IE60450B1 - Lymphokine in pure form, novel monoclonal antibodies, hybridoma cell lines, processes and applications - Google Patents

Abstract

The invention relates to purified human macrophage migration inhibitory factor (human MIF) and its individual proteins, to a process for the purification of human MIF and for the isolation of its individual proteins, to monoclonal antibodies against human MIF, derivatives thereof, processes for the preparation of these antibodies and derivatives thereof, hybridoma cell lines which produce these antibodies, processes for the preparation of said hybridoma cell lines, and to the use of the monoclonal antibodies and derivatives thereof for the qualitative and quantitative determination of human MIF in biological fluids and on cell surfaces, and to the use of the monoclonal antibodies for the purification of human MIF. Human MIF and monoclonal antibodies against human MIF are important for the diagnosis and therapy of immunoregulatory disorders and chronic inflammatory disorders.

Description

(C) Copyright 1994, Government of Ireland.

The invention relates to purified human macrophage migration inhibition factor (human MIF) comprising proteins of human origin that are recognised and bound by the monoclonal antibody 1C5 to human MIF, and its individual proteins, a process for the purification of human MIF and for the isolation of its individual proteins, novel monoclonal antibodies to human MIF, derivatives thereof, processes for the preparation of these antibodies and their derivatives, hybridoma cell lines that produce these antibodies, processes for the preparation of said hybridoma cell lines, the use of the monoclonal antibodies and their derivatives for the qualitative and quantitative determination of human MIF in biological fluids and on cell surfaces, the use of the monoclonal antibodies for the purification of human MIF, and to pharmaceutical preparations comprising purified human MIF, its individual proteins, monoclonal antibodies to human MIF or derivatives thereof.

Background to the invention 2o Although human MIF from human cells is known as an active principle, hitherto it has been described only in the form of a mixture and together with other proteins in biological fluids and has not been characterised from the point of view of structure. MIF belongs to the group of so-called lymphokines which comprises biologically active, soluble polypeptides that are secreted by lymphocytes and monocytes or macrophages when these are stimulated by antigens, mitogens or the like. Other examples of lymphokines are immune interferon (i~ interferon), interleukin 1 and 2 and macrophageactivating factor (MAF). These lymphokines control the differentiation, activation and proliferation of various cell types of the immune system.

According.to the known state of the art, human MIF consists of a group of polypeptides that inhibit the migration ability of macrophages. Human MIF is secreted not only by activated lymphocytes, T- and B-cells, but also by non-lymphoid cells, for example by growing fibroblasts and certain tumour cells. MIF can be clearly differentiated from τ-interferon, macrophage-activating factor (MAF) and other lymphokines. Hitherto, it was not possible, however, to obtain MIF from human cells in pure form and to clarify its structure. It was known of human MIF that it probably consisted of a mixture of structurally related polypeptides having respective molecular weights of approximately 8.5, 18, 27, 36, 45 and 67 kg/mole (Kilodaltons) and an isoelectric point of pH 5.1 and 2„9 [G. Burmeister, Η» Steffen, U. Feige and C. Sorg, Immunobiology 160. 15 (1981)].

Human MIF plays a decisive role in the early phase of an inflammation reaction (’’delayed type hypersensitivity reaction"). It induces the differentiation of monocytes and quiescent tissue macrophages to mature inflammatory macrophages. Purified human MIF and its individual proteins and monoclonal antibodies that bind specifically to human MIF and inhibit its activity are therefore important for the diagnosis and therapy of immune regulation diseases and chronic inflammatory diseases. Monoclonal antibodies that bind to and inhibit human MIF are valuable in the control of contact eczemas, primary chronic polyarthritis and a wide variety of autoimmune diseases. Purified human MIF and its individual proteins can increase resistance to infection, for example resistance to tuberculosis, leprosy or leishmaniosis and to candidosis, and also resistance to tumours, especially j to metastases.

The use of antibodies in diagnosis and therapy was, until recently, severely limited in its range of application. Antibodies were obtained in very small amounts from animal serum in the form of a complex mixture of various proteins. Standardisation of the antibodies was not possible since each immunised animal individual and even a single individual when immunised repeatedly produces a serum with antibodies of a different composition in each case. By using a technique developed by Kohler and Milstein [G. Kohler and C. Milstein, Nature 256, 495 (1975)] it has now become possible to obtain reproducibly in industrial quantities antibodies in homogeneous form from cell cultures, that is to say socalled monoclonal antibodies. By fusion of suitable myeloma cells with antibody-producing lymphocytes from a donor immunised with antigen, hybridoma cells are produced which combine the ability to undergo unlimited cell division and unlimited growth in vitro with the production of a homogeneous antibody. Hence, it is possible to render independent the immune response of an organism to a specific antigen and to prepare monoclonal antibodies by continuous culturing of hybridoma cells.

Although many examples of the preparation of specific antibodies by the hybridoma technique have so far become known and the general procedure has been described in principle, with each new example specific problems arise that require adaptation of the technique to the particular case. Without such adaptation there is no certainty that the desired hybridoma cells will ever be formed, that they will be genetically stable and produce the desired antibodies, and that the antibodies so prepared will have the desired specificity. The degree of success is influenced in principle by the type and purity of the antigen used for immunisation of the donor, the technique of cell fusion, the procedure in the selection of suitable hybridoma cell lines and the way in which the antibodies are isolated and purified.

An important application of antibodies which has as a prerequisite the availability of homogeneous monoclonal antibodies in large quantities, as has now been made possible by the hybridoma technique, is immunoaffinity chromatography. For this, antibodies having a desired specificity are applied to a solid carrier material.

From a solution containing a large number of different compounds there are bound to the antibodies, and hence to the solid carrier material, selectively those compounds having a structural element (determinant, epitope) that is recognised and bound by the antibody. After removing the solution with the undesired compounds, the desired compounds are dissolved out of the carrier material by washing with reagents that break the bond to the antibodies, and are isolated by classical methods.

A problem of the present invention is to provide purified human MIF and its individual proteins. This problem is solved by means of the monoclonal antibodies according to the invention.

Description of the invention The invention relates to purified human macrophage migration inhibition factor (human MIF) comprising only proteins of human origin that have epitopes that are recognised and bound by the monoclonal antibody 1C5 to human MIF, and its individual proteins. Purified human MIF consists of at least four individual proteins of molecular.weight approximately 8, approximately 14, approximately 28 and approximately 45 kg/mole. Purified human MIF is active in standard test procedures in which the migration of macrophages is measured.

Individual proteins of purified human MIF are proteins that are homogeneous according to the customary methods of protein analysis, for example SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) or gel fiitration-HPLC (high pressure liquid chromatography), that are active in standard test procedures in which the migration of macrophages is measured, and that are constituents of purified human MIF. If, during the course of separation and isolation of the individual proteins, detergents or other denaturing reagents are added, the natural tertiary structure of the proteins alters and therewith the property of inhibiting the migration of the macrophages, although the primary structure remains unchanged. The invention relates also to such denatured forms of the individual proteins. Examples of individual proteins of purified human MIF are the two proteins having the respective molecular weights approximately 8 kg/mole and approximately 14 kg/mole and the N-terminal amino acid sequence Xj-Leu-Thr-Glu-Leu10 l 5 Glu~Lys-Ala~Leu-Asn~Ser-Ile~Ile~Asp-VaI~Tyr~His~Lys-Tyr in which the meaning of the amino acid Xj is not fixed, and also proteins having a molecular weight of approximately 28 kg/mole and approximately 45 kg/mole.

The invention relates especially to the individual protein of purified human MIF having the approximate molecular weight 8 kg/mole and the N-terminal amino acid sequence Met-Leu-Thr-Glu-Leu-Glu-Lys-Ala-Leu-Asn-Ser-Ile15 7 Ο 2 5 Ile-Asp-Vai-Tyr-His-Lys-Tyr-Ser-Leu-Ile-Lys-Gly-Asn-Phe30 35 40 His-Aia-Vai-Tyr-Arg-Asp-Asp-Leu-Lys-Lys-Leu-Leu-Glu-T'hr45 5 0 Giu-X42-Pro-Gln-Tyr-Iie-Arg~Lys-Lys-Gly-Aia-Asp~Val-Trp5 5 6 0 6 5 Phe-Lys-Glu-LeU"Asp-Ile"Asn"X62-x63"x64Ala_Val which the meaning of the amino acids X42, x62' x63 and x64 is not fixed but X42 nay represent only Ser or Cys.

The invention relates also to a process for the preparation of purified human MIF comprising proteins of human origin that are recognised and bound by the monoclonal antibody 1C5 to human MIF, and its individual proteins, characterised in that a solution comprising human MIF, for example a cell extract, cell culture supernatant or a cell culture filtrate of human cells, if desired after purification steps that are known per se. a) is brought into contact with a carrier material having the monoclonal antibody 1C5 specific to human MIF, unbound proteins and other foreign substances are removed, the human MIF bound to the antibodies is selectively split off and isolated and, b) if desired, the purified human MIF is separated into its individual proteins.

