MHC multimers will be used interchangeably with the terms MHC'mers and MHCmers, and will include any number, (larger than one) of MHC-peptide complexes, held together in a large complex by covalent or non-covalent interactions between a multimerization domain and one or more MHC-peptide complexes, and will also include the monomeric form of the MHC-peptide complex, i.e. a MHC-peptide complex that is not attached to a multimerization domain.
  • the multimerization domain consists of one or more carriers and/or one or more scaffolds while the MHC-peptide complex consists of MHC molecule and antigenic peptide.
  • MHC-peptide complexes may be attached to the multimerization domain through one or more linkers.
  • a schematic representation of a MHC multimer is presented in figure 1.
  • MHC class 1 protein typically binds octa-, nona-, deca- or ondecamer (8-, 9-, 10,- 1 1 - mer) peptides in their peptide binding groove.
  • the individual MHC class 1 alleles have individual preferences for the peptide length within the given range.
  • MHC class 2 proteins typically bind peptides with a total length of 13-18 amino acids, comprising a 9'-mer core motif containing the important amino acid anchor residues. However the total length is not strictly defined, as opposed to most MHC class 1 molecules.
  • a given peptide is a binder it is not necessarily a functional T-cell epitope. Functionality needs to be confirmed by a functional analysis e.g. ELISPOT, CTL killing assay or flow cytometry assay.
  • the binding affinity of the peptide for the MHC molecules can for some MHC molecules be predicted in databases such as www.syfpeithi.de; http://www- bimas.cit.nih.gov/molbio/hla_bind/; www.cbs.dtu.dk/services/NetMHC/; www.cbs.dtu.dk/services/NetMHCII/
  • binding peptides The first step in the design of binding peptides is obtaining the protein's amino acid seguence.
  • genomic DNA sequences i.e. the reading frame and direction of transcription of the genes is unknown
  • the DNA sequence needs to be translated in all three reading frames in both directions leading to a total of six amino acid sequences for a given genome.
  • binding peptides can then be identified as described below.
  • the present approach must be modified accordingly, to identify peptide sequence motifs that are derived by combination of amino acid sequences derived partly from two separate introns.
  • cDNA sequences can be translated into the actual amino acid sequences to allow peptide identification. In cases where the protein sequence is known, these can directly be used to predict peptide epitopes.
  • Binding peptide sequences can be predicted from any protein sequence by either a total approach, generating binding peptide sequences for potentially any MHC allele, or by a directed approach, identifying a subset of binding peptides with certain preferred characteristics such as affinity for MHC protein, specificity for MHC protein, likelihood of being formed by proteolysis in the cell, and other important characteristics.
  • MHC-peptide complex Many parameters influence the design of the individual binding peptide, as well as the choice of the set of binding peptides to be used in a particular application. Important characteristics of the MHC-peptide complex are physical and chemical (e.g. proteolytic) stability. The relevance of these parameters must be considered for the production of the MHC-peptide complexes and the MHC multimers, as well as for their use in a given application. As an example, the stability of the MHC-peptide complex in assay buffer (e.g. PBS), in blood, or in the body can be very important for a particular application.
  • assay buffer e.g. PBS
  • MHC-peptide complexes In the interaction of the MHC-peptide complex with the TCR, a number of additional characteristics must be considered, including binding affinity and specificity for the TCR, degree of cross-talk, undesired binding or interaction with other TCRs. Finally, a number of parameters must be considered for the interaction of MHC-peptide complexes or MHC multimers with the sample or individual it is being applied to. These include immunogenicity, allergenicity, as well as side effects resulting from un-desired interaction with "wrong" T cells, including cross-talk with e.g. autoimmune diseases and un-desired interaction with other cells than antigen-specific T cells. For some applications, e.g.
  • binding peptides of that antigen are included in the application (i.e. the "total approach” for the design of binding peptides described below).
  • total approach for the design of binding peptides described below.
  • vaccines it may be adequate to include a few or just one binding peptide for each of the HLA-alleles included in the application (i.e. the "directed approach” whereby only the most potent binding peptides can be included).
  • Personalized diagnostics, therapeutics and vaccines will often fall in- between these two extremes, as it will only be necessary to include a few or just one binding peptide in e.g.
  • the MHC class 1 binding peptide prediction is done as follows using the total approach.
  • the actual protein sequence is split up into 8-, 9-, 10-, and 1 1 -mer peptide sequences. This is performed by starting at amino acid position 1 identifying the first 8- mer; then move the start position by one amino acid identifying the second 8-mer; then move the start position by one amino acid, identifying the third 8-mer. This procedure continues by moving start position by one amino acid for each round of peptide identification. Generated peptides will be amino acid position 1 -8, 2-9, 3-10 etc. This procedure can be carried out manually or by means of a software program ( Figure 2). This procedure is then repeated in an identical fashion for 9-, 10 and 1 1 -mers, respectively.
  • the directed approach identifies a preferred subset of binding peptides from the binding peptides generated in the total approach. This preferred subset is of particularly value in a given context.
  • Software programs are available that use neural networks or established binding preferences to predict the interaction of specific binding peptides with specific MHC class I alleles, and/or probability of the binding peptide in question to be generated by the proteolytic machinery of the average individual.
  • the proteolytic activitiy varies a lot among individuals, and for personalized diagnostics, treatment or vaccination it may be desirable to disregard these general proteolytic data.
  • Identified peptides can then be tested for biological relevance in functional assays such as Cytokine release assays, ELISPOT and CTL killing assays or their binding to selected MHC molecules may be determined in binding assays.
  • Prediction of good HLA class 1 peptide binders can be done at the HLA superfamily level even taking the combined action of endosolic, cytosolic and membrane bound protease activities as well as the TAP1 and TAP2 transporter specificities into consideration using the program www.cbs.dtu.dk/services/NetCTL/.
  • consensus sequences for the individual MHC allele can be used to choose a set of relevant binding peptides that will suit the "average" individual.
  • Such consensus sequences often solely consider the affinity of the binding peptide for the MHC protein; in other words, a subset of binding peptides is identified where the designed binding peptides have a high probability of forming stable MHC-peptide complexes, but where it is uncertain whether this MHC-peptide complex is of high relevance in a population, and more uncertain whether this MHC-peptide complex is of high relevance in a given individual.
  • the consensus sequence for a binding peptide is generally given by the formula
  • X1 -X2-X3-X4-....-Xn where n equals 8, 9, 10, or 1 1 , and where X represents one of the twenty naturally occurring amino acids, optionally modified as described elsewhere in this application.
  • XI -Xn can be further defined.
  • certain positions in the consensus sequence are the socalled anchor positions and the selection of useful amino acids for these positions is limited to those able to fit into the corresponding binding pockets in the HLA molecule.
  • X2 and X9 are primary anchor positions and useful amino acids at these two positions in the binding peptide are preferable limited to leucine or methionine for X2 and to valine or leucine at postion X9.
  • the primary anchor positions of peptides binding HLA-B * 08 are X3, X5 and X9 and the corresponding preferred amino acids at these positions are lysine at position X3, lysine or arginine at position X5 and leucine at position X9.
  • MHC class 2 binding peptide sequence Design of MHC class 2 binding peptide sequence.
  • a) Total approach and b) directed approach The approach to predict putative peptide binders for MHC class 2 can be done in a similar way as described for MHC class 1 binding peptide prediction above.
  • the change is the different size of the peptides, which is preferably 13-16 amino acids long for MHC class 2.
  • the putative binding peptide sequences only describe the central part of the peptide including the 9-mer core peptide; in other words, the peptide sequences shown represent the core of the binding peptide with a few important flanking amino acids, which in some cases may be of considerably length generating binding peptides longer than the 13-16 amino acids.
  • simple consensus sequences for the individual MHC allele can be used to choose a set of relevant binding peptides that will suit the "average" individual.
  • consensus sequences often solely consider the affinity of the binding peptide for the MHC protein; in other words, a subset of binding peptides is identified where the designed binding peptides have a high probability of forming stable MHC-peptide complexes, but where it is uncertain whether this MHC-peptide complex is of high relevance in a population, and more uncertain whether this MHC-peptide complex is of high relevance in a given individual.
  • the consensus sequence for the interacting core of a binding peptide is generally given by the formula X1 -X2-X3-X4-....-Xn, where n equals 9, and where X represents one of the twenty naturally occurring amino acids, optionally modified as described elsewhere in this application.
  • XI -Xn can be further defined.
  • certain positions in the consensus sequence are the socalled anchor positions and the selection of useful amino acids for these positions is limited to those able to fit into the corresponding binding pockets in the HLA molecule.
  • HLA-DRB1 * 1501 have X1 , X4 and X7 as primary anchor positions where preferred amino acids at the three positions are as follows, X1 : leucine, valine and isoleucine, X4: phenylalanine, tyrosine or isoleucine, X7: isoleucine, leucine, valine, methionine or phenylalanine.
  • MHC Il binding peptides have much more varied anchor positions than MHC I binding peptides and the number of usefull amino acids at each anchor position is much higher.
  • MHC alleles class 1 and 2
  • Figure 3 presents an updated list of the HLA class 1 alleles.
  • the frequency of the different HLA alleles varies considerably, also between different ethnic groups ( Figure 4). Thus it is of outmost importance to carefully select the MHC alleles that corresponds to the population that one wish to study.
  • binding peptides and which MHC alleles are available.
  • MHC-peptide complex or MHC multimer.
  • characteristics of binding peptides and MHC alleles are important when using the MHC-peptide complex or MHC-multimer for different purposes.
  • a first preferred embodiment employs binding peptides of particularly high affinity for the MHC proteins. This may be done in order to increase the stability of the MHC- peptide complex. A higher affinity of the binding peptide for the MHC proteins may in some instances also result in increased rigidity of the MHC-peptide complex, which in turn often will result in higher affinity and/or specificity of the MHC-peptide complex for the T-cell receptor. A higher affinity and specificity will in turn have consequences for the immunogenicity and allergenicity, as well as possible side-effects of the MHC- peptide complex in e.g. the body.
  • Binding peptides of particularly high affinity for the MHC proteins may be identified by several means, including the following.
  • binding peptides that have most frequently been associated with MHC proteins typically will represent high-affinity binding peptides.
  • the identification of binding peptides with particularly high-affinity may involve enrichment of binding peptides, e.g. incubation of candidate peptides with immobilized MHC molecules, removal of non-binding peptides by e.g. washing, elution of binding peptides.
  • This pool of peptides enriched for binding to the chosen MHC molecules may then be identified e.g. by mass spectrometry or HPLC and amino acid sequencing or the pool can be further enriched by another round of incubation with immobilized MHC.
  • Candidate binding peptides may be compared to consensus sequences for the binding to a specific MHC allele.
  • the consensus 8'mer sequence may be given by the sequence "X1 -X2-X3-X4-X5-X6-X7-X8", where each of the X1 -X8 amino acids can be chosen from a specific subset of amino acids, as described above.
  • binding peptides that correlate the best with the consensus sequence are expected to have particularly high affinity for the MHC allele in question.
  • a second prefered embodiment employs binding peptides with medium affinity for the MHC molecule.
  • a medium affinity of the peptide for the MHC protein will often lead to lower physical and chemical stability of the MHC-peptide complex, which can be an advantage for certain applications.
  • it is often desirable to administer a drug on a daily basis due to convenience.
  • An MHC-peptide complex-based drug with high stability in the body would not allow this.
  • a binding peptide with medium or low affinity for the MHC protein can be an advantage for such applications, since these functional MHC-peptide molecules will be cleared more rapidly from the body due to their lower stability.
  • MHC-peptide complexes containing binding peptides from different strains of a given species
  • a medium or low affinity of the binding peptide for the MHC protein can be an advantage.
  • these MHC-peptide complexes are often more structurally flexible, allowing the MHC-peptide complexes to interact with several structurally related TCRs.
  • the affinity of a given peptide for a MHC protein should only be considered a guideline to its real affinity. Moreover, the affinity can vary a lot depending on the conditions in the o
  • the affinity in blood may be very different from the affinity in a biochemical assay.
  • the flexibility of the MHC-peptide complex can sometimes be an important parameter for overall avidity.
  • the affinity of the binding peptide for MHC protein is preferably high, medium, or low; the physical and/or chemical stability of the MHC-peptide complex is preferably high, medium or low; the binding peptide is preferably a very common or very rare epitope in a given population; etc.
  • binding peptides generated by the total approach have important applications.
  • relevant MHC multimers that suit the different applications with regard to e.g. personalized or general targeting, or with regard to affinity, avidity, specificity, immunogenicity, stimulatory efficiency, or stability, one must be able to choose from the whole set of binding peptides generated by the total approach
  • binding peptides designed by the total approach, homologous peptides and peptides that have been modified in the amino acid side chains or in the backbone can be used as binding peptides.
  • MHC peptide sequences may arise from the existence of multiple strongly homologous alleles, from small insertions, deletions, inversions or substitutions. If they are sufficiently homologous to peptides derived by the total approach, i.e. have an amino acid sequence identity greater than e.g. more than 90%, more than 80%, or more than 70%, or more than 60%, to one or two binding peptides derived by the total approach, they may be good candidates. Identity is often most important for the anchor residues.
  • a MHC binding peptide may be of split- or combinatorial epitope origin i.e. formed by linkage of peptide fragments derived from two different peptide fragments and/or proteins.
  • Such peptides can be the result of either genetic recombination on the DNA level or due to peptide fragment association during the complex break down of proteins during protein turnover. Possibly it could also be the result of faulty reactions during protein synthesis i.e. caused by some kind of mixed RNA handling.
  • a kind of combinatorial peptide epitope can also be seen if a portion of a longer peptide make a loop out leaving only the terminal parts of the peptide bound in the groove.
  • Peptides having un-common amino acids such as selenocysteine and pyrrolysine, may be bound in the MHC groove as well.
  • Artificial amino acids e.g. having the isomeric D-form may also make up isomeric D-peptides that can bind in the binding groove of the MHC molecules.
  • Bound peptides may also contain amino acids that are chemically modified or being linked to reactive groups that can be activated to induce changes in or disrupt the peptide. Example post-translational modifications are shown below. However, chemical modifications of amino acid side chains or the peptide backbone can also be performed.
  • any of the modifications can be found individually or in combination at any position of the peptide, e.g. position 1 , 2, 3, 4, 5, 6, etc. up to n.
  • Tyrosine GFP prosthetic group Thr-Tyr-Gly sequence
  • Lysine tyrosine quinone LTQ
  • TPQ Topaquinone
  • amino acids of the antigenic peptides can also be modified in various ways dependent on the amino acid in question, or the modification can affect the amino- or carboxy-terminal end of the peptide. See table 1. Such peptide modifications are occuring naturally as the result of post tranlational processing of the parental protein. A non-exhaustive description of the major post translational modifications is given below, divided into three main types.
  • alkylation the addition of an alkyl group (e.g. methyl, ethyl).
  • Methylation the addition of a methyl group, usually at lysine or arginine residues is a type of alkylation.
  • Demethylation involves the removal of a methyl-group.
  • glycosylation the addition of a glycosyl group to either asparagine, hydroxylysine, serine, or threonine, resulting in a glycoprotein. Distinct from glycation, which is regarded as a nonenzymatic attachment of sugars.
  • heme moiety may be covalently attached hydroxylation, is any chemical process that introduces one or more hydroxyl groups (-OH) into a compound (or radical) thereby oxidizing it.
  • the principal residue to be hydroxylated is Proline.
  • the hydroxilation occurs at the C ⁇ atom, forming hydroxyproline (Hyp).
  • proline may be hydroxylated instead on its C ⁇ atom.
  • Lysine may also be hydroxylated on its C ⁇ atom, forming hydroxylysine (HyI).
  • isoprenylation the addition of an isoprenoid group (e.g. farnesol and geranylgeraniol)
  • lipoylation attachment of a lipoate functionality, as in prenylation, GPI anchor formation, myristoylation, farnesylation, geranylation
  • nucleotides or derivatives thereof may be covalently attached, as in ADP- ribosylation and flavin attachment
  • Typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine, tyrosine.
  • the N-terminal amino group and the C-terminal carboxylic acid can also be used
  • phosphatidylinositol may be covalently attached
  • phosphorylation the addition of a phosphate group, usually to serine, tyrosine, threonine or histidine
  • ISGylation the covalent linkage to the ISG15 protein (Interferon-Stimulated Gene
  • citrullination or deimination the conversion of arginine to citrulline
  • the peptide modifications can occur as modification of a single amino acid or more than one i.e. in combinations. Modifications can be present on any position within the peptide i.e. on position 1 , 2, 3, 4, 5, etc. for the entire length of the peptide.
  • binding peptides can be obtained from natural sources by enzymatic digestion or proteolysis of natural proteins or proteins derived by in vitro translation of mRNA. Binding peptides may also be eluted from the MHC binding groove.
  • peptides can be produced recombinantly by transfected cells either as monomeric antigenic peptides or as multimeric (concatemeric) antigenic peptides.
  • the Multimeric antigenic peptides are cleaved to form monomeric antigenic peptides before binding to MHC protein.
  • Binding peptides may also constitute a part of a bigger recombinant protein e.g. consisting of,
  • Peptide-linker-MHC class 1 heavy chain the heavy chain being full length or truncated. Most importantly the truncated class I heavy chain will consist of the extracellular part i.e the ⁇ i , ⁇ 2, and ⁇ domains.
  • the heavy chain fragment may also only contain the ⁇ 1 and ⁇ 2 domains, or ⁇ 1 domain alone, or any fragment or full length ⁇ 2m or heavy chain attached to a designer domain(s) or protein fragment(s).
  • the recombinant construction can consist of,
  • Peptide-linker-MHC class 2 ⁇ -chain-linker-MHC class 2 ⁇ -chain both chains can be full length or truncated, truncation may involve, omission of ⁇ - and/or ⁇ -chain intermembrane domain, or omission of ⁇ - and/or ⁇ -chain intermembrane plus cytoplasmic domains.
  • MHC class 2 part of the construction may consist of fused domains from NH2-terminal, MHC class 2 ⁇ 1 domain-MHC class 2 ⁇ 1 domain-constant oc3 of MHC class 1 , or alternatively of fused domains from NH2-terminal, MHC class 2 ⁇ 1 domain-MHC class 2 ⁇ 1 domain-constant ⁇ 3 of MHC class 1 . In both cases ⁇ 2m will be associated non-covalently in the folded MHC complex.
  • ⁇ 2m can also be covalently associated in the folded MHC class 2 complex if the following constructs are used from NH2 terminal, MHC class 2 ⁇ 1domain-MHC class 2 ⁇ 1 domain-constant ⁇ 3 of MHC class 1 -linker- ⁇ 2m, or alternatively of fused domains from NH2-terminal, MHC class 2 ⁇ 1 domain-MHC class 2 ⁇ 1 domain-constant ⁇ 3 of MHC class 1 -linker- ⁇ 2m; the construct may also consist of any of the above MHC class 2 constructs with added designer domain(s) or sequence(s).
  • MHC binding peptide may also be chemically synthesized by solid phase or fluid phase synthesis, according to standard protocols.
  • the protocol for the synthesis of the full-length antigen on solid support is modified by adding a partial cleavage step after each coupling of an amino acid.
  • the starting point for the synthesis is a solid support to which has been attached a cleavable linker.
  • the first amino acid X1 (corresponding to the C-terminal end of the antigen) is added and a coupling reaction performed.
  • the solid support now carries the molecule "Iinker-X1 ".
  • a fraction (e.g. 10%) of the cleavable linkers are now cleaved, to release into solution X1.
  • the supernatant is transferred to a collection container. Additional solid support carrying a cleavable linker is added, e.g. corresponding to 10% of the initial amount of solid support.
  • the second amino acid X2 is added and coupled to X1 or the cleavable linker, to form on solid support the molecules "Iinker-X2" and "Iinker-X1 -X2".
  • a fraction e.g. 10%
  • the cleavable linker is cleaved, to release into solution X2 and X1 -X2.
  • the supernatant is collected into the collection container, which therefore now contains X1 , X2, and X1 -X2.
  • Additional solid support carrying a cleavable linker is added, e.g. corresponding to 10% of the initial amount of solid support.
  • the third amino acid X3 is added and coupled to X2 or the cleavable linker, to form on solid support the molecules "Iinker-X3", “Iinker-X2-X3” and "Iinker-X1 -X2-X3".
  • a fraction e.g. 10%
  • the cleavable linker is cleaved, to release into solution X3, X2-X3 and X1 -X2-X3.
  • the supernatant is collected into the collection container, which therefore now contains X1 , X2, X3, X1 -X2, X2-X3 and X1 -X2-X3.
  • Additional solid support carrying a cleavable linker is added, e.g.
  • the collection container will now contain a large number of peptides of different length and sequence. In the present example where a 10% partial cleavage was employed, a large fraction of the peptides will be 8'-mers, 9'- mers, 10'-mers and 1 1 '-mers, corresponding to class I antigenic peptides.
  • the 8'-mers will consist of the sequences X1 -X2- X3-X4-X5-X6-X7-X8, X2-X3-X4-X5-X6-X7-X8-X9, , X93-X94-X95-X96-X97-X98-
  • the used (inactivated) linkers on solid support can be regenerated, in order to maintain a high fraction of linkers available for synthesis.
  • the collection of antigenic peptides can be used as a pool for e.g. the display by APCs to stimulate CTLs in ELISPOT assays, or the antigenic peptides may be mixed with one or more MHC alleles, to form a large number of different MHC-peptide complexes which can e.g. be used to form a large number of different MHC multimers which can e.g. be used in flow cytometry experiments.
