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WO2003098219A1 - Mesure par dosage immunoenzymatique (elisa) de l'interaction de novo entre un peptide et une molecule mhc - Google Patents

Mesure par dosage immunoenzymatique (elisa) de l'interaction de novo entre un peptide et une molecule mhc Download PDF

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Publication number
WO2003098219A1
WO2003098219A1 PCT/DK2003/000325 DK0300325W WO03098219A1 WO 2003098219 A1 WO2003098219 A1 WO 2003098219A1 DK 0300325 W DK0300325 W DK 0300325W WO 03098219 A1 WO03098219 A1 WO 03098219A1
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Prior art keywords
peptide
mhc
concentration
complexes
binding
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English (en)
Inventor
Søren Buus
Christina Silvester-Hvid
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Københavns Universitet
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Københavns Universitet
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Priority to AU2003223937A priority Critical patent/AU2003223937A1/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules

Definitions

  • the present invention relates to a quantitative assay capable of measuring peptide-MHC interaction.
  • the immune system cannot be manipulated in any rational way (vaccines, treatment, cancer, and autoimmune diseases etc.) without a thorough understanding of the major histocompatibility gene complex (MHC); how it selects and binds peptides.
  • MHC major histocompatibility gene complex
  • the classical MHC molecules can be divided into two groups, class I and class II respectively, both are cell surface glycoproteins with an overall quite similar structure.
  • the class I MHC complex contains a large ⁇ heavy chain associated non-covalently with the much smaller light chain ⁇ 2 - microglobulin ( ⁇ 2 m) and a peptide placed in the peptide binding groove made up by the heavy chain.
  • the class II MHC complex contains two different polypeptide chains, the ⁇ and the ⁇ associated non-covalently in complex with a peptide placed in the peptide binding cleft.
  • the Human MHC project is an endeavour aimed at a detailed mapping of all MHC specificities and the development of quantitative prediction models for the binding of peptides to MHC molecules.
  • the long-term goal is to provide the following resources: Standardised strategies and methods for detection of peptide-MHC binding, detailed description of MHC specificity and quantitative predictions of antigen processing and presentation.
  • affinity purified MHC molecules are incubated with a radio-iodinated indicator peptide, and the resulting peptide-MHC complexes are separated from free peptide allowing an accurate determination of the degree of binding (Buus, S. et al. 1986). Once a binding assay has been established for a particular MHC allotype, the binding of any other peptide can be determined in an appropriately conducted competition assay.
  • the present invention relates to a quantitative method to measure the interaction between a peptide and a MHC molecule.
  • the basis for measuring this interaction is substantially pure denatured MHC heavy chains. These denaturated MHC heavy chains are subsequently renaturated in a buffer containing excess of ⁇ 2 m and/or an increasing concentration of the peptide in question, leading to a de novo folding and generation of peptide-MHC complexes.
  • the present invention is exemplified with reference to MHC class I proteins, but it is envisaged that it is possible in a similar manner to generate data from any type of complex where the de novo folding is peptide dependent, e.g. MHC class II.
  • the concentration of the generated complexes can be measured in a quantitative ELISA.
  • a quantitative ELISA By the preferred conditions of the present invention neither the amount of capturing agents nor detection agents are limited in said quantitative ELISA, thus enabling a person skilled in the art to determine the concentration of the complexes formed using a generated standard curve.
  • the de novo folding of peptide-MHC complexes happens in an entirely peptide dependent manner and thus the present invention measures the amount of de novo folded molecules, not just the amount of peptides bound to a complex.
  • the OD 50 response from said quantitative ELISA vs. the logarithm of the molar concentration of an MHC standard are plotted, and optimally fitted to a sigmoid curve.
  • the OD 450 of any sample can be converted to the concentration of MHC complexes present in that sample.
  • a sample is either de novo folded peptide-MHC complexes in solution or a prefolded peptide-MHC complex.
  • prefolded peptide-MHC complex relates to any peptide- MHC complex soluble in a hydrophilic solution generated either in vitro or in vivo, such as natural peptide-MHC complexes purified from eucaryotic cells or from recombinantly produced compounds in e.g., but not limited, to a prokaryotic cell.
  • the generated peptide-MHC complexes are transferred to a standard ELISA plate coated with a capturing agent, capturing the peptide-MHC complexes.
  • the peptide-MHC complexes are immobilised by the capturing agent and can then be detected by a detection agent.
  • the invention relates to a quantitative method to measure the interaction between peptide and MHC, the method comprising the steps of
  • the term "substantially pure” should be understood as a MHC heavy chain in a solution essentially devoid of contaminating peptides.
  • the term "denaturated MHC” should be understood as any unfolded MHC polypeptide chain, in which the molecular structure of the MHC has been modified by a denaturing agent such as any chaeotrophic compound like urea or guanidine hydrochloride, heat, acid, alkali, or ultraviolet radiation allowing removal of any contaminating peptide.
  • a denaturing agent such as any chaeotrophic compound like urea or guanidine hydrochloride, heat, acid, alkali, or ultraviolet radiation allowing removal of any contaminating peptide.
