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WO2004074840A1 - Procede de mesure quantitative de ligands dans un echantillon de proteines de liaison dissoutes - Google Patents

Procede de mesure quantitative de ligands dans un echantillon de proteines de liaison dissoutes Download PDF

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Publication number
WO2004074840A1
WO2004074840A1 PCT/EP2004/001272 EP2004001272W WO2004074840A1 WO 2004074840 A1 WO2004074840 A1 WO 2004074840A1 EP 2004001272 W EP2004001272 W EP 2004001272W WO 2004074840 A1 WO2004074840 A1 WO 2004074840A1
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WIPO (PCT)
Prior art keywords
antibody
binding protein
ligand
hgh
binding
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PCT/EP2004/001272
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German (de)
English (en)
Inventor
Christian J. Strasburger
Martin Bidlingmaier
Zida Wu
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Klinikum Der Univeristät München Grosshadern- Innenstadt
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Publication of WO2004074840A1 publication Critical patent/WO2004074840A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/02—Drugs for disorders of the endocrine system of the hypothalamic hormones, e.g. TRH, GnRH, CRH, GRH, somatostatin
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the invention relates to a method for the quantitative determination of ligands in the sample of dissolved or suspended binding proteins using specific antibodies, antibodies usable in the method, medicines, diagnostics and (diagnostic) kits containing these
  • Antibodies the use of these antibodies in quantitative analysis and in selected indications for therapeutic purposes, and methods for their production.
  • hGH Human growth hormone
  • immunoassays are used for the analysis of the growth hormone concentration. These are based on the principle of the interaction of specific antibodies with an analyte.
  • Competitive assays are also optionally used (such as radioimmunoassays, RIA), in which labeled growth hormone competes with the growth hormone present in the sample for binding to an antibody and thus produces a signal which is inversely proportional to the concentration of the analyte to be measured.
  • sandwich immunoassays are now most frequently used, in which an immobilized specific antibody binds the analyte (“capture antibody”), and a second labeled antibody directed against a different epitope of the analyte then binds the now immobilized analyte. that is proportional to the amount of bound Analyte is.
  • capture antibody an immobilized specific antibody binds the analyte
  • second labeled antibody directed against a different epitope of the analyte then binds the now immobilized analyte. that is proportional to the amount of bound Analyte is.
  • the best-known example of this measurement method is the "enzyme linked immunosorbent assay” (ELISA) with a colorimetric end point since the photometer required for the measurement is regularly available.
  • ELISA enzyme linked immunosorbent assay
  • Other possibilities for signal generation are radioactivity, chemiluminescence or (time-resolved) fluorescence.
  • the results of various stimulation tests are used to diagnose a growth hormone deficiency.
  • blood is drawn at intervals of 15 to 30 minutes over a period of 1 to 2 hours, and growth hormone secretion is determined from the samples.
  • the growth hormone concentration does not rise above an arbitrary one, but traditionally - e.g. in pediatrics - at a defined cut-off value of approx. 10 ng / ml, the diagnosis “growth hormone manager is considered very likely and the health insurance companies usually reimburse the following therapy costs. These costs of a - potentially lifelong - therapy with recombinant growth hormone are considerable.
  • growth hormone that is produced by the human pituitary gland is not a completely homogeneous substance, but consists of different isoforms.
  • growth hormone that is produced by the human pituitary gland is not a completely homogeneous substance, but consists of different isoforms.
  • the "International Reference Preparation 80/505" which is still used, is a preparation extracted from human pituitary glands, which also consists of various isoforms, while the newer "International Reference Preparation 88/624” is of recombinant origin and consists in pure form of the 22,000 dalton major isoform of growth hormone.
  • the main disruptive factor for the measurement of growth hormone to which the invention described here relates is the presence of a high-affinity growth hormone binding protein (hGH binding protein, hGHBP) in human serum (2-4).
  • binding protein on the measurement result can lead to false high or false low values depending on the assay principle used (competitive assay or sandwich assay).
  • competitive assay on the one hand, labeled hGH (as a so-called “tracer”) can be bound by the binding protein and thus withdrawn from the assay mixture, which leads to incorrectly high concentrations.
  • the binding protein can also sterically interact with the specific antibody with the growth hormone molecules from a serum sample
  • this steric hindrance to the interaction between antibody and hormone in sandwich immunoassay usually leads to false low results because less hormone or fewer detection antibodies are bound this disturbing factor, errors in the measurement results under certain physiological or pathological conditions which are associated with a change in the hGHBP concentration in the blood, such as, for example, pregnancies in the course of which there is an increase in the hGHBP is coming.