  • MHC class 1 or class 2 Loading of the peptides into the MHCmer being either MHC class 1 or class 2 can be performed in a number of ways depending on the source of the peptide and the MHC, and depending on the application. MHC class 2 molecules can in principle be loaded with peptides in similar ways as MHC class 1. However, due to complex instability the most successful approach have been to make the complexes recombinant in toto in eukaryotic cells from a gene construct encoding the following form ⁇ chain-flexible linker- ⁇ chain-flexible linker-antigenic peptide.
  • the antigenic peptide may be added to the other peptide chain(s) at different times and in different forms, as follows. a) Loading of antigenic peptide during MHC complex folding a1) Antigenic peptide is added as a free peptide MHC class I molecules are most often loaded with peptide during assembly in vitro by the individual components in a folding reaction i.e. consisting of purified recombinant heavy chain ⁇ with the purified recombinant ⁇ 2 microglobulin and a peptide or a peptide mix. a2) Antigenic peptide is part of a recombinant protein construct Alternatively the peptide to be folded into the binding groove can be encoded together with e.g. the ⁇ heavy chain or fragment hereof by a gene construct having the structure, heavy chain-flexible linker- peptide. This recombinant molecule is then folded in vitro with ⁇ 2-microglobulin.
  • Loading of desired peptide can also be made by an in vitro exchange reaction where a peptide already in place in the binding groove are being exchanged by another peptide species.
  • Peptide exchange reactions can also take place when the parent molecule is attached to other molecules, structures, surfaces, artificial or natural membranes and nano- particles.
  • Aided exchange reaction This method can be refined by making the parent construct with a peptide containing a meta-stable amino acid analog that is split by either light or chemically induction thereby leaving the parent structure free for access of the desired peptide in the binding groove.
  • Display by in vivo loading Loading of MHC class I and Il molecules expressed on the cell surface with the desired peptides can be performed by an exchange reaction.
  • cells can be transfected by the peptides themselves or by the mother proteins that are then being processed leading to an in vivo analogous situation where the peptides are bound in the groove during the natural cause of MHC expression by the transfected cells.
  • professional antigen presenting cells e.g. dendritic cells, macrophages,
  • the proteins and peptides can be taken up by the cells themselves by phagocytosis and then bound to the MHC complexes the natural way and expressed on the cell surface in the correct MHC context.
  • the MHC multimer is between 50,000 Da and 1 ,000,000 Da, such as from 50,000 Da to 980,000; for example from 50,000 Da to 960,000; such as from 50,000 Da to 940,000; for example from 50,000 Da to 920,000; such as from 50,000 Da to 900,000; for example from 50,000 Da to 880,000; such as from 50,000 Da to 860,000; for example from 50,000 Da to 840,000; such as from 50,000 Da to 50,000 Da to 50,000 Da to 980,000; 50,000 Da to 840,000; such as from 50,000 Da to
  • 820,000 for example from 50,000 Da to 800,000; such as from 50,000 Da to 780,000; for example from 50,000 Da to 760,000; such as from 50,000 Da to 740,000; for example from 50,000 Da to 720,000; such as from 50,000 Da to 700,000; for example from 50,000 Da to 680,000; such as from 50,000 Da to 660,000; for example from 50,000 Da to 640,000; such as from 50,000 Da to 620,000; for example from 50,000 Da to 600,000; such as from 50,000 Da to 580,000; for example from 50,000 Da to 560,000; such as from 50,000 Da to 540,000; for example from 50,000 Da to 520,000; such as from 50,000 Da to 500,000; for example from 50,000 Da to 480,000; such as from 50,000 Da to 460,000; for example from 50,000 Da to 440,000; such as from 50,000 Da to 420,000; for example from 50,000 Da to 400,000; such as from 50,000 Da to 380,000; for example from 50,000 Da to 360,000; such as from 50,000 Da to 340,000; for example from 50,000 Da to 320,000
  • the MHC multimer is between 1 ,000,000 Da and 3,000,000 Da, such as from 1 ,000,000 Da to 2,800,000; for example from 1 ,000,000 Da to 2,600,000; such as from 1 ,000,000 Da to 2,400,000; for example from 1 ,000,000 Da to 2,200,000; such as from 1 ,000,000 Da to 2,000,000; for example from 1 ,000,000 Da to 1 ,800,000; such as from 1 ,000,000 Da to 1 ,600,000; for example from 1 ,000,000 Da to 1 ,400,000.
  • n indicates the number of MHC complexes comprised in the multimer:
  • Dimers, multimerization can be based on IgG scaffold, streptavidin with two MHCs, coiled-coil dimerization e.g. Fos.Jun dimerization
  • Trimers, multimerization can be based on streptavidin as scaffold with three MHCs, TNFalpha-MHC hybrids, triplex DNA-MHC konjugates or other trimer structures
  • Tetramers, multimerization can be based on streptavidin with all four binding sites occupied by MHC molecules or based on dimeric IgA
  • Dextramers as multimerization domain polymers such as polypeptide, polysaccharides and Dextrans can be used.
  • Multimerization can make use of dendritic cells (DC), antigen-presenting cells (APC), micelles, liposomes, beads, surfaces e.g. microtiterplate, tubes, microarray devices, micro-fluidic systems I) 1 ⁇ n, n in billions or trillions or higher, multimerization take place on beads, and surfaces e.g. microtiterplate, tubes, microarray devices, micro-fluidic systems
  • Any of the three components of a MHC complex can be of any of the below mentioned origins.
  • the list is non- exhaustive. A complete list would encompass all Chordate species.
  • origin is meant that the sequence is identical or highly homologous to a naturally occurring sequence of the specific species.
  • MHC multimers can be generated by first expressing and purifying the individual protein components of the MHC protein, and then combining the MHC protein components and the peptide, to form the MHC-peptide complex. Then an appropriate number of MHC-peptide complexes are linked together by covalent or non- covalent bonds to a multimerization domain.
  • reactive groups of the multimerization domain e.g. vinyl sulfone functionalities on a dextran polymer
  • reactive groups on the MHC protein e.g. amino groups on the protein surface
  • non-covalent interaction between a part of the MHC protein e.g. a biotinylated peptide component
  • the multimerization domain e.g. four binding sites for biotin on the strepavidin tetrameric protein.
  • the MHC multimer can be formed by the non-covalent association of amino acid helices fused to one component of the MHC protein, to form a pentameric MHC multimer, held together by five helices in a coiled-coil structure making up the multimerization domain.
  • acylation such as amide formation, pyrazolone formation, isoxazolone formation; alkylation; vinylation; disulfide formation
  • addition to carbon-hetero multiple bonds e.g. alkene formation by reaction of phosphonates with aldehydes or ketones; arylation; alkylation of arenes/hetarenes by reaction with alkyl boronates or enolethers
  • nucleophilic substitution using activation of nucleophiles e.g. condensations; alkylation of aliphatic halides or tosylates with enolethers or enamines
  • cycloadditions e.g. acylation such as amide formation, pyrazolone formation, isoxazolone formation; alkylation; vinylation; disulfide formation
  • carbon-hetero multiple bonds e.g. alkene formation by reaction of phosphonates with aldehydes or ketones; arylation; alkylation of arenes/
  • Appropriate molecules capable of providing non-covalent interactions between the multimerization domain and the MHC-peptide complex, involve the following molecule pairs and molecules: streptavidin/biotin, avidin/biotin, antibody/antigen, DNA/DNA,
  • DNA/PNA, DNA/RNA, PNA/PNA, LNA/DNA leucine zipper e.g. Fos/Jun, IgG dimeric protein, IgM multivalent protein, acid/base coiled-coil helices, chelate/metal ion-bound chelate, streptavidin (SA) and avidin and derivatives thereof, biotin, immunoglobulins, antibodies (monoclonal, polyclonal, and recombinant), antibody fragments and derivatives thereof, leucine zipper domain of AP-1 (jun and fos), hexa-his (metal chelate moiety), hexa-hat GST (glutathione S-transferase) glutathione affinity, Calmodulin-binding peptide (CBP), Strep-tag, Cellulose Binding Domain, Maltose Binding Protein, S-Peptide Tag, Chitin Binding Tag, Immuno-reactive Epitopes, Epitope Tags, E2
  • binding entity can be an "anti-tag".
  • anti-tag is meant an antibody binding to the tag and any other molecule capable of binding to such tag.
  • MHC multimers When employing MHC multimers for diagnostic purposes, it is preferable to use a MHC allele that corresponds to the tissue type of the person or animal to be diagnosed.
  • a MHC allele Once the MHC allele has been chosen, a peptide derived from the antigenic protein may be chosen. The choice will depend on factors such as known or expected binding affinity of the MHC protein and the various possible peptide fragments that may be derived from the full sequence of the antigenic peptide, and will depend on the expected or known binding affinity and specificity of the MHC-peptide complex for the TCR.
  • the affinity of the peptide for the MHC molecule, and the affinity and specificity of the MHC-peptide complex for the TCR should be high.
  • stability it must be decided whether stability is important. For some applications low stability may be an advantage, e.g. when a short-term effect is desired; in other instances, a long-term effect is desired and MHC multimers of high stability is desired. Stabilities of the MHC protein and of the MHC-peptide complex may be modified as described elsewhere herein. Finally, modifications to the protein structure may be advantageous for some diagnostics purposes, because of e.g. increased stability, while for vaccine purposes modifications to the MHC protein structure may induce undesired allergenic responses.
  • MHC class I heavy chain (HC) and ⁇ 2-mircroglobulin ( ⁇ 2m) can be obtained from a variety of sources.
  • Natural sources by means of purification from eukaryotic cells naturally expressing the MHC class 1 or ⁇ 2m molecules in question.
  • the molecules can be obtained by recombinant means e.g. using. a. in vitro translation of mRNA obtained from cells naturally expressing the MHC or ⁇ 2m molecules in question b. by expression and purification of HC and/or ⁇ 2m gene transfected cells of mammalian, yeast, bacterial or other origin. This last method will normally be the method of choice.
  • the genetic material used for transfection/transformation can be: i.
  • MHC class 1 heavy chain fragments consisting of, the complete chain minus the intramembrane domain, a chain consisting of only the extracellular ⁇ 1 and ⁇ 2 class 1 heavy chain domains, or any of the mentioned ⁇ 2m and heavy chain fragments containing modified or added designer domain(s) or sequence(s).
  • MHC class 2 ⁇ - and ⁇ -chains can be obtained from a variety of sources: a) Natural sources by means of purification from eukaryotic cells naturally expressing the MHC class 2 molecules in question. b) By recombinant means e.g. using: a. in vitro translation of mRNA obtained from cells naturally expressing the MHC class 2 molecules in question b. By purification from MHC class 2 gene transfected cells of mammalian, yeast, bacterial or other origin. This last method will normally be the method of choice.
  • the genetic material used for transfection/transformation can be i. of natural origin isolated from cells, tissue or organisms ii. of synthetical origin i.e. synthetic genes identical to the natural
  • DNA sequence or it could be modified to introduce molecular changes or to ease recombinant expression.
  • the genetic material can encode all or only a fragment of MHC class 2 ⁇ - and ⁇ -chains.
  • MHC class 2 ⁇ - and ⁇ -chain_fragments consisting of, the complete ⁇ - and 3-chains minus the intramembrane domains of either or both chains; and o> and 3-chains consisting of only the extracellular domains of either or both, i.e ⁇ 1 plus ⁇ 2 and ⁇ 1 plus ⁇ 2 domains, respectively.
  • the genetic material can be modified to encode the interesting MHC class 2 molecule fragments consisting of domains starting from the amino terminal in consecutive order, MHC class 2 ⁇ 1 plus MHC class 2 ⁇ 1 plus MHC class 1 ⁇ 3 domains or in alternative order, MHC class 2 ⁇ 1 plus MHC class 2 ⁇ 1 plus MHC class 1 ⁇ 3 domains.
  • the gentic material can encode any of the above mentioned MHC class 2 ⁇ - and ⁇ -chain molecules or fragments containing modified or added designer domain(s) or sequence(s).
  • the MHC material may also be of exclusively synthetic origin manufactured by solid phase protein synthesis. Any of the above mentioned molecules can be made this way.
  • MHC I and MHC Il complexes modified in any way as described above, can bind TCR. Modifications include mutations (substitutions, deletions or insertions of natural or non- natural amino acids, or any other organic molecule. The mutations are not limited to those that increase the stability of the MHC complex, and could be introduced anywhere in the MHC complex.
  • One example of special interest is mutations introduced in the ⁇ 3 subunit of MHC I heavy chain. The ⁇ 3-subunit interacts with CD8 molecules on the surface of T cells. To minimize binding of MHC multimer to CD8 molecules on the surface of non-specific T cells, amino acids in ⁇ 3 domain involved in the interaction with CD8 can be mutated. Such a mutation can result in altered or abrogated binding of MHC to CD8 molecules.
  • Another example of special interest is mutations in areas of the ⁇ 2-domain of MHC Il molecules responsible for binding CD4 molecules.
  • MHC complexes where the chemical modification could be introduced anywhere in the complex, e.g. a MHC complex where the peptide in the peptide-binding cleft has a dinitrophenyl group attached.
  • Modified MHC complexes could also be MHC I or MHC Il fusion proteins where the fusion protein is not necessarily more stable than the native protein.
  • This biotinylation sequence could be fused with the COOH-terminal of ⁇ 2m or the heavy chain of MHC I molecules or the COOH-terminal of either the ⁇ - chain or ⁇ -chain of MHC II.
  • other sequences capable of being enzymatically or chemically modified can be fused to the NH 2 or COOH-terminal ends of the MHC complex.
  • Classical MHC complexes are in nature embedded in the membrane.
  • a preferred embodiment includes multimers comprising a soluble form of MHC Il or I where the transmembrane and cytosolic domains of the membrane-anchored MHC complexes are removed. The removal of the membrane-anchoring parts of the molecules can influence the stability of the MHC complexes. The stability of MHC complexes is an important parameter when generating and using MHC multimers.
  • MHC I complexes consist of a single membrane-anchored heavy chain that contains the complete peptide binding groove and is stable in the soluble form when complexed with ⁇ 2m.
  • the long-term stability is dependent on the binding of peptide in the peptide- binding groove. Without a peptide in the peptide binding groove the heavy chain and ⁇ 2m tend to dissociate.
  • peptides with high affinity for binding in the peptide- binding groove will typically stabilize the soluble form of the MHC complex while peptides with low affinity for the peptide-binding groove will typically have a smaller stabilizing effect.
  • MHC Il complexes consist of two membrane-anchored chains of almost equal size. When not attached to the cell membrane the two chains tend to dissociate and are therefore not stable in the soluble form unless a high affinity peptide is bound in the peptide-binding groove or the two chains are held together in another way.
  • MHC I molecules consist of a heavy chain combined with ⁇ 2m, and a peptide of typically 8-1 1 amino acids.
  • MHC I molecules also include molecules consisting of a heavy chain and ⁇ 2m (empty MHC), or a heavy chain combined with a peptide or a truncated heavy chain comprising ⁇ 1 and ⁇ 2 subunits combined with a peptide, or a full-length or truncated heavy chain combined with a full-length or truncated ⁇ 2m chain.
  • empty MHC empty MHC
  • MHC I molecules can be produced in E. coli as recombinant proteins, purified and refolded in vitro (Garboczi et al., (1992), Proc. Natl. Acad.
  • MHC I molecules can be stabilized by introduction of one or more linkers between the individual components of the MHC I complex.
  • This could be a complex consisting of a heavy chain fused with ⁇ 2m through a linker and a soluble peptide, a heavy chain fused to ⁇ 2m through a linker, a heavy chain / ⁇ 2m dimer covalently linked to a peptide through a linker to either heavy chain or ⁇ 2m, and where there can or can not be a linker between the heavy chain and ⁇ 2m, a heavy chain fused to a peptide through a linker, or the ⁇ 1 and ⁇ 2 subunits of the heavy chain fused to a peptide through a linker.
  • each of the heavy chain, ⁇ 2m and the peptide can be truncated.
  • the linker could be a flexible linker, e.g. made of glycine and serine and e.g. between 5-20 residues long.
  • the linker could also be rigid with a defined structure, e.g. made of amino acids like glutamate, alanine, lysine, and leucine creating e.g. a more rigid structure.
  • the COOH terminus of ⁇ 2m can be covalently linked to the NH 2 terminus of the heavy chain, or the NH 2 terminus of ⁇ 2m can be linked to the COOH terminus of the heavy chain.
  • the fusion-protein can also comprise a ⁇ 2m domain, or a truncated ⁇ 2m domain, inserted into the heavy chain, to form a fusion- protein of the form "heavy chain (first part)- ⁇ 2m-heavy chain (last part)".
  • the fusion-protein can comprise a heavy chain domain, or a truncated heavy chain, inserted into the ⁇ 2m chain, to form a fusion-protein of the form
  • the COOH terminus of the peptide is preferable linked to the NH 2 terminus of ⁇ 2m but the peptide can also be linked to the COOH terminal of ⁇ 2m via its NH 2 terminus.
  • heavy chain-peptide fusion proteins it is preferred to fuse the NH 2 terminus of the heavy chain to the COOH terminus of the peptide, but the fusion can also be between the COOH terminus of the heavy chain and the NH 2 terminus of the peptide.
  • the NH 2 terminus of the heavy chain can be fused to the COOH terminus of ⁇ 2m and the NH 2 terminus of ⁇ 2m can be fused to the COOH terminus of the peptide.
  • Non-covalent stabilization by binding to an unnatural component can lead to increased stability.
  • the unnatural component can bind to both the heavy chain and the ⁇ 2m, and in this way promote the assemble of the complex, and/or stabilize the formed complex.
  • the unnatural component can bind to either ⁇ 2m or heavy chain, and in this way stabilize the polypeptide in its correct conformation, and in this way increase the affinity of the heavy chain for ⁇ 2m and/or peptide, or increase the affinity of ⁇ 2m for peptide.
  • unnatural components mean antibodies, peptides, aptamers or any other molecule with the ability to bind peptides stretches of the MHC complex.
  • Antibody is here to be understood as truncated or full-length antibodies (of isotype IgG, IgM, IgA, IgE), Fab, scFv or bi-Fab fragments or diabodies.
  • An example of special interest is an antibody binding the MHC I molecule by interaction with the heavy chain as well as ⁇ 2m.
  • the antibody can be a bispecific antibody that binds with one arm to the heavy chain and the other arm to the ⁇ 2m of the MHC complex.
  • the antibody can be monospecific, and bind at the interface between heavy chain and ⁇ 2m.
  • Another example of special interest is an antibody binding the heavy chain but only when the heavy chain is correct folded. Correct folded is here a conformation where the MHC complex is able to bind and present peptide in such a way that a restricted T cell can recognize the MHC-peptide complex and be activated.
  • This type of antibody can be an antibody like the one produced by the clone W6/32
  • One way to improve stability of a MHC I complex is to increase the affinity of the binding peptide for the MHC complex. This can be done by mutation/substitution of amino acids at relevant positions in the peptide, by chemical modifications of amino acids at relevant positions in the peptide or introduction by synthesis of non-natural amino acids at relevant positions in the peptide. Alternatively, mutations, chemical modifications, insertion of natural or non-natural amino acids or deletions could be introduced in the peptide binding cleft, i.e. in the binding pockets that accommodate peptide side chains responsible for anchoring the peptide to the peptide binding cleft.
  • reactive groups can be introduced into the antigenic peptide; before, during or upon binding of the peptide, the reactive groups can react with amino acid residues of the peptide binding cleft, thus covalently linking the peptide to the binding pocket.
  • Mutations/substitutions, chemical modifications, insertion of natural or non-natural amino acids or deletions could also be introduced in the heavy chain and/or ⁇ 2m at positions outside the peptide-binding cleft.
  • substitution of XX with YY in position nn of human ⁇ 2 m enhance the biochemical stability of MHC Class I molecule complexes and thus may lead to more efficient antigen presentation of subdominant peptide epitopes.
  • a preferred embodiment is removal of "unwanted cysteine residues" in the heavy chain by mutation, chemical modification, amino acid exchange or deletion.
  • "Unwanted cysteine residues” is here to be understood as cysteines not involved in the correct folding of the final MHC I molecule. The presence of cysteine not directly involved in the formation of correctly folded MHC I molecules can lead to formation of intra molecular disulfide bridges resulting in a non correct folded MHC complex during in vitro refolding.
  • Another method for covalent stabilization of MHC I complex am to covalently attach a linker between two of the subunits of the MHC complex. This can be a linker between peptide and heavy chain or between heavy chain and beta2microglobulin.
  • the stability of proteins in aqueous solution depends on the composition of the solution. Addition of salts, detergents organic solvent, polymers ect. can influence the stability.
  • additives that increase surface tension of the MHC molecule without binding the molecule are sucrose, mannose, glycine, betaine, alanine, glutamine, glutamic acid and ammoniumsulfate. Glycerol, mannitol and sorbitol are also included in this group even though they are able to bind polar regions.
  • Another group of additives of special interest are able to increase surface tension of the MHC molecule and simultaneously interact with charged groups in the protein. Examples are MgSO 4 , NaCI, polyethylenglycol, 2-methyl-2,4-pentandiol and guanidiniumsulfate.
  • soluble additives could be added to any solution containing MHC I molecules in order to increase the stability of the molecule. That could be during the refolding process, to the soluble monomer or to a solutions containing MHC I bound to a carrier.
  • MHC Il molecules as used herein are defined as classical MHC Il molecule consisting of a ⁇ -chain and a ⁇ -chain combined with a peptide. It could also be a molecule only consisting of ⁇ -chain and ⁇ -chain ( ⁇ / ⁇ dimer or empty MHC II), a truncated ⁇ -chain
  • a truncated ⁇ -chain e.g. ⁇ 1 domain alone
  • full-length ⁇ -chain either empty or loaded with a peptide
  • a truncated ⁇ -chain e.g. ⁇ 1 domain alone
  • a full-length ⁇ -chain either empty or loaded with a peptide
  • a truncated ⁇ -chain combined with a truncated ⁇ -chain either empty or loaded with a peptide.