  • the denaturated MYC has to be subjected to a folding process where the investigator has full control over the conditions including the peptide identity and amounts offered, the buffer composition, time, temperature, and/or pH.
  • MHC heavy chains generated recombinantly such as described in Pedersen L.0., et al., 2001 are highly active in terms of peptide binding, thus such MHC heavy chains are a presently preferred embodiment of the present invention.
  • a "quantitative ELISA” is the conversion of a given OD value obtained from a given sample, containing de novo folded peptide-MHC complexes in solution, to an accurate determination of the concentration of said peptide-MHC complexes. This conversion is performed by use of a standard curve as described above.
  • curve fitting relates to any function which describes the concentration of bound peptide as a function of the concentration of offered peptide, or vice versa.
  • a non-linear regression such as hyperbola or similar known to a person skilled in the art is used.
  • a proper de novo folding of a peptide-MHC complex into a "native state” all compounds are crucial, said crucial compounds for the class I complex being the ⁇ 2 m, peptide and class I heavy chain, and for the class II molecule the ⁇ and ⁇ -chain and peptide respectively, whether they be natural molecules or recombinant molecules (including engineered truncated, fused molecules etc.).
  • "native state” is used to define a MHC molecule occupied with a peptide in the peptide binding groove.
  • an MHC heavy chain relates to a polypeptide chain encoded by any allele located in loci Mhc-I and Mhc-II, or equivalent polypeptide chains encoded recombinantly, or fragments thereof. Further, since any vertebrae studied to date possesses the major histocompatibility gene complex, the invention relates to any denatured heavy chain MHC encoded by any vertebrae.
  • vertebrae include but are not limited to the Order Rodentia, such as mice; Order Logomorpha, such as rabbits; more particularly the Order Camivora, including Felines (cats) and Canines (dogs); even more particularly the Order Artiodactyla, Bovines (cows) and Suines (pigs); and the Order Perissodactyla, including Equines (horses); and most particularly the Order Primates, Ceboids and Simoids (monkeys) and Anthropoids (humans and apes).
  • the vertebrae are humans.
  • a MHC complex relates to a complex where the various component of MHC class I or MHC class II have been assembled.
  • MHC molecule relates to the same subject matter.
  • a preferred embodiment of the present invention determines peptide affinity (K D ).
  • Nonlinear regression is employed on all data obtained by the present invention, where the data are fitted to an equation describing the binding of a ligand to a receptor that follows the law of mass action.
  • the law of mass action is based on the assumption that equilibrium is reached when the rate at which new peptide-MHC complexes are formed equals the rate at which the peptide-MHC complex dissociates.
  • B max approximates the total number of receptors or MHC heavy chains (maximal binding)
  • peptide affinity, K D can be calculated from a one site binding curve (hyperbola) following the law of mass action.
  • MHC class I heavy chain offered should be at a concentration of 1 nM level or less in the folding buffer.
  • MHC molecule Addressing the affinity of any binders is a preferred embodiment for the detailed mapping of the specificity of any MHC molecule.
  • the specificity of a MHC molecule relates to the precise peptide binding motif represented by important structural requirements needed for peptide binding, such as the presence and proper spacing of particular amino acids in anchor positions.
  • the concentration of denatured MHC class I heavy chain is about 0.1 nM denatured MHC class I heavy chain, about 0.5 nM denatured MHC class I heavy chain, about 0.75 nM denatured MHC class I heavy chain, about 1 nM denatured MHC class I heavy chain, about 1.25 nM denatured MHC class I heavy chain, about 1.5 nM denatured MHC class I heavy chain, about 1.75 nM denatured MHC class I heavy chain, about 2 nM denatured MHC class I heavy chain, about 2.25 nM denatured MHC class I heavy chain, about 2.5 nM denatured MHC class I heavy chain, or about 3 nM denatured MHC class I heavy chain.
  • the concentration of denatured MHC class I heavy chain is above 3 nM, such as about 4 nM denaturered MHC class I heavy chain, about 5 nM denatured MHC class I heavy chain, about 6 nM denatured MHC class I heavy chain, about 7 nM denatured MHC class I heavy chain, about 8 nM denatured MHC class I heavy chain, about 9 nM denatured MHC class I heavy chain, about 10 nM denatured MHC class I heavy chain, about 15 nM denatured MHC class I heavy chain, about 20 nM denatured MHC class I heavy chain, about 25 nM denatured MHC class I heavy chain, about 30 nM denatured MHC class I heavy chain, about 35 nM denatured MHC class I heavy chain, about 40 nM denatured MHC class I heavy chain, about 50 nM denatured MHC class I heavy chain, about 75 nM denatured MHC class I heavy chain, about
  • a de novo folding of peptide-MHC complexes can be detected at the amount of 5 X 10 "15 mol of denatured MHC class I heavy chain.
  • the folding buffer contains a concentration of ⁇ 2 m of 100 nM.
  • said concentration of the ⁇ 2 m could be less than 500 nM, such as 400 nM, 300 nM, 250 nM, 200 nM, 150 nM, 125 nM, 100 nM, 90 nM, 80 nM, 70 nM, 50 nM, 25 nM, 1 nM, 0.5 nM, or 0.1 nM.