Solutions, for example cell extracts, cell culture supernatants or cell culture filtrates of human cells, that comprise MIF are prepared according to methods that are known per se. Suitable human cells are, for example, mononuclear cells that can be obtained from buffy coat", the layer of white blood corpuscles which is deposited in the centrifugation of venous blood to which citrate or heparin has been added, by leucapheresis and/or density gradient centrifugation. The mononuclear cells are Ί stimulated to produce MIF and other lymphokines by suitable adjuncts, for example concanavalin A or phytohaemagglutinin, and are cultured according to the customary methods for from approximately 12 to approxi5 mately 72 hours, preferably between 18 and 36 hours, in suitable culture media, for example RPMI 1640 medium, to which are added, if desired, fetal calf serum, buffer solutions and/or antibiotics, such as penicillin or streptomycin, at approximately 37°C and, if desired, while gassing with CO2. Solutions comprising human MIF are obtained from the cells and/or the cell culture supernatants, for example, by extraction, filtration and/or centrifugation and, if desired, are stabilised by the addition of antibiotics and/or protease-inhibitors.

Such human MIF solutions may be brought into contact with the monoclonal antibody 1C5 bound to the carrier material directly in step a), but preferably are subjected beforehand to preliminary purification by means of ultrafiltration over membranes having separation limits of molecular weight approximately 6 kg/mole or less, concentration, optional dialysis and, if desired, further purification by chromatography, for example over DEAE-cellulose or Sephadex (§)„ In step a), human MIF is separated from other proteins and foreign substances present in the solutions, the separating action based on binding interactions between the monoclonal antibody 1C5 specific to human MIF and the known antigenic determinants of human MIF being utilised. For this purpose, the solutions comprising human MIF are brought into contact with a carrier material to which the monoclonal antibody 1C5 specific to human MIF is bound, according to the immunoaffinity chromatography method known per se.

A suitable carrier material on an inorganic or organic basis, for example silicates, crosslinked agarose, dextran or polyacrylamide in suitably functionalised form, optionally having been activated, is charged in a manner known per se with the monoclonal antibody 1C5 or its derivatives. For example, a carrier material containing activated ester functions, for example N-hydroxysuccinimide ester groups, is suspended in an aqueous buffer solution and mixed with a solution of the monoclonal antibody, and then unbound monoclonal antibodies are washed, out and unoccupied reactive sites of the carrier material are blocked, for example with a primary amine, such, as ethanolamine. The carrier material is suspended in a suitable aqueous solvent, for example a salt solution, such as NaCl solution, or a buffer solution, such as phosphate-buffered NaCl solution, Na.HCO3 solution or 3~(N-morpholino)propanesulphonic acid solution, and brought into contact with the solution comprising human MIF, for example is poured into a chromatography column and the solution comprising human MIF is introduced and pumped through the carrier material, if desired under pressure. Unbound proteins and other impurities are washed away with aqueous solutions, for example buffer solutions in a pH range of from approximately pH 5 to approximately pH 9 and/or salt solutions, for example NaCl solution. The human MIF bound to the antibodies on the carrier material is eluted with suitable aqueous solutions, for example buffer solutions in a pH range of from approximately pH 2 to approximately pH 5, such as glycine buffer, or pH gradients of differing composition or salt solutions, for example concentrated NH^SCN solution. The solutions comprising purified human MIF which are obtained are optionally Q neutralised, and the purified human MIF is isolated therefrom. according to methods that are known per se, for example by chromatography over Sephadex (£) electrodialysis, electrophoretic concentration and/or vacuum centrifugation.

If desired, in step b) , the purified human MIF is separated into its individual proteins, for example by separating the protein mixture by chromatography into fractions of different molecular weight according to methods that are known per se. For example, the purified human MIF is separated by preparative sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and homogeneous molecular weight fractions are eluted from the gel and isolated in pure form, for example by chromatography over Sephadex electrophoretic concentration and/or vacuum centrifugation. The purified human MIF can also be separated by preparative gel filtration-HPLC into homogeneous molecular weight fractions and the individual proteins can be isolated therefrom.

The invention relates also to novel monoclonal antibodies to human macrophage migration inhibition factor (human MIF), and to derivatives thereof.

The monoclonal antibodies according to the invention bind and/or inhibit the biological activity of human MIF. The binding of monoclonal antibodies to human MIF is advantageously determined in an immunoassay, for example with a test procedure in which human MIF is applied to a solid carrier, the coated carrier is incubated with a monoclonal antibody solution and monoclonal antibodies bound thus are developed with a second antibody which carries a radioisotope or enzyme label. Monoclonal antibodies bound to human MIF are thus determined by measuring the radioactivity or by an enzyme substrate reaction. There is also suitable, for example, a test procedure in which the monoclonal antibodies are fixed to a carrier, incubated with a solution comprising human MIF and, finally, the residual content of human MIF activity of that solution is determined.

The human MIF activity of a solution is measured in a manner known per se by determining the inhibiting action on the migration of suitably activated human macrophages. For example, an experimental procedure can be selected in which the migration distance of macrophages applied to titre plates in droplets of agarose is measured in a test solution.

Preferred are monoclonal antibodies to human MIF that are produced by mouse/mouse, rat/rat or rat/mouse hybridoma cells. Examples of such preferred monoclonal antibodies according to the invention are the monoclonal antibody of subclass IgGjte designated 1C5 which is produced by the hybridoma cell line 1C5, and the monoclonal antibody of subclass IgG2a designated 7D10 which is produced by the hybridoma cell line 7D10. The monoclonal antibodies 1C5 and 7D10 bind to human MIF without inhibiting its biological activity.

Derivatives of monoclonal antibodies according to the invention are, for example, fragments, such as Fab, Fab' or F(ab')2 fragments, that retain their specificity for the antigenic determinants of human MIF, radioactively labelled monoclonal antibodies which are labelled, for example, with radioactive iodine (125I, 131I), carbon (14C), sulphur (35S), tritium (3H) or the like, monoclonal antibody conjugates with biotin or avidin or monoclonal antibody conjugates with enzymes, such as horseradish peroxidase, alkaline phosphatase, β-Dgalactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholineesterase, lysozyme, malate dehydrogenase or glucose-6-phosphate dehydrogenase. Preferred derivatives are monoclonal antibodies labelled with 125iodine and antibody conjugates with biotin.

The invention relates also to processes known per se for the preparation of monoclonal antibodies to human MIF and their derivatives, characterised in that hybridoma cells producing said monoclonal antibodies a) are cultured in vitro and the monoclonal antibodies are isolated from the culture supernatants, or b) are multiplied in vivo in a suitable mammal and the monoclonal antibodies are isolated from the body fluids of that mammal, and, c) if desired, a resulting monoclonal antibody is converted into a derivative thereof.

Suitable culture media for the in vitro culturing according to process a) are the customary standard culture media, for example Dulbecco^s Modified Eagle Medium or RPMI 1640 medium, optionally supplemented by fetal calf serum. For isolation of the monoclonal antibodies, the proteins in the culture supernatants are precipitated with ammonium sulphate or the like and purified by the customary chromatography methods, for example gel filtration, ion-exchange chromatography, chromatography over DEAE-cellulose or immunoaffinity chromatography.

Large quantities of the desired antibodies can be obtained by multiplying the hybridoma cells in vivo according to process b). For this purpose, cell clones are injected into mammals, preferably syngeneic mammals, and, after 1-3 weeks, the monoclonal antibodies are isolated from the body fluids of those mammals. For example, hybridoma cells originating from Balb/c mice are injected intraperitoneally into Balb/c mice that have optionally been pre-treated with a hydrocarbon such as pristane, and, after 8-10 days, ascitic fluid is taken from these animals. The desired monoclonal antibodies are isolated from the body fluids according to methods that are known per se. for example by precipitation with ammonium sulphate or the like and purification by chromatography, for example over DEAE-cellulose, hydroxyapatite (HPKT, high performance hydroxyapatite column chromatography), ion-exchange resin, by gel filtration or immunoaffinity chromatography.

Fragments of monoclonal antibodies according to the invention, for example Fab, Fab? or F(ab')2 fragments, that retain their specificity for the antigenic determinants of human MIF are prepared according to methods that are known per se. for example by treating monoclonal antibodies prepared according to process a) or b) with enzymes such as pepsin or papain and/or by cleavage of disulphide bonds by chemical reduction.

Monoclonal antibodies radioactively labelled with iodine (125i 131j) are obtained from the monoclonal antibodies according to the invention by iodination known per se. for example with radioactive sodium or potassium iodide and a chemical oxidising agent, such as sodium hypochlorite, chloramine T or the like, or an enzymatic oxidising agent, such as lactoperoxidase, glucose oxidase and glucose. Radioactively labelled monoclonal antibodies according to the invention are also prepared by adding to the culture media for the in vitro culturing, in a manner known per se. radioactively labelled nutrients containing radioactive carbon (34C), tritium (3H), sulphur (35S) or the like, for example L-(14C)leucine, L-(3H)-leucine or L-(35S)-methionine, and obtaining the monoclonal antibodies according to process a).