  • the invention therefore relates to a method for the quantitative determination of a ligand of a binding protein in a sample containing ligand and binding protein in dissolved or suspended form, in which the sample to be measured has an antibody A against the binding protein which is directed against the binding site of the ligand on the binding protein is added.
  • the sample can advantageously be used exactly as described by the manufacturer for the particular assay.
  • Binding protein in the sense of this invention is to be understood to mean all proteins which can bind a ligand to be measured here with high affinity.
  • the binding proteins relevant here are mostly soluble or are present in the sample as a suspension. Examples are certain soluble receptors or, for example, hormone binding protein, such as growth hormone binding protein, which occurs in the cytosol or blood.
  • Ligand in the sense of this invention is to be understood as meaning compounds which bind to a binding protein with high affinity and whose concentration can be measured in certain assays and, for example, is also measured for medical purposes.
  • messenger substances such as hormones, transmitters, for example. Meurotransmitter, ⁇ i cellular signal peptides or proteins, cylokines, chemokines, lymphokines etc.
  • sample is to be understood as meaning any type of solution to be examined, but in particular solutions of medically relevant substances, such as blood, lymph, serum, urine, liquor, also in a form prepared for sample processing.
  • “in dissolved or suspended form” means any form of solution of the binding protein or of the ligand in the broader sense in the solvent. This also includes situations in which, for example, the binding protein is on the edge or beyond the point of failure , but is still part of the solution.
  • Antibodies in the sense of this invention are vertebrates or artificially produced proteins or possibly other structures which bind with high affinity to a certain surface confirmation (epitope) of an antigen, that is to say another molecule, preferably mono- or polyclonal (partial) structures of immunoglobulins (eg IgG, IgA, IgD, IgE) or also polyclonal monospecific antibodies Typically, such antibodies contain at least the variable part of immunoglobulins, possibly also at least one domain of the constant part of immunoglobulins (ab) 2 - fragments of antibodies in the sense of the present invention.
  • immunoglobulins eg IgG, IgA, IgD, IgE
  • ab constant part of immunoglobulins
  • An antibody against the binding protein which is directed against the binding site of the ligand on the binding protein means that the antibody, as a binding epitope, specifically binds the binding site of the ligand on the binding protein with high affinity.
  • “Quantitative determination” is generally understood to mean any type of measurement of the amount of a dissolved analyte in a sample known to the person skilled in the art. Explicitly, this means, for example, quantification using chromatographic methods with a moving standard and, in particular, using the high affinity interaction between antibody and ligand (Antigen) quantification by competitive assays or binding assays, such as a sandwich assay also in ELISA format It is a particular advantage of this invention that the method of antibody addition presented with practically any known - especially commercially available - test kit or test system for quantitative analysis of the respective analyte (ligand) can be combined with little effort. It is particularly preferred if, in the method according to the invention, the antibody A is added before or during, preferably before, the quantitative determination and / or is incubated with the sample.
  • Incubation is understood to mean a reaction condition in which reaction partners, here antibody A and binding protein and secondarily also the ligand, can react with one another.
  • the incubation is usually limited in time, here for example 6, 12, 18 or 24 h.
  • Incubation is to be understood here in particular to mean the pre-incubation - for example over 6, 12, 18 or 24 h - which takes place before the start of the quantitative measurement (for example with a commercially available test kit).
  • sample measured in the method according to the invention contains body fluid, preferably human body fluid, in particular blood or human blood.
  • Body fluid means any fluid obtained from the body of a vertebra, in particular a mammal, in particular a human. In the case of humans, this would be, for example, blood, urine or lymph, but also cytosolic preparations from human cells.
  • the ligand is a physiological ligand of the physiological binding protein.
  • the method according to the invention is also useful if ligand and / or binding protein has been added (exogenously) from the outside, for example after injection or other absorption of recombinant hGH in patients or test subjects.
  • This makes it possible to use a method according to the invention, for example for analyzing samples if there is a suspicion of ping (eg in high-performance sports) with hGH or other hormones (examples of ligands in the sense of the present invention) to suspect doping after quantifying, for example, the hGH concentration in the sample (without falsifying influences of the binding protein (s) in question) to exclude or justify.
  • binding proteins also interfere in pharmacokinetic analyzes, so that the artificially added hGH is also bound in this regard, and results in, for example, hGH concentration are falsified by the presence of binding protein.