  • ⁇ 2 m As the skilled addressee would recognise the concentration of ⁇ 2 m described above relates to functional ⁇ 2 m molecules.
  • functional ⁇ 2 m molecule relates to a ⁇ 2 m molecule which is able to support peptide binding to the heavy chain to a MHC molecule.
  • the introduction of Lutrol F-68 improves the folding efficiency as depicted in figure 4 and as described in figure 6 of the present application the folding buffer should contain an efficient concentration of Lutrol F-68 of 300 mg/l.
  • any other block copolymers of ethylene oxide (EO) and propylene oxide (PO) with the ability to increase the de novo folding efficiency of peptide-MHC complexes are within the scope of the present invention, such as, but not limited to, Pluronic® surfactants: L10, L31, L35, F38, L42, L43, L44, L61, L62 L62D, L62LF, L63, L64, P65, F68LF, L72, P75, F77, L81, P84, P85, F87, F88, L92, F98, L101, P103, P104, P105, F108, L121, L122, P123, and/or F127 or any Tetronic® Surfactants such as but not limited to 304, 504, 701, 702, 704, 707, 901 1101, 1104, 1302, 1304, 1502, 1504 and/or 1508.
  • the dose-response for a panel of surfactants is shown in figure 14 data.
  • a preferred embodiment of the present invention relates to the introduction of Lutrol F-68 in the folding buffer at a concentration of 300 mg/l, such as lg/l, 500 mg/l, 200 mg/l, 100 mg/l, 75 mg/l, 50 mg/l, 25 mg/l, 20 mg/l, 15 mg/l, 10 mg/l, 5 mg/l, 4 mg/l, 3 mg/l, 2 mg/l, and 1 mg/l.
  • 300 mg/l such as lg/l, 500 mg/l, 200 mg/l, 100 mg/l, 75 mg/l, 50 mg/l, 25 mg/l, 20 mg/l, 15 mg/l, 10 mg/l, 5 mg/l, 4 mg/l, 3 mg/l, 2 mg/l, and 1 mg/l.
  • the renaturation process of the present invention relates to the de novo folding of peptide-MHC complexes, is shown to be entirely peptide dependent.
  • MHC-I complex formation can be observed from around 1 nM, and a plateau is reached around 1 ⁇ M, whereas lower affinity binding peptides would have to be offered at a higher concentration to achieve the same complex formation.
  • the lowest binding affinity measurable thus depends upon the concentration of the peptide that can be achieved for the particular peptide in question i.e its solubility.
  • a preferred embodiment of the present invention relates to a folding buffer containing graded concentration of peptide at a concentration of 0.1 nM up to 1 ⁇ M, such as a folding buffer containing graded concentration of peptide at a concentration of 0.1 nM up to 10 ⁇ M, 0.1 nM up to 25 ⁇ M, 0.1 nM up to 50 ⁇ M, 0.1 nM up to 100 ⁇ M, 0.1 nM up to 250 ⁇ M, 0.1 nM up to 500 ⁇ M, 0.1 nM up to 750 ⁇ M, 0.1 nM up to 1000 ⁇ M, 0.1 nM up to 5 mM, 0.1 nM up to 10 mM, 0.1 nM up to 25 mM, 0.1 nM up to 50 mM, or 0.1 nM up to 100 mM.
  • At least 3 data points generated by the present invention as a result of at least 3 graded concentrations of peptide in question offered to MHC in the renaturation process is preferred in order to determine an accurate fit of the amount of de novo folded complexes generated as a function of how much peptide is offered. It is further contemplated by the present inventors that an optimum of data points would be within 3-24 points. A total number of data points to generate the binding curve is in principle unlimited.
  • a most important aspect of the detection step of the present invention is the capturing of de novo generated peptide-MHC complexes.
  • Said capturing should be generated by at least one agent capable of binding at least one component of the de novo generated peptide-MHC complex.
  • Such capturing agents could be but not limited to antibodies, streptavidin (SA) and avidin and derivatives thereof, biotin immunoglobulins and derivatives thereof, leucine zipper domain of AP-1 (jun and fos), hexa-his (metal chelate moiety) or derivatives thereof , GST (glutathione S-transferase) glutathione affinity, lectins that mediate binding to a diversity of compounds, including carbohydrates, lipids and protein, e.g. Con A ⁇ Canavalia ensiformis) or WGA (whet germ agglutinin) and tetranectin or protein A or G (antibody affinity).
  • the MHC heavy chains may suitably be attached to the binding entity by tags.
  • MHC heavy chains being recombinantly tagged or chemically tagged bind specifically to the binding entity due to high affinity.
  • the recombinant tags of MHC heavy chain allow region- specific attachment sites in the MHC heavy chain.
  • the tags may be located at any part of the peptide-MHC complex.
  • said capturing agent is an antibody that binds to at least one component of the de novo generated peptide-MHC complex.
  • the capturing agent is the pan-specific anti-human MHC-I mouse monoclonal antibody W6/32.
