DK171646B1 - Purified Human Macrophage Migration Inhibition Factor (Human MIF) and Its Single Proteins, Methods of Preparation thereof, Monoclonal Antibodies to Human MIF and Method of Preparation, Hybridoma Cell Lines Producing Monoclonal Antibodies to Human MIF, Their Use and Procedure for their Preparation pharmaceutical preparations containing purified human MIF or - 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

DK 171646 B1

The invention relates to purified human macrophage migration inhibitory factor (human MIF) and its single proteins, to a method of producing purified human MIF and to its single proteins, novel monoclonal antibodies to human MIF, to a method of producing these monoclonal antibodies , hybridoma cell lines producing these monoclonal antibodies, a method for producing the hybridoma cell lines, the use of the monoclonal antibodies for qualitative and quantitative determination of 10 human MIF, the use of the monoclonal antibodies for the purification of human MIF, and pharmaceutical preparations containing the human purifier -MIF, its single proteins or monoclonal antibodies against human MIF.

Human MIF from human cells is known as an active principle, but has so far been described only as a mixture and together with other proteins in biological fluids and is not characterized in terms of structure. MIFs belong to the group of so-called lymphokines which include biologically active, soluble polypeptides secreted by lymphocytes and monocytes or macrophages when stimulated by antigens, mitogens or the like. Other examples of lymphokines are immune interferon (γ-interferon), interleukin 1 and 2, and macrophage activating factor (MAF). These lymphokines regulate the differentiation, activation and proliferation of various cell types in the immune system.

According to the prior art, human MIF consists of a group of polypeptides which inhibit the motility of macrophages. Human MIF 30 is secreted not only by activated lymphocytes, T and B cells, but also by non-lymphoid cells, e.g. of growing fibroblasts and certain tumor cells. MIF is clearly different from γ-interferon, macrophage activating factor (MAF) and other lymphokines. However, to date, it has not been possible to purify MIF from human cells and to clarify its structure. It is known that human MIF is likely to consist of a mixture of structurally related polypeptides having a molecular weight of, respectively. ca. 8.5, 18, 27, 36, 45 and 67 kg / mol (kilo-daltons) and an isoelectric point at pH 5.1 and 2.9, cf. G. Burmeister, H. Steffen, U. Feige and C. Care, Immunobiology 160, 15 (1981).

5 Human MIF plays a crucial role in the early phase of an inflammatory reaction ("delayed type hypersensitivity reaction"). It induces the differentiation of monocytes and quiescent tissue macrophages into functional inflammatory macrophages. Purified human MIF and its single proteins and monoclonal antibodies, which specifically bind human MIF and inhibit its activity, are therefore important for the diagnosis and treatment of immunoregulatory and chronic inflammatory diseases. Monoclonal antibodies that bind and inhibit human MIF are valuable in fighting contact eczema, primary chronic polyarthritis and various autoimmune diseases. Purified human MIF and its single proteins may increase infection resistance e.g. against tuberculosis, leprosy or leishmaniasis and against can-didosis, as well as resistance to tumors, especially against metastases.

Until recently, the use of antibodies for diagnosis and treatment has been severely limited in scope. Antibodies have been extracted in small amounts from animal serum as a complex mixture of various proteins. It has not been possible to standardize the antibodies, as each individual immunized animal and also the individual at 25 repeated immunizations each time form a serum with antibodies of different composition. Using the technique developed by Kohier and Milstein, cf. G. Kohier and C. Milstein, Nature 256, 495 (1975), it has now become possible to extract antibodies in homogeneous form, i.e. so-called monoclonal antibodies in reproducible manner in industrial quantities from cell cultures. With fusion of suitable myeloma cells with antibody-producing lymphocytes from an antigen-immunized donor, hybridoma cells are formed, which combine the ability of unlimited cell division and unlimited growth in vitro with the production of a homogeneous antibody. Thus, it is possible to make an organism's immune response specific to a particular antigen and to produce antibodies by permanently culturing hybridoma cells.

Although many examples of the preparation of specific antibodies by hybridoma technique are known today and the general method is described in principle, however, with each new example there are specific problems which require the development of the technique for the particular case. Without such a development, it is not certain that the desired hybridoma cells are formed at all, that they are genetically stable, form the desired antibodies and that the antibodies thus produced have the desired specificity. The result is influenced in principle by the nature and purity of the antigen used for immunization of the donor, the cell fusion technique, the method of selection of suitable hybridoma cell lines, and the manner and nature of the isolation and purification of the antibodies.

An important use of antibodies that require the availability of homogeneous monoclonal antibodies in large quantities, which can now be prepared by the hybridoma technique, is immunoaffinity chromatography. In this technique, antibodies having a desired specificity are applied to a solid support material. From a solution containing several different compounds, those compounds are selectively bound to the antibodies and thus to the solid support containing a structural element (determinant, epitope) which is recognized and bound by the antibody. After removing the solution with the undesirable compounds, the desired compounds are released from the support material by leaching with reagents which break the binding to the antibody and the desired compounds are isolated by classical methods.

An object of the present invention is to provide human MIF and its single proteins. This is achieved by the monoclonal antibodies of the invention.

This invention relates to purified human macrophage migration inhibitory factor (human MIF) and its single proteins.

4 DK 171646 B1

The purified human MIF of the invention is characterized in that it contains only proteins of human origin which exhibit epitopes that are recognized and bound by the monoclonal antibody 1C5 to human MIF, and are active in standard test methods where the migration of macrophages is measured.

Purified human MIF consists of at least four single proteins with a molecular weight of respectively. ca. 8, ca. 14, ca. 28 and approx. 45 kg / mol 10 and optionally other single proteins or oligomeric protein aggregates having a molecular weight of more than ca. 45 kg / mol.

Single purified human MIF proteins are proteins that are homogeneous by conventional protein analysis methods, e.g. SDS-PAGE 15 (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) or gel filtration HPLC (high pressure liquid chromatography), which are active by standard test methods for measuring macrophage migration or movement, and which are constituents of purified human MIF.

If detergents or other denaturing reagents are added during the separation and isolation of the individual proteins, a change in the natural tertiary structure of the proteins and thus their ability to inhibit the movement of macrophages, even if the primary structure remains unchanged. Such denatured forms of single proteins are also encompassed by the invention. As examples of single purified human MIF proteins, the two molecular weight proteins are respectively. ca.

8 kg / mol and approx. 14 kg / mol and the N-terminal amino acid sequence X X-leu-thr-glu-leu-glu-lys-ala-leu-asn-ser-ile-ile-asp-vα-tyr-his-lys-tyr, wherein the importance of the amino acid is not determined, as well as proteins having a molecular weight of approx. 28 kg / mol and approx. 45 kg / mol.

First, the invention relates to the single protein of purified human MIF having an approximate molecular weight of 8 kg / mol and the 35 N-terminal amino acid sequence met-leu-thr-glu-leu-glu-lys-ala, 10 to 20 leu-asn -ser-ile-ile-asp-val-tyr-his-lys-tyr-ser-leu-ile-lys-25 35 gly-asn-phe-his-ala-val-tyr-arg-asp-asp leu-lys-lys-leu-leu-40, 45, 50 glu-thr-glu-X42-pro-gln-tyr-ile-arg-lys-lys-gly-ala-asp-55 60 6J5 trp-phe-lys-glu-leu-asp-ile-asn-X ^ -Xg 3-X64-aIa-val, where the meaning of amino acids X42, X62 # X63 X64 is added, but where X42 can only mean look or cys.