  • a procedure according to the invention can also remedy this in these cases.
  • a physiological ligand is to be understood as a ligand in the sense given above, which occurs in the body of a vertebrate, in particular in a body fluid, without being added from outside.
  • the binding protein used in the method according to the invention is soluble, preferably a soluble receptor or a hormone binding protein, in particular a hormone binding protein.
  • the ligand is a peptidic compound and / or a hormone, preferably a peptidic hormone, in particular a growth hormone.
  • Hormone means any compound whose components are predominantly linked to one another via a peptide bond R1-NH-C (O) -R2.
  • Hormone is a chemical messenger that acts at a distance from its place of synthesis and release. Hormones are preferably produced by endocrine glands, for example the pituitary gland, the gonads or the epiphysis. Examples are growth hormone or luteinizing hormone LTH, insulin, melatonin, glucagon, gastrin, angiotensin, substance P, interleukins, vasopressin, endorphins, enkephalins, relaxax or the atrionatriuretic factor. Hormone binding proteins are binding proteins (see above) that bind affine hormones.
  • the ligand is human growth hormone (hGH) and the binding protein human growth hormone binding protein (hGHBP) and the antibody A is against the binding site of the hGH on the hGHBP - judges.
  • the antibody A used in the method according to the invention is a monoclonal antibody or a single-chain antibody, preferably a monoclonal antibody. It is particularly preferred if the antibody A used is ZMC1.
  • ZMC1 is a monoclonal antibody which is directed against the binding site of the human growth hormone (hGH) on the human growth hormone binding protein (hGHBP) and was optimized within the scope of the invention to achieve the object.
  • DSM ACC2582 reference number assigned by the depositor: ZMC1 in accordance with the Budapest Treaty at the DSMZ (German Collection for Microorganisms and Cell Cultures GmbH) in Braunschweig (Germany, Maschenroder Weg 1b , 38124 Braunschweig) in viable form.
  • monoclonal means in particular the product of an artificial construct in which an antibody-producing cell (B cell) is fused with an immortalized cancer cell (hybridoma), resulting in a hybridoma cell.
  • B cell an antibody-producing cell
  • hybridoma an immortalized cancer cell
  • the quantitative determination of the ligand is carried out using the binding of the ligand as antigen to a, preferably monoclonal, antibody B.
  • the quantitative determination is carried out using a competitive binding test, preferably a “radio-immunoassay” (RIA).
  • RIA radio-immunoassay
  • the quantitative determination by measuring a function of the concentration of the measurement parameters to be measured increasing ligand, preferably by an enzyme-linked immunosorbent assay (ELISA) and / or by an appropriate sandwich assay.
  • the binding protein is not separated from the sample to be measured before the quantitative determination.
  • the ligand to be measured - such as the hormone - remains in the sample while the binding protein is being separated, for example by precipitation and / or filtration with certain molecular weight cut-off limits, chromatographic methods such as Affinity chromatography or HPLC, dialysis for certain ligand sizes etc.
  • This advantage occurs in particular in a particularly favorable and preferred method according to the invention, in which the antibody A is added such that the antibody A is present in the sample after the addition in a higher concentration than the binding protein, preferably in an at least 50%, in particular at least 100%, preferably at least 200%, in particular at least 400% higher concentration.
  • the person skilled in the art can determine the exact, correct and sufficient amount of specific antibody A that must or can be added to the sample for quantitative measurement of the ligand, which is as interference-free as possible, at most, at most, in a few simple preliminary experiments.
  • the optimal addition naturally depends on the amount of binding protein in the sample and possibly the quantitative test system used as well as possibly on the amount of ligand to be measured.
  • the particularly preferred form of the method according to the invention is a method for the quantitative determination of hGH using the affine binding of hGH as an antigen to a monoclonal antibody.
  • B in a sample containing human blood, in which hGH and hGHBP are contained in dissolved or suspended form, in which the sample to be measured is antibody A directed against the binding site of hGH on hGHBP, preferably the deposited monoclonal antibody ZMC 1 , in a clear excess over the amount of hGHBP before or during, preferably before, the quantitative determination of the hGH by means of antibody B is added and incubated.
  • the invention prevents the interference of the growth hormone binding protein by adding a specific excess of a specific, preferably monoclonal antibody, specifically produced against the growth hormone binding protein and characterized by specific experiments, to the sample to be measured.