  • any antibody specific to any compound within the peptide-MHC complex from any vertebrae can be useful in the method of the present invention.
  • complexes as defined above further comprising one or more labelling compounds.
  • the labelling compound of the present invention is preferably a compound, which can be detected directly or indirectly.
  • the labelling compound may be any compound suitable for direct or indirect detection.
  • direct is meant that the labelling compound can be detected per se without the need for a secondary compound, i.e. a "primary” labelling compound.
  • indirect is meant that the labelling compound can be detected by using one or more "secondary” compounds, i.e. the detection is performed by detecting the binding of the secondary compound to the primary compound.
  • suitable compounds are antibodies, fluorescent labels, enzyme labels, radioisotopes, chemituminescent labels, bioluminescent labels, polymers, metal particles, haptens, and dyes.
  • the labelling compound may suitably be selected from fluroscent labels such as 5-(and 6) -carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein) - 5- (and 6) -carboxamido hexanoic acid, fluorescein isothiocyanate, rhodamine, tetramethylrhodamine, and dyes such as Cy2, Cy3, and Cy5, optinally 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.
  • fluroscent labels such as 5-(and 6) -carboxyfluorescein, 5- or 6-carboxyfluorescein, 6-(fluorescein) - 5- (and
  • Cy5 or Texas Red and inorganic fluorescent labels such as particles based on semiconductor material like coated CdSe Nanocystallites, from haptens such as DNP, fluorescein isothiocyanate (FITC), biotin, and digoxinin,
  • HRP horse radish peroxidase
  • AP alkaline phosphatase
  • ⁇ -GAL beta-galactosidase
  • glucose-6-phosphate dehydrogenase beta_N-acetyl- glucosaminidase
  • ⁇ -glucuronidase invertase
  • Xanthine Oxidase firefly luciferase and glucose oxidase
  • luminescence labels such as iuminol, isoluminol, acridinium esters, 1,2-dioxetanes and pyridopyridazines, and
  • radioactivity labels such as incorporated isotopes of iodide, cobalt, selenium, tritium and phosphor.
  • the detection step could be mediated by an antibody specific to any compound of the peptide-MHC complex, said antibody could be prelabelled with any of the above mentioned labelling agents.
  • the detection agent is a horse radish peroxidase (HRP) labelled anti-human ⁇ 2 m rabbit polyclonal antisera P0174.
  • the determination of the concentration of complexes formed using the generated standard curve can be amplified by an amplification step improving the sensitivity of the determination.
  • an amplification agent could be any of the above mentioned labelling agents further improving the sensitivity of the detection step. It is contemplated that a person skilled in the art would perform a determination of the optimal dilution factor of any of the above mentioned amplification agents e.g. analysis of the sensitivity by adding graded concentrations of the amplification agent investigated.
  • the amplification step is provided by a polyclonal goat anti-rabbit IgG, HRP conjugated dextran polymer.
  • the amplification obtained by the polyclonal goat anti-rabbit IgG, HRP conjugated dextran polymer improves the sensitivity about tenfold.
  • the determination of the optimal dilution factor for the polyclonal goat anti-rabbit IgG, HRP conjugated dextran polymer is by the present inventors optimised to a dilution of 1: 15. However, results can be obtained by any dilution within the range of 1:2 to 1: 100.000.
  • a blocking agent should be included in the detection step of the ELISA assay.
  • a blocking agent could be any serum obtained from any animal producing antibodies or compounds with similar blocking capabilities, such as but not limited to pig, mouse, horse and bovine serum ( Figure 11).
  • the serum blocks any cross-reaction between any detection agent or amplification agent and the capturing agent.
  • the blocking agent is a serum which blocks any cross-reaction between said polyclonal goat anti-rabbit IgG, HRP conjugated dextran polymer and the capturing agent.
  • the blocking agent is a mouse serum which blocks any cross-reaction between said polyclonal goat anti-rabbit IgG, HRP conjugated dextran polymer and the capturing agent.
  • the present inventors have shown that an amount of 1% mouse serum is preferred for blocking the cross-reaction between said polyclonal goat anti-rabbit IgG, HRP conjugated dextran polymer and the capturing agent.
  • the concentration of any peptide-MHC complex of interest within vertebrae can be determined by the highly specific quantitative data obtainable by the invention.
  • the present invention discloses a new and surprisingly sensitive quantitative ELISA capable of detecting de novo folded peptide-MHC complexes at concentrations below 100 nM, such as concentrations below 90 nM, 80 nM, 75 nM, 50 nM, 25 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM. Determination of the accurate concentration of peptide-MHC complexes can be used for diagnostic purposes in any disease and/or syndromes leading to an increased level of said complexes in e.g. serum, urine, blood, saliva, lymph fluid, or other body fluids or secretes. Increased levels of soluble MHC complexes have been found in patients with e.g.
  • rheumatoid-like inflammatory joint diseases melanoma
  • Non Hodgkin's Lymphoma type I diabetes patients
  • SLE systemic lupus erythematosis
  • acute myeloid leukaemia and sarcoidosis.
  • the present invention further enables determination of the peptide binding affinity to the MHC molecule, the dissociation equilibrium constant.