The method of the invention for the preparation of purified human MIF and its single proteins is characterized in that a human MIF-containing solution, optionally after known purification steps, a) is contacted with carrier material on human-MIF 10 specific monoclonal antibodies, unbound proteins and other foreign substances are removed and the human MIF bound to the antibodies is selectively cleaved and isolated and b) if desired, the purified human MIF is separated into its single proteins.

15

Solutions, e.g. cell extracts, cell culture supernatants or cell culture filtrates from human cells containing MIF are prepared using methods known per se. Suitable human cells are, for example, mononuclear cells that can be recovered from "buffy coat", which is the white blood cell layer that is secreted by centrifugation of citrate or heparin-added venous blood, by leukapheresis and / or centrifugation in a density gradient. The mononuclear cells are provoked by suitable adjuvants, e.g.

25 concanavalin A or phytohemagglutinin, for the production of MIF and other lymphokines and grown using conventional methods for from ca. 12 to approx. 72 hours, preferably from 18 to 36 hours, in suitable culture media, for example, the medium RPMI 1640, if desired, fetal calf serum, buffer solutions and / or antibiotics such as penicillin or streptomycin are added, at approx. 37 ° C and if desired under CC 2 aeration. Solutions containing human MIF are recovered from the cells and / or cell culture supernatants, e.g. by extraction, filtration and / or centrifugation and, if desired, stabilized by the addition of antibiotics and / or protease inhibitors. Such human MIF solutions can be directly contacted with antibodies bound to carrier material in step a), but they are preferably pre-purified by ultrafiltration with mem- branes having a separation limit at a molecular weight of approx.

6 kg / mol or less are concentrated and then optionally dialyzed and, if desired, further purified by chromatography, e.g. on DEAE cellulose or "Sephadex" ®.

In step a), human MIF is separated from other proteins and foreign substances present in the solution, the separation effect due to alternating binding between two monoclonal antibodies specifically utilized on human MIF and the recognized antigenic determinants on human MIF. To this end, the solution containing human MIF is contacted in a known manner by immunoaffinity chromatography with a carrier to which is bound human MIF-specific monoclonal antibodies.

A suitable carrier material on an inorganic or organic basis, e.g. 15 silicates, cross-linked agarose, dextran or polyacrylamide in a suitable functionalized form are coated in a manner known per se, possibly after prior activation, with the monoclonal antibodies of the invention described below or their derivatives. For example, one may suspend a carrier material containing activated after-functions, e.g.

N-hydroxy succinimide ester groups, in an aqueous buffer solution, to which is added a solution of the monoclonal antibody, leaching unbound monoclonal antibodies and blocking unoccupied reactive sites on the support material, e.g. a primary amine such as ethanolamine. The carrier material is suspended in a suitable aqueous solvent, e.g. a saline solution such as a NaCl solution, or a buffer solution such as phosphate-buffered NaCl solution, NaHCO3 solution or 3- (N-morpholino) propanesulfonic acid solution, and contacted with the 30 human MIF-containing solution, for example. placed in a chromatography column where the human MIF-containing solution is applied and pumped through the support material, optionally using pressure. Unbound proteins and other impurities are washed away with aqueous solutions, e.g. buffer solutions in the pH range from about 35 5 to approx. 9 and / or saline solutions, e.g. NaCl solution. The human MIF bound to the antibody on the carrier

B1 is eluted with suitable aqueous solutions, e.g. buffer solutions in the pH range from ca. 2 to approx. 5, such as glycine buffer, or pH gradients of different composition or saline solutions, e.g. The resulting 5 solutions containing purified human MIF are optionally neutralized, followed by isolation of the purified human MIF using methods known per se, e.g. by chromatography on "Sephadex" ®, electrodialysis, electrophoretic concentration and / or vacuum centrifugation.

10

Optionally, in step b), the purified human MIF is separated into single proteins, for example by chromatographically separating the protein mixture into fractions of different molecular weights in a manner known per se. For example, the purified human MIF is partitioned by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), after which homogeneous molecular weight fractions are eluted from the gel and isolated in pure form, e.g. by chromatography on "Sephadex" ®, electrophoretic concentration and / or vacuum centrifugation. The purified human MIF can also be separated or cleaved by preparative gel filtration HPLC in homogeneous molecular weight fractions from which the single proteins can be isolated.

Furthermore, as previously stated, the invention relates to novel monoclonal antibodies against human macrophage migration inhibition factor (human MIF).

The monoclonal antibodies of the invention bind and / or inhibit the biological activity of human MIF. The binding of 30 monoclonal antibodies to human MIF is advantageously determined by an immunoassay, e.g. with a test equipment in which human MIF is placed on a solid support, the coated carrier is incubated with a monoclonal antibody solution, whereby bound monoclonal antibodies are elicited with another antibody labeled with a radioactive isotope or enzyme. Monoclonal antibodies bound to human MIF are thus determined by measuring the radioactivity or by an enzyme substrate reaction. For example, one can also use a test equipment where the monoclonal antibodies are fixed on a carrier, incubated with a human-MIF-containing solution, and then the residual content of human-MIF activity in this solution is determined.

5

Human MIF activity in a solution is measured in a manner known per se by determining the inhibitory effect on the movement or migration of appropriately activated human macrophages. For example, an experimental set-up can be chosen where one measures the migration trajectory of macrophages placed in agar droplets on titre plates in a sample solution.

Monoclonal antibodies against human MIF produced by mouse / mouse, rat / rat or rat / mouse hybridoma cells are preferred. Examples of such preferred monoclonal antibodies of the invention may be mentioned the monoclonal antibody belonging to the subclass IgG1k designated 1C5 produced by the hybridoma cell line 1C5, and the monoclonal antibody belonging to the subclass IgG2a labeled 7D10ac line produced by . The monoclonal antibodies 1C5 and 7D10 bind to human MIF without inhibiting its biological activity.

Derivatives of monoclonal antibodies of the invention are, for example, fragments such as fab, fab 'or f (ab') 2 fragments which retain their specificity for the antigenic determinants of human MIF, radiolabelled monoclonal antibodies which for example, are labeled by radioactive iodine (I, 131 14 35 3 I), carbon (C), sulfur (S), tritium (JH) or the like, monoclonal antibody conjugates with biotin or avidin or monoclonal antibody conjugates with enzymes such as horseradish peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucoamylase, carbohydrate, acetylcholine esterase, lysozyme, malate dehydrogenase or glucose-6-phosphate dehydrogenase. Preferred derivatives are monoclonal antibodies labeled with iodine and antibody conjugates with biotin.

9 DK 171646 B1

The invention also relates, as mentioned, to a method known per se for producing the monoclonal antibodies against human MIF, and the method of the invention is characterized in that hybridoma cells which produced these monoclonal antibodies a) are grown in vitro, and the monoclonal antibodies are isolated from the culture supernatants or b) propagated in vivo in a suitable mammal, and the monoclonal antibodies are isolated from the mammalian fluid.

10

Suitable culture media for in vitro culture according to method a) are the standard standard culture media, for example Dulbecco's Modified Eagles medium or RPMI 1640 medium, optionally with total calf serum added. For isolation of the 15 monoclonal antibodies, the proteins are found in the culture supernatants with ammonium sulphate or the like, and purified using the usual chromatographic methods, e.g. gel filtration, ion exchange chromatography, DEAE cellulose chromatography or immunoaffinity chromatography.