Enzyme-labelled monoclonal antibodies according to the invention are obtained according to methods that are known per se by reacting monoclonal antibodies prepared according to process a) or b) and the desired enzyme with a coupling reagent, for example glutaraldehyde, periodate, N,N/-o-phenylenedimaleimide, N-(m-maleimidobenzoyloxy)-succinimide, N-(3-(2f-pyridvl-dithio)propionoxy)-succinimide or the like. Conjugates of monoclonal antibodies according to the invention with avidin are obtained in like manner. Conjugates with biotin are obtained according to methods that are known per se by reacting monoclonal antibodies according to the invention, for example, with biotin N~hydroxysuccinimidyl ester.

The invention relates also to hybridoma cell lines, characterised in that they produce monoclonal antibodies to human macrophage migration inhibition factor (human MIF).

Preferred hybridoma cell lines that produce monoclonal antibodies directed against human MIF are those which are hybrids of mouse myeloma cell lines and mouse or rat lymphocytes.

Most especially preferred is the hybridoma cell line designated 1C5 which was deposited on 13.7.1984 at the Collection Nationale de Cultures de Microorganismes of the Institut Pasteur in Paris under number 1-316. The cell line 1C5 is a hybrid of the mouse myeloma cell line P3-X63-Ag8„653 and a B-lymphocyte of the spleen of a Balb/c mouse. Likewise preferred is the hybridoma cell line designated 7D10 which was deposited on 29.1.1985 at the Collection Nationale de Cultures de Microorganismes of the Institut Pasteur in Paris under number 1-418. The cell line 7D10 is a hybrid of the mouse myeloma cell line P3-X63-Ag8.653 and a B-lymphocyte of the spleen of a DA rat. Both cell lines are genetically stable, secrete monoclonal antibodies of constant specificity and can be activated from deep-frozen cultures by thawing and recloning.

The invention relates also to processes that are known per se for the preparation of hybridoma cells that produce monoclonal antibodies to human MIF, characterised in that suitable mammals are immunised with MIF or MIF conjugates, the antibody-producing cells taken from the mammal are fused with myeloma cells, the resulting hybridoma cells are cloned and those cell clones which produce the desired monoclonal antibodies are selected.

As antigens there may be used any MIF-containing protein fractions or MIF conjugates, for example MIF-containing protein fractions or conjugates of these protein fractions which have been obtained as described above from human mononuclear cells with suitable immunogenic carriers, for example proteins, polysaccharides, latex particles or cells. A pre-requisite for the use of animal, for example murine, MIF-containing protein fractions is that the animal MIF in question and human MIF have identical epitopes. Conjugates are prepared according to methods that are known per se. for example by coupling with carbodiimides, periodate, glutaraldehyde, N^'-o-phenylenedimaleimide, N-(m-maleimidobenzoyloxy)-succinimide, M-(3-(2'-pyridyldithio)propionoxy)-succinimide or the like. There are preferably used for immunisation conjugates of sheep erythrocytes pre-treated with glutaraldehyde and MIFcontaining protein fractions from human or mouse cells.

Preferred mammals for immunisation with human MIF are mice or rats, especially Balb/c mice. For immunisation with mouse MIF, rats, for example DA rats, are preferably used. The immunisations are performed in a manner known per se. for example by administering parenterally, for example intraperitoneally and subcutaneously, three to eight injections of antigenic human MIF conjugates at intervals of from one to ten days, if desired together with an adjunct that stimulates the production of lymphocytes, for example complete or incomplete Freund's adjuvant. It is also possible to pre-immunise the animals by two to four injections and administer further injections only after an interval of from 8 to 12 months.

Antibody-producing cells of the immunised animals, preferably spleen cells, are taken from the animals two to six days after the last immunisation and fused with myeloma cells of a suitable cell line in the presence of a fusion-promoter. Several different myeloma cell lines and cell lines derived therefrom are known as suitable fusion partners. Preferred are myeloma cells that lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) or the enzyme thymidine kinase (TK) and that, for that reason, do not survive in a selective culture medium containing hypoxanthine, aminopterin and thymidine (HAT medium) or hypoxanthine and azaserine.

Especially preferred are myeloma cells and cell lines prepared therefrom that do not survive in HAT medium or hypoxanthine/azaserine medium and do not secrete any immunoglobulins or parts thereof, for example the cell lines X63-Ag8.653 and Sp2/O-Agl4. The cell line X63Ag8.653 originating from Balb/c mice is suitable for fusion not only with lymphocytes of mice, for example Balb/c mice, but also with lymphocytes of rats, such as DA rats. i As fusion-promoters there come into consideration Sendai virus or other paramyxoviruses, optionally in UV-inactivated form, calcium ions, surface-active lipids, such as lysolecithin, or polyethylene glycol. Myeloma cells are preferably fused with a two- to ten-fold excess of spleen cells from immunised animals in a solution containing from approximately 30 to approximately 50 % polyethylene glycol of molecular weight from approximately 1000 to approximately 6000.

After fusion, the cells are portioned out and cultured in selective HAT medium or hypoxanthine/azaserine medium, with only hybridoma cells surviving since these combine, from the myeloma cells, the ability to grow in vitro and, from the antibody-producing cells of the immunised animals, the missing HGPRT or TK genes and, therewith, 30 the ability to survive in selective media.

Suitable culture media for the growth of the hybridoma cells are the customary standard culture media, for example Dulbecco^s Modified Eagle Medium or RPMI 1640 medium. At the beginning of cell growth, there are preferably added so-called feeder cells, for example normal mouse peritoneal exudate cells, spleen cells, bone marrow macrophages or the like. At regular intervals, said culture media are supplemented by selective HAT medium or hypoxanthins/azaserine medium to prevent the hybridoma cells from being overgrown by ordinary myeloma cells.

The cell culture supernatants of the hybridoma cells are examined to see whether they contain the desired monoclonal antibodies. There is preferably used for this purpose a radioimmunoassay, an enzyme-immunoassay and/or a determination of the MIF activity, as are described above. The cell clones selected in such a manner are cultured in customary standard media and# if desired, in a manner known oar se deep-frozen and/or recloned by limiting dilution or by application to agar.

The invention relates also to the use of the monoclonal antibodies to human macrophage migration inhibition factor (human MIF) and their derivatives for the qualitative and quantitative determination of human MIF, especially in biological fluids and on cell surfaces. For example, the monoclonal antibodies according to the invention can be used in any of the immunoassays known per se that utilise the binding interactions between antigen (human MIF) and monoclonal antibody. Examples of such assays are radioimmunoassays (RIA), enzyme-immuno30 assays, immuno-fluorescence tests, latex agglutination or haemagglutination.

IS The antibodies according to the invention can be used as such or in the form of radioactively labelled derivatives optionally in combination with other labelled antibodies and/or proteins in a radioimmunoassay (RIA). Any of the known modifications of an RIA can be used, for example RIA in homogeneous phase, solid phase RIA or heterogeneous RIA, single RIA or double (sandwich) RIA with direct or indirect (competitive) determination of human MIF.

There is preferred an RIA in which a suitable carrier, for example the plastic surface of a titre plate or of a test tube, for example of polystyrene, polypropylene or polyvinyl chloride, glass or plastic beads, filter paper, or dextran, cellulose acetate or nitrocellulose sheets or the like, is coated with a human MIF-containing test solution or standard solution by simple adsorption or optionally after activation of the carrier, for example with glutaraldehyde or cyanogen bromide, and incubated with a solution of a monoclonal antibody according to the invention and then with a solution of a second antibody, the second antibody, for example a rabbit-anti-mouse immunoglobulin, recognising and binding to the monoclonal antibody according to the invention. The amount of second antibody bound is determined by measuring the bound radioactivity, either directly if the second antibody is radioactively labelled, or after development with a radioactively labelled protein with a high affinity for that second antibody, for example 125I-labelled protein A from Staphylococcus aureus.

The monoclonal antibodies according to the invention can be used as such or as enzyme-labelled derivatives in an enzyme-immunoassay. Such immunoassays are, for example, test procedures in which enzyme-labelled antibodies known per se that recognise and bind epitopes of the monoclonal antibodies according to the invention, or enzyme-labelled monoclonal antibody derivatives according to the invention are used. Instead of enzyme-labelled antibodies, it is also possible to use antibody-biotin conjugates together with avidin-enzyme conjugates. Examples of enzymes used in enzyme-immunoassays are horseradish peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carbonic anhydrase, acetylcholineesterase, lysozyme, malate dehydrogenase or glucose-6-phosphate dehydrogenase.