  • a truncated ⁇ -chain either empty or loaded with a peptide or a truncated ⁇ -chain combined with a truncated ⁇ -chain (e.g. ⁇ 1 and ⁇ 1 domain) either empty or loaded with a peptide.
  • MHC Il molecules are not easily refolded in vitro. Only some MHC Il alleles may be produced in E. coli followed by refolding in vitro. O--
  • MHC Il molecules are eukaryotic systems where refolding after expression of protein is not necessary.
  • Such expression systems could be stable Drosophila cell transfectants, baculovirus infected insect cells, CHO cells or other mammalian cell lines suitable for expression of proteins.
  • Stabilization of soluble MHC Il molecules is even more important than for MHC I molecules since both ⁇ - and ⁇ -chain are participants in formation of the peptide binding groove and tend to dissociate when not embedded in the cell membrane.
  • MHC Il complexes can be stabilized by introduction of one or more linkers between the individual components of the MHC Il complex.
  • This can be a ⁇ / ⁇ dimer with a linker between ⁇ -chain and ⁇ -chain; a ⁇ / ⁇ dimer covalently linked to the peptide via a linker to either the ⁇ -chain or ⁇ -chain; a ⁇ / ⁇ dimer, covalently linked by a linker between the ⁇ -chain and ⁇ -chain, and where the dimer is covalently linked to the peptide; a ⁇ / ⁇ dimer with a linker between ⁇ -chain and ⁇ -chain, where the dimer is combined with a peptide covalently linked to either ⁇ -chain or ⁇ -chain.
  • the linker can be a flexible linker, e.g. made of glycine and serine, and is typically between 5-20 residues long, but can be shorter or longer.
  • the linker can also be more rigid with a more defined structure, e.g. made of amino acids like glutamate, alanine, lysine, and leucine.
  • the peptides can be linked to the NH 2 - or COOH-terminus of either ⁇ -chain or ⁇ - chain.
  • peptides linked to the NH 2 -terminus of the ⁇ -chain via their COOH-terminus since the linker required is shorter than if the peptide is linked to the COOH-terminus of the ⁇ -chain.
  • Linkage of ⁇ -chain to ⁇ -chain can be via the COOH-terminus of the ⁇ -chain to the NH 2 -terminus of the ⁇ -chain or from the COOH-terminus of the ⁇ -chain to the NH 2 - terminus of the ⁇ -chain.
  • a preferred construct is where one linker connect the COOH-terminus of the ⁇ -chain with the NH 2 -terminus of the ⁇ -chain and another linker connects the COOH- terminal of the peptide with the NH 2 -terminal of the ⁇ -chain.
  • one linker joins the COOH-terminus of the ⁇ -chain with the NH 2 -terminus of the ⁇ -chain and the second linker joins the NH 2 -terminus of the peptide with the COOH-terminus of the ⁇ -chain.
  • the three peptides of the MHC complex can further be linked as described above for the three peptides of the MHC complex, including internal fusion points for the proteins.
  • Non-covalent binding of ligands to the MHC Il complex can promote assembly of ⁇ - and ⁇ -chain by bridging the two chains, or by binding to either of the ⁇ - or ⁇ -chains, and in this way stabilize the conformation of ⁇ or ⁇ , that binds ⁇ or ⁇ , respectively, and/or that binds the peptide.
  • Ligands here mean antibodies, peptides, aptamers or any other molecules with the ability to bind proteins.
  • a particular interesting example is an antibody binding the MHC complex distal to the interaction site with TCR, i.e. distal to the peptide-binding cleft.
  • An antibody in this example can be any truncated or full length antibody of any isotype (e.g. IgG,
  • the antibody could be bispecific with one arm binding to the ⁇ -chain and the other arm binding to the ⁇ -chain.
  • the antibody could be monospecific and directed to a sequence fused to the ⁇ -chain as well as to the ⁇ -chain.
  • Another example of interest is an antibody binding more central in the MHC Il molecule, but still interacting with both ⁇ - and ⁇ -chain.
  • the antibody binds a conformational epitope, thereby forcing the MHC molecule into a correct folded configuration.
  • the antibody can be bispecific binding with one arm to the ⁇ - chain and the other arm to the ⁇ -chain.
  • the antibody is monospecific and binds to a surface of the complex that involves both the ⁇ - and ⁇ -chain, e.g. both the ⁇ 2- and ⁇ 2- domain or both the ⁇ 1 - and ⁇ 1 - domain.
  • the antibodies described above can be substituted with any other ligand that binds at the ⁇ -/ ⁇ -chain interface, e.g. peptides and aptamers.
  • the ligand can also bind the peptide, although, in this case it is important that the ligand does not interfere with the interaction of the peptide or binding cleft with the TCR.
  • MHC Il molecules are incorporated into artificial membrane spheres like liposomes or lipospheres. MHC Il molecules can be incorporated as monomers in the membrane or as dimers like the MHC II- antibody constructs describes above. In addition to stabilization of the MHC Il complex an increased avidity is obtained. The stabilization of the dimer will in most cases also stabilize the trimeric MHC-peptide complex.
  • Induced multimerization can also be achieved by biotinylation of ⁇ - as well as ⁇ - chain and the two chains brought together by binding to streptavidin.
  • Long flexible linkers such as extended glycine-serine tracts can be used to extend both chains, and the chains can be biotinylated at the end of such extended linkers.
  • streptavidin can be used as a scaffold to bring the chains together in the presence of the peptide, while the flexible linkers still allow the chains to orientate properly.
  • Stability of MHC Il complexes can be increased by covalent modifications of the protein.
  • One method is to increase the affinity of the peptide for the MHC complex.
  • Mutations, chemical modifications, insertion of natural or non-natural amino acids or deletions can alternatively be introduced in ⁇ - and/or ⁇ - chain at positions outside the peptide-binding cleft.
  • a preferred embodiment is to replace the hydrophobic transmembrane regions of ⁇ -chain and ⁇ -chain by leucine zipper dimerisation domains (e.g. Fos-Jun leucine zipper; acid-base coiled-coil structure) to promote assembly of ⁇ -chain and ⁇ -chain.
  • leucine zipper dimerisation domains e.g. Fos-Jun leucine zipper; acid-base coiled-coil structure
  • Another preferred embodiment is to introduce one or more cysteine residues by amino acid exchange at the COOH-terminal of both ⁇ -chain and ⁇ -chain, to create disulfide bridges between the two chains upon assembly of the MHC complex.
  • Another embodiment is removal of "unwanted cysteine residues" in either of the chains by mutation, chemical modification, amino acid exchange or deletion.
  • "Unwanted cysteine residues” is here to be understood as cysteines not involved in correct folding of the MHC ll-peptide complex. The presence of cysteines not directly involved in the formation of correctly folded MHC Il complexes can lead to formation of intra molecular disulfide bridges and incorrectly folded MHC complexes.
  • MHC Il complexes can also be stabilized by chemically linking together the subunits and the peptide. That can be a linker between peptide and ⁇ -chain, between peptide and ⁇ -chain, between ⁇ -chain and ⁇ -chain, and combination thereof.
  • Such linkages can be introduced prior to folding by linking two of the complex constituents together, then folding this covalent hetero-dimer in the presence of the third constituent.
  • An advantage of this method is that it only requires complex formation between two, rather than three species.
  • Another possibility is to allow all three constituents to fold, and then to introduce covalent cross-links on the folded MHC-complex, stabilizing the structure.
  • An advantage of this method is that the two chains and the peptide will be correctly positioned relatively to each other when the cross linkages are introduced.
  • Salts, detergents, organic solvent, polymers and any other soluble additives can be added to increase the stability of MHC complexes.
  • additives that increase surface tension of the MHC complex are sucrose, mannose, glycine, betaine, alanine, glutamine, glutamic acid and ammonium sulfate.
  • Glycerol, mannitol and sorbitol are also included in this group even though they are able to bind polar regions.
  • Another group of additives of special interest increases surface tension of the MHC complex and simultaneously can interact with charged groups in the protein.
  • Examples are MgSO 4 , NaCI, polyethylenglycol, 2-methyl-2,4-pentanediol and guanidiniumsulphate.
  • All of the above mentioned soluble additives could be added to any solution containing MHC complexes in order to increase the stability of the molecule. This can be during the refolding process, to the formed MHC complex or to a solution of MHC multimers comprising several MHC complexesThat could be to the soluble monomer, to a solution containing MHC Il bound to a carrier or to solutions used during analysis of MHC Il specific T cells with MHC Il multimers.
  • soluble additives could be added to any solution containing MHC Il molecules in order to increase the stability of the molecule. That could be to the soluble monomer, to a solution containing MHC Il bound to a carrier or to solutions used during analysis of MHC Il specific T cells with MHC Il multimers.
  • Chemically modified MHC I and Il complexes There are a number of amino acids that are particularly reactive towards chemical cross linkers. In the following, chemical reactions are described that are particularly preferable for the cross-linking or modification of MHC I or MHC Il complexes.
  • the amino group at the N-terminal of both chains and of the peptide, as well as amino groups of lysine side chains, are nucleophilic and can be used in a number of chemical reactions, including nucleophilic substitution by activation of electrophiles (e.g. acylation such as amide formation, pyrazolone formation, isoxazolone formation; alkylation; vinylation; disulfide formation), addition to carbon-hetero multiple bonds (e.g.
  • Example reagents that can be used in a reaction with the amino groups are activated carboxylic acids such as NHS-ester, tetra and pentafluoro phenolic esters, anhydrides, acid chlorides and fluorides, to form stable amide bonds.
  • sulphonyl chlorides can react with these amino groups to form stable sulphone-amides.
  • Iso-Cyanates can also react with amino groups to form stable ureas, and isothiocyanates can be used to introduce thiourea linkages.
  • Aldehydes such as formaldehyde and glutardialdehyde will react with amino groups to form shiff's bases, than can be further reduced to secondary amines.
  • the guanidino group on the side chain of arginine will undergo similar reactions with the same type of reagents.
  • cysteine Another very useful amino acid is cysteine.
  • the thiol on the side chain is readily alkylated by maleimides, vinyl sulphones and halides to form stable thioethers, and reaction with other thiols will give rise to disulphides.
  • Carboxylic acids at the C-terminal of both chains and peptide, as well as on the side chains of glutamic and aspartic acid, can also be used to introduce cross-links. They will require activation with reagents such as carbodiimides, and can then react with amino groups to give stable amides.
  • GMBS MesetrachloroButyryloxy-Succinimide ester
  • GMBS can be used to introduce maleimides on the ⁇ -chain
  • iminothiolane can be used to introduce thiols on the ⁇ -chain; the malemide and thiol can then form a thioether link between the two chains.
  • the folded MHC-complex can be reacted with dextrans possessing a large number (up to many hundreds) of vinyl sulphones. These can react with lysine residues on both the ⁇ and ⁇ chains as well as with lysine residues on the peptide protruding from the binding site, effectively cross linking the entire MHC-complex.
  • Such cross linking is indeed a favored reaction because as the first lysine residue reacts with the dextran, the MHC-complex becomes anchored to the dextran favoring further reactions between the MHC complex and the dextran multimerization domain.
  • dextran chemistry Another great advantage of this dextran chemistry is that it can be combined with fluorochrome labelling; i.e. the dextran is reacted both with one or several MHC- complexes and one or more fluorescent protein such as APC.
  • Another valuable approach is to combine the molecular biological tools described above with chemical cross linkers.
  • one or more lysine residues can be inserted into the ⁇ -chain, juxtaposed with glutamic acids in the ⁇ -chain, where after the introduced amino groups and carboxylic acids are reacted by addition of carbodiimide.
  • Such reactions are usually not very effective in water, unless as in this case, the groups are well positioned towards reaction. This implies that one avoids excessive reactions that could otherwise end up denaturing or changing the conformation of the MHC-complex.
  • a dextran multimerization domain can be cross-linked with appropriately modified MHC-complexes; i.e. one or both chains of the MHC complex can be enriched with lysine residues, increasing reactivity towards the vinylsulphone dextran.
  • the lysine's can be inserted at positions opposite the peptide binding cleft, orienting the MHC-complexes favorably for T-cell recognition.
  • An extended linker will allow the two chains to interact with little or no strain resulting from the linker that connects them, while keeping the chains in the vicinity of each other should the complex dissociate. An excess of peptide should further favor reformation of dissociated MHC-complex.
  • MHC I and MHC Il complexes bind to TCRs. However, other molecules also bind TCR. Some TCR-binding molecules are described in the following. MHC I and MHC Il complexes binding to TCRs may be substituted with other molecules capable of binding TCR or molecules that have homology to the classical MHC molecules and therefore potentially could be TCR binding molecules. These other TCR binding or MHC like molecules include:
  • Non-classical MHC complexes and other MHC-like molecules include protein products of MHC Ib and MHC Nb genes.
  • MHC Ib genes encode ⁇ 2m-associated cell-surface molecules but show little polymorphism in contrast to classical MHC class I genes.
  • Protein products of MHC class Ib genes include HLA-E, HLA-G, HLA-F, HLA-H, MIC A, MIC B, ULBP-1 , ULBP- 2, ULBP-3 in humans and H2-M, H2-Q, H2-T and Rae1 in mice.
  • Non-classical MHC Il molecules include HLA-DM, HLA-DO in humans and H2-DM and H2-DO in mice that are involved in regulation of peptide loading into MHC Il molecules.
  • Another MHC-like molecule of special interest is the MHC l-like molecule CD1 .
  • CD1 is similar to MHC I molecules in its organization of subunits and association with ⁇ 2m but presents glycolipids and lipids instead of peptides.
  • Antibodies herein include full length antibodies of isotype IgG, IgM, IgE, IgA and truncated versions of these, antibody fragments like Fab fragments and scFv.
  • Antibodies also include antibodies of antibody fragments displayed on various supramolecular structures or solid supports, including filamentous phages, yeast, mammalian cells, fungi, artificial cells or micelles, and beads with various surface chemistries.
  • Peptide binding TCR Another embodiment of special interest is peptides that bind TCRs.
  • Peptides herein include peptides composed of natural, non-natural and/or chemically modified amino acids with a length of 8-20 amino acid. The peptides could also be longer than 20 amino acids or shorter than 8 amino acids. The peptides can or can not have a defined tertiary structure.
  • Aptamers are another preferred group of TCR ligands.
  • Aptamers are herein understood as natural nucleic acids (e.g. RNA and DNA) or unnatural nucleic acids (e.g. PNA, LNA, morpholinos) capable of binding TCR.
  • the aptamer molecules consist of natural or modified nucleotides in various lengths.
  • TCR-binding molecules can be ankyrin repeat proteins or other repeat proteins, Avimers, or small chemical molecules, as long as they are capable of binding TCR with a dissociation constant smaller than 10 '3 M..
  • the fraction or amount of functional and/or correctly folded MHC can be tested in a number of different ways, including: • Measurement of correctly folded MHC in a quantitative ELISA, e.g. where the MHC bind to immobilized molecules recognizing the correctly folded complex.
  • Multimerization domain A number of MHC complexes associate with a multimerization domain to form a MHC multimer.
  • the size of the multimerization domain spans a wide range, from multimerisation domains based on small organic molecule scaffolds to large multimers based on a cellular structure or solid support.
  • the multimerization domain may thus be based on different types of carriers or scaffolds, and likewise, the attachment of MHC complexes to the multimerization domain may involve covalent or non-covalent linkers. Characteristics of different kinds of multimerization domains are described below.
  • the multimerization domain(s) in the present invention is preferably less than 1 ,000 Da (small molecule scaffold).
  • small molecule scaffold examples include short peptides (e.g. comprising 10 amino acids), and various small molecule scaffolds (e.g. aromatic ring structures).
  • the multimerization domain(s) is preferably between 1 ,000 Da and 10,000 Da (small molecule scaffold, small peptides, small polymers).
  • small molecule scaffold, small peptides, small polymers examples include polycyclic structures of both aliphatic and aromatic compounds, peptides comprising e.g. 10-100 amino acids, and other polymers such as dextran, polyethylenglycol, and polyureas.
  • the multimerization domain(s) is between 10,000 Da and 100,000 Da (Small molecule scaffold, polymers e.g. dextran, streptavidin, IgG, pentamer structure). Examples include proteins and large polypeptides, small molecule scaffolds such as steroids, dextran, dimeric streptavidin, and multi-subunit proteins such as used in Pentamers.
  • the multimerization domain(s) is preferably between 100,000 Da and 1 ,000,000 Da (Small molecule scaffold, polymers e.g. dextran, streptavidin, IgG, pentamer structure). Typical examples include larger polymers such as dextran (used in e.g. Dextramers), and streptavidin tetramers.
  • the multimerization domain(s) is preferably larger than 1 ,000,000 Da (Small molecule scaffold, polymers e.g. dextran, streptavidin, IgG, pentamer structure, cells, liposomes, artificial lipid bilayers, polystyrene beads and other beads. Most examples of this size involve cells or cell-based structures such as micelles and liposomes, as well as beads and other solid supports.
  • multimerisation domains can comprise carrier molecules, scaffolds or combinations of the two.
  • any kind of carrier or scaffold can be used as multimerization domain, including any kind of cell, polymer, protein or other molecular structure, or particles and solid supports. Below different types and specific examples of multimerization domains are listed.
  • Cells can be used as carriers. Cells can be either alive and mitotic active, alive and mitotic inactive as a result of irradiation or chemically treatment, or the cells may be dead.
  • the MHC expression may be natural (i.e. not stimulated) or may be induced/stimulated by e.g. lnf- ⁇ .
  • natural antigen presenting cells APCs
  • APCs antigen presenting cells
  • Cell-like carriers include membrane-based structures carrying MHC-peptide complexes in their membranes such as micelles, liposomes, and other structures of membranes, and phages such as filamentous phages.
  • Solid support includes beads, particulate matters and other surfaces.
  • a preferred embodiment include beads (magnetic or non-magnetic beads) that carry electrophilic groups e.g. divinyl sulfone activated polysaccharide, polystyrene beads that have been functionalized with tosyl- activated esters, magnetic polystyrene beads functionalized with tosyl-activated esters), and where MHC complexes may be covalently immobilized to these by reaction of nucleophiles comprised within the MHC complex with the electrophiles of the beads.
  • electrophilic groups e.g. divinyl sulfone activated polysaccharide, polystyrene beads that have been functionalized with tosyl- activated esters, magnetic polystyrene beads functionalized with tosyl-activated esters
  • MHC complexes may be covalently immobilized to these by reaction of nucleophiles comprised within the MHC complex with the electrophiles of
  • Beads may be made of sepharose, sephacryl, polystyrene, agarose, polysaccharide, polycarbamate or any other kind of beads that can be suspended in aqueous buffer.
  • Another embodiment includes surfaces, i.e. solid supports and particles carrying immobilized MHC complexes on the surface.
  • surfaces i.e. solid supports and particles carrying immobilized MHC complexes on the surface.
  • Multimerization domains may also be molecules or complexes of molecules held together by non-covalent bonds.
  • the molecules constituting the multimerization domain can be small organic molecules or large polymers, and may be flexible linear molecules or rigid, globular structures such as e.g. proteins. Different kinds of molecules used in multimerization domains are described below.
  • Small organic molecules here includes steroids, peptides, linear or cyclic structures, and aromatic or aliphatic structures, and many others.
  • the prototypical small organic scaffold is a functionalized benzene ring, i.e. a benzene ring functionalized with a number of reactive groups such as amines, to which a number of MHC molecules may be covalently linked.
  • Typical scaffolds include aromatic structures, benzodiazepines, hydantoins, piperazines, indoles, furans, thiazoles, steroids, diketopiperazines, morpholines, tropanes, coumarines, qinolines, pyrroles, oxazoles, amino acid precursors, cyclic or aromatic ring structures, and many others.
  • Typical carriers include linear and branched polymers such as peptides, polysaccharides, nucleic acids, and many others.
  • Multimerization domains based on small organic or polymer molecules thus include a wealth of different structures, including small compact molecules, linear structures, polymers, polypeptides, polyureas, polycarbamates, cyclic structures, natural compound derivatives, alpha-, beta-, gamma-, and omega-peptides, mono-, di- and tri-substituted peptides, L- and D-form peptides, cyclohexane- and cyclopentane-backbone modified beta- peptides, vinylogous polypeptides, glycopolypeptides, polyamides, vinylogous sulfonamide peptide, Polysulfonamide-conjugated peptide (i.e., having prosthetic groups), Polyesters, Polysaccharides such as dextran and aminodextran, polycarbamates, polycarbon
  • Monofunctional, Difunctional, Trifunctional and Oligofunctional open-chain hydrocarbons Monofunctional, Difunctional, Trifunctional and Oligofunctional Nonaromat Carbocycles, Monocyclic, Bicyclic, Tricyclic and Polycyclic Hydrocarbons, Bridged Polycyclic Hydrocarbones, Monofunctional, Difunctional, Trifunctional and Oligofunctional
  • Nonaromatic, Heterocycles Monocyclic, Bicyclic, Tricyclic and Polycyclic Heterocycles, bridged Polycyclic Heterocycles, Monofunctional, Difunctional, Trifunctional and Oligofunctional Aromatic Carbocycles, Monocyclic, Bicyclic, Tricyclic and Polycyclic Aromatic Carbocycles, Monofunctional, Difunctional, Trifunctional and Oligofunctional Aromatic
  • Hetero-cycles Monocyclic, Bicyclic, Tricyclic and Polycyclic Heterocycles. Chelates, fullerenes, and any combination of the above and many others.
  • Biological polymers include peptides, proteins (including antibodies, coiled-coil helices, streptavidin and many others), nucleic acids such as DNA and RNA, and polysaccharides such as dextran.