20

Large amounts of the desired antibodies can be prepared by propagating the hybridoma cells in vivo according to method b). In this method, cell clones are injected into mammals, preferably into singular mammals, and after 1-3 weeks, the monoclonal antibodies from the body fluids of these mammals are isolated. For example, hybridoma cells derived from Balb / c mice can be injected intraperitoneally, optionally with a hydrocarbon such as pristane, pretreated Balb / c mice and after 8-10 days, ascite fluid is withdrawn from these animals. The desired monoclonal antibodies are isolated from the body fluids using known methods, e.g. by precipitation with ammonium sulphate or the like and chromatographic purification, e.g. over DEAE cellulose, hydroxylapatite (HPHT, high performance hydroxylapatite column chromatography), ion exchange resin, by gel filtration or by immunoaffinity chromatography.

10 DK 171646 B1

Fragments of monoclonal antibodies, e.g. fab, fab 'or f (ab1) 2 fragments which retain their specificity for the antigenic determinants of human MIF are prepared in a manner known per se, e.g. by treatment of monoclonal antibodies prepared according to process a) or b) with enzymes such as pepsin or papain, and / or by cleavage of disulfide bonds by chemical reduction.

125 131

Monoclonal antibodies labeled with radioactive iodine (I, I) 10 are obtained from the subject monoclonal antibodies by known iodine, for example, with radioactive sodium or potassium iodide and a chemical oxidant such as sodium hypochlorite, chloramine T or the like, or a enzymatic oxidizing agents such as lactoperoxidase, glucose oxidase and glucose. Radioactively labeled monoclonal antibodies of the invention are also prepared by adding, in known manner to the culture media for in vitro culture, radiolabelled nutrients containing radioactive carbon (C), tritium (3 H), sulfur (3 S) or the like. for example. L - (3C) -leu-3-cin, L- (H) -leucine or L- (S) -methionine, and isolates the monoclonal antibodies by method a).

Enzyme-labeled monoclonal antibodies of the invention are prepared in a manner known per se, by reacting monoclonal antibodies prepared according to process a) or b) and the desired enzyme with coupling reagent, e.g., glutaraldehyde, periodate, Ν, Ν'-o -phenylenedimaleimide, N- (m-maleimido-benzoyloxy) -succinimide, N- (3- (2'-pyridyldithio) -propionoxy) -succinimide or the like. Similarly, conjugates of monoclonal antibodies of the invention are obtained with avidine. Biotin conjugates are prepared using known methods, reacting monoclonal antibodies of the invention with e.g. Biotin-N-hydroxysuccinimi-dylester.

The hybridoma cell lines of the invention are peculiar in that they produce monoclonal antibodies against human MIF.

35 11 DK 171646 B1

Monoclonal antibody-producing hybridoma cell lines that are hybrids of mouse myeloma cell lines and mouse or rat lymphocytes are preferred.

5 In particular, the hybridoma cell line designated 1C5, deposited on July 13, 1984 in the "Collection Nationale de Cultures de Microorganismes" collection at the Pasteur Institute in Paris under number 1-316 is preferred. Cell line 1C5 is a hybrid of the mouse myeloma cell line P3-X63-Ag8.653 and a B-lymphocyte from the spleen of a balb / c mouse. Also preferred is the hybridoma cell line, designated 7D10, which was deposited on January 29, 1985 in the collection "Collection Nationale de Cultures de Microorganisms" in the Pasteur Institute in Paris under deposit number 1-418. The cell line 7D10 is a hybrid of the 15 mouse myeloma cell line P3-X63-Ag8.653 and a B lymphocyte from the spleen of a DA rat. The two cell lines are genetically stable, secrete monoclonal antibodies of constant specificity and can be activated from deep-frozen cultures by thawing and re-cloning.

20

The method of the invention for the preparation of hybridoma cells producing monoclonal antibodies against human MIF is characterized by immunizing suitable mammals with MIF or MIF conjugates, the antibody producing cells taken from the mammal fuse with myeloma cells, clones the hybridoma cells and select the cell clones that produce the desired monoclonal antibodies.

As antigens can be used any MIF-containing protein fraction or MIF conjugate, e.g. from human mononuclear cells such as MIF-containing protein fractions or conjugates of these protein fractions recovered as described above with suitable immunogenic carriers, e.g. proteins, polysaccharides, latex particles or cells. It is a prerequisite for the use of animals, e.g. murine, MIF-containing protein fractions that the animal MIF and human MIF in question have identical epitopes. Conjugates are prepared using methods known per se, e.g. by coupling with carbodiimides, periodate, glutaraldehyde, Ν, Ν'-o-phenylene dimaleimide, N- (m-maleimidobenzoyloxy) succinimide, N- (3- (2'-pyridyldithio) propionoxy) succinimide or the like. For immunization, conjugates of glutaraldehyde pretreated sheep erythrocytes and MIF-containing protein fractions are preferably used from human cells or from mouse cells.

The preferred mammals for immunization with human MIF are mice or rats, especially balb / c mice. For the immunization with 10 mouse MIF, rats, e.g. DA rats. The immunization is carried out in a manner known per se, e.g. administering three to eight injections parenterally, e.g. intraperitoneally and subcutaneously with antigenic human MIF conjugates at intervals of one to ten days, if desired, together with an adjuvant which promotes lymphocyte production, e.g. complete or incomplete Freund's Adjuvant. It is also possible to pre-immunize the animals by two to four injections and only after an 8 to 12 month break to make further injections.

Antibody-producing cells from the immunized animals, preferably spleen cells, are taken two to six days after the last immunization of the animals and the sampled cells are fused with myeloma cells by a suitable cell line in the presence of a fusion promoter. As distinct fusion partners, several different myeloma cell lines and derived cell lines are known. Myeloma cells which lack the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT) or the enzyme thymidine kinase (TK) are preferred and therefore do not survive in a selective culture medium containing hypoxanthine, aminopterin and thymidine (HAT). medium) or hypoxanthine and azaserin. Particularly preferred are myeloma cells and derived cell lines which do not survive in HAT medium or hypoxanthine / azaserin medium and do not secrete immunoglobulins or portions thereof, e.g. cell lines X63-Ag8.653 and Sp2 / 0-Agl4. The cell line 35 X63-Ag8.653, which is derived from balb / c mice, is not only suitable for the fusion with mouse lymphocytes, e.g. balb / c mice, but also with lymphocytes from rats, such as DA rats.

As fusion promoters may be used sendai virus or other paramyxoviruses, optionally in UV-inactivated form, calcium ions, surfactant lipids such as lysolecithin, elites piolyethyl anglycol. Preferably, myeloma cells are fused with a two to ten fold excess of spleen cells from immunized animals in a solution containing from 30 to approx. 50% polyethylene glycol having a molecular weight of between approx. 1000 and approx. 6000th

Following the fusion, the cells are then subdivided into selective HAT medium or hypoxanthine / azaserin medium, leaving only hybridoma cells because they combine the in vitro growth ability of the myeloma cells and the missing HGPRT or TK genes. from antibody producing cells of the immunized animals and thus the ability to survive in the selective medium.

Suitable culture media for the expansion of the hybridoma cells are the usual standard culture media, e.g. Dulbecco's Modified Eagles medium or RPMI 1640 medium. To initiate cell growth, so-called feeder cells, e.g. normal peritoneal mouse exudate cells, spleen cells, bone marrow macrophages and the like. At regular intervals, the respective culture medium is supplemented with selective HAT medium or hypoxanthine / azaserin medium to prevent the overgrowth of the hybridoma cells with usual myeloma cells.

The cell culture supernatants of the hybridoma cells are assayed for content of the desired monoclonal antibodies. For this purpose, preferably, a radioimmunoassay, an enzyme immunoassay and / or a determination of the MIF activity is used as described above. The cell clones thus selected are grown in the usual standard media and, if desired, deep-frozen in a manner known per se and / or reclaimed by limiting dilution and / or application to agar.