  • peptide binding affinities of the highest of the known affinities of peptide-MHC interactions corresponding to a K D at the 0.5 nM level or better can be determined. It is known to a person skilled in the art that high affinities correspond to K D values between 0.5-50 nM, whereas intermediate affinities correspond to values between 250-500 nM, and poor affinities relates to values above 1 ⁇ M.
  • a preferred embodiment of the present invention relates to affinities from 5 ⁇ M to 0.5 nM, such as, but not limited to 5 ⁇ M, 2.5 ⁇ M, 1.25 ⁇ M, 750 nM, 500 nM, 250 nM, 125 nM, 100 nM, 50 nM, 25 nM, 10 nM, 1 nM, 0.75 nM or 0.5 nM.
  • the present invention enables a precise and accurate determination of the in vitro stability of any peptide-MHC complex in question, obtained by removal of excess ⁇ 2 m and peptide by any purification method known to the person skilled in the art, such as, but not limited to, gel filtration.
  • de novo folded peptide-MHC-I complexes can be purified by ge! filtration, and the excess of peptide and ⁇ 2 m can be removed. Since the excess of ⁇ 2 m, needed to support peptide binding, is removed no dissociated peptide from the MHC heavy chain can reassociate. Therefore, any peptide dissociation will rapidly lead to ⁇ 2 m dissociation, and the quantitative ELISA is therefore an indirect measurement of the rate of peptide dissociation.
  • one embodiment of the present invention relates to a method wherein the data generated enable measurement of the stability of the interaction between the peptide and the MHC heavy chains.
  • said stability of the interaction between the peptide and the MHC heavy chains can determine where the half-life (T 0 . 5 ) is between 5 seconds and 5 days, such as, but not limited to, between 10 seconds and 5 days, 30 seconds and 2 days, 1 minute and 1 day, 5 minutes and 18 hours, 10 minutes and 12 hours, 30 minutes and 6 hours, or 2 hours and 4 hours.
  • the dissociation rate is the result of a simple first order decay of a homogeneous peptide-MHC complex, and said person would expect that all the dissociation curves would intersect the Y-axis at 100% (the amount of peptide-MHC complex at time zero equals 100%).
  • the amount of peptide-MHC complex at time zero equals 100%.
  • the term "maturation” is used to define at least one of the following events leading to a de novo folding of the peptide-MHC complex.
  • a maturation event is one that eventually leads to peptide-MHC complex becoming stable when expressed on the cell surface and/or any event leading to a stable peptide-MHC complex, which will be soluble in a hydrophilic solution.
  • the present invention thus relates to situations for generating quantitative data for both the maturation and the stability of the generated complexes at temperatures from 45° to 4°C, such as, but not limited to, 42°C, 41°C, 40°C, 39°C, 38°C, 37.5°C, 37°C, 36.5°C, 35°C, 30°C, 25°C, 20°C, 18°C, 16°C, 14°C, 12°C, 10°C, or 4°C.
  • the maturation is correlated to the fraction being resistant to immediate dissociation at 37°C.
  • the peptide-MHC complexes are liberated from the transmembrane region of the MHC by physiological processes such as the proteolytic activity of members of complement factor 1 and present free in serum [Eriksson, H & Nissen MH 1992].
  • the binding of an appropriate peptide to MHC is the single most selective event in antigen presentation.
  • mapping e.g. human or mouse specificity
  • a person skilled in the art is able to generate quantitative prediction of peptide binding to the MHC molecules.
  • the prediction could be matrix-generated or performed by artificial neural networks (bioinfomatics) or by other means of prediction known to a person skilled in the art.
  • these predictions would need to be validated by a highly sensitive peptide- binding assay such as by the method of the present invention. Once this analysis has been completed, different assays could be performed to further analysis the immunogenicity of a peptide predicted and/or confirmed to bind to a MHC molecule.
  • HLA transgenic mice mouse expressing human MHC molecules
  • PBMC peripheral blood mononuclear cells
  • these assays which have been used to demonstrate that the peptides that have been selected on the basis of their sequence characteristics and their capacity to bind to MHC in vitro are indeed also immunogenic.
  • This analysis also assists in the selection of epitopes (peptides) that are the most potent in terms of inducing immune responses.
  • the chosen epitopes can also be rationally optimised to increase both their MHC binding and immunogenicity.
  • said epitopes should be confirmed by a highly sensitive peptide binding assay and their capacity to react to T cell receptors.
  • peptides binding with intermediary affinity to a MHC molecule featuring sub-optimal primary anchor residues could be altered by replacement of the sub-optimal amino acids with optimal anchor residues.
  • proteins encoded by the HIV virus derived proteins could be scanned to identify peptide sequences of 8-11 residues in length containing the HLA-A2 supertype motif. conserveed and identified motif-bearing peptides could be tested and the binding affinity could be determined.
  • Peptides found to bind the HLA-A2 supertypic alleles with high affinity by the present invention could be tested for recognition of cytotoxic T cells by HIV-infected individuals.