  • This monoclonal antibody (ZMC 1) was selected on the basis of its particularly preferred property that it binds to the hGHBP molecule exactly where the growth hormone molecule also binds.
  • ZMC 1 was selected on the basis of its particularly preferred property that it binds to the hGHBP molecule exactly where the growth hormone molecule also binds.
  • the “anti-binding protein antibody” itself does not interfere with the “anti-growth hormone antibodies” used to measure hGH, there is also no need to remove the antibody-hGHBP complexes from the sample beforehand.
  • the special anti-hGHBP antibody ZMC 1 with the hGH-displacing property of the sample is added before the analysis and the sample is used after preincubation, as described for the respective assay by the manufacturer.
  • Another object of the invention is an antibody A against a physiological binding protein of a physiological ligand dissolved or suspended in body fluids, the antibody A being is directed to the binding site of the ligand on the binding protein.
  • the antibody A according to the invention is a monoclonal antibody and / or a single-chain antibody, preferably a monoclonal antibody.
  • antibody encompasses both polyclonal, in particular polyclonal, monospecific antibodies (ie antibodies with different variable regions, but all of which recognize a specific epitope), as well as monoclonal, chimeric antibodies, anti-idiotypic antibodies (directed against antibodies according to the invention ), which are all present in bound or soluble form and can optionally be labeled by “labels” (for example fluorescent markers, gold markers, coupled enzymes), and also fragments of the abovementioned antibodies.
  • labels for example fluorescent markers, gold markers, coupled enzymes
  • antibodies according to the invention can also occur in recombinant form as fusion proteins with other (protein) components.
  • antibodies means both polyclonal, monoclonal, human or humanized or recombinant antibodies or fragments thereof, single chain antibodies or synthetic antibodies.
  • the polyclonal antibodies are heterogeneous mixtures of antibody molecules which are produced from sera from animals which have been immunized with an antigen.
  • a monoclonal antibody contains an essentially homogeneous population of anti bodies that are specifically directed against antigens, the antibodies having essentially the same or the same epitope binding sites.
  • the different antibody variants with monospecificity can belong to the immunoglobulin classes described below, so they can be mixtures of different main or subclasses, preferably a homogeneous mixture of IgG antibodies. This homogeneity can be achieved by an additional purification step (immunoprecipitation, chromatography, for example using antibodies directed against IgG).
  • Monoclonal antibodies can be obtained by methods known in the art (e.g., Koehler and Milstein, Nature, 256, 495-397, (1975); U.S. Patent 4,376,110; Ausübel et al., Harlow and Lane “Antibodies” : Laboratory Manual, Cold Spring, Harbor Laboratory (1988); Ausubel et al., (Eds), 1998, Current Protocols in Moleeular Biology, John Wiley & Sons, New York)).
  • the description contained in the abovementioned references is included as part of the present invention in the disclosure of the present invention.
  • Antibodies according to the invention which have been genetically engineered can also be produced by methods as described in the abovementioned publications.
  • Antibodies according to the invention can belong to one of the following immunoglobulin classes: IgG, IgM, IgE, IgA, GILD and possibly a subclass of the aforementioned classes, such as the subclasses of the IgG or their mixtures.
  • IgG and its subclasses such as, for example, IgG1, IgG2, IgG2a, IgG2b, IgG3 or IgGM are preferred.
  • the IgG subtypes IgG1 / k or IgG2b / k are particularly preferred.
  • a hybridoma cell clone that produces monoclonal antibodies according to the invention can be cultivated in vitro or in vivo.
  • chimeric antibodies according to the invention are molecules which contain different constituents, and these are derived from different animal species (e.g. antibodies which have a variable region which is derived from a mouse monoclonal antibody and a constant region of which) human immunoglobulin). Chimeric antibodies are preferably used, on the one hand, to reduce the immunogenicity during use and, on the other hand, to increase the yields in production, for example murine monoclonal antibodies give higher yields from hybridoma cell lines, but also lead to higher immunogenicity in humans, so that human / murine chimeric antibodies are preferably used.
  • An anti-idiotypic antibody according to the invention is an antibody which recognizes a determinant which is generally associated with the antigen binding site of an antibody according to the invention.
  • an anti-idiotypic antibody By immunizing an animal of the same type and the same genetic type (eg a mouse strain), an anti-idiotypic antibody can be used as the starting point for a monoclonal antibody against which an anti-idiotypic antibody according to the invention is directed. getting produced.