  • the biological polymers may be reacted with MHC complexes (e.g. a number of MHC complexes chemically coupled to e.g. the amino groups of a protein), or may be linked through e.g.
  • DNA duplex formation between a carrier DNA molecule and a number of DNA oligonucleotides each coupled to a MHC complex Another type of multimerization domain based on a biological polymer is the streptavidin-based tetramer, where a streptavidin binds up to four biotinylated MHC complexes, as described above (see Background of the invention). o Self-assembling multimeric structures.
  • MHC multimers exist where the multimer is formed through self- assembling.
  • the Pentamers are formed through formation of a coiled-coil structure that holds together 5 MHC complexes in an apparently planar structure.
  • the Streptamers are based on the Streptactin protein which oligomerizes to form a MHC multimer comprising several MHC complexes (see Background of the invention).
  • MHC multimers based on a molecule multimerization domain involve one or more of the abovementoned types of multimerization domains.
  • MHC dextramers can be made by coupling MHC complexes to dextran via a streptavidin-biotin interaction.
  • biotin-streptavdin can be replaced by any dimerization domain, where one half of the dimerization domain is coupled to the MHC- peptide complex and the other half is coupled to dextran.
  • an acidic helix one half of a coiled-coil dimer
  • a basic helix other half of a coiled-coil dimmer
  • Antibodies can be used as scaffolds by using their capacity to bind to a carefully selected antigen found naturally or added as a tag to a part of the MHC molecule not involved in peptide binding. For example, IgG and IgE will be able to bind two MHC molecules, IgM having a pentameric structure will be able to bind 10 MHC molecules.
  • the antibodies can be full-length or truncated; a standard antibody-fragment includes the Fab2 fragment.
  • Peptides involved in coiled-coil structures can act as scaffold by making stable dimeric, trimeric, tetrameric and pentameric interactions. Examples hereof are the Fos-Jun heterodimeric coiled coil, the E. coli homo-trimeric coiled-coil domain Lpp-56, the engineered Trp-zipper protein forming a discrete, stable, ⁇ -helical pentamer in water at physiological pH.
  • suitable scaffolds, carriers and linkers are streptavidin (SA) and avidin and derivatives thereof, biotin, immunoglobulins, antibodies (monoclonal, polyclonal, and recombinant), antibody fragments and derivatives thereof, leucine zipper domain of AP- 1 (jun and fos), hexa-his (metal chelate moiety), hexa-hat GST (glutathione S-tranf erase), glutathione, Calmodulin-binding peptide (CBP), Strep-tag, Cellulose Binding Domain, Maltose Binding Protein, S-Peptide Tag, Chitin Binding Tag, Immuno-reactive Epitopes, Epitope Tags, E2Tag, HA Epitope Tag, Myc Epitope, FLAG Epitope, AU1 and AU5 Epitopes, GIu-GIu Epitope, KT3 Epitope, IRS Epitope, Btag Epitope, Protein Kinase-
  • binding entity may be an "anti-tag".
  • anti-tag is meant an antibody binding to the tag, or any other molecule capable of binding to such tag.
  • MHC complexes can be multimerized by other means than coupling or binding to a multimerization domain.
  • the multimerization domain may be formed during the multimerization of MHCs.
  • One such method is to extend the bound antigenic peptide with dimerization domains.
  • One end of the antigenic peptide is extended with dimerization domain A (e.g. acidic helix, half of a coiled-coil dimer) and the other end is extended with dimerization domain B (e.g. basic helix, other half of a coiled-coil dimer).
  • dimerization domain A e.g. acidic helix, half of a coiled-coil dimer
  • dimerization domain B e.g. basic helix, other half of a coiled-coil dimer
  • the antigenic peptides in the mixture can either be identical or a mixture of peptides with comparable extended dimerization domains. Alternatively both ends of a peptide are extended with the same dimerization domain A and another peptide (same amino acid sequence or a different amino acid sequence) is extended with dimerization domain B.
  • Multimerization of MHC complexes by extension of peptides are restricted to MHC Il molecules since the peptide binding groove of MHC I molecules is typically closed in both ends thereby limiting the size of peptide that can be embedded in the groove, and therefore preventing the peptide from extending out of the groove.
  • Another multimerization approach applicable to both MHC I and MHC Il complexes is based on extension of N- and C-terminal of the MHC complex.
  • the N- terminal of the MHC complex is extended with dimerization domain A and the C- terminal is extended with dimerization domain B.
  • MHC complexes are incubated together they pair with each other and form multimers like: A-MHC-BA-MHC-BA-MHC- BA-MHC-B etc.
  • the N-terminal and the C-terminal of a MHC complex are both extended with dimerization domain A and the N-terminal and C-terminal of another preparation of MHC complex (either the same or a different MHC) are extended with dimerization domain B.
  • multimers will be formed: A-MHC-AB-MHC-BA-MHC-AB-MHC-B etc.
  • extension can be either chemically coupled to the peptide/MHC complex or introduced as extension by gene fusion.
  • Dimerization domain AB can be any molecule pair able to bind to each other, such as acid/base coiled-coil helices, antibody-antigen, DNA-DNA, PNA-PNA, DNA-PNA, DNA- RNA, LNA-DNA, leucine zipper e.g. Fos/Jun, streptavidin-biotin and other molecule pairs as described elsewhere herein.
  • MHC complexes associate with a multimerization domain to form a MHC multimer.
  • the attachment of MHC complexes to the multimerization domain may involve covalent or non-covalent linkers, and may involve small reactive groups as well as large protein-protein interactions.
  • the coupling of multimerization domains and MHC complexes involve the association of an entity X (attached to or part of the multimerization domain) and an entity Y (attached to or part of the MHC complex).
  • the linker that connects the multimerization domain and the MHC complex comprises an XY portion.
  • the XY linkage can be covalent, in which case X and Y are reactive groups.
  • X can be a nucleophilic group (such as -NH 2 , - OH, -SH, -NH-NH 2 ), and Y an electrophilic group (such as CHO, COOH, CO) that react to form a covalent bond XY; or Y can be a nucleophilic group and X an electrophilic group that react to form a covalent bond XY.
  • the reactive groups can be a radical, capable of reacting with the other reactive group.
  • X and Y can be reactive groups naturally comprised within the multimerization domain and/or the MHC complex, or they can be artificially added reactive groups.
  • linkers containing reactive groups can be linked to either of the multimerization domain and MHC complex; subsequently the introduced reactive group(s) can be used to covalently link the multimerization domain and MHC complex.
  • Example natural reactive groups of MHC complexes include amino acid side chains comprising -NH 2 , -OH, -SH, and -NH-.
  • Example natural reactive groups of multimerization domains include hydroxyls of polysaccharides such as dextrans, but also include amino acid side chains comprising -NH 2 , -OH, -SH, and -NH- of polypeptides, when the polypeptide is used as a multimerization domain.
  • polysaccharides such as dextrans
  • amino acid side chains comprising -NH 2 , -OH, -SH, and -NH- of polypeptides, when the polypeptide is used as a multimerization domain.
  • one of the polypeptides of the MHC complex one of the polypeptides of the MHC complex
  • Example MHC multimers where the bond between the multimerization domain and the MHC complex is covalent and results from reaction between natural reactive groups, include MHC-pentamers (described in US patent 2004209295) and MHC-dimers, where the linkage between multimerization domain and MHC complex is in both cases generated during the translation of the fusion protein.
  • Example artificial reactive groups include reactive groups that are attached to the multimerization domain or MHC complex, through association of a linker molecule comprising the reactive group.
  • dextran multimerization domain involves a multistep reaction that results in the decoration of the dextran with maleimide groups, as described in the patent Siiman et al. US 6,387,622.
  • the amino groups of MHC complexes are converted to -SH groups, capable of reacting with the maleimide groups of the activated dextran.
  • both the reactive group of the multimerization domain (the maleimide) and the reactive group of the MHC complex (the thiol) are artificially introduced.
  • activating reagents are used in order to make the reactive groups more reactive.
  • acids such as glutamate or aspartate can be converted to activated esters by addition of e.g. carbodiimid and NHS or nitrophenol, or by converting the acid moiety to a tosyl-activated ester.
  • the activated ester reacts efficiently with a nucleophile such as -NH 2 , -SH, -OH, etc.
  • the multimerization domains (including small organic scaffold molecules, proteins, protein complexes, polymers, beads, liposomes, micelles, cells) that form a covalent bond with the MHC complexes can be divided into separate groups, depending on the nature of the reactive group that the multimerization domain contains.
  • One group comprise multimerization domains that carry nucleophilic groups (e.g. -NH 2 , -OH, -SH, -
  • CN -NH-NH 2
  • polysaccharides polypeptides containing e.g. lysine, serine, and cysteine
  • another group of multimerization domains carry electrophilic groups (e.g. -COOH, -CHO, -CO, NHS-ester, tosyl-activated ester, and other activated esters, acid-anhydrides)
  • polypeptides containing e.g. glutamate and aspartate, or vinyl sulfone activated dextran yet another group of multimerization domains carry radicals or conjugated double bonds.
  • the multimerization domains appropriate for this invention thus include those that contain any of the reactive groups shown in Figure 5 or that can react with other reactive groups to form the bonds shown in Figure 5.
  • MHC complexes can be divided into separate groups, depending on the nature of the reactive group comprised within the MHC complex.
  • One group comprise MHCs that carry nucleophilic groups (e.g. -NH 2 , -OH, -SH, -CN, -NH- NH 2 ), e.g. lysine, serine, and cysteine; another group of MHCs carry electrophilic groups (e.g. -COOH, -CHO, -CO, NHS-ester, tosyl-activated ester, and other activated esters, acid-anhydrides), exemplified by e.g. glutamate and aspartate; yet another group of MHCs carry radicals or conjugated double bonds.
  • nucleophilic groups e.g. -NH 2 , -OH, -SH, -CN, -NH- NH 2
  • electrophilic groups e.g. -COOH, -CHO, -CO, NHS-ester, tosy
  • the reactive groups of the MHC complex are either carried by the amino acids of the MHC-peptide complex (and may be comprised by any of the peptides of the MHC-peptide complex, including the antigenic peptide), or alternatively, the reactive group of the MHC complex has been introduced by covalent or non- covalent attachment of a molecule containing the appropriate reactive group.
  • Preferred reactive groups in this regard include -CSO 2 OH, phenylchloride, -SH, -SS, aldehydes, hydroxyls, isocyanate, thiols, amines, esters, thioesters, carboxylic acids, triple bonds, double bonds, ethers, acid chlorides, phosphates, imidazoles, halogenated aromatic rings, any precursors thereof, or any protected reactive groups, and many others.
  • Example pairs of reactive groups, and the resulting bonds formed, are shown in figure 5.
  • Reactions that may be employed include acylation (formation of amide, pyrazolone, isoxazolone, pyrimidine, comarine, quinolinon, phthalhydrazide, diketopiperazine, benzodiazepinone, and hydantoin), alkylation, vinylation, disulfide formation, Wittig reaction, Horner-Wittig-Emmans reaction, arylation (formation of biaryl or vinylarene), condensation reactions, cycloadditions ((2+4), (3+2)), addition to carbon-carbon multiplebonds, cycloaddition to multiple bonds, addition to carbon-hetero multiple bonds, nucleophilic aromatic substitution, transition metal catalyzed reactions, and may involve formation of ethers, thioethers, secondary amines, tertiary amines, beta-hydroxy ethers, beta-hydroxy thioethers, beta-hydroxy amines, beta-amino ethers, amides, thioamide
  • MHC dextramers can be made by covalent coupling of MHC complexes to the dextran backbone, e.g. by chemical coupling of MHC complexes to dextran backbones.
  • the MHC complexes can be coupled through either heavy chain or ⁇ 2-microglobulin if the MHC complexes are MHC I or through ⁇ -chain or ⁇ - chain if the MHC complexes are MHC II.
  • MHC complexes can be coupled as folded complexes comprising heavy chain/beta2microglobulin or ⁇ -chain/ ⁇ - chain or either combination together with peptide in the peptide-binding cleft.
  • either of the protein chains can be coupled to dextran and then folded in vitro together with the other chain of the MHC complex not coupled to dextran and together with peptide.
  • Direct coupling of MHC complexes to dextran multimerization domain can be via an amino group or via a sulphide group. Either group can be a natural component of the MHC complex or attached to the MHC complex chemically. Alternatively, a cysteine may be introduced into the genes of either chain of the MHC complex.
  • Another way to covalently link MHC complexes to dextran multimerization domains is to use the antigenic peptide as a linker between MHC and dextran.
  • Linker containing antigenic peptide at one end is coupled to dextran.
  • Antigenic peptide here means a peptide able to bind MHC complexes in the peptide- binding cleft.
  • 10 or more antigenic peptides may be coupled to one dextran molecule.
  • MHC complexes When MHC complexes are added to such peptide- dextran construct the MHC complexes will bind the antigenic peptides and thereby MHC-peptide complexes are displayed around the dextran multimerization domain.
  • the antigenic peptides can be identical or different from each other.
  • MHC complexes can be either identical or different from each other as long as they are capable of binding one or more of the peptides on the dextran multimerization domain.
  • Non-covalent linker The linker that connects the multimerization domain and the MHC complex comprises an XY portion. Above different kinds of covalent linkages XY were described. However, the XY linkage can also be non- covalent. Non-covalent XY linkages can comprise natural dimerization pairs such as antigen-antibody pairs, DNA-DNA interactions, or can include natural O
  • Artificial XY examples include XY pairs such as His 6 tag (X) interacting with Ni-NTA (Y) and PNA-PNA interations.
  • the non-covalent linker may comprise a complex of two or more polypeptides or proteins, held together by non-covalent interactions.
  • Example polypeptides and proteins belonging to this group include Fos/Jun, Acid/Base coiled coil structure, antibody/antigen (where the antigen is a peptide), and many others.
  • Another preferred embodiment involves the use of antibodies, with affinity for the surface of MHC opposite to the peptide-binding groove.
  • an anti-MHC antibody with its two binding site, will bind two MHC complexes and in this way generate a bivalent MHC multimer.
  • the antibody can stabilize the MHC complex through the binding interactions. This is particularly relevant for MHC class Il complexes, as these are less stable than class I MHC complexes.
  • the non-covalent linker may comprise nucleotides that interact non-covalently.
  • Example interactions include PNA/PNA, DNA/DNA, RNA/RNA, LNA/DNA, and any other nucleic acid duplex structure, and any combination of such natural and unnatural polynucleotides such as DNA/PNA, RNA/DNA, and PNA/LNA.
  • the non-covalent linker may comprise a macromolecule (e.g. protein, polynucleotide) and a small molecule ligand of the macromolecule.
  • the interaction may be natural (i.e., found in Nature, such as the Streptavidin/biotin interaction) or non-natural (e.g. His-tag peptide/Ni-NTA interaction).
  • Example interactions include Streptavidin/biotin and anti-biotin antibody/biotin.
  • Combinations - non-covalent linker molecules Other combinations of proteins, polynucleotides, small organic molecules, and other molecules, may be used to link the MHC to the multimerization domain. These other combinations include protein-DNA interactions (e.g. DNA binding protein such as the gene regulatory protein CRP interacting with its DNA recognition sequence), RNA aptamer- protein interactions (e.g. RNA aptamer specific for growth hormone interacting with growth hormone)
  • the non-covalent linker may comprise a complex of two or more organic molecules, held together by non- covalent interactions.
  • Example interactions are two chelate molecules binding to the same metal ion (e.g. E DTA- N i ++ - NTA), or a short polyhistidine peptide (e.g. HiS 6 ) bound to NTA-Ni ++ .
  • the multimerization domain is a bead.
  • the bead is covalently or non-covalently coated with MHC multimers or single MHC complexes, through non-cleavable or cleavable linkers.
  • the bead can be coated with streptavidin monomers, which in turn are associated with biotinylated MHC complexes; or the bead can be coated with streptavidin tetramers, each of which are associated with 0, 1 , 2, 3, or 4 biotinylated MHC complexes; or the bead can be coated with MHC-dextramers where e.g. the reactive groups of the MHC-dextramer (e.g. the divinyl sulfone-activated dextran backbone) has reacted with nucleophilic groups on the bead, to form a covalent linkage between the dextran of the dextramer and the beads.
  • the reactive groups of the MHC-dextramer e.g. the divinyl
  • the MHC multimers described above (e.g. where the multimerization domain is a bead) further contains a flexible or rigid, and water soluble, linker that allows for the immobilized MHC complexes to interact efficiently with cells, such as T-cells with affinity for the MHC complexes.
  • the linker is cleavable, allowing for release of the MHC complexes from the bead. If T-cells have been immobilized, by binding to the MHC complexes, the T-cells can very gently be released by cleavage of this cleavable linker. Appropriate cleavable linkers are shown in Figure 6.
  • linker is cleaved at physiological conditions, allowing for the integrity of the isolated cells.
  • linker molecules include Calmodulin-binding peptide (CBP), 6xHIS, Protein A, Protein G, biotin, Avidine, Streptavidine, Strep-tag, Cellulose Binding Domain, Maltose Binding Protein, S-Peptide Tag, Chitin Binding Tag, Immuno-reactive Epitopes, Epitope Tags, GST tagged proteins, E2Tag, HA Epitope Tag, Myc Epitope, FLAG Epitope, AU1 and AU5
  • Epitopes GIu-GIu Epitope, KT3 Epitope, IRS Epitope, Btag Epitope, Protein Kinase-C Epitope, VSV Epitope.
  • dimerization- and multimerization domains define alternative non-covalent linkers between the multimerization domain and the MHC complex.
  • the abovementioned dimerization- and multimerization domains represent specific binding interactions.
  • Another type of non-covalent interactions involves the non-specific adsorption of e.g. proteins onto surfaces.
  • the non-covalent adsorption of proteins onto glass beads represents this class of XY interactions.
  • the interaction of MHC complexes (comprising full-length polypeptide chains, including the transmembrane portion) with the cell membrane of for example dendritic cells is an example of a non-covalent, primarily non-specific XY interaction.
  • multimerization domains e.g. streptavidin tetramers bound to biotinylated MHC complexes
  • another multimerization domain e.g. the bead
  • Additional components may be coupled to carrier or added as individual components not coupled to carrier
  • T-cells normally respond to a single signal stimulus by going into apoptosis. T cells needs a second signal in order to become activated and o
  • start development into a specific activation state e.g. become an active cytotoxic T cell, helper T cell or regulatory T cell.
  • the MHC multimer of the invention may further comprise one or more additional substituents.
  • additional substituents include one or more additional substituents.
  • Such biologically active molecules may be attached to the construct in order to affect the characteristics of the constructs, e.g. with respect to binding properties, effects, MHC molecule specificities, solubility, stability, or detectability. For instance, spacing could be provided between the MHC complexes, one or both chromophores of a Fluorescence Resonance Energy Transfer (FRET) donor/acceptor pair could be inserted, functional groups could be attached, or groups having a biological activity could be attached.
  • FRET Fluorescence Resonance Energy Transfer
  • MHC multimers can be covalently or non-covalently associated with various molecules: having adjuvant effects; being immune targets e.g. antigens; having biological activity e.g. enzymes, regulators of receptor activity, receptor ligands, immune potentiators, drugs, toxins, co-receptors, proteins and peptides in general; sugar moieties; lipid groups; nucleic acids including siRNA; nano particles; small molecules. In the following these molecules are collectively called biologically active molecules.
  • Such molecules can be attached to the MHC multimer using the same principles as those described for attachment of MHC complexes to multimerisation domains as described elsewhere herein.
  • attachment can be done by chemical reactions between reactive groups on the biologically active molecule and reactive groups of the multimerisation domain and/or between reactive groups on the biologically active molecule and reactive groups of the MHC-peptide complex.
  • attachment is done by non- covalent interaction between part of the multimerisation domain and part of the biological active molecule or between part of the MHC-peptide complex and part of the biological active molecule.
  • a linker molecule can connect the two.
  • the linker molecule can be covalent or non-covalent attached to both molecules. Examples of linker molecules are described elsewhere herein. Some of the MHCmer structures better allows these kind of modifications than others.
  • Biological active molecules can be attached repetitively aiding to recognition by and stimulation of the innate immune system via Toll or other receptors. o
  • MHC multimers carrying one or more additional groups can be used as therapeutic or vaccine reagents.
  • the biologically active molecule may be selected from
  • MHC Class l-like proteins like MIC A, MIC B, CD1 d, HLA E, HLA F, HLA G, HLA H, ULBP-1 , ULBP-2, and ULBP-3,
  • co-stimulatory molecules such as CD2, CD3, CD4, CD5, CD8, CD9, CD27, CD28, CD30, CD69, CD134 (OX40), CD137 (4-1 BB), CD147, CDw150 (SLAM), CD152
  • CTLA-4 CD153 (CD30L), CD40L (CD154), NKG2D, ICOS, HVEM, HLA Class II, PD- 1 , Fas (CD95), FasL expressed on T and/or NK cells, CD40, CD48, CD58, CD70, CD72, B7.1 (CD80), B7.2 (CD86), B7RP-1 , B7-H3, PD-L1 , PD-L2, CD134L, CD137L, ICOSL, LIGHT expressed on APC and/or tumour cells,
  • cell modulating molecules such as CD16, NKp30, NKp44, NKp46, NKp ⁇ O, 2B4, KIR, LIR, CD94/NKG2A, CD94/NKG2C expressed on NK cells, IFN-alpha, IFN-beta, IFN- gamma, IL-1 , IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-10, IL-1 1 , IL-12, IL-15, CSFs (colony- stimulating factors), vitamin D3, IL-2 toxins, cyclosporin, FK-506, rapamycin, TGF-beta, clotrimazole, nitrendipine, and charybdotoxin,
  • accessory molecules such as LFA-1 , CD1 1 a/18, CD54 (ICAM-1 ), CD106 (VCAM), and CD49a,b,c,d,e,f/CD29 (VLA-4),
  • adhesion molecules such as ICAM-1 , ICAM-2, GlyCAM-1 , CD34, anti-LFA-1 , anti- CD44, anti-beta7, chemokines, CXCR4, CCR5, anti-selectin L, anti-selectin E, and anti-selectin P,
  • toxic molecules selected from toxins, enzymes, antibodies, radioisotopes, chemi- luminescent substances, bioluminescent substances, polymers, metal particles, and haptens, such as cyclophosphamide, methrotrexate, Azathioprine, mizoribine, 15- deoxuspergualin, neomycin, staurosporine, genestein, herbimycin A, Pseudomonas exotoxin A, saporin, Rituxan, Ricin, gemtuzumab ozogamicin, Shiga toxin, heavy metals like inorganic and organic mercurials, and FN18-CRM9, radioisotopes such as OO
  • Antibody derivatives or fragments thereof may also be used.