14 DK 171646 B1

The invention also relates, as mentioned, to the use of the monoclonal antibodies against human MIF for the qualitative and quantitative determination of human MIF, especially in biological fluids and on cell surfaces. The monoclonal antibodies of the invention may be used, for example, in any of the known immunoassays based on the binding interaction between antigen (human MIF) and monoclonal antibodies. Examples of such assays include radioimmunoassay (RIA), enzyme immunoassay, immunofluorescent assay, latex agglutination or hemagglutination.

The antibodies of the invention can be used as such or as radiolabelled derivatives optionally in combination with other labeled antibodies and / or proteins in a radioimmunoassay (RIA). Any of the known embodiments of RIA may be used, for example, RIA in homogeneous phase, RIA in solid or heterogeneous phase, single RIA or double (sandwich) RIA with direct or indirect (competitive) determination of human MIF.

20

Preferably, an RIA is used in which a suitable carrier is, for example, plastic surface of a titer plate or test tube, e.g. of polystyrene, polypropylene or polyvinyl chloride, glass or plastic beads, filter paper, dextran, cellulose acetate or nitrocellulose sheets or the like are coated with a human MIF-containing sample or standard solution by simple adsorption or optionally after activation of the carrier, e.g. with glutaraldehyde or bromocyan, then incubated with a solution of a monoclonal antibody of the invention and then with a solution of another antibody, the second antibody, e.g. a rabbit anti-mouse immunoglobulin, recognizes and binds the monoclonal antibody of the invention. The amount of the bound second antibody is determined by measuring the bound radioactivity either directly when the second antibody is radiolabeled, or upon development with a radiolabeled protein with high affinity for this second antibody, e.g. 125I-labeled protein A from DK 171646 B1

Staphylococcus aureus.

The monoclonal antibodies of the invention can be used as such or as enzyme labeled derivatives in an enzyme immunoassay. Such immunoassays are, for example, test devices utilizing known enzyme-labeled antibodies which recognize and bind the epitopes of the monoclonal antibodies of the invention, or enzyme-labeled monoclonal antibody derivatives of the invention. Instead of enzyme-labeled antibodies, antibody-biotin conjugates can also be used in conjunction with avidin-enzyme conjugates. Examples of enzymes that can be used in enzyme immunoassays include horseradish peroxidase, alkaline phosphatase, β-D-galactoside ase, glucose oxidase, glucoamylase, carbohydrate, acetylcholinesterase, lysocyme, malate dehydrogenase or glucose-6. phosphate that gives dehydrogenase.

An ELISA (enzyme-linked immunosorbent assay) test is preferred in which a carrier described above in conjunction with a single RIA test and optionally coated with erythrocytes is either coated with a human MIF. containing sample or standard solution by simple adsorption or, optionally, after activation of the carrier or carrier-bound erythrocytes with glutaraldehyde or coated with cells to be assayed for human MIF, which carrier is incubated with a solution of a monoclonal antibody of the invention and then with a solution of an enzyme-labeled second antibody that recognizes and binds the monoclonal antibody of the invention. The amount of the bound second antibody, e.g. a peroxidase-labeled rabbit anti-mouse immunoglobulin is visualized and determined by enzyme substrate development.

Particularly preferred is an ELISA (enzyme-linked immunosorbent assay) test, in which a carrier as described above is preferred for a single RIA test with a solution of a monoclonal antibody of the invention by simple adsorption or, optionally, after activation. of the carrier, e.g. with glutaraldehyde or bromocyan on which this carrier is incubated with a human MIF-containing sample or standard solution and then either with a solution of an enzyme-labeled antibody of the invention, which may recognize another epitope from human MIF, or preferably with a solution of a biotin-conjugated antibody of the invention followed by a solution of an avidin-enzyme conjugate. Thereby, the amount of bound enzyme or biotin-labeled antibody is visualized and determined by development with enzyme substrate.

Preferred enzymes in the enzyme immunoassays of the invention are horseradish peroxidase, which can be induced, for example, with the enzyme substrates 5-amino salicylic acid, o-phenylenediamine, 3,3'-dimethoxybenzidine, 3,3 ', 5,51-tetramethylbenzidine, 2, 2'-azino-bis- (3-ethylbenzthiazoline-6-sulfonic acid) or the like and hydrogen peroxide, and alkaline phosphatase which can, for example, release p-nitrophenol from the enzyme substrate p-nitrophenyl phosphate.

The use of the invention of monoclonal antibodies 20 to human MIF for qualitative and quantitative determination of human MIF also includes other known immunoassays, e.g., immunofluorescent assays using antibody or antigen conjugates with fluorescent, latex -agglutination with antibody or anti-gene coated latex particles or hemagglutination with antibody or antigen-coated red blood cells or the like.

The described immunoassays can be used to determine the amounts of human MIF present in biological fluids, especially in human blood, or on cell surfaces in fixed form, thus facilitating the diagnosis of diseases caused by immune regulation disorders. For example, at low infection resistance, an associated immune regulation disorder can be diagnosed when there is no human MIF or amounts below the blood in the blood. Finally, the determination of human MIF in pathological tissues, e.g. In melanomas, granulomas and hyperplasia, B1 may be a simple diagnosis, as MIF occurs only in certain tissue types and only in certain pathological conditions, cf. the table below.

Table 5: Binding of the monoclonal antibody 1C5 to frozen tissue sections.

Normal tissues: A) Liver 10 B) Spleen + (few cells) C) Skin + (few macrophages) D) Lung -

Pathological tissues: 15 e) Primary melanoma (very cell infiltrate) ++ (cell infiltrate)

Melanoma metastases (little cell infiltrate) + (few cells)

Melanoma micrometastases (in lymph nodes) 20 f) BCG granuloma (1, 3 or 5 weeks after grafting) + G) Sézary syndrome + H) Acid oligomphoid hyperplasia + I) Subacute eczema 25 J) Hypereosinophilic syndrome + Binding detected, - none binding detected

The test equipment of the invention for the qualitative and quantitative determination of human MIF is peculiar in that it contains monoclonal antibodies against human MIF and, optionally, aids.

Test equipment of the invention for a radioimmunoassay 35 contains, for example, a suitable carrier, optionally freeze-dried or concentrated solutions of an antibody of the invention, solutions of any radioactively labeled antibody and / or optionally solutions of radiolabeled protein A from Staphylococcus aureus, standard solutions of purified human MIF or single proteins thereof, buffer solutions, fixing solutions containing glutaraldehyde, detergents to prevent nonspecific adsorption and aggregate formation, pipettes, reaction vessels, adjustment curves and the like.

Test equipment of the invention for an enzyme immunoassay, for example, contains a suitable carrier, optionally freeze-dried or concentrated solutions of a monoclonal antibody of the invention, of an enzyme-labeled or biotin-labeled antibody derivative of the invention, optionally freeze-dried or concentrated solutions of an enzyme, recognizing and binding the monoclonal antibody of the invention, optionally freeze-dried or concentrated solutions of avidin-enzyme conjugates, solid or dissolved enzyme substrates, standard solutions of purified human MIF or of single proteins thereof, buffer solutions, fixed solution-20 are containing glutaraldehyde, detergents, pipettes, reaction vessels, adjustment curves and the like.

The invention also relates, as mentioned, to the use of the human MIF monoclonal antibodies for the purification of human MIF. For example, human MIF can be isolated using known separation methods, the separation effect of which is based on the alternate binding between monoclonal antibodies and antigenic determinants of human MIF. A preferred separation method is immunoaffinity chromatography as described above.