  • the immunized animal will recognize the idiotypic determinants of the immunizing antibody by producing an antibody which is directed against the idiotypic determinants (namely an anti-idiotypic antibody according to the invention) (US Pat. No. 4,699,880).
  • An anti-idiotypic antibody according to the invention can also be used as an immunogen to elicit an immune response in another animal and to produce an anti-idiotypic antibody there.
  • the anti-anti-idiotypic antibody can, but need not, be identical in terms of its epitope construction to the original monoclonal antibody which caused the anti-idiotypic reaction. In this way, by using antibodies directed against idiotypic determinants of a monoclonal antibody, other clones which express antibodies of identical specificity can be identified.
  • Monoclonal antibodies which are directed against a dissolved or suspended physiological binding protein of a physiological ligand in body fluids, can be used to bind anti- ⁇ diotypic antibodies in corresponding animals, such as. B. the BALB / c mouse. Spleen cells from such an immunized mouse can be used to produce anti-idiotypic hybridoma cell lines that secrete anti-idiotypic monoclonal antibodies. Furthermore, anti-idiotypic monoclonal antibodies can also be coupled to a carrier (KLH, "keyhole limpet hemocyanin”) and then used to immunize further BALB / c mice.
  • KLH "keyhole limpet hemocyanin
  • the sera of these mice then contain anti-anti-idiotypic antibodies which have the binding properties of the original monoclonal antibodies and are specific for a physiological binding protein of a physiological ligand dissolved or suspended in body fluids (see preferred examples below).
  • the anti-idiotypic monoclonal antibodies thus have their own idiotypic epitopes or "idiotopes" which are structurally similar to the epitope to be examined.
  • the term "antibody” is intended to include both intact molecules and fragments thereof. Fragments include all shortened or modified antibody fragments with one or two binding sites complementary to the antigen, such as antibody parts with a binding site formed by light and heavy chains corresponding to the antibody, such as Fv, Fab or F (ab ') 2 fragments or Single strand fragments, called.
  • Shortened double-strand fragments such as Fv, Fab or F (ab ') 2-Fab and F (ab') 2 fragments are preferably devoid of an Fc fragment, such as present in an intact antibody, so that they are transported faster in the bloodstream can and have comparatively less non-specific tissue binding than intact antibodies.
  • Fab and F (ab ') 2 fragments of antibodies according to the invention can be used in a method according to the invention as defined by the present invention.
  • Such fragments are typically produced by proteolytic cleavage using enzymes such as. B. papain (for the production of Fab fragments) or pepsin (for the production of F (ab ') 2 , fragments) can be used, or can be obtained by chemical oxidation or by genetic engineering of the antibody genes.
  • an antibody A according to the invention can also have further covalently (or not covalently) coupled molecules or groups, for example a fluorescent label or a label, for example a gold label, or specific epitopes that can be recognized by third-party molecules.
  • an antibody A according to the invention can also be bispecific, that is to say it can recognize different epitopes with its two paratopes, preferably two different epitopes of the same protein or peptide (see above), but the structure of the two paratopes may also be different, but the same Tie epitope or at least overlapping areas.
  • the present invention also relates to mixtures of antibodies according to the invention in the abovementioned sense, for example mixtures of monoclonal antibodies or mixtures of monoclonal antibodies with antibody fragments, mixtures of anti-idiotypic antibodies, etc.
  • Ligand at the binding site of which the antibody A according to the invention binds, is a peptide and / or hormone and / or preferably of human origin, may also be directed against fragments of native poly- or oligopeptides.
  • the antibody A is directed against the binding site of the human growth hormone (hGH) on the human growth hormone binding protein (hGHBP).
  • the antibody A according to the invention is the antibody ZMC1, for example the antibody DSM ACC2582 deposited with the DSMZ.
  • Another object of the invention is a medicament containing an antibody A according to the invention and optionally further active ingredients and additives and / or auxiliaries.
  • This medicinal product is particularly suitable for treating a deficiency state of one of the ligands already mentioned, since the ligand can be released by the antibody A or its binding can be prevented by binding protein, so that, for example, the plasma level of free ligand increases, for example in the event of a desired increase in the plasma titer of hGH.
  • the hGH-binding binding protein corresponds to an extracellular fragment of the hGH receptor.
  • hGHBP binding antibodies which are directed against its binding site for hGH can also bind to the membrane-bound hGH receptor in a manner such that the hGH receptor is blocked for the attachment of hGH.