  • Design and generation of product to be used for immune monitoring, diagnosis, therapy or vaccination The product of the present invention may be used forjmmune monitoring, diagnosis, therapy and/or vaccination.
  • the generation of product may follow some or all of the following general steps.
  • Production of a MHC multimer diagnostic or immune monitoring reagent may follow some or all of the following steps.
  • Protein antigen(s) This may be individual proteins, a group of proteins from a given tissue or subgroups of proteins from an organism.
  • MHC allele(s) Decide on needed MHC allele population coverage. If a broad coverage of a given population is needed (i.e. when generally applicable reagents are sought) the most frequently expressed MHC alleles by the population of interest may be chosen e.g. using the database http://www.allelefrequencies.net/test/default1.asp or http://epitope.liai.org:8080/tools/population/iedb_input.
  • the patient is tissue typed (HLA type) and then MHC alleles may be selected according to that.
  • HLA type tissue typed
  • MHC alleles may be selected according to that.
  • step 5 Run the general peptide epitope generator program described elsewhere herein on all selected amino acid sequences from step 3, thereby generating all possible epitopes of defined length (8'-, 9'-, 10'-, 1 1 '-, 13-, 14'-, 15'-, and/or 16'- mers). 6. If searching for broadly applicable epitope sequences, a good alternative to step 5 is to run the "intelligent" peptide epitope prediction programs on the selected amino acid sequences of step 3 using the selected MHC alleles from step 4 e.g.
  • This step can also be used supplementary to step 5 by running selected or all epitopes from the general peptide epitope generator program through one or more of the intelligent peptide epitope prediction programs.
  • epitopes with highest binding score, or the most likely proteolytic products of the species in question, for the chosen MHC alleles and run them through the BLAST program (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) to validate the uniqueness of the peptides. If the peptide sequences are present in other species, evaluate the potential risk of disease states caused by the non- relevant species in relation to causing false positive results. If considered being a potential problem for evaluating the future analysis outcome, leave out the peptide. Preferably, choose unique peptide sequences only present in the selected protein.
  • the MHC multimer reagents may be used in a diagnostic procedure or kit for testing patient and control samples e.g. by flow cytometry, immune histochemistry, Elispot or other methods as described herein.
  • Production of a MHC multimer therapeutic reagent may follow some or all of the following steps. 1. As step 1 - 8 above for diagnostic reagent.
  • additional molecules e.g. biologically active molecules, toxins
  • the additional molecules can have different functionalities as e.g. adjuvants, specific activators, toxins etc. 10. Test the therapeutic reagent following general guidelines
  • the present invention relates to methods for detecting the presence of MHC recognising cells in a sample comprising the steps of
  • Binding indicates the presence of MHC recognising cells.
  • Such methods are a powerful tool in diagnosing various diseases. Establishing a diagnosis is important in several ways. A diagnosis provides information about the disease, thus the patient can be offered a suitable treatment regime. Also, establishing a more specific diagnosis may give important information about a subtype of a disease for which a particular treatment will be beneficial (i.e. various subtypes of diseases may involve display of different peptides which are recognised by MHC recognising cells, and thus treatment can be targeted effectively against a particular subtype). In this way, it may also be possible to gain information about aberrant cells, which emerge through the progress of the disease or condition, or to investigate whether and how T- cell specificity is affected. The binding of the MHC multimer makes possible these options, since the binding is indicative for the presence of the MHC recognising cells in the sample, and accordingly the presence of MHC multimers displaying the peptide.
  • the present invention also relates to methods for monitoring MHC recognising cells comprising the steps of
  • Such methods are a powerful tool in monitoring the progress of a disease, e.g. to closely follow the effect of a treatment.
  • the method can i.a. be used to manage or control the disease in a better way, to ensure the patient receives the optimum treatment regime, to adjust the treatment, to confirm remission or recurrence, and to ensure the patient is not treated with a medicament which does not cure or alleviate the disease.
  • it may also be possible to monitor aberrant cells, which emerge through the progress of the disease or condition, or to investigate whether and how T- cell specificity is affected during treatment.
  • the binding of the MHC multimer makes possible these options, since the binding is indicative for the presence of the MHC recognising cells in the sample, and accordingly the presence of MHC multimers displaying the peptide.
  • the present invention also relates to methods for establishing a prognosis of a disease involving MHC recognising cells comprising the steps of (a) providing a sample suspected of comprising MHC recognising cells,
  • Such methods are a valuable tool in order to manage diseases, i.a. to ensure the patient is not treated without effect, to ensure the disease is treated in the optimum way, and to predict the chances of survival or cure. In this way, it may also be possible to gain information about aberrant cells, which emerge through the progress of the disease or condition, or to investigate whether and how T-cell specificity is affected, thereby being able to establish a prognosis.
  • the binding of the MHC multimer makes possible these options, since the binding is indicative for the presence of the MHC recognising cells in the sample, and accordingly the presence of MHC complexs displaying the peptide.
  • the present invention also relates to methods for determining the status of a disease involving MHC recognising cells comprising the steps of
  • a disease could, e.g. change from one stage to another, and thus it is important to be able to determine the disease status. In this way, it may also be possible to gain information about aberrant cells which emerge through the progress of the disease or condition, or to investigate whether and how T-cell specificity is affected, thereby determining the status of a disease or condition.
  • the binding of the MHC complex makes possible these options, since the binding is indicative for the presence of the MHC recognising cells in the sample, and accordingly the presence of MHC complexs displaying the peptide.
  • the present invention also relates to methods for the diagnosis of a disease involving MHC recognising cells comprising the steps of (a) providing a sample suspected of comprising MHC recognising cells, (b) contacting the sample with a MHC multimer as defined above, and
  • Such diagnostic methods are a powerful tool in the diagnosis of various diseases. Establishing a diagnosis is important in several ways. A diagnosis gives information about the disease, thus the patient can be offered a suitable treatment regime. Also, establishing a more specific diagnosis may give important information about a subtype of a disease for which a particular treatment will be beneficial (i.e. various subtypes of diseases may involve display of different peptides which are recognised by MHC recognising cells, and thus treatment can be targeted effectively against a particular subtype). Valuable information may also be obtained about aberrant cells emerging through the progress of the disease or condition as well as whether and how T-cell specificity is affected. The binding of the MHC multimer makes possible these options, since the binding is indicative for the presence of the MHC recognising cells in the sample, and accordingly the presence of MHC multimers displaying the peptide.
  • the present invention also relates to methods of correlating cellular morphology with the presence of MHC recognising cells in a sample comprising the steps of (a) providing a sample suspected of comprising MHC recognising cells, (b) contacting the sample with a MHC multimer as defined above, and
  • Such methods are especially valuable as applied in the field of histochemical methods, as the binding pattern and distribution of the MHC multimers can be observed directly.
  • the sample is treated so as to preserve the morphology of the individual cells of the sample. The information gained is important i.a. in diagnostic procedures as sites affected can be observed directly.
  • the methods may be used to determine whether a treatment is effectively combating the disease.
  • the method may also provide information about aberrant cells which emerge through the progress of the disease or condition as well as whether and how T-cell specificity is affected, thereby providing information of the effectiveness of a medicament in question.
  • the binding of the MHC multimer makes possible these options, since the binding is indicative for the presence of the MHC recognising cells in the sample, and accordingly the presence of MHC multimers displaying the peptide.
  • the present invention also relates to methods for manipulating MHC recognising cells populations comprising the steps of
  • ex vivo methods are a powerful tool to generate antigen-specific, long-lived human effector T-cell populations that, when re-introduced to the subject, enable killing of target cells and has a great potential for use in immunotherapy applications against various types of cancer and infectious diseases.
  • MHC recognising cells are intended to mean such which are able to recognise and bind to MHC multimers.
  • MHC recognising cells may also be called MHC recognising cell clones, target cells, target MHC recognising cells, target MHC molecule recognising cells, MHC molecule receptors, MHC receptors, MHC peptide specific receptors, or peptide-specific cells.
  • MHC recognising cells is intended to include all subsets of normal, abnormal and defect cells, which recognise and bind to the MHC molecule. Actually, it is the receptor on the MHC recognising cell that binds to the MHC molecule.
  • MHC recognising cells As described above, in diseases and various conditions, peptides are displayed by means of MHC multimers, which are recognised by the immune system, and cells targeting such MHC multimers are produced (MHC recognising cells). Thus, the presence of such MHC protein recognising cells is a direct indication of the presence of MHC multimers displaying the peptides recognised by the MHC protein recognising cells.
  • the peptides displayed are indicative and may involved in various diseases and conditions. For instance, such MHC recognising cells may be involved in diseases of inflammatory, auto-immune, allergic, viral, cancerous, infectious, allo- or xenogene (graft versus host and host versus graft) origin.
  • the MHC multimers of the present invention have numerous uses and are a valuable and powerful tool e.g. in the fields of therapy, diagnosis, prognosis, monitoring, stratification, and determining the status of diseases or conditions.
  • the MHC multimers may be applied in the various methods involving the detection of MHC recognising cells.
  • the present invention relates to compositions comprising the MHC multimers in a solubilising medium.
  • the present invention also relates to compositions comprising the MHC multimers immobilised onto a solid or semi-solid support.
  • the MHC multimers can be used in a number of applications, including analyses such as flow cytometry, immunohistochemistry (IHC), and ELISA-like analyses, and can be used for diagnostic, prognostic or therapeutic purposes including autologous cancer therapy or vaccines such as CMV or HIV vaccine or cancer vaccine.
  • analyses such as flow cytometry, immunohistochemistry (IHC), and ELISA-like analyses
  • IHC immunohistochemistry
  • ELISA-like analyses can be used for diagnostic, prognostic or therapeutic purposes including autologous cancer therapy or vaccines such as CMV or HIV vaccine or cancer vaccine.
  • the MHC multimers are very suitable as detection systems.
  • the present invention relates to the use of the MHC multimers as defined herein as detection systems.
  • the present invention relates to the general use of MHC peptide complexes and multimers of such MHC peptide complexes in various methods. These methods include therapeutic methods, diagnostic methods, prognostic methods, methods for determining the progress and status of a disease or condition, and methods for the stratification of a patient.
  • the MHC multimers of the present invention are also of value in testing the expected efficacy of medicaments against or for the treatment of various diseases.
  • the present invention relates to methods of testing the effect of medicaments or treatments, the methods comprising detecting the binding of the MHC multimers to MHC recognising cells and establishing the effectiveness of the medicament or the treatment in question based on the specificity of the MHC recognising cells.
  • the present invention also relates generally to the field of therapy.
  • the present invention relates per se to the MHC multimer as defined herein for use as medicaments, and to the MHC multimers for use in in vivo and ex vivo therapy.
  • the present invention relates to therapeutic compositions comprising as active ingredients the MHC multimers as defined herein.
  • compositions comprising as active ingredients effective amounts of MHC recognising cells obtained using the MHC multimers as defined herein to isolate relevant MHC recognising cells, and expanding such cells to a clinically relevant number.
  • the present invention further relates to methods for treating, preventing or alleviating diseases, methods for inducing anergy of cells, as well as to methods for up-regulating, down-regulating, modulating, stimulating, inhibiting, restoring, enhancing and/or otherwise manipulating immune responses.
  • the invention also relates to methods for obtaining MHC recognising cells by using the MHC multimers as described herein.
  • Also encompassed by the present invention are methods for preparing the therapeutic compositions of the invention.
  • the present invention is also directed to generating MHC multimers for detecting and analysing receptors on MHC recognising cells, such as epitope specific T-cell clones or other immune competent effector cells.
  • the ability to generate and optionally attach recombinant MHC multimers to multimerization domains, such as scaffolds and/or carrier molecules, will enable the development of a novel analytical and therapeutical tool for monitoring immune responses and contribute to a rational platform for novel therapy and "vaccine" applications.
  • compositions that stimulate specific T-cell proliferation by peptide-specific stimulation is indeed a possibility within the present invention.
  • quantitative analysis and ligand-based detection of specific T-cells that proliferate by the peptide specific stimulation should be performed simultaneously to monitoring the generated response.
  • MHC multimers as described herein can be used to identify and isolate specific T cells in a wide array of applications. In principle all kind of samples possessing T cells can be analyzed with MHC multimers.
  • T cells are pivotal for mounting an adaptive immune response. It is therefore of importance to be able to measure the number of specific T cells when performing a monitoring of a given immune response.
  • the adaptive immune response is monitored by measuring the specific antibody response, which is only one of the effector arms of the immune system. This can lead to miss-interpretation of the actual clinical immune status.
  • intruders of the organism can hide away inside the cells, which often does not provoke a humoral response.
  • the intruder mutates fast, particularly in the genes encoding the proteins that are targets for the humoral response. Examples include the influenza and HIV viruses.
  • the high rate of mutagenesis renders the humoral response unable to cope with the infection.
  • the immune system relies on the cellular immune response. When developing vaccines against such targets one needs to provoke the cellular response in order to get an efficient vaccine.
  • MHC multimers can be used for monitoring immune responses elicited by vaccines
  • One preferred embodiment of the present invention is monitoring the effect of vaccines against infectious disease, e.g. CMV infection.
  • CMV is a member of the herpes virus familily and is particularly abundant, with 50-80 % of all individuals world-wide being infected.
  • CMV infection is asymptomatic. An equilibrium is achieved where CMV-specific T-cells control the persisting virus.
  • AIDS patients in patients rcieving immune suppressive treatment or in others with compromisedimmune system the latent infections may be reactivated if the immune system is suppressed too much, leading to serious complications and death.
  • MHC multimers of the present invention can be useful during development of CMV vaccine's and later to monitor the outcome of a vaccination.
  • MHC multimers are used as components of a CMV vaccine.
  • An example of usefull MHC multimers are cells expressing MHC-peptide complexes where the antigenic peptides are derived from proteins of CMV. Such cells if used as a vaccine may be able to induce a cellular immune response generating T cells specific for the protein from which the antigenic peptides are derived and thereby generate an immune respons against the CMV.
  • T cell stimulatory molecules can be coupled to the multimerisation domain together with MHC or may be added as soluble adjuvant together with the MHC multimer.
  • Example T cell stimulatory molecules include but are not limited to IL-2, CD80 (B7.1 ), CD86 (B7.2), anti-CD28 antibody, CD40, CD37ligand (4-1 BBL), IL-6, IL-15JL-21 , IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , CD27 ligand, CD30 ligand, IL-23, IL-1 ⁇ and IL-1 ⁇ .
  • One or more T cell stimulatory molecules may be added together with or coupled to the MHC multimer.
  • adjuvants or molecules enhancing or otherwise affecting the cellular, humoral or innate immune response may be coupled to or added together with the MHC multimer vaccine.
  • MHC multimers as described elsewhere herein may also be usefull as vaccines against CMV or other infectious diseases by eliciting pathogen-specific immune responses.
  • any MHC multimer or derivatives of MHC multimers can be useful as vaccines, as vaccine components or as engineered intelligent adjuvant.
  • the possibility of combining MHC multimers that specifically bind certain T cells with molecules that trigger, e.g. the humoral response or the innate immune response, can accelerate vaccine development and improve the efficiency of vaccines.
  • the number of antigen-specific cytotoxic T cells can be used as surrogate markers for the overall wellness of the immune system.
  • the immune system can be compromised severely by natural causes such as HIV infections, big traumas, by immuno suppressive therapy in relation to transplantation or due to treatment with chemotherapy.
  • the efficacy of an anti HIV treatment can be evaluated by studying the number of common antigen-specific cytotoxic T cells, specific for e.g. Cytomegalovirus (CMV) and Epstein- Barr virus. In this case the measured T cells can be conceived as surrogate markers. The treatment can then be corrected accordingly and a prognosis can be made.
  • CMV Cytomegalovirus
  • Epstein- Barr virus Epstein- Barr virus
  • a similar situation is found for patients undergoing transplantation as they are severely immune compromised due to pharmaceutical immune suppression to avoid organ rejection.
  • the suppression can lead to outbreak of opportunistic infections caused by reactivation of otherwise dormant viruses residing in the transplanted patients or the grafts. This can be the case for CMV and EBV viruses. Therefore measurement of the number of virus-specific T cells can be used to give a prognosis for the outcome of the transplantation and adjustment of the immune suppressive treatment.
  • the BK virus has been implied as a causative reagent for kidney rejection. Therefore measurement of BK-virus specific T cells can have prognostic value.
  • MHC multimers can be of importance in diagnosis of infections caused by bacteria, virus and parasites that hide away inside cells. Serum titers can be very low and direct measurement of the disease-causing organisms by PCR or other methods directly detecting the presence of pathogen can be very difficult because the host cells are not identified or are inaccessible. Other clinical symptoms of a chronical infection can be unrecognizable in an otherwise healthy individuals, even though such persons still are disease-carriers and at risk of becoming spontaneously ill if being compromised by other diseases or stress.
  • Antigen-specific T helper cells and regulatory T cells have been implicated in the development of autoimmune disorders. In most cases the timing of events leading to autoimmune disease is unknown and the exact role of the immune cells not clear. Use of MHC multimers to study these diseases will lead to greater understanding of the disease-causing scenario and make provisions for development of therapies and vaccines for these diseases.
  • MHC multimers are possible, either directly or as part of therapeutic vaccines.
  • therapies involving T cells e.g. treatment with in vitro amplified antigen- specific effector T cells
  • the T cells often do not home effectively to the correct target sites but ends up in undesired parts of the body.
  • the molecules responsible for interaction with the correct homing receptor can be identified these can be added to the MHC multimer making a dual, triple or multiple molecular structure that are able to aid the antigen-specific T cells home to the correct target, as the MHC multimer will bind to the specific T cell and the additional molecules will mediate binding to the target cells.
  • MHC multimers bound to other functional molecules are employed to directly block, regulate or kill the targeted cells.
  • modulation of regulatory T cells could be part of a treatment.
  • diseases where the function of regulatory T cells it may be possible to directly block, regulate or kill these regulatory cells by means of MHC multimers that besides MHC-peptide complexes also features other functional molecules.
  • the MHC multimers specifically recognize the target regulatory T cells and direct the action of the other functional molecules to this target T cell.
  • MHCmers can be used in immune monitoring, diagnostics, prognostics, therapy and vaccines for many different diseases, including but not limited to the diseases listed in the following.
  • Adenovirus (subgropus A-F), BK-virus, CMV (Cytomegalo virus, HHV-5), EBV (Epstein Barr Virus, HHV-4), HBV (Hepatitis B Virus), HCV (Hepatitis C virus), HHV-6a and b
  • HSV-6a and b Human Herpes Virus-6a and b
  • HHV-7, HHV-8, HSV- 1 Herpes simplex virus-1 , HHV- 1
  • HSV-2 HSV-2
  • JC-virus SV-40 (Simian virus 40)
  • VZV Varizella-Zoster- Virus, HHV-3
  • Parvovirus B19 Haemophilus influenza, HIV-1 (Human immunodeficiency Virus-1 ), HTLV-1 (Human T-lymphotrophic virus-1 ), HPV (Human Papillomavirus giving rise to clinical manifestions such as Hepatitis, AIDS, Measles, Pox, Chicken pox, Rubella, Herpes and others
  • Infectious diseases caused by bacteria such as, Gram positive bacteria, gram negative bacteria, intracellular bacterium, extracellular bacterium, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium avium subsp.
  • Plasmodium falciparum Plasmodium vivax
  • Plasmodium malariae Plasmodium mansoni
  • Schistosoma japonicum Schistosoma haematobium
  • Trypanosoma cruzi Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Schistosoma mansoni, Schistosoma japonicum, Schistosoma haematobium, Trypanosoma cruzi,
  • Trypanosoma rhodesiense Trypanosoma gambiense, Leishmania donovani, and
  • Transplantation-related diseases caused by reactions to minor histocompatibility antigens such as HA-1 , HA-8, USP9Y, SMCY, TPR-protein, HB-1 Y and other antigens in relation to, Graft-versus-host-related disease, allo- or xenogene reactions i.e. graft-versus-host and host-versus-graft disease.
  • minor histocompatibility antigens such as HA-1 , HA-8, USP9Y, SMCY, TPR-protein, HB-1 Y and other antigens in relation to, Graft-versus-host-related disease, allo- or xenogene reactions i.e. graft-versus-host and host-versus-graft disease.
  • Cancerous diseases associated with antigens such as Survivin, Survivin-2B, Livin/ML-IAP, Bcl-2, Mcl-1 , BcI-X(L), Mucin-1 , NY-ESO-1 , Telomerase, CEA, MART-1 , HER-2/neu, bcr-abl, PSA, PSCA, Tyrosinase, p53, hTRT, Leukocyte Proteinase-3, hTRT, gpl OO, MAGE antigens, GASC, JMJD2C, JARD2 (JMJ), JHDM3a, WT- 1 ,CA 9, Protein kinases, where the cancerous diseases include malignant melanoma, renal carcinoma, breast cancer, lung cancer, cancer of the uterus, cervical cancer, prostatic cancer, pancreatic cancer, brain cancer, head and neck cancer, leukemia, cutaneous lymphoma, hepatic carcinoma, colorectal cancer, bladder cancer.