The pharmaceutical compositions of the invention are characterized in that they contain a therapeutically effective amount of purified human MIF or its single proteins of the invention and a significant amount of a pharmaceutical aid.

DK 171646 B1 19

ISLAND

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

The amount of therapeutically active compounds to be administered depends on the condition of the warm-blooded animal or human, e.g. the body weight, the nature and severity of the disease and the general condition, furthermore by the method of administration, and the dosage is made according to the instructions of the physician treating the disease. An effective dose of human MIF and its active single proteins is of the order of 0.001-1 pg per day. kg body weight per per day, and for human anti-human MIF and its derivatives in the range of 0.001-1 mg per day. kg body weight per day.

20

The pharmaceutical compositions of the invention contain the usual inorganic or organic, solid or liquid pharmaceutically usable carriers, optionally together with other therapeutically active compounds and / or excipients. Preferably, solutions or suspensions of the active substance, especially isotonic aqueous solutions or suspensions, are also employed, and also lyophilized preparations which dissolve in water shortly before use. The pharmaceutical compositions may be sterilized and / or contain preservatives, stabilizers, wetting agents, emulsifiers, solubilizers, viscosity enhancers, salts for regulating the osmotic pressure and / or buffers, as well as other proteins, e.g. human serum albumin or human blood plasma preparations.

35 20 DK 171646 B1

Pharmaceutical preparations in the form of liposomes in aqueous dispersion containing a therapeutically effective amount of purified human MIF or single proteins thereof are preferred. Particularly suitable are liposomes with a population of the greatest possible homogeneous size - 8 ~ c size and a diameter of ca. 2.0 x 10 to 5.0 x 10 ~ m consisting of one or more bilayer lipid components, e.g. amphipathic lipids such as phospholipids, e.g. lecithin, caphaline or phosphatidic acid, and optionally neutral lipids, e.g. cholesterol which encloses an aqueous internal compartment with purified human MIF of the invention or single proteins thereof. Liposomes are preferred of a mixture of synthetic phosphatidyl serine and phosphatidylcholine.

The invention is further illustrated in the following examples.

In the examples, the abbreviations used have the following meanings: ELISA Enzyme Assay (Enzyme-linked Immunosorbent Assay) -PAGE SDS-polyacrylamide gel electrophoresis SRBC Sheep erythrocytes

Tris Tris (hydroxymethyl) aminomethane rpm Rpm minute.

Example 1

Extraction of a protein fraction containing human MIF

Mononuclear cells are recovered from so-called "buffy coat", which is the white blood cell layer that is secreted by centrifugation of citrate or heparin-treated venous blood, and the recovered mononuclear cells are purified by a two-step method of leukapheresis and continuous centrifugation over a "Ficoll" ® gradient in an IBM blood cell separator (IBM 2997), cf. U. Feige and C. Sorg # J. Immunol. Methods _66, 161 (1984).

The mononuclear cells are washed in a spinner medium: (seromed) and then stimulated at a concentration of 5 x 10 6 cells in 2 0.17 ml RPMI 1640 medium per ml. 2 cm culture container area for two hours with 0.67 µg concanavalin A per day. 10 5 cells. The RPMI 1640 medium is replaced with a new portion and then incubated for 20 hours at 37 ° C with 5% CC 2 aeration to give a culture supernatant containing the lymphokines. The culture supernatant is centrifuged for 30 minutes at 17,000 rpm. per minute (SS34 rotor, Sorvall centrifuge) at 4 ° C. The cell-free supernatant is desalted over "Sephadex" ® G25, which is equilibrated in 0.05 M NH 2 HCO 3 and the proteinaceous fraction is lyophilized. The lyophilisate is taken up in 0.01M sodium phosphate buffer pH 7.5 with 0.1M NaCl and chromatographed in the same buffer over "Sephadex" ® G100. The human MIF containing fractions comprising proteins in the molecular weight range of 8-14 kg / mol 20 (kilo-Dalton) are pooled and concentrated by a factor of 16 by evaporation on a rotary evaporator.

Example 2

Preparation of the hybridoma cells 2.1 Conjugation of human MIF containing protein fractions with 25 sheep erythrocytes.

2.5 ml packed sheep red blood cells (SRBC, Behringwerke) are suspended in 20 ml PBS. 100 µl of this SRBC suspension is incubated with 900 μΐ glutaraldehyde solution in PBS for 5 minutes, so that the final concentration of glutaraldehyde 30 is 0.05%. The SRBC thus pretreated is washed twice with ice-cold distilled water, then centrifuged and incubated for one hour at 20 ° C with 300 μ 300 of the human MIF-containing fraction of Example 1. This suspension is used for the immunization.

2.2 Immunization

Balb / c mice are immunized by 3 injections of 0.5 ml each of the human-MIF-coupled SRBC suspension of Example 2.1 together with 0.5 ml of complete Freund's adjuvant on day 0, 7 days 10. Half of 5 the injection amount is injected intraperitoneally (ip) and the other half is injected subcutaneously (sc) divided into four portions. Two further boosters (0.5 ml each) of conjugate suspension without adjuvant are administered intraperitoneally on day 13 and day 14. On day 18, the spleen is taken from the treated 10 mice.

2.3 Cell Fusion In accordance with the method of Kohier and Milstein (G. Kohier and C. Milstein, Nature 256, 495 (1975)), 108 7 spleen lymphocytes are mixed with 3.3 x 10 6 mouse myeloma cells P3-X63-Ag8.653 (JF Kearney, A. Radbruch, B. Liesegang and K. Rajewsky, J.

Immunol. 123, 1548 (1979)) in 1.5 ml of 35% polyethylene glycol solution 4000 (Merck, Darmstadt) and 9.7% dimethyl sulfoxide in Dulbecco's Modified Eagle Medium. Following the fusion, cells are placed in 600 holes on falcon 3040 96-hole plates and cultured in Littlefield's HAT medium (hypoxanthine / aminopterin / thymidine standard medium) (JW Littlefield, Science 145, 709 (1974)) together with bone marrow macrophages such as Feeder. cells. After 10 days in HAT medium, culture is continued in RPMI 1640 HT medium.

Example 3 Testing of the hybridoma cells for antibody specificity.

3.1 γ-globulin detection

The supernatants from the hybridoma cell cultures are tested in an ELISA (enzyme-linked immunosorbent assay) using a rabbit second peroxidase conjugated antibody that recognizes and binds mouse γ-globulin. Out of 103 hybridoma cell lines, 72 clones produce γ-globulins.

23 DK 171646 B1 3.2 Specificity to proteins in the desired molecular guard region g

Singular mouse erythrocytes (1 x 10 erythrocytes in 100 µl PBS per hole) are placed on 96-hole plates (Dynatech Microtiter) coated with poly-L-lysine (25 mg / ml) / and incubated overnight at 4 ° C. . Unbound erythrocytes are removed by multiple washing with PBS. The erythrocytes thus bound fix ace. as described in Example 2.1 in glutaraldehyde is washed, then incubated with the human MIF-containing fractions in the molecular weight range of 8 to 14 kg / Mol (Example 1). Unbound protein is removed by washing several times and the plates are placed in PBS containing 0.1% NaN 2. The plates are incubated with the culture supernatant of the hybridoma cells and then with alkaline phosphate-coupled rabbit anti-mouse immunoglobulin second antibody and eluted with p-nitrophenyl phosphate in 2-amino-2-ethyl-1,3-propanediol. buffer (serva). The released p-nitrophenol is photometrically detected at 405 nm. Using the described ELISA, monoclonal antibodies that bind to the erythrocyte-coupled proteins in the human MIF-containing molecular weight fraction of Example 1. are recognized from Example 1. Of the 72 cell clones secreting γ-globulins, 15 clones produce monoclonal antibodies. which is bound by this analysis.