  • Antibodies A according to the invention can in principle be used therapeutically as growth hormone receptor antagonists. An antibody according to the invention can thus be used to produce a medicament which inhibits the physiological effect of human growth hormone (hGH).
  • a medicament containing one or more antibodies A according to the invention can be used therapeutically in all those diseases in which the effect of hGH is increased in an unphysiological, for example pathological, manner .
  • this also results in the use of corresponding antibodies A according to the invention for the treatment (or for the production of a medicament for the treatment) of all those disorders, diseases or pathophysiologies for which hGH excess is etiological, for example.
  • a medicament containing at least one antibody A according to the invention or an antibody A according to the invention can be used for the treatment of acromegaly.
  • therapy for affected patients with pituitary adenoma can be carried out in combination with drugs or active substances that reduce the secretion of hGH.
  • drugs or active substances that reduce the secretion of hGH can it is, for example, treatment with somatostatin analogs (for example octreotide) and / or dopamine agonists (for example bromocriptine, cabergoline).
  • somatostatin analogs for example octreotide
  • dopamine agonists for example bromocriptine, cabergoline
  • conventional methods such as neurosurgical intervention or radiation, can also be used therapeutically in connection with the administration of medicaments according to the invention or antibodies.
  • other anti-tumor substances chemotherapy drugs
  • a medicament according to the invention containing antibody A according to the invention, can also contain other substances, in particular somatostatin analogs, dopamine antagonists and / or antineoplastic substances, or can be used separately, for example therapeutically combined in a kit.
  • a therapeutic combination of antibodies according to the invention with pegvisomant is also conceivable, as is a medicament according to the invention containing antibodies A and pegvisomant according to the invention and, if appropriate, further active substances (for example one of the active ingredients mentioned above).
  • a pharmaceutical containing at least one antibody A and pegvisomant according to the invention
  • the long-term effect of antibody A according to the invention could be combined with the short half-life of the receptor antagonist pegvisomant due to their longer half-life.
  • a medicament according to the invention or an antibody according to the invention can be used or could be used to produce a medicament are in particular diabetes mellitus (for example with regard to the retinopathy mediated indirectly by hGH in diabetes mellitus) or also as antitumor agent in hGH-dependent tumors.
  • An antibody according to the invention will typically be in the form of a lyophilized powder which contains between 0.5 mg and 100 mg of antibody A according to the invention and further additives, for example glycine, manitol and / or sodium phosphate monohydrate. This lyophilized powder is provided in a suitable aqueous solution and then administered, for example subcutaneously, once or several times a day.
  • the pharmaceuticals according to the invention can be administered as liquid pharmaceutical forms, in particular in the form of injection solutions.
  • Suitable additives and / or auxiliary substances are e.g. Solvents or diluents, stabilizers, suspending agents, buffer substances, preservatives, as well as dyes, fillers and / or binders.
  • solvents or diluents e.g. Solvents or diluents, stabilizers, suspending agents, buffer substances, preservatives, as well as dyes, fillers and / or binders.
  • the choice of excipients and the amounts to be used depend on whether the drug is to be administered parenterally, intrvasally, intravenously, intraperitoneally or intramuscularly. Preparations in the form of suspensions and solutions as well as easily reconstitutable dry preparations are suitable for all parenteral applications.
  • Another object of the invention is a diagnostic agent containing at least one antibody according to the invention and optionally additives and / or auxiliaries.
  • a diagnostic is understood to mean a preparation or aids with the help of which, for example, a specific illness can be diagnosed.
  • the invention furthermore relates to a kit comprising, separated from one another, at least one first preparation comprising an antibody according to the invention and a finished test assay which functions on the basis of an antigen / antibody reaction for the quantitative determination of a ligand which serves as an antigen in this test.
  • a kit is to be understood as a jointly occurring form of different components in a packaging form.
  • a diagnostic kit that contains the various components necessary for the quantitative analysis of a ligand.
  • kits in which the first preparation contains an antibody which is directed against the binding site of the human growth hormone (hGH) on the human growth hormone binding protein (hGHBP) and / or in addition to the first preparation and the one based on of an antigen / antibody reaction functioning finished test assay for the quantitative determination of a ligand also contains a preparation for calibration.
  • hGH human growth hormone
  • hGHBP human growth hormone binding protein
  • a particularly preferred embodiment is a kit according to the invention, in which the first preparation contains the antibody ZMC1 (deposited above) and / or the preparation for calibration “international reference preparation 88/624” or “international reference preparation”
  • hGH ligand
  • Another object of the invention is the use of an antibody for, preferably quantitative, determination of a physiological ligand of a physiological binding protein.