There is preferred an ELISA test (enzyme-linked immunosorbent assay) in which a carrier as described above for a single RIA test, which, if desired, is charged with erythrocytes, is either coated with a human MIF-containing test or standard solution by simple adsorption or, if desired, after activation of the carrier or of the carrier-bound erythrocytes with glutaraldehyde, or is charged with cells to be examined for human MIF, and then this carrier is incubated with a solution of a monoclonal antibody according to the invention and finally with a solution of an enzyme-labelled second antibody that recognises and binds to the monoclonal antibody according to the invention. In so doing, the amount of second antibody bound, for example a peroxidase-labelled rabbitanti-mouse immunoglobulin, is made visible and determined by development with enzyme substrate.

There is especially preferred an ELISA test (enzymelinked immunosorbent assay) in which a carrier as described above for a single RIA test is coated with a solution of a monoclonal antibody according to the invention by simple adsorption or optionally after activation of the carrier, for example with glutaraldehyde or cyanogen bromide, this carrier is then incubated with a human MIF-containing test or standard solution and finally either with a solution of an enzymelabelled antibody according to the invention that optionally recognises another epitope of human MIF, or preferably with a solution of an antibody according to the invention conjugated with biotin, followed by a solution of an avidin-enzyme conjugate. In so doing, the quantity of the bound enzyme- or biotin-labelled antibody is made visible and determined by development with enzyme substrate.

Preferred enzymes in the enzvme-immunoassays according to the invention are horseradish peroxidase which can be developed, for example, with the enzyme substrates 5aminosalicylic acid, o-phenylenediamine, 3,3'-dimethoxybenzidine, 3,3',5,5'-tetramethylbenzidine, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) or the like and hydrogen peroxide, and alkaline phosphatase which, for example, releases p-nitrophenol from the enzyme substrate p-nitrophenyl phosphate.

The use according to the invention of monoclonal antibodies to human MIF and their derivatives for the qualitative and quantitative determination of human MIF also includes other immunoassays known per se. for example immunofluorescence tests using antibody conjugates or antigen conjugates with fluorescing substances, latex agglutination with antibody-coated or antigencoated latex particles or haemagglutination with antibody-coated or antigen-coated red blood corpuscles or the like.

The immunoassays described can be used to determine the quantity of human MIF in biological fluids, especially in human blood, or on cell surfaces in fixed form and therefore facilitate the diagnosis of diseases caused by immune regulation disorders. For example, in the case of low resistance to infection, a causative immune regulation disorder can be diagnosed if no human MIF or lower than average amounts thereof are found in the blood. Furthermore, the determination of human MIF in patholog10 ical tissues, for example in melanomas, granulomas and hyperplasias, can render a simple diagnosis possible since MIF occurs only in certain types of tissue and only in certain pathological conditions (Table).

Table: Binding of the monoclonal antibody 1C5 to frozen tissue sections J Stormal tissue: i A) liver - B) spleen + (only few cells) 2) skin + (only few macrophages) D) lung — Pathological tissue: E) primary melanoma (much cell infiltrates) ++ (cell infiltrates) melanoma metastases (little cell infiltrates) + (only few cells) melanoma micro- metastases (in lymph nodes) F) BCG granuloma (1, 3 or 5 weeks after inoculation)+ G) Sezary syndrome + H) syringolymphoid hyper- plasia + I) subacute eczema J) hypereosinophilic syndrome 1 + binding detected, - no binding detected The invention relates also to test kits for the qualitative and quantitative determination of human macrophage migration inhibition factor (human MIF), characterised in that they comprise monoclonal antibodies to human MIF and/or derivatives thereof and, optionally, adjuncts.

Test kits, according to the invention for a radioimmunoassay comprise, for example, a suitable carrier, optionally freeze-dried or concentrated solutions of an antibody according to the invention or of a radioactively labelled antibody derivative according to the invention, solutions of an optionally radioactively labelled second antibody and/or optionally solutions of radioactively labelled protein A from Staphylococcus aureus, standard solutions of purified human MIF or its individual proteins, buffer solutions, fixing solutions containing glutaraldehyde, detergents for preventing nonspecific adsorption and aggregate formation, pipettes, reaction vessels, calibration curves and the like.

Test kits according to the invention for an enzymeimmunoassay comprise, for example, a suitable carrier, optionally freeze-dried or concentrated solutions of a monoclonal antibody according to the invention, of an enzyme-labelled or biotin-labelled antibody derivative according to the invention, optionally freeze-dried or concentrated solutions of an enzyme-labelled antibody that recognises and binds to the monoclonal antibodies according to the invention, optionally freeze-dried or concentrated solutions of avidin-enzvme conjugates, enzyme substrates in solid or dissolved form, standard solutions of purified human MIF or its individual proteins, buffer solutions, fixing solutions containing glutaraldehyde, detergents, pipettes, reaction vessels, calibration curves and the like.

The invention, relates also to the use of the monoclonal antibodies to human macrophage migration inhibition factor (human MIF) and their derivatives for the purifi2 4 cation of human MIF. For example, human MIF can be separated.using separating methods known per se, the separating action of which is based on binding interactions between monoclonal antibody and antigenic deter5 minants of human MIF. A preferred separating method is immunoaffinity chromatography as described above.

The invention relates also to pharmaceutical preparations comprising a therapeutically effective amount of purified human MIF, its individual proteins, monoclonal antibodies o to human MIF or derivatives of such monoclonal antibodies and a significant amount of a pharmaceutical adjunct. Suitable derivatives of monoclonal antibodies are fragments, for example Fab, Fab' or F(abz)2 fragments, that have retained their specificity for the antigenic determinants of human MIF.

The pharmaceutical preparations according to the invention are those for enteral, for example nasal, rectal or oral, administration and preferably for parenteral, for example intramuscular, subcutaneous or intravenous, administration to warm-blooded animals, for example humans. Depending on the intended method of administration, the pharmaceutical preparations may be in unit dose form, for example in ampoules, vials, suppositories, dragees, tablets, capsules or nasal sprays in liquid or solid form.

The amount of the therapeutically effective compounds to be administered depends on the condition of the warmblooded animal, for example the human, such as the body weight, the nature and severity of the disease and the general condition and also on the mode of administration, and is carried out in accordance with the assessment of the doctor giving the treatment. The effective dose of human MIF. and its active individual proteins is in the order of magnitude of from 0.001 to 1 ng per kg of body weight per day, and of monoclonal antibodies to human MIF and derivatives thereof in the order of magnitude of from 0.001 to 1 mg per kg of body weight per day.

The pharmaceutical preparations according to the invention comprise the customary inorganic or organic, solid or liquid pharmaceutically acceptable carriers, option10 ally together with other therapeutically effective compounds and/or adjuncts. There are preferably used solutions or suspensions of the active ingredient, especially isotonic aqueous solutions or suspensions, or also lyophilised preparations which are dissolved in water shortly before use. The pharmaceutical preparations may be sterilised and/or comprise preservatives, stabilisers, wetting agents, emulsifiers, solubilisers, viscosity-increasing substances, salts for regulating the osmotic pressure and/or buffers, and also other proteins, for example human serum albumin or human blood plasma preparations.

Preferred are pharmaceutical preparations in the form of liposomes in aqueous dispersion comprising a therapeutically effective amount of purified human MIF or its individual proteins. There are suitable, in particular, liposomes having a population of as homogeneous a size as possible and a diameter of approximately from 2.0 x IO8 to 5.0 x 10"6 m consisting of one or more double layers of lipid components, for example amphipatic lipids, for 3q example phospholipids, such as lecithin, cephalin or phosphatidic acid, and optionally neutral lipids, for example cholesterol, enclosing an aqueous interior containing purified human MIF according to the invention or its individual proteins. Preferred are liposomes consisting of a mixture of synthetic phosphatidylserine and phosphatidylcholine.

The following Examples illustrate the invention without, however, limiting the scope thereof in any way.