  • cancerous diseases include malignant
  • autoimmune and inflammatory diseases associated with antigens such as GAD64, Collagen, human cartilage glycoprotein 39, ⁇ -amyloid, A ⁇ 42, APP, Presenilin 1 , where the autoimmune and inflammatory diseases include Diabetes type 1 , Rheumatoid arthritis, Alzheimer, chronic inflammatory bowel disease, Crohn's disease, ulcerative colitis uterosa, Multiple Sclerosis, Psoriasis
  • A. Disease to be e.g. treated, prevented, diagnosed, monitored.
  • Label e.g. should the MHC multimer be labelled with a fluorophore or a chromophore
  • Biologically active molecule e.g. should a biologically active molecule such as an interleukin be added or chemically linked to the complex
  • E. Peptide e.g. decide on a peptide to be complexed with MHC
  • MHC e.g. use a MHC allele that does not interfere with the patient's immune system in an undesired way.
  • a number of diseases A 1 -A n relevant in connection with MHC multimers, have been described herein; a number of applications B 1 -B n , relevant in connection with MHC multimers, have been described herein; a number of Labels C 1 -C n , relevant in connection with MHC multimers, have been described herein; a number of biologically active molecules D 1 -D n , relevant in connection with MHC multimers, have been described herein; a number of peptides ErE n , relevant in connection with MHC multimers, have been described herein; and a number of MHC molecules FrF n , relevant in connection with MHC multimers, have been described herein.
  • n is different for different choices A, B, C, D, E, or F. Consequently, the present invention describes a large number of approaches to the diagnosis, monitoring, prognosis, therapeutic or vaccine treatment of diseases.
  • the total number of approaches, as defined by these parameters, are
  • n(A) describes the number of different diseases A described herein
  • n(B) describes the number of different applications B described herein, etc.
  • diagnostic procedures, immune monitoring and some therapeutic processes all involve identification and/or enumeration and/or isolation of antigen-specific T cells. Identification and enumeration of antigen-specific T cells may be done in a number of ways, and several assays are currently employed to provide this information. In the following it is described how MHC multimers as described in the present invention can be used to detect specific T cell receptors (TCRs) and thereby antigen- specific T cells in a variety of methods and assays.
  • detection includes detection of the presence of antigen-specific T cell receptors/ T cells in a sample, detection of and isolation of cells or entities with antigen-specific T cell receptor from a sample and detection and enrichment of cells or entities with antigen- specific T cell receptor in a sample.
  • the sample may be a biological sample including solid tissue, solid tissue section or a fluid such as, but not limited to, whole blood, serum, plasma, nasal secretions, sputum, urine, sweat, saliva, transdermal exudates, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, cerebrospinal fluid, synovial fluid, fluid from joints, vitreous fluid, vaginal or urethral secretions, or the like.
  • disaggregated cellular tissues such as, for example, hair, skin, synovial tissue, tissue biopsies and nail scrapings are also considered as biological samples.
  • Blood samples are whole blood samples or blood processed to remove erythrocytes and platelets (e.g., by Ficoll density centrifugation or other such methods known to one of skill in the art) and the remaining PBMC sample, which includes the T-cells of interest, as well as B-cells, macrophages and dendritic cells, is used directly.
  • labels and marker molecules can be used.
  • Marker molecules are molecules or complexes of molecules that bind to other molecules. Marker molecules thus may bind to molecules on entities, including the desired entities as well as undesired entities. Labeling molecules are molecules that may be detected in a certain analysis, i.e. the labeling molecules provide a signal detectable by the used method. Marker molecules, linked to labeling molecules, constitute detection molecules. Likewise labeling molecules linked to MHC multimers also constitute detection molecules but in contrast to detection molecules made up of marker and lebelling molecule labeled MHC multimers are specific for TCR. Sometimes a marker molecule in itself provides a detectable signal, wherefore attachment to a labeling molecule is not necessary.
  • Marker molecules are typically antibodies or antibodyfragments but can also be aptamers, proteins, peptides, small organic molecules, natural compounds (e.g. steroids), non-peptide polymers, or any other molecules that specifically and efficiently bind to other molecules are also marker molecules.
  • Labelling molecules are molecules that can be detected in a certain analysis, i.e. the labelling molecules provide a signal detectable by the used method. The amount of labelling molecules can be quantified.
  • the labelling molecule is preferably such which is directly or indirectly detectable.
  • the labelling molecule may be any labelling molecule suitable for direct or indirect detection.
  • direct is meant that the labelling molecule can be detected per se without the need for a secondary molecule, i.e. is a "primary” labelling molecule.
  • indirect is meant that the labelling molecule can be detected by using one or more "secondary” molecules, i.e. the detection is performed by the detection of the binding of the secondary molecule(s) to the primary molecule.
  • the labelling molecule may further be attached via a suitable linker.
  • Linkers suitable for attachment to labelling molecules would be readily known by the person skilled in the art and as described elsewhere herein for attachment of MHC molecules to multimerisation domains.
  • labelling compounds examples include fluorescent labels, enzyme labels, radioisotopes, chemiluminescent labels, bioluminescent labels, polymers, metal particles, haptens, antibodies, and dyes.
  • fluorescent labels such as 5-(and 6)-carboxyfluorescein, 5- or 6-carboxy- fluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexanoic acid, fluorescein isothio- cyanate (FITC), rhodamine, tetramethylrhodamine, and dyes such as Cy2, Cy3, and Cy5, optionally substituted coumarin including AMCA, PerCP, phycobiliproteins including R-phycoerythrin (RPE) and allophycoerythrin (APC), Texas Red, Princeston Red, Green fluorescent protein (GFP) and analogues thereof, and conjugates of R- phycoerythrin or allophycoerythrin and e.g.
  • fluorescent labels such as 5-(and 6)-carboxyfluorescein, 5- or 6-carboxy- fluorescein, 6-(fluorescein)-5-(and 6)-carboxamido hexa
  • Cy5 or Texas Red and inorganic fluorescent labels based on semiconductor nanocrystals (like quantum dot and QdotTM nanocrystals), and time-resolved fluorescent labels based on lanthanides like Eu3+ and Sm3+,
  • haptens such as DNP, biotin, and digoxiginin
  • HRP horse radish peroxidase
  • AP alkaline phosphatase
  • GAL beta-galactosidase
  • glucose-6-phosphate dehydrogenase beta-N-acetyl- glucosaminidase
  • ⁇ -glucuronidase invertase
  • Xanthine Oxidase firefly luciferase
  • glucose oxidase GO
  • lumiscence labels such as luminol, isoluminol, acridinium esters, 1 ,2-dioxetanes and pyridopyridazines, and
  • radioactivity labels such as incorporated isotopes of iodide, cobalt, selenium, tritium, and phosphor.
  • Radioactive labels may in particular be interesting in connection with labelling of the peptides harboured by the MHC multimers.
  • labelling Different principles of labelling and detection exist, based on the specific property of the labelling molecule.
  • Examples of different types of labelling are emission of radioactive radiation (radionuclide, isotopes), absorption of light (e.g. dyes, chromophores), emission of light after excitation (fluorescence from fluorochromes), NMR (nuclear magnetic resonance form paramagnetic molecules) and reflection of light (scatter from e.g. such as gold-, plastic- or glass -beads/particles of various sizes and shapes).
  • the labelling molecules can have an enzymatic activity, by which they catalyze a reaction between chemicals in the near environment of the labelling molecules, producing a signal, which include production of light (chemi-luminescence), precipitation of chromophor dyes, or precipitates that can be detected by an additional layer of detection molecules.
  • the enzymatic product can deposit at the location of the enzyme or, in a cell based analysis system, react with the membrane of the cell or diffuse into the cell to which it is attached. Examples of labelling molecules and associated detection principles are shown in table 2 below.
  • Table 2 Examples of labelling molecules and associated detection principles.
  • Photometry is to be understood as any method that can be applied to detect the intensity, analyze the wavelength spectra, and or measure the accumulation of light derived form a source emitting light of one or multiple wavelength or spectra.
  • Labelling molecules can be used to label MHC multimers as well as other reagents used together with MHC multimers, e.g. antibodies, aptamers or other proteins or molecules able to bind specific structures in another protein, in sugars, in DNA or in other molecules.
  • molecules able to bind a specific structure in another molecule are named a marker.
  • Labelling molecules can be attached to a given MHC multimer or any other protein marker by covalent linkage as described for attachment of MHC multimers to multimerization domains elsewhere herein.
  • the attachment can be directly between o
  • reactive groups in the labelling molecule and reactive groups in the marker molecule or the attachment can be through a linker covalently attached to labelling molecule and marker, both as described elsewhere herein.
  • the label can be attached either to the MHC complex (heavy chain, ⁇ 2m or peptide) or to the multimerization domain.
  • one or more labelling molecules may be attached to the carrier molecule, or one or more labelling molecules may be attached to one or more of the scaffolds, or one or more labelling compounds may be attached to one or more of the MHC complexes, or one or more labelling compounds may be attached to the carrier molecule and/or one or more of the scaffolds and/or one or more of the MHC complexes, or one or more labelling compounds may be attached to the peptide harboured by the MHC molecule.
  • a single labelling molecule on a marker does not always generate sufficient signal intensity.
  • the signal intensity can be improved by assembling single label molecules into large multi-labelling compounds, containing two or more label molecule residues.
  • Generation of multi-label compounds can be achived by covalent or non-covalent, association of labelling molecules with a major structural molecule. Examples of such structures are synthetic or natural polymers (e.g. dextramers), proteins (e.g. streptavidin), or polymers.
  • the labelling molecules in a multi-labelling compound can all be of the same type or can be a mixture of different labelling molecules.
  • MHC complexs it may be advantageous to apply different MHC complexs, either as a combination or in individual steps.
  • Such different MHC multimers can be differently labelled (i.e. by labelling with different labelling compounds) enabling visualisation of different target MHC recognising cells.
  • labelling compounds i.e. by labelling with different labelling compounds
  • Detection principles can be applied to flow cytometry, stationary cytometry, and batch-based analysis. Most batch-based approaches can use any of the labelling substances depending on the purpose of the assay.
  • Flow cytometry primarily employs fluorescence
  • stationary cytometry primarily employs light absorption, e.g. dyes or chromophore deposit from enzymatic activity.
  • principles involving fluorescence detection will be exemplified for flow cytometry, and principles involving chromophore detection will be exemplified in the context of stationary cytometry.
  • the labelling molecules can be applied to any of the analyses described in this invention.
  • the typical label is detected by its fluorescence. Most often a positive detection is based on the presents of light from a single fluorochrome, but in other techniques the signal is detected by a shift in wavelength of emitted light; as in FRET based techniques, where the exited fluorochrome transfer its energy to an adjacent bound fluorochrome that emits light, or when using Ca 2+ chelating fluorescent props, which change the emission (and absorption) spectra upon binding to calcium.
  • labelling molecules employed in flowcytometry are illustrated in Table 3 and 4 and described in the following.
  • the marker is not identified by a single fluorochrome but by a code of identification being a specific combination of fluorochromes, as well as inter related ratio of intensities.
  • Antibody Ab1 and Ab2 are conjugated to both. FITC and BP but Ab1 have 1 FITC to 1 BP whereas Ab2 have 2 FITC to 1 BP. Each antibodymay then be identified individually by the relative intensity of each fluorochrome. Any such combinations of n fluorochromes with m different ratios can be generated.
  • AlexaFluor® (AF) AF ⁇ 350, AF405, AF430, AF488.AF500, AF514, AF532,
  • DyLightTM Dyes DL549, DL649, DL680, DL800 Small fluorescing dyes FITC, Pacific BlueTM, Pacific OrangeTM, Cascade YellowTM,
  • E Fluorescent Proteins
  • GFP (enhanced) green fluorescent protein) derived mutant proteins
  • Calcium dyes Indo-1 -Ca2+ lndo-2-Ca2+ Preferably labelling molecules employed in stationary cytometry and IHC
  • Enzymatic labelling as exemplified in Table 5: o Horse radish peroxidase; reduces peroxides (H 2 O 2 ), and the signal is generated by the Oxygen acceptor when being oxidized.
  • Precipitating dyes Dyes that when they arereduced they are soluble, and precipitate when oxidized, generating a coloured deposit at the site of the reaction.
  • Precipitating agent carrying a chemical residue, a hapten, for second layer binding of marker molecules, for amplification of the primary signal.
  • Luminol reaction generating a light signal at the site of reaction.
  • Other enzymes such as Alkaline Phosphatase, capable of converting a chemical compound from a non-detectable molecule to a precipitated detectable molecule, which can be coloured, or carries a hapten as described above.
  • Fluorescent labels as exemplified in Table 3 and 4; as those described for Flow cytometry are likewise important for used in stationary cytometry, such as in fluorescent microscopy.
  • Detection of TCRs with multimers may be direct or indirect.
  • Direct detection of TCRs is detection directly of the binding interaction between the specific T cell receptor and the MHC multimer. Direct detection includes detection of TCR when TCR is attached to lipid bilayer, when TCR is attached to or in a solid medium or when TCR is in solution.
  • TCRs attached to lipid bilayer including but is not limited to naturally occurring T cells (from blood, spleen, lymphnode, brain or any other tissue containing T cells), TCR transfected cells, T cell hybridomas, TCRs embedded in liposomes or any other membrane structure.
  • T cells any entity consisting of TCR attached to lipid bilayer will be referred to as T cells.
  • T cells can be directly detected either when in a fluid solution or when immobilized to a support.
  • T cells can be detected in fluid samples as described elsewhere herein and in suspension of disrupted tissue, in culture media, in buffers or in other liquids.T cells in fluid samples can be detected individually or deteced as populations of T cells. In the following different methods for direct detction of T cells in fluid samples are shown.
  • Direct detection of individual T cells o Direct detection of individual T cells using flow cytometry.
  • a suspension of T cells are added MHC multimers, the sample washed and then the amount of MHC multimer bound to each cell are measured. Bound
  • MHC multimers may be labelled directly or measured through addition of labelled marker molecules.
  • the sample is analyzed using a flow cytometer, able to detect and count individual cells passing in a stream through a laser beam.
  • cells are stained with fluorescently labeled MHC multimer by incubating cells with MHC multimer and then forcing the cells with a large volume of liquid through a nozzle creating a stream of spaced cells.
  • Each cell passes through a laser beam and any fluorochrome bound to the cell is excited and thereby fluoresces.
  • Sensitive photomultipliers detect emitted fluorescence, providing information about the amount of MHC multimer bound to the cell.
  • MHC multimers can be used to identify specific T cell populations in liquid samples such as synovial fluid or blood.
  • lymphocytes can be purified before flow cytometry analysis using standard procedures like a Ficoll-Hypaque gradient.
  • Another possibility is to isolate T cells from the blood sample for example by binding to antibody coated plastic surfaces, followed by elution of bound cells. This purified T cell population can then be used for flow cytometry analysis together with MHC multimers.
  • unwanted cells like B cells and NK cells can be removed prior to the analysis.
  • affinity chromatography using columns coated with antibodies specific for the unwanted cells.
  • specific antibodies can be added to the blood sample together with complement, thereby killing cells recognized by the antibodies.
  • Gating reagents here means labeled antibodies or other labeled markers identifying subsets of cells by binding to unique surface proteins.
  • Preferred gating reagents when using MHC multimers are antibodies directed against CD3, CD4, and CD8 identifying major subsets of T cells.
  • Other preferred gating reagents are antibodies against CD14, CD15, CD19, CD25, CD56, CD27, CD28, CD45, CD45RA, CD45RO,
  • CCR7, CCR5, CD62L, Foxp3 recognizing specific proteins unique for different lymphocytes of the immune system.
  • the number of cells in a sample can vary. When the target cells are rare, it is preferable to analyze large amounts of cells. In contrast, fewer cells are required when looking at T cell lines or samples containing many cells of the target cell type.
  • the flow cytometer can be equipped to separate and collect particular types of cells. This is called cell sorting. MHC multimers in combination with sorting on a flowcytometer can be used to isolate specific T cell populations. Isolated specific T cell populations can then be expanded in vitro. This can be useful in autologous cancer therapy.
  • Direct determination of the concentration of MHC-peptide specific T cells in a sample can be obtained by staining blood cells or other cell samples with MHC multimers and relevant gating reagents followed by addition of an exact amount of counting beads of known concentration.
  • Counting beads is here to be understood as any fluorescent bead with a size that can be visualized by flow cytometry in a sample containing T cells.
  • the beads could be made of polystyrene with a size of about 1 -10 ⁇ m. They could also be made of agarose, polyacrylamide, silica, or any other material, and have any size between 0,1 ⁇ m and 100 m.
  • the counting beads are used as reference population to measure the exact volume of analyzed sample.
  • Amounts of MHC-peptide specific T cells in a blood sample can be determined by flow cytometry by calculating the amount of MHC'mer labeled cells in a given volumen of sample with a given cell density and then back calculate. Exact enumeration of specific T cells is better achieved by staining with MHC'mers together with an exact amount of counting beads followed by flow cytometry analysis. The amount of T cells detected can then be correlated with the amount of counting beads in the same volume of the sample and an exact number of MHC-peptide specific T cells determined:
  • MHC multimers Direct detection of individual T cells in fluid sample by microscopy
  • a suspension of T cells are added MHC multimers, the sample washed and then the amount of MHC multimer bound to each cell are measured.
  • Bound MHC multimers may be labelled directly or measured through addition of labelled marker molecules.
  • the sample is then spread out on a slide or similar in a thin layer able to distinguish individual cells and labelled cells identified using a microscope.
  • microscopes e.g. if fluorescent labels are used a fluorescent microscope is used for the analysis.
  • MHC multimers can be labeled with a flourochrome or bound MHC multimer detected with a fluorescent antibody. Cells with bound fluorescent MHC multimers can then be visualized using an immunofluorescence microscope or a confocal fluorescence microscope.
  • MHC multimers are immobilized to a support e.g. beads, immunotubes, wells of a microtiterplate, CD, mircrochip or similar and as decribed elsewhere herein, then a suspension of T cells are added allowing specific T cells to bind MHC multimer molecules. Following washing bound T cells are recovered/eluted (e.g. using acid or competition with a competitor molecules) and counted.
  • a support e.g. beads, immunotubes, wells of a microtiterplate, CD, mircrochip or similar and as decribed elsewhere herein.
  • MHC multimer Direct detection of populations of T cells o Cell suspensions are added labeled MHC multimer, samples are washed and then total signal from label are measured.
  • the MHC multimers may be labeled themselves or detected through a labeled marker molecule.
  • T cells may be immobilized and then detected directly. Immobilization can be on solid support, in solid tissue or in fixator (e.g. paraffin, a sugar matrix or another medium fixing the T cells). Direct detection of T cells immobilized on solid support.
  • fixator e.g. paraffin, a sugar matrix or another medium fixing the T cells.
  • Such support may be any which is suited for immobilisation, separation etc.
  • Non-limiting examples include particles, beads, biodegradable particles, sheets, gels, filters, membranes (e. g. nylon membranes), fibres, capillaries, needles, microtitre strips, tubes, plates or wells, combs, pipette tips, micro arrays, chips, slides, or indeed any solid surface material.
  • the solid or semi-solid support may be labelled, if this is desired.
  • the support may also have scattering properties or sizes, which enable discrimination among supports of the same nature, e.g. particles of different sizes or scattering properties, colour or intensities.
  • the support may be made of glass, silica, latex, plastic or any polymeric material.
  • the support may also be made from a biodegradable material.
  • the nature of the support is not critical and a variety of materials may be used.
  • the surface of support may be hydrophobic or hydrophilic.
  • Such supports may be for example be porous or particulate e.g. particles, beads, fibres, webs, sinters or sieves. Particulate materials like particles and beads are generally preferred due to their greater binding capacity. Particularly polymeric beads and particles may be of interest.
  • a particulate support e.g. beads or particles
  • the size of the particulate support is not critical, but it may for example have a diameter of at least 1 ⁇ m and preferably at least 2 ⁇ m, and have a maximum diameter of preferably not more than 10 ⁇ m and more preferably not more than 6 ⁇ m.
  • particulate supports having diameters of 2.8 ⁇ m and 4.5 ⁇ m will work well.
  • a particulate support is monodisperse particles, i.e. such which are substantially uniform in size (e. g. size having a diameter standard deviation of less than 5%). Such have the advantage that they provide very uniform reproducibility of reaction.
  • Monodisperse particles e.g. made of a polymeric material, produced by the technique described in US 4,336,173 (ref. 25) are especially suitable.
  • Non-magnetic polymer beads may also be applicable. Such are available from a wide range of manufactures, e.g.
  • Magnetic beads or particles Another example of a suitable support is magnetic beads or particles.
  • the term "magnetic" as used everywhere herein is intended to mean that the support is capable of having a magnetic moment imparted to it when placed in a magnetic field, and thus is displaceable under the action of that magnetic field.
  • a support comprising magnetic beads or particles may readily be removed by magnetic aggregation, which provides a quick, simple and efficient way of separating out the beads or particles from a solution.
  • Magnetic beads and particles may suitably be paramagnetic or superparamagnetic.
  • Superparamagnetic beads and particles are e.g. described in EP 0 106 873 (Sintef, ref. 26). Magnetic beads and particles are available from several manufacturers, e.g. Dynal Biotech ASA (Oslo, Norway, previously Dynal AS, e.g. Dynabeads ® ).
  • the support may suitably have a functionalised surface.
  • functionalisation include making the surface of the support positively or negatively charged, or hydrophilic or hydrophobic. This applies in particular to beads and particles.