  • Another object of the invention is the use of an antibody according to the invention for the manufacture of a medicament for the treatment of growth disorders.
  • Another object of the invention is a method for producing an antibody according to the invention, ie an antibody which is directed against a binding protein for a ligand, with the following steps: (a) immunization of animals with recombinantly produced binding protein, (b) isolation of Animal immune cells, (c) fusion with myeloma cell lines to hybridoma cell cultures, (d) selection of clones with high specificity for the binding protein.
  • the immunization can be carried out in all animals which are suitable for such purposes, for example in mice, rabbits, pigs, horses, etc.
  • binding protein for example GHBP
  • suitable media eg wells of microtiter plates, polystyrene beads, plastic tubes
  • the binding protein eg GHBP
  • ligand eg hGH
  • the ligand should be labeled (for example by biotinylation, label (radioactive label, fluorescent marker, enzyme marker (sea radical peroxidase etc.)) or be detectable via an appropriate anti-ligand antibody to determine the property of the ligand on the binding protein in the container coated with catch antibody.
  • alternative methods are, for example, radioactivity, chemiluminescence, colorimetric methods or enzyme reaction. After a washing step, such "wells" or "beads" can be identified that have no signal after ligand addition. In this case, the capture antibody blocks the ligand binding site, the binding site is therefore no longer available for ligand binding, and the capture antibody interferes with the ligand.
  • an antibody according to the invention can be identified by competitive binding assays. For this purpose, as described above, coated, however, each “well” with an anti-binding protein antibody that does not specifically recognize the ligand binding site. After adding binding protein, a potentially interesting antibody (with the property of specifically recognizing the ligand binding site) is marked in each “well” Ligands added. The signal intensity of the ligand in the “well” decreases with increasing concentration of the ligand binding site-specific antibody. In this way, antibodies according to the invention (for example against GHBP) can be selected.
  • phage display methods can also be used to generate antibodies according to the invention and then to select them, as described above.
  • Figure 1 shows the results of the investigation in embodiment example 2.
  • the results show that the addition of the antibody according to the invention reduces the dose-dependent influence of the negative interference that hGHBP has on the results of the hGH measurement (false low values in the sandwich assays tested) can cancel.
  • Balb / c mice were repeatedly immunized with recombinantly produced hGHBP using the generally known method (see package insert Titermax®).
  • package insert Titermax® the generally known method
  • the spleen was removed from the animals, and according to the method described by Koehler and Milstein (Continuous cultures of fused cells secreting antibody of predefined specificity, Nature, 1975, Aug. 7; 256 (5517): 495- 7) described method produced by fusion with a mouse myeloma cell line hybridoma cell cultures.
  • Clones which produced monoclonal antibodies of high affinity and specificity for hGHBP were also selected according to generally known methods (limited dilution, screening of the hybrid cell culture supernatants with labeled antigen).
  • GHBP GHBP
  • binds to the coating antibody - in an initially still unknown orientation binding depending on the epitope inside or outside the hormone-receptor interaction site.
  • labeled (in our case biotinylated) growth hormone was added, which can now only bind to the GHBP molecules whose binding site is freely accessible (ie, where the coating antibody does not interfere with the growth hormone binding).
  • Bound antibodies were measured by adding streptavidin-europium, which binds to bound biotinylated hGH and can be measured in a fluorometer (time-resolved fluorescence after addition of enhancement solution). Alternatively, this step can also be carried out using the analog methods (radioactivity, enzyme reaction / colorimetric method or chemiluminescence).
  • a microtiter plate was coated with one of the above-selected methods against GHBP, which does not interfere with the binding of hGH. Addition of GHBP caused the GHBP molecules to be bound in a directional manner, that is to say with a freely accessible binding site for hGH. Now biotinylated hGH was added and at the same time all other monoclonal antibodies against GHBP (of course each antibody in a separate well of the microtiter plate).
  • Sheep serum - which, in contrast to the sera of other species, does not contain any high-affinity growth hormone binding protein and thus releases the optimal "control medium” - was mixed with various concentrations of growth hormone (preparation 80/505, final concentration 0.3 / 0.6 / 4 and 9 ng / ml) Then aliquots of these sera were mixed with different concentrations of recombinant human GHBP and incubated for 24 hours at 4 ° C.