The abbreviations used in the Examples have the following meanings: ELISA 0 HAT HPLC HT MIF PBS RIA SDS SDS-PAGE 20 SRBC tris rpm enzyme-assay (enzyme-linked immunosorbent assay) hypoxanthine/aminopterin/thymidine high pressure liquid chromatography hypoxanthine/thymidine macrophage migration inhibition factor phosphate-buffered physiological saline solution radioimmunoassay sodium dodecyl sulphate SDS-polyacrylamide gel electrophoresis sheep erythrocytes tris (hydroxymethyl) aminomethane revolutions per minute Example 1 Obtaining a protein fraction containing human MIF Mononuclear cells are obtained from Slbuffy coat", the layer of white blood corpuscles that is deposited in the centrifugation of venous blood to which citrate or heparin has been added, and purified by a two-step method consisting of leucapheresis and continuous centrifugation over a Ficoll gradient in an IBM blood cell separator (IBM 2997) [U. Feige and C. Sorg, J. Immunol. Methods 66, 161 (1984)]. The mononuclear cells are washed in Spinner medium (Seromed) and then stimulated at a concentration of 5 x 106 cells in 0.17 ml of RPMI 1640 medium per cm2 of culture vessel base for 2 hours with 0.67 μα of concanavalin A per 106 cells. Renewal of the RPMI 1640 medium and subsequent incubation for 20 hours at 37"C while gassing with 5 % CO3 yields the culture supernatant containing lymphokines. This is centrifuged for 30 minutes at 17 000 rpm (SS34 Rotor, Sorvall centrifuge) and 4°C. The cell-free supernatant is desalted over Sephadex ® G25 that has been equilibrated in 0.05M NH4HCO3 and the protein-containing fractions are then lyophilised. The lyophilisate is taken up in 0.01M sodium phosphate buffer pH 7.5 to which 0.1M NaCl has been added and chromatographed in the same buffer over Sephadex® G100. The human MIF-containing fractions comprising proteins in a molecular weight range of from 8 to 14 kg/mole (Kilodaltons) are combined and concentrated 16-fold in a Rotavapor.

Example 2 Manufacture of the hybridoma cells 2.1 Conjugation of human MIF-containing protein fractions to sheen erythrocytes 2.5 ml of packed sheep erythrocytes (sheep red blood cells, SRBC, Behringwerke) are suspended in 20 ml of PBS. 100 μΐ of this SRBC suspension are incubated with 900 μΐ of a glutaraldehyde solution in PBS for 5 minutes so that the final concentration of the glutaraldehyde is 0.05 %. The SRBC so pre-treated are washed twice with ice-cold distilled water, centrifuged off and then incubated for 1 hour at 20°C with 300 μΐ of the human MIF-containing fractions from Example 1. This suspension is used for immunisation. 2.2 Immunisation Balb/c mice are immunised on days 0, 7 and 10 by 3 injections each of 0.5 ml of the SRBC suspension coupled with human MIF from Example 2.1 together with 0.5 ml of complete Freund's adjuvant. In so doing, half of the injection quantity is injected intraperitoneally (i.p.) and the other half in four portions subcutaneously (s.c.). Two further (5abooster") injections each of 0.5 ml of conjugate suspension without adjuvant are administered i.p. on days 13 and 14. The spleen of the treated mice is removed on day 18. 2,. 3 Cell fusion In accordance with the method of Kohler and Milstein [G. Kohler and C. Milstein, Nature 256. 495 (1975)], 108 spleen lymphocytes are mixed with 3.3 x 107 mouse myeloma cells P3-X63-Ag8.653 [J.F. Kearney, A. Radbruch, B. Liesegang and Κ» Rajewsky, J. Immunol. 123, 1548 (1979)] in 1.5 ml of a solution of 35 % polyethylene glycol 4000 (Merck, Darmstadt) and 9.7 % dimethyl sulphoxide in Dulbecco's Modified Eagle Medium. After fusion, the cells are plated out in 600 wells of Falcon 3040 96-well plates and cultured in Littlefield's HAT medium (hypoxanthine/aminopterin/thymidine standard medium) [J.W. Littlefield, Science 145, 709 (1974)] together with bone marrow macrophages as feeder cells. After 10 days in HAT medium, culturing is continued in RPMI 1640 HT medium.

Example 3 Testing of the hybridoma cells for antibody . specificity 3.1 T-globulin detection The supernatants of the hybridoma cell cultures are tested in an ELISA (enzyme-linked immunosorbent assay) in which there is used a second antibody that is obtained from rabbits, is conjugated with peroxidase and that recognises and binds to mouse Ύ-globulin. Of 103 hybridoma cell lines 72 clones produce T-globulins. 3.2 Specificity for proteins in the desired molecular weight range Syngeneic mouse erythrocytes (1 χ 106 erythrocytes in 100 μΐ of PBS per hole) are applied to 96-well plates (Dynatech Microtiter) coated with poly-L-lysine (25 mg/ml) and incubated overnight at 4°C. Unbound erythrocytes are removed by repeated washing with PBS.

The erythrocytes so bound are, as described in Example 2.1, fixed in glutaraldehyde, washed and then incubated with the human MIF-containing fractions in the molecular weight range of from 8 to 14 kg/mole (Example 1).

Unbound protein is removed by repeated washing and the plates are stored in PBS containing 0.1 % ΝβΝβ. The plates are incubated with culture supernatant of the hybridoma cells and then with a rabbit-anti-mouse immunoglobulin second antibody coupled with alkaline phosphatase and are developed with p-nitrophenyl phosphate in 2-amino-2-ethyl-l,3-propanediol buffer (Serva). The p-nitrophenol released is detected photometrically at 405 nm. The described ELISA identifies monoclonal antibodies that bind to the erythrocyte-coupled proteins of the human MIF-containing molecular weight fractions from Example 1. Of the 72 cell clones that secrete T-globu30 lins, 15 clones produce monoclonal antibodies that are bound in this assay. 3.3 Specificity for human MIF ϊ-Globulins from the culture supernatants of the clones identified according to Example 3.2 are precipitated with ammonium sulphate at 50 % saturation, and the precipitates are taken up in PBS and coupled to Af f i-Gel ® 10 (Bio-Rad) as directed by the manufacturer. The τ-globulins so immobilised are incubated overnight at 4°C with human MIF-containing supernatants of mononuclear cells stimulated with concanavalin A (Example 1) and then centrifuged at 3000 rpm and 4°C for 10 minutes in an IEC centrifuge. The supernatant solution is then examined for its residual content of human MIF in the MIF test according to Example 4. In this manner, a cell line designated 1C5 is identified that produces monoclonal antibodies to human MIF.

Example 4 Test for human macrophage migration inhibition factor (MIF test) 4.1 Production of the Percoll gradient parts of Percoll1®’of density 1.007 (Pharmacia), 1 part of 10-fold concentrated Earl's MEM (Seromed) and 10 parts of Spinner medium (Seromed) are mixed for 12 minutes at 12 000 rpm and 20°C in a Sorvall centrifuge (Du Pont). 4.2 Obtaining target cell for the MIF test -Buffy coat·’, that is to say cell concentrate of white blood corpuscles, from 9-12 donors is diluted with Spinner medium (Seromed) in a ratio of 1:2, heated to 20°C and separated on Ficoll paque® (Pharmacia) at 20°C as directed by the manufacturer. The mononuclear cells of the interphase are washed twice with the same medium after centrifugation and then separated into monocytes and lymphocytes in a Percoll gradient, prepared according to Example 4.1, by centrifugation over a period of 40 minutes at 20°C and 1600 rpm in an IEC centrifuge.

The monocytes are washed three times with Spinner medium, taken up in McCoy's medium (Seromed) to which 20 % horse serum has been added and cultured overnight at 37°C while gassing with 7 % C02 in the same medium in Teflon bags. 4.3 Carrying out the MIF test The cultured monocytes from Example 4.2 are washed twice in Dulbecco's MEM (Seromed) and taken up in a mixture of one part double-concentrated Dulbecco's MEM and one part 0.4 % agarose (Miles) in such a manner that the cell con15 centration is 5 x 10® cells per ml. This cell suspension is pipetted in droplets of 1 μΐ into the inner 60 wells of a 96-well plate (Falcon, Microtest III) by means of a Hamilton syringe. After 15 minutes at 4°C the agarose has solidified. 100 μΐ of the sample solutions to be tested are then added to each well. As a control solution there is used Dulbecco's MEM to which 1 % horse serum has been added. Dilutions of the sample solutions to be tested are prepared in the same medium. The plates are incubated for 15 hours at 37°C in a humid atmosphere and while gassing with 7 % CO2.

The migration of the monocytes out of the agarose droplets is measured with the aid of a graduated reticule in the ocular of a microscope. The measurement is carried out by placing one axis of the reticule tangentially to the edge of the droplet and determining the distance from the edge of the droplet to the migration limit of the cells using the axis perpendicular thereto. The migra32 tion of the cells in the control solution, measured in graduations, is set as 100 % migration or 0 % migration inhibition. The migration distance achieved in the sample solutions is expressed with reference thereto as % inhibition of migration. The biological activity of a sample solution is expressed in MIF units. An MIF unit is defined as the biological activity that causes 30 % migration inhibition in the described test procedure.

The number of MIF units of a sample solution corresponds accordingly to the factor by which that solution must be diluted to cause exactly 30 % migration inhibition.

Example 5 Isolation and purification of the monoclonal antibodies from ascitic fluid Balb/c mice are pre-treated intraperitoneallv with 0.4 ml of pristane (Carl Roth). After one week, from 2 to 5 x 106 cloned hybridoma cells are injected intraperitoneally. Ascitic fluid is taken repeatedly from each mouse and frozen at ~80°c. The collected fluid is thawed and centrifuged for 30 minutes at 4°C and 16 000 rpm. The fat is filtered off with suction, and a saturated ammonium sulphate solution is slowly added dropwise to the remaining debris-free supernatant, while stirring at 0°C, until a concentration of 50 % is reached. The crude immunoglobulin fraction so precipitated is chromato25 graphed with 0.1M tris-HCl (pH 8.2) over DEAE Affi-Gel Blue © (Bio-Rad) as specified by the manufacturer.