3.3 Specificity to human MIF

γ-Globulins from the culture supernatants of the clones identified in Example 3.2 are precipitated with ammonium sulfate at 50% saturation, the precipitates are taken up in PBS, then coupled to "Affi-Gel" ® 10 (Bio-Rad) using the manufacturer's instructions. The thus-immobilized γ-globulins are incubated overnight at 4 ° C with human MIF-containing supernatants from 30 with concanavalin A stimulated mononuclear cells (Example 1), then centrifuged at 3000 rpm. per minute and 4 ° C for 10 minutes in an IEC centrifuge. The supernatant is then tested for residual human MIF content using the MIF test of Example 4. In this way, a cell line labeled 1C5 which produces monoclonal antibodies against human MIF is identified.

Example 4

Human Roacrophage Migration Inhibition Factor Test (MIF Test) 4.1 Preparation of Percoll Gradent 5 9 Parts "Percoll" · with a density of 1.007 (pharmacia) 1 part 10 double concentrated MEM Earl's Medium (seromed) and 10 parts spinner medium ( seromed) is mixed for 12 minutes at 12,000 rpm. per minute in a sorvall centrifuge (DuPont) at 20 ° C.

4.2 Target Cell Extraction for the MIF Test 10 So-called "Buffy coat", a white blood cell concentrate, from 9-12 donors is diluted with 1: 2 spinner medium, the mixture is heated to 20 ° C and separated at Ficoll paque "® (pharmacia) at 20 ° C using the manufacturer's instructions. The interphase 15 mononuclear cells are washed twice after centrifugation with the same medium and then separated into monocytes and lymphocytes in a percoll gradient prepared according to Example 4.1 by centrifugation for 40 minutes at 20 ° C and 1600 rpm. per minute in an IEC centrifuge. The monocytes are washed three times with spinning medium, taken up in McCoy's medium (seromed) with 20% horse serum and grown overnight at 37 ° C and a CO 2 aeration of 7% in the same medium in Teflon bags.

4.3 Conducting the MIF test

The cultured monocytes of Example 4.2 are washed twice in MEM-dulbecco medium (seromed) and taken up in a mixture of part double-concentrated MEM-dulbecco medium and part 0.4% agarose 5 (miles), so that the cell concentration is 5x10 per. ml. This cell suspension is pipetted dropwise to 1 μΐ using a Hamilton syringe for the inner 60 holes 30 of a 96-hole plate (Falcon, Microtest III). After 15 minutes at 4 ° C, the agarose is solidified. Then add 100 μΐ of the sample solution to each hole. As a control solution, MEM-dulbecco medium is used as a: 1% horse serum added. Dilutions of the sample solutions are prepared in the same medium. The plate 5 is incubated for 15 hours at 37 ° C in a humid atmosphere at a CO 2 aeration of 7%.

The migration of the monocytes from the agarose drops is measured by a scale cross-section of the eyepiece in a microscope. The measurement is carried out such that one axis of the wire cross 10 is tangentially applied to the droplet edge and with the axis perpendicular to it then determines the distance from the droplet edge to the migration limit of the cells. The cells' migration in control solution, measured in scaled sections, is defined as 100% migration or 0% migration inhibition. According to this, the migration directions obtained in the sample solutions are expressed as percent inhibition of the migration. The biological activity of a sample solution is expressed in 1 MIF units. An MIF unit is defined as the biological activity which, in the test described, induces a migration inhibition of 30%. Accordingly, the number of MIFs in a sample solution 20 corresponds to the factor by which this solution must be diluted to achieve a migration inhibition of 30%.

Example 5

Isolation and purification of monoclonal antibodies from ascites.

Balb / c mice are pretreated intraperitoneally with 0.4 ml pristane 25 (Carl Roth). After a week, intraperitoneally 2 - 5 x 10 5 cloned hybridoma cells are injected. Ascite fluid is repeatedly withdrawn from each mouse and the fluids are frozen at -80 ° C.

The total liquid is thawed and centrifuged for 30 minutes at 4 ° C at 16,000 rpm. minute. The fat is suctioned off and 30 to the remaining debris-free supernatant is slowly and dropwise added with stirring at 0 ° C a saturated ammonium sulfate solution until the concentration is 50%. The crude immunoglobulin fraction thus precipitated is chromatographed at 0.1M tris-HCl (pH 8.2) over DEAE "Affi-Gel Blue" ® (Bio-Rad) using the manufacturer's specifications. Active fractions are pooled and concentrated on an amicon XM50 (amicon) filter.

Example 6

Preparation of an antibody column 5 "Affi-Gel" ® 10 (Bio-Rad) is washed using the manufacturer's prescription with cold distilled water and coupling buffer pH 7.5 (MOPS, 3- (N-morpholino) propanesulfonic 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 monoclonal antibody 1C5) and the tube is rotated for 4 hours at room temperature. The gel is then washed with coupling buffer. To block the still free active sites, the gel is treated with 0.1 ml of 1M ethanolamine-HCl (pH 8.0) per ml. ml of gel for 2 hours at room temperature, then washed with PBS containing 10 mM sodium acid per ml. ml of gel and storage in the same liquid at 4 ° C. The coupling rate is determined by measuring the extinction at 280 nm and amounts to 12 to 30 mg of monoclonal antibody per ml. ml of gel.

Example 7

20 Isolation and Purification of Human MIF Proteins 7.1 Production of Human MIF

1011 mononuclear cells are isolated using the procedure described in Example 1 from so-called "buffy coat" and stimulation is done with concanavalin A (conA) for 25 lymphokine production. The stimulated cells are grown in nunc container stacks with a culture area of 6,000. at a cell concentration of 5 x 10 6 cells in 0.25 ml medium RPMI 1640 2 o per cm surface at 37 C for 24 hours and with a CO 2 aeration of 5%. Then, the cell culture supernatant is centrifuged for 30 minutes at 30 ° C. To the clear supernatant is added phenylmethanesulfonyl fluoride, a serine protease inhibitor (final concentration: 50 μΜοΙ / l) and sodium azide (final concentration: 0.05%).

7.2 Concentration of the cell culture supernatant.

The cell culture supernatants of Example 7.1 are concentrated 5 by a factor of 15 by ultrafiltration in an amicon tube cell over a YMS membrane (nominal separation limit: molecular weight 5 kg / Mol). To avoid loss by adsorption and to counteract any aggregation of the proteins, ultrafiltration is performed with the addition of "Triton" ® X-100 (alkylphenyl-10 polyethylene glycol, Rohm & Haas), the final concentration of "Triton" ® X-100 being about . 0.2% (w / r). The concentrate is centrifuged at 35,000 G and filtered through a 0.25 µm filter (millipore).

7.3 Immunoaffinity Chromatography

15 l of the lymphokine concentrate of Example 7.2 is pumped at 4 ° C

with a flow rate of approx. 10 ml per per hour through an antibody column (Example 6) containing 4 ml of gel with a coupling rate of 12 mg antibody per hour. ml of gel. Non-specifically bound proteins and accompanying substances are eluted from the column by washing 20 with 100 ml PBS / 0.5 M NaCl / 0.2% Triton® X-100 / 0.02% sodium azide, pH 7.3, then with 20 ml PBS and finally with 10 ml of 0.1 M NaCl at a flow rate of 15 ml per ml. hour. The specifically bound human MIF proteins are eluted with a solution of 0.1 M glycine hydrochloride (0.1 M NaCl, pH 25 2.6, followed by elution by automatically measuring the absorbance at 280 nm (Uvicord S (LKB Instruments) To avoid adsorption losses, the eluate is fractionally captured (3 ml) in polypropylene tubes, each containing 100 μΐ 3% SDS solution.