  • Ebdrup L Fisker S, Sorensen HH, Ranke MB, Orskov H. Variety in growth hormone determinations due to use of different immunoassays and to the Interference of growth hormone-binding protein, Horm Res 1999; 51 (SuppM); 20-6,

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Abstract

L'invention concerne un procédé d'analyse quantitative de ligands dans un échantillon de protéines de liaison dissoutes ou en suspension au moyen d'anticorps spécifiques. L'invention concerne également des anticorps pouvant être utilisés dans ce procédé, des médicaments, des moyens diagnostiques et des nécessaires (de diagnostic) contenant ces anticorps, l'utilisation de ces anticorps lors de l'analyse quantitative et pour des indications spécifiques, ainsi qu'un procédé de production de ces anticorps.
PCT/EP2004/001272 2003-02-19 2004-02-11 Procede de mesure quantitative de ligands dans un echantillon de proteines de liaison dissoutes WO2004074840A1 (fr)

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DE10306992A DE10306992A1 (de) 2003-02-19 2003-02-19 Verfahren zur quantitativen Messung von Liganden in der Probe gelöster Bindungsproteine
DE10306992.5 2003-02-19

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Citations (5)

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US5506107A (en) * 1991-05-10 1996-04-09 Genentech, Inc. Selecting ligand agonists and antagonists
WO1998041872A1 (fr) * 1997-03-19 1998-09-24 Pharmacia & Upjohn Ab Procede pour determiner l'activation de recepteurs de cytokines au moyen d'un anticorps
WO1999046597A1 (fr) * 1998-03-09 1999-09-16 Diagnostic Systems Laboratories, Inc. Dosage biologique de complexe d'igfbp
US6387879B1 (en) * 1997-12-15 2002-05-14 Dgi Biotechnologies, Inc. Compounds that bind growth to hormone receptor

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DE4328070C1 (de) * 1993-08-20 1994-11-24 Henning Berlin Gmbh Verfahren zur Bestimmung eines Analyten in einem Volumen einer flüssigen Probe sowie seine Anwendung zur Bestimmung von anti-TSH-Rezeptor-Autoantikörpern in einem Patientenserum

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WO1992008986A1 (fr) * 1990-11-16 1992-05-29 Societe Alcyon Analyzer S.A. Analyseur automatique d'echantillons par colorimetrie
US5506107A (en) * 1991-05-10 1996-04-09 Genentech, Inc. Selecting ligand agonists and antagonists
WO1998041872A1 (fr) * 1997-03-19 1998-09-24 Pharmacia & Upjohn Ab Procede pour determiner l'activation de recepteurs de cytokines au moyen d'un anticorps
US6387879B1 (en) * 1997-12-15 2002-05-14 Dgi Biotechnologies, Inc. Compounds that bind growth to hormone receptor
WO1999046597A1 (fr) * 1998-03-09 1999-09-16 Diagnostic Systems Laboratories, Inc. Dosage biologique de complexe d'igfbp

Non-Patent Citations (4)

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Title
BIDLINGMAIER M ET AL: "A method to eliminate the interference of human growth hormone binding protein (hGHBP) in immunoassays for human growth hormone (hGH).", GROWTH HORMONE & IGF RESEARCH, vol. 14, no. 2, April 2004 (2004-04-01), & SECOND INTERNATIONAL GH-IGF SYMPOSIUM; QUEENSLAND, AUSTRALIA; APRIL 18-22, 2004, pages 133 - 134, XP008031083, ISSN: 1096-6374 *
KHOSRAVI J ET AL: "The high molecular weight insulin-like growth factor-binding protein complex: Epitope mapping, immunoassay, and preliminary clinical evaluation", JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM 1999 UNITED STATES, vol. 84, no. 8, 1999, pages 2826 - 2833, XP001191030, ISSN: 0021-972X *
WANG B S ET AL: "Functional characterization of monoclonal antibodies specific to growth hormone receptor.", MOLECULAR IMMUNOLOGY. OCT 1996, vol. 33, no. 15, October 1996 (1996-10-01), pages 1197 - 1202, XP002282994, ISSN: 0161-5890 *
WANG B S ET AL: "Promotion of animal growth with a monoclonal antibody specific to growth hormone receptor.", MOLECULAR AND CELLULAR ENDOCRINOLOGY. 5 FEB 1996, vol. 116, no. 2, 5 February 1996 (1996-02-05), pages 223 - 226, XP002282908, ISSN: 0303-7207 *

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