  • Various methods therefore are e.g. described in US 4,336,173 (ref. 25), US 4,459,378 (ref. 27) and US 4,654,267 (ref. 28).
  • Immobilized T cells may be detected in several ways including:
  • T cells may be directly immobilized on solid support e.g. by non-specific adhesion. Then MHC multimers are added to the immobilized T cells thereby allowing specific T cells to bind the MHC multimers. Bound MHC multimer may be measured through label directly attached to the multimer or through labeled marker molecules. Individual T cells may be detected if the method for analysis is able to distinguish individual labelled cells, e.g. cells are immobilized in a monolayer on a cell culture well or a glass slide. Following staining with labelled multimer a digital picture is taken and labelled cells identified and counted. Alternatively a population of T cells is detected by measurement of total signal from all labelled T cells, e.g. cells are plated to wells of a microtiter plate, stained with labelled MHC multimer and total signal from each well are measured.
  • T cells can also be immobilized to solid support through a linker molecule.
  • the linker molecule can be an antibody specific for the T cell, a MHC multimer, or any molecule capable of binding T cells.
  • the linker may be attached directly to the solid support, to the solid support through another linker, or the linker may be embedded in a matrix, e.g. a sugar matrix.
  • MHC multimers are added to the immobilized T cells thereby allowing specific T cells to bind the MHC multimers.
  • Bound MHC multimer may be measured through label directly attached to the multimer or through labeled marker molecules. Individual T cells may be detected if the method for analysis is able to distinguish individual labelled cells, e.g. a digital picture is taken and labelled cells identified and counted.
  • a population of T cells is detected by measurement of total signal from all labeled T cells.
  • o lmmuno profiling Phenotyping T cell sample using MHC multimer beads or arrays.
  • Different MHC multimers are immobilized to different beads with different characteristics (e.g. different size, different fluorophores or different fluorescence intensities) where each kind of bead has a specific type of MHC multimer molecule immobilized.
  • the immobilization may be direct or through a linker molecule as described above.
  • the amount of bound T cells to a specific population of beads can be analyzed, thereby phenotyping the sample.
  • the TCR on the T cell is defined by the MHC multimer and hence the bead to which it binds.
  • MHC multimers can be immobilized in an array, e.g. on a glass plate or pin array so that the position in the array specifies the identity of the MHC multimer.
  • the immobilization may be direct or through a linker molecule as described above.
  • the amount of bound T cells at a specified position in the array can be determined by addition of a label or labelled marker that binds to cells in general, or that binds specifically to the cells of interest.
  • the cells may be generally labelled by the addition of a labelled molecule that binds to all kinds of cells, or specific cell types, e.g. CD4+ T-cells, may be labelled with anti-CD4 antibodies that are labelled with e.g. a chromophore or fluorophore. Either of these approaches allow a phenotyping of the sample.
  • Mass profiling of the T-cells of an individual may be done by first immobilizing specific MHC multimers (e.g. 10-10 6 different MHC multimers, each comprising a specific MHC-peptide combination) in an array (e.g. a glass plate), adding e.g. a blood sample from the individual, and then after washing the unbound cells off, label the immobilized cells. Positions in the array of particularly high staining indicate MHC-peptide combinations that recognize specific T-cells of particularly high abundance or affinity. Thus, an immuno profiling of the individual with regard to the tested MHC-peptide combinations is achieved. A similar profiling of an individuals disease may be made using MHC multimers immobilized to different beads as described above.
  • the profiling may entail a number of diseases, a specific disease, a set of specific antigens implicated in one or more diseases, or a specific antigen (e.g. implicated in a specific disease or set of diseases).
  • an individual's immuno profile for a particular antigen is obtained.
  • peptides corresponding to all possible 8'-, 9'- 10'- and 1 1 '-mer peptide sequences derived from the peptide antigen sequence are generated, for example by standard organic synthesis or combinatorial chemistry, and the corresponding MHC multimers are produced, using one or more of the class I MHC-alleles of the individual in question.
  • peptides of e.g. 13, 14, 15, 16 and upto 25 amino acids length may be generated, for example by organic synthesis or combinatorial chemistry, corresponding to all
  • HLA-A * 02, HLA-A * 03, HLA-B * 08 and HLA-B * 07 all these HLA class I alleles should be combined with every tested peptide and similarly for all HLA class Il alleles of the given individual. Based on the profile, a personalized drug, -vaccine or -diagnostic test may be produced.
  • the principle described above may also be employed to distinguish between the immune response raised against a disease (e.g. an infection with a bacterium or the formation of a tumour), and the immune response raised against a vaccine for the same disease (in the example, a vaccine against the bacterium or the tumour).
  • Most vaccines consists of subcomponents of the pathogen /tumour they are directed against and/or are designed to elicit an immune response different from the natural occuring immune response i.e. the T cell epitopes of the two immune reponses differs.
  • a comprehensive array i.e.
  • an array that comprises all possible epitopes from one or more antigen(s)) or a subset of these epitopes, it is possible to deduce whether the immune response has been generated against the disease or the vaccine, or against both the disease and the vaccine. If the vaccine raises a response against a particular epitope or a particular set of epitopes, the corresponding positions in the array will give rise to high signals (compared to the remaining positions). Similarly a natural generated immune response will be directed against other and/or more particular epitopes and therefore give rise to high signals in other positions and/or more positions in the array.
  • the immuno profile will reflect the effect of the vaccination on the immune response, and even if the individual has encountered the disease before and has generated a general immune response towards this disease, it will still be possible to deduce from the profiling whether this individual also has generated a specific response against the vaccine.
  • an individual's immuno profile for a set of antigens implicated in a specific disease is obtained.
  • a subset of epitopes from a number of antigens is used.
  • this is not a comprehensive profiling of this individual with regard to these antigens, but careful selection of the epitopes used may ensure that the profiling data can be used afterwards to choose between e.g. a limited set of vaccines available, or the data can be used to evaluate the immune response of the individual following an infection, where the epitopes used have been selected in order to avoid interference from related infectious diseases.
  • a personalized drug, -vaccine or -diagnostic test may be produced, based on the information obtained from the immuno profiling.
  • the array comprising all possible 8'-, 9'- 10'- and 1 1 '-mer peptide sequences derived from a given peptide antigen, and all 13, 14, 15 and 16'-mers of the same antigen, are synthesized and assembled in MHC multimers, and immobilized in an array. Then, the ability of the individual peptide to form a complex with MHC is tested.
  • W6/32 antibody an antibody that binds correctly folded MHC I heavy chain, when this heavy chain is assembled together with antigenic peptide and beta2microglobulin, and which therefore can be used to detect formation of MHC-peptide complex, as binding of W6/32 antibody is usually considered a strong indication that the MHC-peptide complex has been formed.
  • the ability of different peptides to enter into a MHC-peptide complex may also be promoted by the addition to the array of T-cells. Specific T-cells will drive the formation of the corresponding specific MHC-peptide complexes.
  • the MHC-peptide complex integrity can be examined by addition of the labelled W6/32 antibody or other antibodies specific for correct conformation. Positions on the array that have strong signals indicate that the peptide that was added to MHC and immobilized at this position, was capable of forming the MHC-peptide complex in the presence of specific T-cells. Alternatively, the binding of the specific T-cells to the corresponding MHC-peptide complexes may be detected directly through a labbelled antibody specific for the T cell.
  • T cells immobilized to solid support in either of the ways described above can following washing be eluted from the solid support and treated further.
  • Specific T-cells can e.g. be isolated through the use of bead-based MHC multimers. Bead-based MHC multimers are beads whereto monomer MHC-peptide complexes or MHC multimers are immobilized.
  • the cells After the cells have been isolated they can be manipulated in many different ways.
  • the isolated cells can be activated (to differentiate or proliferate), they can undergo induced apoptosis, or undesired cells of the isolated cell population can be removed. Then, the manipulated cell population can be re-introduced into the patient, or can be introduced into another patient.
  • a typical cell sorting experiment based on bead-based MHC multimers, would follow some of the steps of the general procedure outlined in general terms in the following: Acquire the sample, e.g. a cell sample from the bone marrow of a cancer patient. Block the sample with a protein solution, e.g. BSA or skim milk. Block the beads coated with MHC complexes, with BSA or skim milk. Mix MHC-coated beads and the cell sample, and incubate. Wash the beads with washing buffer, to remove unbound cells and non-specifically bound cells.
  • a protein solution e.g. BSA or skim milk
  • BSA or skim milk Block the beads coated with MHC complexes, with BSA or skim milk. Mix MHC-coated beads and the cell sample, and incubate. Wash the beads with washing buffer, to remove unbound cells and non-specifically bound cells.
  • Isolate the immobilized cells by either cleavage of the linker that connects MHC complex and bead; or alternatively, release the cells by a change in pH, salt- concentration addition of competitive binder or the like.
  • the cells are released under conditions that do not disrupt the integrity of the cells.
  • Manipulate the isolated cells induce apoptosis, proliferation or differentiation
  • solid tissue includes tissue, tissue biopsies, frozen tissue or tissue biopsies, paraffin embedded tissue or tissue biopsies and sections of either of the above mentioned.
  • sections of fixed or frozen tissues are incubated with MHC multimer, allowing MHC multimer to bind to specific T cells in the tissue section.
  • the MHC multimer may be labeled directly or through a labeled marker molecule.
  • the MHC multimer can be labeled with a tag that can be recognized by e.g. a secondary antibody, optionally labeled with HRP or another label.
  • the bound MHC multimer is then detected by its fluorescence or absorbance (for fluorophore or chromophore), or by addition of an enzyme- labeled antibody directed against this tag, or another component of the MHC multimer (e.g. one of the protein chains, a label on the multimerization domain).
  • the enzyme can be Horse Raddish Peroxidase (HRP) or Alkaline Phosphatase (AP), both of which convert a colorless substrate into a colored reaction product in situ. This colored deposit identifies the binding site of the MHC multimer, and can be visualized under a light microscope.
  • the MHC multimer can also be directly labeled with e.g. HRP or AP, and used in IHC without an additional antibody.
  • the tissue sections may derive from blocks of tissue or tissue biopsies embedded in paraffin, and tissue sections from this paraffin-tissue block fixed in formalin before staining. This procedure may influence the structure of the TCR in the fixed T cells and thereby influence the ability to recognize specific MHC complexes. In this case, the native structure of TCR needs to be at least partly preserved in the fixed tissue. Fixation of tissue therefore should be gentle.
  • the staining is performed on frozen tissue sections, and the fixation is done after MHC multimer staining.
  • MHC multimers are injected in to the body of the individual to be investigated.
  • the MHC multimers may be labeled with e.g. a paramagnetic isotope.
  • MRI magnetic resonance imaging
  • ESR electron spin resonance
  • MHC multimer binding T cells can then be measured and localized.
  • any conventional method for diagnostic imaging visualization can be utilized.
  • gamma and positron emitting radioisotopes are used for camera and paramagnetic isotopes for MRI.
  • T cells using MHC multimers also applies to detection of TCR in solution and detection of TCR attached to or in a solid medium. Though detection of individual TCRs may not be possible when TCR is in solution.
  • Indirect detection of TCR is primarily usefull for detection of TCRs embedded in lipid bilayer, preferably natural occurring T cells, T cell hybridomas or transfected T cells.
  • indirect detection the number or activity of T cells are measured, by detection of events that are the result of TCR-MHC-peptide complex interaction. Interaction between MHC multimer and T cell may stimulate the T cell resulting in activation of T cells, in cell division and proliferation of T cell populations or alternatively result in inactivation of T cells. All these mechanism can be measured using various detection methods.
  • MHC multimers e.g. antigen presenting cells
  • T cells can stimulate T cells resulting in activation of the stimulated T cells.
  • Activation of T cell can be detected by measurement of secretion of specific soluble factor from the stimulated T cell, e.g. secretion of cytokines like INF ⁇ and IL2.
  • Stimulation of T cells can also be detected by measurement of changes in expression of specific surface receptors, or by measurement of T cell effector functions.
  • MHC multimer To a sample of T cells, preferably a suspension of cells, is added MHC multimer to induce either secretion of soluble factor, up- or down-regulation of surface receptor or other changes in the T cell.
  • a sample of T cells containing antigen presenting cells is added antigenic peptide or protein/protein fragments that can be processed into antigenic peptides by the antigen presenting cell and that are able to bind MHC I or MHC Il molecules expressed by the antigen presenting cells thereby generating a cell based MHC multimer in the sample.
  • antigenic peptide or protein/protein fragments that can be processed into antigenic peptides by the antigen presenting cell and that are able to bind MHC I or MHC Il molecules expressed by the antigen presenting cells thereby generating a cell based MHC multimer in the sample.
  • the peptide-loaded antigen presenting cells can then stimulate specific T cells, and thereby induce the secretion of soluble factor, up- or down-regulation of surface receptors, or mediate other changes in the T cell, e.g. enhancing effector functions. --O
  • a second soluble factor e.g. cytokine and/or growth factor(s) may be added to facilitate continued activation and expansion of T cell population, b) Detect the presence of soluble factor, the presence/ absence of surface receptor or detect effector function c) Correlate the measured result with presence of T cells.
  • the measured signal/response indicate the presence of specific T cells that have been stimulated with particular MHC multimer.
  • the signal/response of a T lymphocyte population is a measure of the overall response.
  • the frequency of specific T cells able to respond to a given MHC multimer can be determined by including a limiting-dilution culture in the assay also called a Limiting dilution assay.
  • the limiting-dilution culture method involves the following steps: a) Sample of T cells in suspension are plated into culture wells at increasing dilutions b) MHC multimers are added to stimulate specific T cells . Alternatively antigen presenting cells are provided in the sample and then antigenic peptide I added to the sample as descrinbed above. Optionally growth factors, cytokines or other factors helping T cells to proliferate are added.
  • c) Cells are allowed to grow and proliferate (V2- several days). Each well that initially contained a specific T cell will make a response to the MHC multimer and divide. d) Wells are tested for a specific response e.g. secretion of soluble factors, cell proliferation, cytotoxicity or other effector function. The assay is replicated with different numbers of T cells in the sample, and each well that originally contained a specific T cell will make a response to the MHC multimer. The frequency of specific T cells in the sample equals the reciprocal of the number of cells added to each well when 37% of the wells are negative, because due to Poisson distrubtion each well then on average contained one specific T cell at the beginning of the culture.
  • Indirect detection of T cells by measurement of secretion of soluble factors Indirect detection of T cells by measurement of extracellular secreted soluble factors.
  • Indirect detection of T cells by measurement of extracellular secreted soluble factors Secreted soluble factors can be measured directly in fluid suspension, captured by immobilization on solid support and then detected or an effect of the secreted soluble factor can be detected.
  • a sample of T cells are added MHC multimer or antigenic peptide as described above to induce secretion of soluble factors from antigen-specific T cells.
  • the secreted soluble factors can be measured directly in the supernatant using e.g. mass spectrometry.
  • T cells o Indirect detection of T cells by capture of extracellular secreted soluble factor on solid support.
  • a sample of T cells are added MHC multimer or antigenic peptide as described above to induce secretion of soluble factors from antigen-specific T cells.
  • Secreted soluble factors in the supernatant are then immobilized on a solid support either directly or through a linker as described for immobilization of T cells elsewhere herein. Then immobilized soluble factors can be detected using labeled marker molecules.
  • Soluble factors secreted from individual T cells can be detected by capturing of the secreted soluble factors locally by marker molecules, e.g antibodies specific for the soluble factor. Soluble factor recognising marker molecules are then immobilised on a solid support together with T cells and soluble factors secreted by individual T cells are thereby captured in the proximity of each T cell. Bound soluble factor can be measured using labelled marker molecule specific for the captured soluble factor. The number of T cells that has given rise to labelled spots on solid support can then be enumerated and these spots indicate the presence of specific T cells that may be stimulated with particular
  • Soluble factors secreted from a population of T cells are detected by capture and detection of soluble factor secreted from the entire population of specific T cells.
  • soluble factor do not have to be captured locally close to each T cell but the secreted soluble factors my be captured and detected in the same well as where the T cells are or transferred to another solid support with marker molecules for capture and detection e.g. beads or wells of ELISA plate.
  • Different MHC multimers og MHC-peptide complexes are immobilized to a support to form a spatial array in a defined pattern, where the position specifies the identity of the MHC multimer/MHC-peptide complex immobilized at this position.
  • Marker molecules able to bind T cell secreted soluble factors are co- spotted together with MHC multimer/MHC-peptide complex.
  • Such marker molecules can e.g. be antibodies specific for cytokines like INF ⁇ or IL-2.
  • the immobilization may be direct or through a linker molecule as described above.
  • a suspension of labeled T cells are added or passed over the array of MHC multimers/MHC-peptide complexes and specific T cells will bind to the immobilized MHC multimers/MHC-peptide complexes and upon binding be stimulated to secrete soluble factors e.g. cytokines like INF ⁇ ord IL-2.
  • Soluble factors secreted by individual T cells are then captured in the proximity of each T cell and bound soluble factor can be measured using labelled marker molecule specific for the soluble factor.
  • the number and position of different specific T cells that has given rise to labelled spots on solid support can then be identified and enumerated. In this way T cells bound to defined areas of the support are analyzed, thereby, phenotyping the sample.
  • Each individual T cell is defined by the TCR it expose and depending on these TCRs each entity will bind to different types of MHC multimers/MHC-peptide complexes immobilized at defined positions on the solid support.
  • Secreted soluble factors can be measured and quantified indirectly by measurement of the effect of the soluble factor on other cell systems. Briefly, a sample of T cells are added MHC multimer or antigenic peptide as described above to induce secretion of soluble factors from antigen-specific T cells. The supernatant containing secreted soluble factor are transferred to another cell system and the effect measured. The soluble factor may induce proliferation, secretion of other soluble factors, expression/downregulation of receptors, or the soluble factor may have cytotoxic effects on these other cells,. All effects can be measured as described elsewhere herein.
  • Soluble factor production by stimulated T cells can be also be measured intracellular by e.g. flow cytometry. This can be done using block of secretion of soluble factor (e.g. by monensin), permeabilization of cell (by e.g. saponine) followed by immunofluorescent staining.
  • the method involves the following steps: 1 ) Stimulation of T cells by binding specific MHC multimers, e.g. antigen presenting cells loaded with antigenic peptide.
  • An reagent able to block extracellular secretion of cytokine is added, e.g. monensin that interrupt intracellular transport processes leading to accumulation of produced soluble factor, e.g.
  • cytokine in the Golgi complex During stimulation other soluble factors may be added to the T cell sample during stimulation to enhance activation and/or expansionn This other soluble factor can be cytokine and or growth factors. 2) addition of one or more labelled marker able to detect special surface receptors (e.g. CD8,
  • labelled marker specific for the produced soluble factor to be determined e.g. INF ⁇ , IL-2, IL-4, IL-10.
  • Activation of T cells can be detected by measurement of expression and/or down regulation of specific surface receptors.
  • the method includes the following steps.
  • a sample of T cells are added MHC multimer or antigenic peptide as described above to induce expression or downregulation of specific surface receptors on antigen-specific T cells.
  • These receptors include but are not limited to CD28, CD27, CCR7, CD45RO, CD45RA, IL2-receptor, CD62L, CCR5.
  • Their expression level can be detected by addition of labelled marker specific for the desired receptor and then measure the amount of label using flow cytometry, microscopy, immobilization of activated T cell on solid support or any other method like those decribed for direct detection of TCR in lipid bilayer.
  • T cells Indirect detection of T cells by measurement of effector function Activation of T cells can be detected indirectly by measurement of effector functions.
  • a sample of T cells are added MHC multimer or antigenic peptide as described above to induce the T cell to be able to do effector function.
  • the effector function is then measured.
  • activation of antigen-specific CD8 positive T cells can be measured in a cytotoxicity assay.
  • T cells can be stimulated to proliferate upon binding specific MHC multimers. Proliferation of T cells can be measured several ways including but not limited to: o Detection of mRNA
  • Proliferation of T cells can be detected by measurement of mRNA inside cell.
  • Cell division and proliferation requires production of new protein in each cell which as an initial step requires production of mRNA encoding the proteins to be synthesized.
  • a sample of T cells are added MHC multimer or antigenic peptide as described above to induce proliferation of antigen-specific T cells.
  • Detection of levels of mRNA inside the proliferating T cells can be done by quantitative PCR and indirectly measure activation of a T cell population as a result of interaction with MHC multimer.
  • An example is measurement of cytokine mRNA by in sity hybridization.
  • the proliferative capacity of T cells in response to stimulation by MHC multimer can be determined by a radioactive assay based on incorporation of [ 3 H]thymidine
  • Detection of incorporation of BrdU T cell proliferation can also be detected by of incorporation of bromo-2'- deoxyuridine (BrdU) followed by measurement of incorporated BrdU using a labeled anti-BrdU antibody in an ELISA based analysis.
  • BrdU bromo-2'- deoxyuridine
  • Viability of cells may be measured by measurement ATP in a cell culture. Indirect detection of T cells by measurement of inactivation
  • MHC multimers will lead to activation of the T cells they bind. Under certain circumstances some MHC multimers may rather inactivate the T cells they bind to.
  • Inactivation of T cells by MHC multimers may be measured be measuring the effect of blocking TCR on antigen-specific T cells.
  • MHC multimers e.g. MHC-peptide complexes coupled to IgG scaffold can block the TCR of an antigen-specific T cell by binding the TCR, thereby prevent the blocked T cell receptor interacting with e.g. antigen presenting cells.
  • Blockade of TCRs of a T cell can be detected in any of the above described medthods for detection of TCR by addition of an unlabeled blocking MHC multimer together with the labelled MHC multimer and then measuring the effect of the blockade on the readout.