  • Peptides able to be recognised as T cell epitopes could be included in a vaccine where one seeks the induction of a specific T cell response
  • tumour associated peptides Another example could be the binding affinity of tumour associated peptides to be determined by the present invention.
  • Peptides identified as high affinity binders could be potential candidates for peptide or DNA based vaccines against malignant diseases.
  • peptides derived from tumour associated proteins found to bind to MHC with a high affinity could be analysed for the ability to elicit a cytotoxic response against a given tumour.
  • Proteins often associated with the mutation of a normal cell into a cancer cell e.g. oncogenic proteins could be scanned to identify peptide sequences of 8-11 residues in length containing a given MHC molecule binding motif. Identified motif-bearing peptides could be tested and the binding affinity could be determined. Peptides found to bind with high affinity to the MHC molecule could be tested for recognition of cytotoxic T cells. Even peptides binding with intermediate affinity could be included, in particular the affinity could be improved by anchor-optimisation. Tumour associated peptides recognised as T cell epitopes could be included in the development of anti cancer vaccines or in the treatment of already established malignant diseases.
  • Step I renaturation of the MHC class I molecules "in solution” (in a V-shaped folding well). "Empty" heavy chains are diluted into a buffer containing peptide and ⁇ 2 m and incubated at 18°C for 48 hours.
  • Step II The MHC class I complexes generated in step I are transferred from the folding well to a W6/32 coated ELISA plate and the concentration of MHC class I molecules are subsequently measured in a quantitative step.
  • MHC Class I complexes were captured by W6/32 and detected with P0174 antibody with or without Envision® enhancement. Note that on this figure the standard deviations (SD) of the triplicates are indicated. Since the SD tends to be smaller than the figure symbols, they have been omitted from all other figures ( ⁇ ) HLA-A2 + Envision®; (D) HLA-A2 - Envision®; (•) HLA-A11 + Envision®; (O) HLA-A11 - Envision®.
  • SD standard deviations
  • Lutrol-F68 Graded concentrations of Lutrol-F68 were diluted into fixed concentrations of 3 nM MHC-I HC, 100 nM h ⁇ 2 m, with or without 10,000 nM peptide. Under these conditions, Lutrol F-68 strongly supported complex formation. (•) + Peptide, (O) - Peptide.
  • Y-axis de novo folded peptide-MHC-I complexes (OD450).
  • Lutrol F-68 with or without 10,000 nM peptide.
  • a peptide specific signal was observed, when MHC-I HC was offered at a concentration around 1 nM. ( ⁇ ) + Peptide ( -*• ) - Peptide.
  • Y-axis de novo folded peptide-MHC-I complexes (OD450).
  • Graded concentrations of h ⁇ 2 m were diluted into 3 nM A*6901 or A*0201 HC, 0.3 mg/ml Lutrol-F68, with or without an excess of 10,000 nM peptide.
  • h ⁇ 2 m supported peptide binding from around 10 nM and a plateau is reached around 300 nM.
  • A*6901 ( ⁇ ) + Peptide (•) - Peptide; A*0201 ( ⁇ ) + peptide (O) - Peptide.
  • Y-axis de novo folded peptide-MHC-I complexes (OD450).
  • Graded concentrations of a panel of surfactants chemically related to Lutrol F68 were examined for the ability to facilitate peptide binding to the MHC class I molecule in the presence of ⁇ 2m.
  • Graded concentrations of surfactant were diluted into fixed concentrations of 3 nM MHC-I HC, 100 nM h ⁇ 2 m, with or without 10,000 nM peptide.
  • all of the tested surfactants supported complex formation, as seen as an increase in OD450 value of approximately 1,000.
  • pan-specific mouse anti-HLA class I antibody W6/32 (from ATCC # HB-95), was purified from ascites by protein A affinity chromatography.
  • the E.coli strain XA90 was transformed with the vector pHNl containing a HLA-A*0201 (1- 20 275) gene under T7 polymerase control.
  • the gene encoding the HLA-A*1101, A*3101, A*6901, A*0204, B*2705, B*0702, A*0101, A*0301 and B*5801 (1-276) was PCR amplified from a cDNA library from the EBV transformed cell line, KHAGNI (IHW 9248), inserted into pET28 (Novagen, Madison, Wisconsin), codon optimised for E.coli expression using QuickChange (Stratagene, La Jolla, California), verified by sequencing (ABI 310, 25 Perkin Elmer), and finally transformed into E.coli strain BL21(DE3) (Novagen).
  • Protein expression and purification were performed as follows. Briefly, inclusion bodies containing the recombinant HLA protein product were extracted into 8M Urea, 20 mM Tris, pH 8 under non-reducing conditions. They were subsequently purified by ion-exchange, 30 hydrophobicinteraction chromatography and gel filtration chromatography, concentrated by diafiltration (Amicon YM10) and stored at 20°C until use. Human ⁇ 2 -microglobulin was obtained from the urine of ureamic patients and purified by chromatofocusing, or generated recombinantly. ELISA assay of peptide-MHC complex formation
  • the plate was incubated for 1 h at 4°C with 50 ⁇ l/well of a horseradish peroxidase (HRP)-conjugated, polyclonal rabbit anti-human ⁇ 2 m antibody (DAKO #0174) diluted 1:2500 into 2% SMP-PBS, and then washed 4 x 600 ⁇ l/well at room temperature.