Active fractions are combined and concentrated with Amicon XM50 filter (Amicon).

Example 6 Preparation of an antibody column Affi-Gel® 10 (Bio-Rad) is washed as specified by the manufacturer with cold distilled water and coupling buffer pH 7.5 (MOPS, 3-(N-morpholino)propanesulphonic acid). A 50 % suspension of the gel in coupling buffer (1 ml) is. transferred to a plastic tube, mixed with the same amount of purified antibody solution (20 mg of monoclonal antibodies 1C5) and rotated for 4 hours at room temperature. The gel is then washed with coupling buffer. In order to block the active sites that are still free, the gel is treated with 0.1 ml of IM et’nanolamine-HCl (pH 8.0) per ml of gel for two hours at room temperature, then washed with PBS containing 10 mmol of sodium azide per ml of gel and kept therein at 4°C. The degree of coupling is determined by measuring the extinction at 280 nm and is from 12 to 30 mg of monoclonal antibodies per ml of gel.

Example 7 Isolation and purification of human MIF proteins 7.1 Production of human MIF 1011 mononuclear cells are isolated from ’’buffy coat" and stimulated to produce lymphokines with concanavalin A (conA) according to the method described in Example 1.

The stimulated cells are cultured for 24 hours at 37°C and while gassing with 5 % CO2 in Nunc stacked tanks having a culturing area of 6000 cm2 in each case at a cell concentration of 5 x 106 cells in 0.25 ml of RPMI 1640 medium per cm2 of surface. The cell culture supernatant is then centrifuged for 30 minutes at 35 000 g and 4°C. There is then added to the clear supernatant phenylmethanesulphonyl fluoride, a serine-protease inhibitor, (final concentration: 50 /imol/litre) and sodium azide (final concentration: 0.05 %). 7.2 Concentration of the cell culture supernatant The cell culture supernatants from Example 7.1 are concentrated 15-fold by means of ultrafiltration over a YMS membrane (nominal separation limit; molecular weight kg/mole) in an Amicon stirring cell. In order to avoid losses by adsorption and counteract possible aggregation of the proteins, the ultrafiltration is carried out with the addition of Triton X-100® (alkylphenylpolyethylene glycol, Rohm & Haas); the final concentration of Triton X-100 ® is approximately 0.2 % (w/v). The concentrate is centrifuged at 35 000 g and filtered through a 0.25 gm filter (Millipore). 7.3 Immunoaffinity chromatography litre of the lymphokine concentrate from Example 7.2 is pumped at 4°C and at a flow rate of approximately 10 ml/hour through an antibody column (Example 6) containing 4 ml of gel having a degree of coupling of 12 mg of antibodies per ml of gel. Non-specifically bound proteins and accompanying substances are eluted from the column by washing with 100 ml of PBS/0.5M NaCl/0.2 % Triton X-100 ©/0.02 % sodium azide, pH 7,3, then 20 ml of PBS and finally 10 ml of 0.1M NaCl at a flow rate of 15 ml/hour. The specifically bound human MIF proteins are eluted with a solution of 0.1M glycine hydrochloride/0.IM NaCl, pH 2.6, the elution being monitored by automatic measurement of the absorption at 280 nm (Uvicord S, LKB Instruments). In order to avoid losses by adsorption, the eluate is collected in fractions (each of 3 ml) in polypropylene tubes each containing 100 μΐ of 3 % SDS solution. The proteincontaining fractions are combined and neutralised by the 30 addition of IM tris solution. 7.4 Electrodialysis,.,electrophoretic concentration The neutralised eluate from Example 7.3 is dialysed against 25 mmol of ammonium acetate/0.01 % SDS, pH 8.3, by means of an "ISCO Electrophoretic Concentrator Model 1750 (Isco. Inc.) and a Spectrapor <9membrane (Spectrum Medical industries) having a nominal separation limit of molecular weight 3.5 kg/mole, and simultaneously concentrated to a volume of 0.2 ml. The dialysed concentrate is concentrated to dryness by evaporation in a vacuum centrifuge (Speed Vac Concentrator, Savant Inc.) in order to remove the ammonium acetate. 7.5 Analvsis of the MIF proteins bv SDS-polyacrvlamide gel electrophoresis (SDS-PAGE) An aliquot (approximately 2 %) of the dialysed concentrate (Example 7.4) is subjected to electrophoresis over 15 % polyacrylamide slab gel according to the Laemmli method [U„K. Laemmli, Nature 227. 680-685 (1970)]. The protein bands are made visible by staining with Comassie brilliant blue (Fluka) and with the silver staining method according to C. R. Merril et al. [Anal. Biochem. 110. 201 (1981)]. According to this, the material eluted from the antibody column contains proteins of molecular weight approximately 8 kg/mole and approximately 14 kg/mole, and comparatively very small amounts of molecular weight approximately 28 kg/mole and approximately 45 kg/mole. 7.6 Characterisation of,_the_MIF.activity of .the _p.rptei.ns isolated by immunoaffinitv chromatography A low-loss preparative separation and isolation of the individual proteins of human MIF is advantageously effected in the presence of a detergent, for example SDS (see Examples 7.3, 7.4 and 7.7). The MIF activity of the proteins is destroyed by the interaction with SDS. The MIF activity is assigned to the individual molecular weight fractions with the aid of biosynthetically radiop actively -labelled lymphokines as follows: 10° human mononuclear cells are suspended in 5 ml of RPMI 1640 medium containing 3 pig of concanavalin A per ml and incubated for 2 hours at 37‘C while gassing with 5 % CO2 in a cell culture vessel having a surface of 25 cm'1 (Nuncolon®TC 25). The medium is then removed and the cells are cultured for 20 hours in leucine-free RPMI 1640 medium to which 10 μθί of L-[U-14C]leucine (Amersham) per ml are added. The radioactive cell culture supernatant is combined with 500 ml of unlabelled, human MIFcontaining cell culture supernatant (Example 7.1) and concentrated 15-fold as described in Example 7.2. Analogously to Example 7.3, the human MIF proteins are isolated by immunoaffinity chromatography using a column containing 0.5 ml of gel, the eluate being collected without the addition of SDS. After neutralisation with IM tris solution, the eluate is transferred by chromatography over a Sephadex ® G 25 column (Pharmacia) in 50 mmol of ammonium bicarbonate solution and lyophilised. The residue is dissolved in 0.5 ml of PBS and centrifuged at approximately 35 000 g. An aliquot of 0.1 ml is fractionated by means of HPLC over a Bio-Sil® TSK-125 column (7.5 x 300 mm, Bio-Rad). The fractions are characterised by measuring the radioactivity (as a measure of labelled proteins) and by determining the MIF activity (Example 4). The radioactivity and MIF activity are in the same fractions whose position corresponds to molecular weight regions of approximately 8 kg/mole and approximately 14 kg/mole. A further aliquot of the radioactive eluate is subjected to SDS-PAGE analogously to Example 7.5. The radioactive proteins are made visible by autoradiography. Strong bands appear in molecular weight regions of approximately 8 kg/mole and approximately 14 kg/mole, and weaker bands in the regions of approximately 28 kg/mole and approximately 45 kg/mole. 7.7 Preparative separation of the human MIF proteins bv SDS-PAGE and isolation by electroelution Material prepared according to Example 7.4, consisting of approximately 50 litres of cell culture supernatants is separated by SDS-PAGE in a discontinuous buffer system according to the Laemmli method [Nature 227. 680 (1970)] in a vertical slab gel electrophoresis system. The sample is dissolved in 300 μΐ of a buffer solution of composition 0.05M tris-HCl/3 % (w/v) SDS/0.02M dithiothreitol/10 % (v/v) glycerine, pH 6.8, and applied in a width of 1.5 cm to the 1.5 mm thick gel containing 15 % acrylamide. In order to prevent possible derivatisation of the proteins by free radicals and oxidants in the gel, sodium thioglycolate is added in a concentration of 0.1 mmol to the cathode buffer [see M. W. Hunkapiller et al. , Methods in Enzymology 91. 227 (1983)]. To make the proteins visible, the gel is placed in an ice-cold 0.25M KC1 solution for 5 minutes [see D.A. Hager and R. R. Burgess, Anal. Biochem. 109. 76 (1980)]. Visible bands in the molecular weight region of 8 kg/mole and 14 kg/mole are cut out and the proteins are eluted from the gel using a technique described by Bhown et al.