  • Inactivation of T cells by MHC multimers may be measured be measuring apoptosis of the antigen-specific T cell. Binding of some MHC multimers to specific T cells may lead to induction of apoptosis. Inactivation of T cells by binding MHC multimer may therefore be detected by measuring apoptosis in the T cell population. Methods to measure apoptosis in T cells include but are not limited to measurement of the following:
  • positive and negative control reagents can also be used to evaluate the quality (e.g. specificity and affinity) and stability (e.g. shelf life) of produced MHC multimers.
  • the quality and stability of a given MHC multimer can be tested in a number of different ways, including: • Measurement of specific MHC multimer binding to beads, other types of solid support, or micelles and liposomes, to which TCR's have been immobilized. Other kinds of molecules that recognize specifically the MHC-peptide complex can be immobilized and used as well. Depending on the nature of the solid support or membrane structure to which the TCR is immobilized, the TCR can be full-length (i.e. comprise the intracellular- and intra-membrane domains), or can be truncated (e.g. only comprise the extracellular domains). Likewise, the TCR can be recombinant, and can be chemically or enzymatically modified. • Measurement of MHC multimer binding to beads, other types of solid support, or micelles and liposomes, to which aptamers, antibodies or other kinds of molecules that recognize correctly folded MHC-peptide complexes have been immobilized.
  • a biotinylated fluorophore in cases where the MHC multimer contains streptavidin proteins, not fully loaded with biotin.
  • the secondary component is unlabelled, and a labelled second component-specific compound is employed (e.g. EnVision System, Dako) for visualization.
  • This solid surface can be beads, immunotubes, microtiterplates act.
  • the principle for purification are basically the same I.e. T cells are added to the solid with immobilized MHC'mer, non-binding T cells are washed away and MHC-peptide specific T cells can be retrieved by elution with mild acid or a competitive binding reagent.
  • a secondary component specific to MHC multimer e.g. TCRs, aptamers, antibodies, streptavidin, or other MHC-peptide binding complex-binding molecules.
  • the secondary receptor is unlabelled, and a labelled second receptor-specific compound is employed (e.g. EnVision System, Dako) before visualization.
  • positive control reagents include MHC multimers comprising correctly folded MHC, complexed with an appropriate peptide that allows the MHC multimer to interact specifically and efficiently with its cognate TCR.
  • Negative control reagents include empty MHC multimers, or correctly folded MHC multimers complexed with so-called nonsense peptides that support a correct conformation of the MHC-peptide complex, but that do not efficiently bind TCRs through the peptide- binding site of the MHC complex.
  • Negative control reagents and negative control experiments for the use of MHC multimers in flow cytometry and related techniques require a negative control in order to determine background staining with MHC multimer.
  • Background staining can be due to unwanted binding of any of the individual components of the MHC multimer, e.g., MHC complex or individual components of the MHC complex, multimerization domain or label molecules.
  • the unwanted binding can be to any surface or intracellular protein or other cellular structure of any cell in the test sample, e.g. undesired binding to B cells, NK cells or T cells.
  • Negative control reagents include the following: • MHC complexes or MHC multimers comprising MHC complexes carrying nonsense peptides.
  • a nonsense peptide is here to be understood as a peptide that binds the MHC protein efficiently, but that does not support binding of the resultant MHC- peptide complex to the desired TCR.
  • An example nonsense peptide is a peptide with an amino acid sequence different from the linear sequence of any peptide derived from any known protein. When choosing an appropriate nonsense peptide the following points are taken into consideration.
  • the peptide should ideally have appropriate amino acids at relevant positions that can anchor the peptide to the peptide-binding groove of the MHC.
  • the remaining amino acids should ideally be chosen in such a way that possible binding to TCR (through interactions with the peptide or peptide-binding site of MHC) are minimized.
  • the peptide should ideally be soluble in water to make proper folding with MHC alpha chain and ⁇ 2m possible in aqueous buffer.
  • the length of the peptide should ideally match the type and allele of MHC complex.
  • the final peptide sequence should ideally be taken through a blast search or similar analysis, to ensure that it is not identical with any peptide sequence found in any known naturally occurring proteins.
  • MHC complexes or MHC multimers comprising MHC complexes carrying a chemically modified peptide in the peptide-binding groove.
  • the modification should ideally allow proper conformation of the MHC-peptide structure, yet should not allow efficient interaction of the peptide or peptide-binding site of MHC with the
  • MHC complexes or MHC multimers comprising MHC complexes carrying a naturally occurring peptide different from the peptide used for analysis of specific T cells in the sample.
  • the peptide in complex with the MHC protein should ideally not be likely to bind a TCR of any T cell in the sample with such an affinity that it can be detected with the applied analysis method.
  • the peptide should ideally be soluble in water to make proper folding with MHC alpha chain and ⁇ 2m possible in aqueous buffer.
  • the length of the peptide should match the type and allele of MHC complex.
  • MHC heavy chain or MHC multimers comprising MHC heavy chain, where MHC heavy chain should be understood as full-length MHC I or MHC Il heavy chain or any truncated version of MHC I or MHC Il heavy chain.
  • the MHC heavy chains can be either folded or unfolded.
  • MHC Il ⁇ chains containing the ⁇ 2 domain that binds CD4 on the surface of helper T cells are also be understood as full-length MHC I or MHC Il heavy chain or any truncated version of MHC I or MHC Il heavy chain.
  • the MHC heavy chains can be either folded or unfolded.
  • MHC Il ⁇ chains containing the ⁇ 2 domain that binds CD4 on the surface of helper T cells are also be understood as full-length MHC I or MHC
  • Beta2microglobulin or subunits of beta2microglobulin, or MHC multimers comprising Beta2microglobulin or subunits of beta2microglobulin, folded or unfolded.
  • MHC-like complexes or MHC multimers comprising MHC-like complexes, folded or unfolded.
  • An example could be CD1 molecules that are able to bind peptides in a peptide-binding groove that can be recognized by T cells (Russano et al. (2007). CD1 -restricted recognition of exogenous and self-lipid antigens by duodenal gammadelta ⁇ T lymphocytes. J Immunol. 178(6):3620-6 )
  • Multimerization domains without MHC or MHC-like molecules e.g. dextran, streptavidin, IgG, coiled-coil-domain liposomes.
  • Labels e.g. FITC, PE, APC, pacific blue, cascade yellow, or any other label listed elsewhere herein.
  • Negative controls 1 -4 can provide information about potentially undesired binding of the MHC multimer, through interaction of a surface of the MHC-peptide complex different from the peptide-binding groove and its surroundings.
  • Negative control 5 and 6 can provide information about binding through interactions through the MHC I or MHC Il proteins (in the absence of peptide).
  • Negative control 7 can provide information about binding through surfaces of the MHC complex that is not unique to the MHC complex.
  • Negative controls 8 and 9 provide information about pontential undesired interactions between non-MHC-peptide complex components of the MHC multimer and cell constituents.
  • MHC-peptide specific T cells can give rise to background signals due to unwanted binding to cells that do not carry TCRs. This undesired binding can result from binding to cells or other material, by various components of the MHC multimer, e.g. the dextran in a MHC dextramer construct, the labelling molecule (e.g. FITC), or surface regions of the MHC-peptide complex that do not include the peptide and the peptide-binding cleft.
  • the MHC multimer e.g. the dextran in a MHC dextramer construct, the labelling molecule (e.g. FITC), or surface regions of the MHC-peptide complex that do not include the peptide and the peptide-binding cleft.
  • MHC-peptide complexes bind to specific T cells through interaction with at least two receptors in the cell membrane of the T-cell. These two receptors are the T-cell receptor (TCR) and CD8 for MHC l-peptide complexes and TCR and CD4 receptor protein for MHC I l-peptide complexes. Therefore, a particularly interesting example of undesired binding of a MHC multimer is its binding to the CD8 or CD4 molecules of T cells that do not carry a TCR specific for the actual MHC-peptide complex.
  • TCR T-cell receptor
  • CD8 or CD4 molecules with the MHC is not very strong; however, because of the avidity gained from the binding of several MHC complexes of a MHC multimer, the interaction between the MHC multimer and several CD8 or CD4 receptors potentially can result in undesired but efficient binding of the MHC multimer to these T cells. In an analytical experiment this would give rise to an unwanted background signal; in a cell sorting experiment undesired cells might become isolated.
  • Other particular interesting examples of undesired binding is binding to lymphoid cells different from T cells, e.g. NK-cells, B-cells, monocytes, dendritic cells, and granulocytes like eosinophils, neutrophils and basophiles.
  • MHC complex Apart from the MHC complex, other components in the MHC multimer can give rise to unspecific binding. Of special interest are the multimerization domain, multimerization domain molecules, and labelling molecules.
  • One way to overcome the problem with unwanted binding is to include negative controls in the experiment and subtract this signal from signals derived from the analyzed sample, as described elsewhere in the invention.
  • Mutations in areas of the MHC complex responsible for binding to unwanted cells can be introduced. Mutations here mean substitution, insertion, or deletion of natural or non-natural amino acids. Sub-domains in the MHC complex can be responsible for unwanted binding of the MHC multimer to cells without a TCR specific for the MHC-peptide complex contained in the MHC multimer.
  • One example of special interest is a small region in the oc3-domain of the ⁇ -chain of MHC I molecules that is responsible for binding to CD8 on all cytotoxic T cells.
  • Another embodiment is to mutate other areas of MHC I /MHC Il complexes that are involved in interactions with T cell surface receptors different from TCR, CD8 and CD4, or that bind surface receptors on B cells, NK cells, Eosiniophils, Neutrophils, Basophiles, Dendritic cells or monocytes.
  • Chemical alterations in areas of the MHC complex responsible for binding to unwanted cells can be employed in order to minimize unwanted binding of MHC multimer to irrelevant cells.
  • Chemical alteration here means any chemical modification of one or more amino acids.
  • Regions in MHC complexes that are of special interest are as mentioned above the ⁇ 3 domain of the ⁇ -chain in MHC I molecules and ⁇ 2 domains in the ⁇ -chain of MHC Il molecules.
  • Other regions in MHC I /MHC Il molecules that can be chemically modified to decrease the extent of undesired binding are regions involved in interaction with T cell surface receptors different from TCR, CD8 and CD4, or that bind surface receptors on B cells, NK cells, Eosiniophils, Neutrophils, Basophiles, Dendritic cells or monocytes.
  • Another method to minimize undesired binding involves the addition of one or more components of a MHC multimer, predicted to be responsible for the unwanted binding.
  • the added component is not labeled, or carries a label different from the label of the MHC multimer used for analysis.
  • MHC multimers that contain nonsense peptides, i.e. peptides that interact efficiently with the MHC protein, but that expectably do not support specific binding of the MHC multimer to the TCR in question.
  • Another example of interest is addition of soluble MHC complexes not coupled to a multimerization domain, and with or without peptide bound in the peptide binding cleft.
  • individual components of the MHC complex can be added to the sample, e.g.
  • Reagents able to identify specific cell types either by selection or exclusion can be included in the analysis to help identify the population of T cells of interest, and in this way deselect the signal arising from binding of the MHC multimer to undesired cells.
  • Reagents able to identify specific cell types either by selection or exclusion can be included in the analysis to help identify the population of T cells of interest, and in this way deselect the signal arising from binding of the MHC multimer to undesired cells.
  • fluorescent antibodies directed against specific surface markers can be used for identification of specific subpopulations of cells, and in this way help to deselect signals resulting from MHC multimers binding to undesired cells.
  • Gating reagents of special interest that helps identify the subset of T cells of interest when using MHC I multimers are reagents binding to CD3 and CD8 identifying all cytotoxic T cells.
  • These reagents are preferably antibodies but can be any labeled molecule capable of binding CD3 or CD8. Gating reagents directed against CD3 and CD8 are preferably used together. As they stain overlapping cell populations they are preferably labeled with distinct fluorochromes. However, they can also be used individually in separate samples. In experiments with MHC Il multimers reagents binding to CD3 and CD4 identifying T helper cells can be used. These reagents are preferably antibodies but can be any labeled molecule capable of binding CD3 or CD4. Gating reagents directed against CD3 and CD4 are preferable used together. As they stain overlapping cell populations they are preferably labeled with distinct fluorochromes. However, they can also be used individually in separate samples.
  • gating reagents of special interest in experiments with any MHC multimer are reagents binding to the cell surface markers CD2, CD27, CD28, CD45RA, CD45RO, CD62L and CCR7. These surface markers are unique to T cells in various differentiation states. Co staining with either of these reagents or combinations thereof together with MHC multimers helps to select MHC multimer binding T cells expressing a correct TCR. These reagents can also be combined with reagents directed against CD3, CD4 and/or CD8. Another flow cytometric method of special interest to remove signals from MHC multimer stained cells not expressing the specific TCR, is to introduce an exclusion gate.
  • Antibodies or other reagents specific for surface markers unique to the unwanted cells are labeled with a fluorochrome and added to the test sample together with the MHC multimer.
  • the number of antibodies or surface marker specific reagents are not limited to one but can be two, three, four, five, six, seven, eight, nine, ten or more individual reagents recognizing different surface markers, all of which are unique to the unwanted cells.
  • all events representing cells labeled with these antibodies are dumped in the same gate and removed from the dataset. This is possible because all the antibodies/reagents that bind to the wrong cells are labeled with the same fluorochrome.
  • Reagents of special interest that exclude irrelevant cells include reagents against CD45 expressed on red blood cells, CD19 expressed on B cells, CD56 expressed on NK cells, CD4 expressed on T helper cells and CD8 expressed on cytotoxic T cells, CD14 expressed on monocytes and CD15 expressed on granulocytes and monocytes.
  • MHC multimers that comprise a polymer such as dextran, or that are cell-based (e.g. specialized dendritic cells such as described by Banchereau and Palucka, Nature Reviews, Immunology, 2005, vol. 5, p. 296-306).
  • Preventive vaccination leading to prophylaxis/sterile immunity by inducing memory in the immune system may be obtained by immunizing/vaccinating an individual or animal with MHC alone, or with MHC in combination with other molecules as mentioned elsewhere in the patent.
  • o Vaccine antigens can be administered alone
  • o Vaccine can be administered in combination with adjuvant(s).
  • D Adjuvant can be mixed with vaccine component or administered alone, simultaneously or in any order.
  • D Adjuvant can be administered by the same route as the other vaccine components o Vaccine administered more than once may change composition from 1 st administration to the 2 nd , 3 rd , etc.
  • o Vaccine administered more than once can be administered by alternating routes o Vaccine components can be administered alone or in combinations by the same route or by alternating/mixed routes o Vaccine can be administered by the following routes D Cutaneously
  • D Pulmonally D Vaginally D Rectal Iy D Therapeutic vaccination i.e. vaccination "teaching" the immune system to fight an existing infection or disease may be obtained by immunizing/vaccinating an individual or animal with MHC alone, or with MHC in combination with other molecules as mentioned elsewhere in the patent.
  • o Vaccine antigens can be administered alone
  • o Vaccine can be administered in combination with adjuvant(s).
  • D Adjuvant can be mixed with vaccine component or administered alone, simultaneously or in any order.
  • D Adjuvant can be administered by the same route as the other vaccine components o Vaccine administered more than once may change composition from 1 st administration to the 2 nd , 3 rd , etc.
  • o Vaccine administered more than once can be administered by alternating routes
  • o Vaccine components can be administered alone or in combinations by the same route or by alternating/mixed routes
  • o Vaccine can be administered by the following routes
  • D Theraoeutic treatment includes the i any molecular combination mentioned elsewhere in the patent application for the purpose of treating a disease in any state. Treatment may be in the form of o Per-orally intake
  • D Treatment can be performed as o Single intake, injection, application, washing o Multiple intake, injection, application, washing D On single day basis D Over prolonged time as days, month, years D
  • Personalized medicine takes advantage of the large diversity of peptide epitopes that may be generated from a given antigen.
  • the immune system is very complex. Each individual has a very large repertoire of specific T cells (on the order of 10 6 -10 9 different T cell specificities), which again is only a small subset of the total T cell repertoire of a population of individuals. It is estimated that the Caucasian population represents a T cell diversity of 10 10 -10 12 . MHC allele diversity combined with large variation among individuals' proteolytic metabolism further enhances the variation among different individuals' immune responses. As a result, each individual has its own characteristic immune response profile.
  • MHC multimer-based immune monitoring reagent or immunotherapeutic agent This is important when designing a MHC multimer-based immune monitoring reagent or immunotherapeutic agent. If an agent is sought that should be as generally applicable as possible, one should try to identify peptide epitopes and MHC alleles that are common for the majority of individuals of a population. As described elsewhere in this application, such peptide epitopes can be identified through computerized search algorithms developed for that same purpose, and may be further strengthened by experimental testing of a large set of individuals.
  • this may involve testing a large number of different epitopes from a given antigen, in order to find peptide epitopes that may provide MHC multimers with efficiency for a given individual.
  • personalized medicine takes advantage of the wealth of peptide epitopes that may be generated from a given antigen.
  • a large number of the e.g. 8-, 9-, 10-, and 1 1 - mer epitopes that may be generated from a given antigen to be included in a class 1 MHC multimer reagent, for use in immune monitoring or immunotherapy, are therefore of relevance in personalized medicine.
  • Only in the case where one wants to generate a therapeutic agent or diagnostic reagent that is applicable to the majority of individuals of a population can the large majority of epitope sequences be said to be irrelevant, and only those identified by computerized search algorithms and experimental testing be said to be of value.
  • these disregarded peptide epitopes may be the epitopes that provide an efficient diagnostic reagent or cures that individual from a deadly disease.
  • the present invention relates in one embodiment to antigenic peptides derived from CMV antigens.
  • the one or more antigenic peptides can in one embodiment comprise one or more fragments from one or more CMV antigens capable of interacting with one or more MHC class 1 molecules.
  • the one or more antigenic peptides can in another embodiment comprise one or more fragments from one or more CMV antigens capable of interacting with one or more MHC class 2 molecules.
  • the antigenic peptides can be generated from any CMV antigen such as the CMV antigens listed in Table 6.
  • MHC Class I and MHC Class Il molecules have different structures, as described above, and therefore have different restrictions on the size of the peptide which may be accommodated.
  • MHC Class I molecules will accommodate peptides of from about 8 amino acids in length to about 1 1 amino acids.
  • MHC Class Il molecules will in general accommodate peptides of from about 13 amino acids in length to about 16 amino acids.
  • Peptides derived from the sequences shown in Table 6, for use preferably with MHC Class I or ll-based multimers are shown in Table 9.
  • the antigenic peptides can in one embodiment be generated by computational prediction using NetMHC (www.cbs.dtu.dk/services/NetMHC/) or by selected of specific 8, 9, 10, 1 1 , 13, 14, 15 or 16 amino acid sequences.
  • the present invention relates to one or more MHC multimers and/or one or more MHC complexes comprising one or more antigenic peptides such as the antigenic peptides listed in Table 7, Table 8 and/or Table 9 (SEQ ID NO 1 to SEQ ID NO 9697) and/or the antigenic peptides characterized by item 1 to 735 herein below.
  • the one or more antigenic peptides can in one embodiment comprise or consist of a fragment of one or more antigenic peptides listed in Table 7, Table 8 and/or Table 9 (SEQ ID NO 1 to SEQ ID NO 9697) and/or the antigenic peptides characterized by item 1 to 735 herein below, such as a fragment consisting of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, or 15 amino acids.
  • the antigenic peptide listed in Table 7, Table 8 and/or Table 9 (SEQ ID NO 1 to SEQ ID NO 9697) and/or the antigenic peptides characterized by item 1 to 735 herein below can be part of a larger peptide/protein, wherein the larger peptide/protein may be of a total length of 17, such as 18, for example 19, such as 20, for example 21 , such as 22, for example 23, such as 24, for example 25, such as 26, for example 27, such as 28, for example 29, such as 30, for example 31 , such as 32, for example 33, such as 34, for example 35, such as 36, for example 37, such as 38, for example 39, such as 40 amino acids, wherein 8 to 16 of said amino acids are defined in the items below.
  • the larger peptide/protein may be of a total length of 17, such as 18, for example 19, such as 20, for example 21 , such as 22, for example 23, such as 24, for example 25, such as 26, for example 27, such as 28, for example 29, such as 30, for example 31 , such as 32, for example
  • the larger protein may be of a total length of between 20 to 30, such as 30-40, for example 40-50, such as 50-60, for example 60-70, such as 70-80, for example 80-90, such as 90-100, for example 100- 150, such as 150-200, for example 200-250, such as 250-300, for example 300-500, such as 500-1000, for example 1000-2000, such as 2000-3000, for example 3000- 4000, such as 4000-5000, for example 5000-10,000, such as 10,000-20,000, for example 20,000-30,000, such as 30,000-40,000, for example 40,000-50,000, such as 50,000-75,000, for example 75,000-100,000, such as 100,000-250,000, for example 250,000-, 500, 000, such as 500,000-1 ,000,000 amino acids.
  • the antigenic peptides listed in Table 7, Table 8 and/or Table 9 are modified by one or more type(s) of post- translational modifications such as one or more of the post-translational modifications listed in the items (item 1 to 735) herein below.
  • the same or different types of post- translational modification can occur on one or more amino acids in the antigenic peptide such as on 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15 or 16 amino acids.
  • Table 7 Prediction of cancer antigen BcIX(L) specific MHC class 1 , 8- , 9- ,10- ,11-mer peptide binders.

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Abstract

La présente invention concerne des complexes MHC (complexe majeur d'histocompatibilité)-peptide et des utilisations de ceux-ci dans le diagnostic de, le traitement de ou la vaccination contre une maladie chez un individu. Plus spécifiquement, l’invention concerne des complexes de MHC comprenant des peptides antigéniques du CMV et des utilisations de ceux-ci.
PCT/DK2009/050257 2008-10-01 2009-10-01 Multimères de mhc dans la surveillance immunitaire contre le cmv WO2010037397A1 (fr)

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