The proteinaceous fractions are pooled and neutralized by the addition of 1 M tris solution.

7.4 Electrodialysis, electrophoretic concentration

The neutralized eluate of Example 7.3 is eluted on an "ISCO

28 DK 171646 B1

Electrophoretic Concentrator "model 1750 (Isco. Inc.) and a" Spectrapor "® membrane (Spectrum Medical Industries) with a nominal separation limit of molecular weight 3.5 kg / Mol against 25 mM ammonium acetate / 0.1% SDS, pH 8 , 3, and concurrently concentrated to a volume of 0.2 ml. The dialyzed concentrate is evaporated to dryness in a vacuum centrifuge (Speed Vac Concentrator, Savant Inc.) to remove the ammonium acetate.

7.5 Analysis of the MIF proteins by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

A portion (about 2%) of the dialyzed concentrate (Example 7.4) is subjected to an electrophoresis of 15% polyacrylamide plate using the method of Laemmli (U.K. Laemmli, Nature 227, 680-685 (1970)). The protein bands are made visible by staining with comassie brilliant blue (Fluka) and by 15 silver staining method according to C.R. Merril et al. (Anal. Biochem. 110, 201 (1981)). The material eluted from the antibody column then contains proteins having a molecular weight of approx. "8 kg / Mol and approx. 14 kg / mole, furthermore relatively small amounts having a molecular weight of approx. 28 kg / Mol and approx. 45 kg / mol.

7.6 Characterization of the MIF activity of the proteins isolated by immunoaffinity chromatography.

Advantageously, a loss of preparative separation and isolation of the single proteins in human MIF is carried out in the presence of a detergent, e.g. of SDS (see Examples 7.3, 7.4 and 7.7).

The MIF activity of the proteins is degraded by interaction with SDS. The MIF activity is added to the individual molecular weight fractions by biosynthetic radiolabeled lymphokines as follows: 10 human mononuclear cells are suspended in 5 ml of RPMI 1640 medium containing 3 µg concanavulin 30 A per ml. and incubate in a cell culture vessel with a surface of 25 cm ("Nuncolon" ®TC 25) for 2 hours at 37 ° C under a 5% CC aeration. Then, the medium is removed and the cells grown for 20 hours in leucine-free RPMI 1640 medium, 14 added with 10 µCi of L- [U-C] leucine (amersham) per ml. ml. The 29 DK 171646 B1 radioactive cell culture supernatant is mixed with 500 ml of unlabeled cell culture supernatant / containing human MIF (Example 7.1) and concentrated to a factor of 15 as described in Example 7.2. By analogy to the procedure described in Example 7.3, the human MIF proteins are isolated by immunoaffinity chromatography with a column containing 0.5 ml of gel, the eluate being captured without SDS addition. After neutralization with 1 M Tris solution, the eluate is chromatographed on a "Sephadex" G 25 column (pharmacia) in 50 mM ammonium hydrocarbonate solution and lyophilized. The residue is dissolved in 0.5 ml PBS and centrifuged at ca. 35,000 g. A 0.1 ml portion is fractionated by HPLC on a "Bio-Sil" ® TSK-125 column (7.5 x 300 mm, biorad). The fractions are characterized by measuring the radioactivity (as an expression of labeled 15). proteins) and in determining MIF activity (Example 4). The radioactivity and MIF activity are found in the same fractions whose position corresponds to the molecular weight range of about 8 kg / Mol and about 14 kg / Mol respectively. The radioactive eluate is subjected to SDS-PAGE by analogy to the procedure described in Example 7.5 The radioactive proteins are made visible by autoradiography, strong bands appear in the molecular weight ranges of about 8 kg / Mol and about 14 kg / Mol respectively. weaker bands in the ranges of about 28 kg / Mol and about 45 kg / Mol respectively.

7.7 Preparative separation of the human MIF proteins by SDS-25 PAGE and isolation by electroelution.

A material prepared according to Example 7.4 from approx. 50 L of cell culture supernatants are separated by SDS-PAGE in a discontinuous buffer system using the method of Laemmli (Nature 227, 680 (1970)) in a vertical slab gel electrophoresis system. The sample is dissolved in 300 μΐ of a buffer solution of the composition 0.05 M tris.HCl / 3% (v / r) SDS / 0.02 M dithiothreitol / 10% (r / r) glycerol, pH 6.8, and is applied to a 1.5 cm width of the 1.5 mm thick gel containing 15% acrylamide. In order to prevent any derivatization of the proteins by free radicals and oxidants in the gel, sodium thioglycolate is added to the cathode buffer at a concentration of 0.1 mM, cf. M.W. Hunkapiller et al.,

Methods in Enzymology 91, 227 (1983). To visualize the proteins, the gel is placed in an ice-cold 0.25 M KCL solution for 5 minutes, cf. D.A. Hager and R.R. Burgess, Anal. Biochem.

5, 109, 76 (1980). Visible bands are cut into molecular weight range, respectively. 8 kg / Mol and 14 kg / Mol, and the proteins are eluted from the gel using that of Bhown et al. described technique, cf. Anal. Biochem 103, 184 (1980). For this, the proteins are eluted in an "ISCO Electrophoretic Concentrator", model 1750 10 (Isco Inc.) with a "Spectropro" ® membrane (Spectrum Medical

Industries, nominal separation limit 3.5 kg / Mol) at a capacity of 2 watts with 0.05 M ammonium acetate / 0.01% SDS for 8 hours. The eluted proteins are found in the so-called "sampling cup" in a volume of approx. 150 μΐ and is free of buffer substances 15 (glycine, tris). To remove the ammonium acetate, the electroeluate is evaporated to dryness in a vacuum centrifuge (Speed Vac Concentrator, Savant Inc.).

The homogeneity of the eluted proteins and the yields is tested, with approx. 5% of the eluate is subjected to an analytical SDS-20 PAGE (Example 7.5) electrophoresis of defined amounts of proteins with known molecular weights, in parallel with the samples. SDS-PAGE shows in the eluate from the molecular weight range 8 kg / mol a homogeneous protein band and in the eluate from the molecular weight range 14 kg / mol a homogeneous band.

7.8 Preparative separation and isolation of the human MIF proteins by gel filtration HPLC.

2 liters of human MIF-containing culture supernatant from Example 7.2 are pumped at a flow rate of approx. 15 ml per per hour through an antibody column (Example 6) containing 5 ml of 30 gel with a coupling rate of approx. 12 mg of 1C5 antibody per ml of gel. The column is washed analogously to the procedure described in Example 7.3 and the specifically bound human MIF proteins are eluted with 0.1 M glycine hydrochloride / 0.1 M NaCl, pH 2.6, with the eluate being fractionally captured in polypropylene tubes without addition of SDS. The elution is followed by measuring the absorbance at 280 nm and determining the MIF activity (Example 4).