[Anal. Biochem. 103. 184 (1980)]. For this purpose, the proteins are eluted with 0.05M ammonium acetate/0.01 % SDS in an "ISCO Electrophoretic Concentrator88, Model 1750 (Isco Inc.) having a Spectropor(£)membrane (Spectrum Medical Industries, nominal separation limit 3.5 kg/mole) over a period of 8 hours at an output of 2 watts. The eluted proteins are found in the sampling cup in a volume of approximately 150 //1 and are free of buffer substances (glycine, tris). In order to remove the ammonium - 38 acetate, the electroeluates are concentrated to dryness by evaporation in a vacuum centrifuge (Speed Vac Concentrator, Savant Inc.).

The homogeneity of the eluted proteins and the yields are examined by subjecting approximately 5 % of the eluates in each case to analytic SDS-PAGE (Example 7.5), defined amounts of proteins of known molecular weights being subjected to electrophoresis parallel to the samples. SDS-PAGE shows a homogeneous protein band in the eluate 0 from the molecular weight region of 8 kg/mole and a homogeneous band in the eluate from the molecular weight region of 14 kg/mole. 7.8 Preparative separation and isolation of the human MIF proteins bv gel fileration-HPLC 2 litres of a concentrate of human MIF-containing culture supernatants from Example 7.2 are pumped at a flow rate of approximately 15 ml/hour through an antibody column (Example 6) containing 5 ml of gel having a degree of coupling of approximately 12 mg of 1C5 antibody per ml 2q of gel. The column is washed analogously to Example 7.3 and the specifically bound human MIF proteins are eluted with 0.1M glycine hydrochloride/ο.IM NaCl, pH 2.6, the eluate being collected in fractions in polypropylene tubes without the addition of SDS. The elution is monitored by measuring the absorption at 280 nm and by determining the MIF activity (Example 4).

The combined eluates from 4 batches (consisting of a total of 8 litres of culture supernatant concentrate) are dialysed against 0.005M ammonium acetate, pH 7.5, and 3Q simultaneously concentrated to a volume of 0.2 ml analogously to Example 7.4. The concentrate is introduced into an HPLC column (9.5 mm x 500 mm) equilibrated with 0.05M ammonium acetate, pH 7.5, containing Si 300 Polyol (ϊχ 0.003 mm (Serva, Heidelberg, West Germany) and eluted with 0.05M ammonium acetate, pH 7.5, at a pressure of 40 bar and with a through-flow speed of 0.3 ml/minute. The elution is monitored by measuring the absorption at 280 nm, by determining the MIF activity (Example 4) and by the enzyme-immunoassay of Example 10. The main portions in terms of protein content and MIF activity are found in fractions whose position corresponds to molecular weight regions of approximately 8 kg/mole and approximately 14 kg/mole; smaller portions are determined in fractions of molecular weight regions of approximately 28 kg/mole and approximately 45 kg/mole. In order to remove the ammonium acetate, the fractions containing human MIF proteins are repeatedly lyophilised.

Example 8 Amino acid sequence analysis 8·1 MIF protein of molecular weight 8 kg/mole The purified protein of molecular weight 8 kg/mole from Example 7.7 is sequenced with a 9SGas-phase Protein Sequencer Model 470'" (Applied Biosystems) according to the method described by M. W. Hunkapiller and L. E. Hood [Methods in Enzymology 91, 399 (1983)]. The rearrangement of the anilino-thiazolinone derivatives to phenylthiohydantoin- (PTH) -amino acids is effected by treatment with 25 % trifluoroacetic acid at 50°C. The PTH amino acids are analysed on a Zorbax CN® column (Du Pont, 200 x 4.6 mm) [see R, Knecht et al.. Anal. Biochem. 130. 65 (1983)]. The following well-defined N-terminal amino acid sequence is found: Met-Leu-Thr-Glu-Leu-Glu-Lys-Ala-Leu-Asn-Ser-Ile-Ile15 20 2 5 , Asp-Val-Tyr-His-Lys-Tyr-Ser-Leu-Ile-Lys-Gly-Asn-Pne-His3 0 3 5 6 0 Ala-Val-Tyr-Arg-Asp-Asp-Leu-Lys-Lys-Leu-Leu-Glu-Thr-Glu4 5 S θ 5 X42-Pro-Gln-Tyr-Ile-Arg-Lys-Lys-Gly-Ala-Asp-Val-Trp-Phe«Ο 6 5 Lys-Glu-Leu™Asp-Ile-Asn"X62"x63x64Ala_Val· X42 represents Ser or Cys. x62' x63 an^ x64 represent unspecified amino acids. 8.2 MIF protein of molecular weight 14 kg/mole The purified protein of molecular weight 14 kg/mole from Example 7.7 is sequenced analogously to Example 8.1. The following well-defined N-terminal amino acid sequence is found: Xl-Leu-Thr-Glu-Leu-Glu-Lys-Ala-Leu-Asn-Ser-Ile-Ile 1 5 Asp-Val-Tyr-His-Lyr-Tvr.

Xi represents an unspecified amino acid.

Example 9 Preparation of monoclonal antibodies to mouse MIF 9.1 Obtaining protein fractions containing mouse MIF In accordance with the process described by C. Sorg [Molecular Immunology 17 . 565 (1980)] there is obtained by stimulating spleen cells from 100 Balb/c mice with concanavalin A a cell supernatant which, after chromatography over Sephadex ® G 100, yields approximately 50 ml of a solution containing mouse MIF proteins of molecular weight range from 40 to 70 kg/mole. 9.2 Conjugation of mouse MIF-containing protein fractions to sheep erythrocytes The fractions from Example 9.1 are concentrated to a total volume of 4.5 ml and coupled analogously to Example 2.1 to sheep erythrocytes (SRBC) pre-treated with glutaraldehyde . 9.3 Immunisation of rats DA rats are immunised on days 0, 10, 43 and 309 by 7 injections each of 0.5 ml of the SRBC suspension coupled with mouse MIF from Example 9.2 together with 0.5 ml of complete Freund's adjuvant. In so doing, half of the injection quantity is injected intraperitoneally (i.p.) and the other half in four portions subcutaneously (s.c.). Further (-boosters?) injections each of 0.5 ml of conjugate suspension without adjuvant are administered i.p. on day 312, 313 and 314. The spleen of the treated rats is removed on day 317. 9.4 Cell fusion Analogously to Example 2.3, 1.14 x 108 spleen lymphocytes of immunised rats are fused with 3.5 x 107 mouse myeloma cells P3-X63-Ag8.653 and cultured in HAT medium. 9.5 Selection of hybridoma cells that secrete antibodies of desired specificity Analogously to the process described in Examples 3.1 and 3.2, there are selected from the total of 244 hybridoma cell lines obtained according to Example 9.4 49 clones that secrete monoclonal antibodies that bind to proteins from fractions containing mouse MIF coupled with rat erythrocytes (coupled analogously to Example 9.2).

I-Globulins from the culture supernatants of these 49 cell clones are coupled to Affi-Gel® 10 (Bio-Rad) according to the process of Example 3.3 and incubated with mouse MIF-containing supernatants of spleen cells stimulated with concanavalin A (Example 9.1). The supernatant solution is then examined for its residual content of mouse MIF. For this purpose, the content of mouse MIF is determined in an MIF test carried out analogously to Example 4.3, there being used, instead of the cultured human monocytes, however, peritoneal mouse macrophages which are taken up in Dulbecco's MEM containing 10 % FCS and fixed in agarose droplets.

Using this selection process, 6 hybridoma cell lines, inter alia the cell clone designated 7D10, that produce monoclonal antibodies to mouse MIF are identified.

These antibodies also bind human MIF in an MIF test according to Example 4 and, therefore, exhibit crossactivity . 9.6 Isolation and purification of the monoclonal antibodies from ascitic fluid Analogously to Example 5, Balb/c mice (nu/nu) pretreated with pristane are injected intraperitoneally with 1 χ 107 cloned hybridoma cells and the antibodies are obtained from ascitic fluid.

Example 10 Enzvme-immunoassav for the determination of human MIF (MIF-ELISA) 100 μΐ of a purified antibody solution 1C5 (anti-human MIF) of a concentration of 10 μ9/πι! are incubated for one hour at 37"C in each well of a 96-well microtitre plate (Costar? Technorama). The plates are washed three times with PGT-20~buffer (PBS to which 0.2 % gelatine (Merck) and 0.05 % Tween 20 have been added) and protein-reactive binding sites of the plate base that are still present 3 are saturated by incubation with 250 μΐ of PGT-20-buffer per well for one hour at 37°C. 50 μΐ of a dilution series of a test solution or a standard solution containing human MIF are incubated for one hour at 37"C in the wells of the microtitre plates. Unbound portions are washed away three times with PGT-20-buffer and then 50 μΐ in each case of a solution of a conjugate (10 μg/ml) prepared from antibody 7D10 (anti-mouse MIF, crossreactive with human MIF) and biotin N-hydroxysuccinimidyl ester (Medac, molar ratio of biotin to antibody 7D10 = 4-7 : 1) in PBS and purified over Sephadex ® G 25 are incubated in the wells for 30 minutes at 37°C. After washing the plate three times with PGT-20-buffer, the biotin-antibody conjugates bound to the plate are reacted by incubation (30 minutes at 37°C) with 100 μΐ of a l:3000-times diluted solution of an avidin-horseradishperoxidase conjugate (0.3 μg/ml) (Sigma). The amount of enzyme taken up is determined after washing the plate by development (30 minutes at 37°C) with a solution of 2,2fasino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS, Boehringer Mannheim, 55 mg in 100 ml of citrate/phosphate buffer, 0.05M citrate, 0.1M Na2HP04, pH 4.0, containing 16 μΐ of 30 % H2O2) and by a photometric measurement at 405 nm.