  • HRP horseradish peroxidase
  • DAKO #0174 polyclonal rabbit anti-human ⁇ 2 m antibody
  • the plate was subsequently incubated for 30 minutes at RT with a dextran polymer conjugated with goat anti-rabbit IgG and HRP (DAKO EnVision+TM, Peroxidase, Rabbit, #K4003) diluted 1: 15 in 2 % SMP-PBS containing 1% normal mouse serum, and then washed 4 x 600 ⁇ l/well at room temperature.
  • a dextran polymer conjugated with goat anti-rabbit IgG and HRP diluted 1: 15 in 2 % SMP-PBS containing 1% normal mouse serum, and then washed 4 x 600 ⁇ l/well at room temperature.
  • TMB-one 3,3' 5,5'-tetramethylbenzidine hydrogenperoxide
  • Specific peptide-MHC-I complexes were generated as above.
  • the resulting complexes were 30 purified by Sephadex G50M spun column chromatography to remove free peptide and free ⁇ 2 m thereby preventing re-association of any dissociated peptide. Dissociation was initiated by incubating the complexes at 37 °C for the time periods indicated. The remaining peptide-MHC-I complexes were measured by the above ELISA.
  • MHC-I class I molecules were diluted into a 0.1 M Tris-maleat buffer, pH 6.6 containing 1 ⁇ M human ⁇ 2 m, 1 g/L % Lutrol-F68, a trace amount of radiolabelled indicator peptide, and a graded dose of the unlabelled test peptide.
  • the reaction was incubated at 18°C for 48 hours. Binding of the labelled peptide to MHC-I was examined by Sephadex G25 spun column chromatography, as previously described, followed by gamma spectrometry (Packard).
  • the fraction of ligand bound to MHC class I relative to the total amount of offered ligand was calculated, and corrected by subtracting the fraction of ligand (usually less than 1%) that appeared in the void volume in the absence of MHC.
  • the concentration of test peptide needed to inhibit the binding of the indicator peptide by 50% was determined. Since the receptor concentration had been adjusted to prevent ligand depletion, the IC 50 reflects the equilibrium dissociation constant, K D .
  • Binding data were analysed by using Prism ® 3.0, GraphPad, San Diego, CA.
  • This K D is the concentration of ligand required to reach half-maximal binding. In other words, when the concentration of ligand equals the K D , half the receptors will be occupied at equilibrium. If the receptors have a high affinity for the ligand, the K D will be low, as it will take a low concentration of ligand to bind half the receptors.
  • Prism ® generates the curve seen in figure la and defines K D including 95% confidence interval intervals and R 2 which is precision of the fit. However, for illustration purposes we use the semi log transformation of this curve to depict the regression line as seen in figure 2b.
  • the assay will be conducted in two steps (figure 2).
  • denatured and purified (“empty") MHC-I heavy chain (HC) would be diluted into a buffer containing excess ⁇ 2 m and graded concentrations of peptide to initiate an "in solution” MHC renaturation step (the “renaturation step”).
  • the concentration of MHC complexes generated in the first step would be measured (the “quantitation step”).
  • This step should be able to measure the concentration of MHC complexes at the 1 nM level or better. This level of sensitivity is required to measure affinities in the low nM range, which corresponds to the highest of the known affinities of peptide-MHC-I interactions.
  • Example 1 is required to measure affinities in the low nM range, which corresponds to the highest of the known affinities of peptide-MHC-I interactions.
  • the W6/32 appeared to bind human MHC-I pan-specifically; at least, the dose-response for recombinant HLA-A2 and HLA-A11 were identical (figure 3).
  • the recombinant HLA-A2(l-275) and natural HLA-A2 had identical dose-response curves (data not shown) showing that the truncated transmembrane region does not contribute significantly to the ELISA signal.
  • the OD 450 response vs. the logarithm of the molar concentration of an MHC standard was plotted and optimally fitted to a sigmoid curve (Prism® 3.0, GraphPad). This allows the OD 450 of any sample to be converted to the concentration of MHC-I complexes present in that sample.
  • a sigmoid curve Prism® 3.0, GraphPad.
  • Lutrol F-68 the surfactant, Lutrol F-68, supported MHC-I renaturation and peptide binding (Pedersen, L. 0. et al. 2001). Lutrol F-68 assists in keeping the reactants soluble (data not shown) and this might be particularly beneficial during renaturation, where folding intermediates otherwise are prone to aggregation. Adding Lutrol F-68 to the renaturation step did improve the sensitivity about 3-fold; an improvement, which came at the cost of a slightly reduced signal to noise ratio (figure 4).
  • Lutrol F-68 appears to substantially reduce the need for ⁇ 2 m without compromising the peptide dependency of the renaturation.
  • Lutrol F-68 Determination of the optimal concentration of Lutrol F-68.
  • Graded concentrations of Lutrol F-68 were diluted into a fixed concentration of 3 nM MHC-I HC and 100 nM ⁇ 2 m with, or without, an excess of peptide, and the resulting complexes were assayed. Under these conditions, Lutrol F-68 strongly supported complex formation (figure 6). Support of peptide binding could be detected already at 1 mg/l and it reached a plateau around 300 mg/l. Background signals (i.e. in the absence of peptide) were barely detectable even at concentrations as high as 300 mg/ml. Tentatively, a Lutrol F-68 concentration of 300 mg/l was chosen.
  • Table II Dissociation of peptide-MHC-I complexes, said recombinant de novo folded peptide-HLA*A1101 complexes.
  • Peptide-MHC-I complexes were generated. Excess peptide and ⁇ 2 nn were removed by gel filtration. The resulting complexes were incubated at 37°C and the concentrations of intact complexes were measured by the ELISA assay at various time points. The data were plotted as in figure 9 and the stability's, precessions and Y- intercepts were determined. * is from (Kubo, R.T. et al., 1994). ** is from (Stryhn, A. et al., 1996).

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Abstract

L'invention concerne un procédé quantitatif permettant de mesurer l'interaction entre un peptide et une molécule MHC. La base de mesure de cette interaction est constituée sensiblement de chaînes lourdes de MHC dénaturées et pures. Ces chaînes lourdes de MHC dénaturées et pures sont ensuite renaturées dans un tampon renfermant une quantité excédentaire de ?2m et une concentration en augmentation du peptide en question, entraînant un repliement de novo et une production de complexes de peptide-MHC. Le procédé selon l'invention est présenté avec une référence aux protéines de classe I de MHC, mais on peut envisager une utilisation possible de celui-ci de manière similaire aux fins de production de données issues d'un type quelconque de complexe où le repliement de novo est dépendant du peptide, par exemple la classe II de MHC.
PCT/DK2003/000325 2002-05-17 2003-05-15 Mesure par dosage immunoenzymatique (elisa) de l'interaction de novo entre un peptide et une molecule mhc Ceased WO2003098219A1 (fr)

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WO2008128201A1 (fr) 2007-04-12 2008-10-23 Siemens Medical Solutions Usa, Inc. Système de radiosynthèse microfluidique pour biomarqueurs de tomographie par émission de positrons
US7829032B2 (en) 2007-01-23 2010-11-09 Siemens Medical Solutions Usa, Inc. Fully-automated microfluidic system for the synthesis of radiolabeled biomarkers for positron emission tomography
US8071035B2 (en) 2007-04-12 2011-12-06 Siemens Medical Solutions Usa, Inc. Microfluidic radiosynthesis system for positron emission tomography biomarkers
US8075851B2 (en) 2005-09-29 2011-12-13 Siemens Medical Solutions Usa, Inc. Microfluidic chip capable of synthesizing radioactively labeled molecules on a scale suitable for human imaging with positron emission tomography

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8075851B2 (en) 2005-09-29 2011-12-13 Siemens Medical Solutions Usa, Inc. Microfluidic chip capable of synthesizing radioactively labeled molecules on a scale suitable for human imaging with positron emission tomography
US8658112B2 (en) 2005-09-29 2014-02-25 Siemens Medical Solutions Usa, Inc. Microfluidic chip capable of synthesizing radioactively labeled molecules on a scale suitable for human imaging with positron emission tomography
US7829032B2 (en) 2007-01-23 2010-11-09 Siemens Medical Solutions Usa, Inc. Fully-automated microfluidic system for the synthesis of radiolabeled biomarkers for positron emission tomography
WO2008128201A1 (fr) 2007-04-12 2008-10-23 Siemens Medical Solutions Usa, Inc. Système de radiosynthèse microfluidique pour biomarqueurs de tomographie par émission de positrons
JP2010531295A (ja) * 2007-04-12 2010-09-24 シーメンス メディカル ソリューションズ ユーエスエー インコーポレイテッド 陽電子放出断層撮影法バイオマーカーのためのマイクロ流体放射合成システム（関連出願）本出願は、２００７年４月１２日に出願された米国特許仮出願第６０／９２３，０８６号明細書、２００７年４月１３日に出願された米国特許仮出願第６０／９２３，４０７号明細書、２００７年８月２３日に出願された米国特許非仮出願第１１／８９５，６３６号明細書、及び２００８年１月１１日に出願された米国特許仮出願第６１／０１０，８２２号明細書に基づく優先権を主張し、それらの各々の内容が参照により全体として本明細書に組み込まれる。
US8071035B2 (en) 2007-04-12 2011-12-06 Siemens Medical Solutions Usa, Inc. Microfluidic radiosynthesis system for positron emission tomography biomarkers
US8173073B2 (en) 2007-04-12 2012-05-08 Siemens Medical Solutions Usa, Inc. Portable microfluidic radiosynthesis system for positron emission tomography biomarkers and program code
KR101176710B1 (ko) 2007-04-12 2012-08-23 지멘스 메디컬 솔루션즈 유에스에이, 인크. 양전자 방출 단층촬영용 바이오마커를 위한 미세유체 방사합성 시스템

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