Instead of the biotin-antibody 7D10 conjugate, an analogously prepared biotin-antibody 1C5 conjugate can be used, but an ELISA carried out in such a manner has a lower sensitivity to human MIF.

Example 11 Test kit for MIF-ELISA A test kit for the enzyme-immunoassay described in Example 10 comprises: polypropylene microtitre plates 20 ml of solution of the monoclonal antibody 1C5 (10 Mg/ml) ml of solution of a biotin conjugate of the monoclonal antibody 7D10 (molar ratio of biotin to antibody 7D10 = 5 : 1, 10 Mg/ml) ml of solution of an avidin-horseradish peroxidase conjugate (0.3 Mg/ml) mg of 2,2-azino-bis(3-ethylbenzothiazoline-6~ sulphonic acid) diammonium salt ml of citrate/phosphate buffer (0.05M citrate/O.lM Na2HPO4) ml of 30 % H2O2 100 ml of PBS 200 ml of PGT-20-buffer (PBS containing 0.2 % gelatine and 0.05 % Tween 20).

Claims (38)

1. Patent claims

1. Purified human MIF, characterised in that it comprises only proteins of human origin that have epitopes that are recognised and bound by the monoclonal antibody 1C5 to human MIF, and is active in standard test procedures in which the migration of macrophages is measured, and its individual proteins.

2. Purified human MIF according to claim 1, characterised, in that it consists of at least four individual proteins of molecular weight approximately 8, approximately 14, approximately 28 and approximately 45 kg/mole.

3. A protein according to claim 1, characterised in that the N-terminal amino acid sequence is Xi-Leu-Thr-Glu-Leu10 15 Glu-Lys-Ala-Leu-Asn-Ser-Ile-Ile-Asp-Val-Tyr-His-Lys-Tyr in which the meaning of the amino acid Xj is not fixed.

4. A protein according to claim 1 or 3, characterised in that the N-terminal amino acid sequence is Met-Leu-Thr5 10 15 Glu-Leu-GlU“Lys-Ala-Leu-Asn-Ser~Ile-Ile-Asp--Val-Tyr-His~ 2 0 2 r 3 0 Lys-Tyr-Ser-Leu-Ile-Lys-Gly-Asn-Phe-His-Ala-Val-Tyr-Arg35 40 45 Asp-Asp“Leu™Lys-Lys-Leu-Leu-Glu-Thr“Glu-X42-Pro~Gln-Tyr50 55 Ile-Arg-Lys~Lys~Gly-Ala-Asp-Val-Trp“Phe~Lys-Glu~Leu-Asp60 65 Ile-Asn“Xg2“ x 63 x 64“' Ala “ Va l i n which the meaning of the amino acids X42/ Xg2/ x 63 an< ^ x 64 i s not fixed, but X42 may represent only Ser or Cys.

5. A protein according to claim 1, 3 or 4 having an approximate molecular weight of 8 kg/mole.

6. A protein according to claim 1 or 3 having an approximate molecular weight of 14 kg/mole.

7. A process for the manufacture of purified human MIF and its individual proteins, characterised in that a solution comprising human MIF, if desired after purifica5 tion steps that are known per se, a) is brought into contact with a carrier material having monoclonal antibodies 1C5 specific to human MIF, unbound proteins and other foreign substances are removed, the human MIF bound to the antibodies is -) q selectively split off and isolated and, b) if desired, the purified human MIF is separated into its individual proteins.

8. A process according to claim 7 in which the solution comprising human MIF is a cell culture supernatant of q 5 mononuclear cells that have been stimulated and cultured with concanavalin A..

9. A process according to claim 7 or 8 in which the solution comprising human MIF is subjected beforehand to ultrafiltration and concentration. 20

10. A process according to claim 7, 8 or 9 in which the human MIF purified in step a) is separated into its individual proteins by preparative sodium dodecyl sulphate polyacrylamide gel electrophoresis.

11. A process according to claim 7, 8 or 9 in which the 25 human MIF purified in step a) is separated into its individual proteins by preparative gel filtration-HPLC (high pressure liquid chromatography).

12. A monoclonal antibody to human macrophage migration - 47 inhibition factor (human MIF), or a derivative thereof.

13. A monoclonal antibody or a derivative thereof according to claim 12, characterised in that it is produced by mouse/mouse hybridoma cells.

14. A monoclonal antibody or a derivative thereof according to claim 12, characterised in that it is produced by rat/mouse hybridoma cells.

15. The monoclonal antibody 1C5 according to claim 12.

16. The monoclonal antibody 7D10 according to claim 12.

17. A process for the preparation of a monoclonal antibody or a derivative thereof according to claim 12, characterised in that hybridoma cells producing the monoclonal antibody a) are cultured in vitro and the monoclonal antibody is isolated from the culture supernatants, or b) are multiplied in vivo in a suitable mammal and the monoclonal antibody is isolated from the body fluids of that mammal, and, c) if desired, a resulting monoclonal antibody is converted into a derivative thereof.

18. A process according to claim 17, characterised in that hybridoma cells originating from Balb/c mice are injected into Balb/c mice that have optionally been pretreated with a hydrocarbon, after 8-10 days ascitic fluid is taken from these animals and the antibody is isolated therefrom by precipitation with ammonium sulphate and purification by chromatography.

19. A hybridoma cell line, characterised in that it produces a monoclonal antibody to human MIF.

20. A hybridoma cell line according to claim 19, characterised in that it is a hybrid of a mouse myeloma cell and a mouse lymphocyte.

21. A hybridoma cell line according to claim 19, characterised in that it is a hybrid of a mouse myeloma cell and a rat lymphocyte.

22. The hybridoma cell line 1C5 according to claim 19 which is deposited at the ’’Collection Rationale de Cultures de Microorganismes 8 of the Institut Pasteur in Paris under number 1-316.

23. The hybridoma cell line 7D10 according to claim 19 which is deposited at the ’’Collection Rationale de Cultures de Microorganismes” of the Institut Pasteur in Paris under number 1-418.

24. A process for the preparation of hybridoma cells according to claim 19, characterised in that a suitable mammal is immunised with MIF or an MIF conjugate, antibody-producing cells taken from the mammal are fused with myeloma cells, the resulting hybridoma cells are cloned and those cell clones which produce the desired monoclonal antibodies are selected.

25. A process according to claim 24, characterised in that antibody-producing cells of Balb/c mice are fused with myeloma cells of the cell lines X63-Ag8.653 or Sp2/0—Agl4.

26. A process according to claim 24, characterised in that antibody-producing cells of DA rats are fused with myeloma cells of the cell line X63-Ag8.653.

27. A process according to claim 24, characterised in that, for immunisation, a conjugate of a human MIFcontaining protein fraction with an immunogenic carrier is used.

28. A process according to claim 24, characterised in that, for immunisation, a conjugate of a mouse MIFcontaining protein fraction with an immunogenic carrier is used.

29. Use of a monoclonal antibody or a derivative according to claim 12 for the qualitative and quantitative determination of human MIF.

30. Use of a monoclonal antibody or a derivative in a radioimmunoassay according to claim 29.

31. Use of a monoclonal antibody or a derivative in an enzyme-immunoassav according to claim 29.

32. A test kit for the qualitative and quantitative determination of human MIF, characterised in that it comprises a monoclonal antibody and/or a derivative according to claim 12 and optionally adjuncts.

33. A test kit according to claim 32 for a radioimmunoassay.

34. A test kit according to claim 32 for an enzymeimmunoassay. 50

35. Use of a monoclonal antibody or a derivative according to claim 12 for the purification of human MIF.

36. A pharmaceutical preparation comprising a therapeutically effective amount of purified human MIF or its 5 individual proteins according to claim 1 and a significant amount of a pharmaceutical adjunct.

37. A pharmaceutical preparation comprising a therapeutically effective amount of a monoclonal antibody to human MIF or a derivative thereof according to claim 12 10. And a significant amount of a pharmaceutical adjunct.

38. A pharmaceutical preparation according to claim 36 in the form of liposomes consisting of a mixture of synthetic phosphatidylserine and phosphatidylcholine in aqueous dispersion.