The mixed eluates from 4 mixtures (from a total of 8 liters of culture supernatant concentrate) are dialyzed analogously to the procedure described in Example 7.4 against 0.005 M ammonium acetate, pH 7.5 and simultaneously concentrated to a volume of 0.2 ml. The concentrate is placed on a 0.05 M ammonium acetate, pH 7.5, equilibrated HPLC column (9.5 mm x 500 mm) containing "Si 300 Polyol" ® 0.003 ram (Serva, Heidelberg, BRD) and eluted at a pressure of 40 bar and a flow rate of 0.03 ml per liter. minute with 0.05 M ammonium acetate, pH 7.5. The elution is followed by measuring the absorbance at 280 nm, by determining the MIF activity (Example 4) and by the enzyme immunoassay of Example 10. The majority of the protein content and MIF activity occur in fractions whose position corresponds to molecular weight ranges of respectively. ca.

8 kg / Mol and approx. 14 kg / mol. Smaller amounts are determined in fractions in the molecular weight range, respectively. ca. 28 kg / Mol and approx. 45 kg / mol. To remove the ammonium acetate, the lyophilization of the human MIF protein-containing fractions is repeated.

Example 8

Amino acid sequence analysis 8.1 MIF protein with molecular weight of 8 kg / Mol

The purified protein at a molecular weight of 8 kg / Mol from Example 7.7 is sequenced with a "Gas-phase Protein Sequencer Model 25 470" (Applied Biosystems) using the method of M.W. Hunkapiller and L.E. Hood, cf. Methods in Enzymology 9_1, 399 (1983). The conversion of the anilino-thiacolinone derivatives to phenylthiohydantoin (PTH) amino acids is accomplished by treatment with 25% trifluoroacetic acid at 50 ° C. The PTH amino acids are analyzed on a "Zorbax CN" ® column (Du Pont, 200 x 4.6 mm), cf. R. Knecht et al., Anal. Biochem. 130, 65 (1983). The following unambiguous N-terminal amino acid sequence is determined: 32 DK 171646 B1 5 10 15

Met-leu-thr-glu-leu-glu-lys-ala-leu-asn-ser-ile-ile-asp-val-20 30 tyr-his-lys-tyr-ser-leu-ile-lys-gly -asn-phe-his-ala-val-tyr- 40 45 arg-aspen-aspen-leu-lys-lys-leu-leu-glu-thr-glu-X ^ 2 ”Pro ~ 9ln“ tyr ”ile- arg-lys-lys-gfy-ala-asp-val-trp-jMe-lys-glu-leu-asp-ife- 5 asn-X.-o-Xc -.- X ^ .- afa-val.

62 63 64 X ^ 2 means ser or cys. X ^ xg3 ° 9 X64 and9Five indefinite amino acids.

8.2 MIF protein with a molecular weight of 14 kg / Mol

The purified protein having a molecular weight of 14 kg / Mol from Example 7.7 is sequenced analogously to the procedure described above in Example 8.1. The following unambiguous N-terminal amino acid sequence is determined: 5 X 15 -leu-thr-glu-leu-glu-lys-ala-leu-asn-ser-ile-ile-asp-val-tyr-his-lys-tyr 15 X 1 represents an indefinite amino acid.

Example 9

Preparation of mouse MIF monoclonal antibodies.

9.1 Extraction of protein fractions containing mouse MIF.

Using the method described by C. Sorg (Molecular Immunology 17, 20565 (1980)), by stimulating spleen cells from 100 balb / c mice with concanavalin A, a cell supernatant which, after chromatography on "Sephadex" ® G 100, was prepared. gives approx. 50 ml of a solution containing mouse MIF proteins in the molecular weight range of 40 to 70 kg / Mol.

9.2 Conjugation of mouse MIF containing protein fractions with sheep erythrocytes.

The fractions from Example 9.1 are evaporated to a total volume of 4.5 ml and coupled analogously to the procedure described in Example 2.1 with glutaraldehyde treated sheep erythrocyte (SRBC).

9.3 Immunization of rats.

DA rats are immunized by seven injections of 0.5 ml of the mouse-MIF-coupled SRBC suspension of Example 9.2 together with 0.5 ml of complete Freund's adjuvant on days 0, 10, 43 and; 309. Half of the injection is administered intraperitoneally (i.p) and the other half is administered subcutaneously in four portions (s.c). Further (booster) injections of 0.5 ml conjugate suspension without adjuvant are administered intraperitoneally on day 312, day 10 313 and day 314. On day 317, the spleen is taken from the treated rats.

9.4 Cell fusion.

Analogous to the procedure described in Example 2.3, 1.14 x 10 4 spleen lymphocytes from immunized rats are fused with 7 15 3.5 x 10 6 mouse myeloma cells P3-X63-Af8.653 and cultured in HAT medium.

9.5 Selection of hybridoma cells secreting antibodies of desired specificity.

Analogous to the procedure described in Examples 3.1 and 3.2, from the total 244 hybridoma cell lines recovered from Example 9.4, 49 clones are selected which secrete monoclonal antibodies that bind to rat erythrocytes proteins (coupling analogous Example 9.2) from mouse MIF-containing fractions . γ-Globulins from the culture supernatants of these 25 49 cell clones are coupled using the procedure of Example 3.3 on "Affi-Gel" ® 10 (bio-rad) and incubated with mouse MIF-containing supernatants from concanavalin A stimulated spleen cells (Example 9.1 ). The supernatant is then tested for residual content of mouse MIF. The content of mouse MIF is determined by an MIF test analogously to Example 4.3, however, instead of the cultured human monocytes, peritoneal mouse macrophages which are taken up in MEM-dulbecco medium are used in 34 DK 171646 B1 containing 10% FCS and fixed in agarase drops.

Using this selection method, 6 hybridoma cell lines are identified, including the cell clone designated 7D10, which produces monoclonal antibodies against the mouse MIF.

These antibodies also bind human MIF in an MIF test of Example 4, i. they exhibit cross-activity.

9.6 Isolation and purification of the monoclonal antibodies from ascites.

Analogously to the procedure described in Example 5, the prab-pretreated balb / c mice (nu / nu) are injected intraperitoneally with 1 x 10 5 cloned hybridoma cells and the antibodies are recovered from the ascites fluid.

Example 10

Enzyme immunoassay to determine human MIF (MIF-ELISA).

15 aliquots of 100 μΐ of a purified antibody solution 1C5 (anti-human MIF) at a concentration of 10 µg / ml are incubated in the holes of a 96-well microtiter plate (Costar, Technorama) for 1 hour at 37 ° C. The plates are washed three times with PGT-20 buffer (PBS added 0.2% gelatin (Merck) and 0.05% Tween® 20) and 20 saturated for still protein-reactive binding sites in the plate bottom by incubation with 250 μΐ PGT-20 buffer per hole for 1 hour at 37 ° C. Aliquots of 50 μΐ of dilution series of a sample solution and a standard solution containing human MIF are incubated in the microtiter plates for 1 hour at 37 ° C. Unbonded parts are washed away by washing three times with PGT-20 buffer, then incubating with 50 µl of a solution of one of antibody 7D10 (anti-mouse MIF, cross-reactive with human MIF) and biotin-N-hydroxysuccinimidyl ester (Medac , molt ratio of biotin to antibody 7D10 = 4-7: 1) in PBS prepared and 30 above "Sephadex" ® G 25 purified conjugate (10 µg / ml in the wells for 30 minutes at 37 ° C. After washing the plates three times with PGT-20 buffer, the biotin-bound antibody conjugates bound to the plates are reacted by incubation (30 minutes at 37 ° C) with 100 µl of a 1: 3000-fold diluted solution of an avidin horseradish peroxidase conjugate. (0.3 μΐ / ml) (sigma) The amount of enzyme taken up is determined after washing the plate by induction (30 minutes at 37 ° C) with a solution of 2,2'-aci-no-bis- 3-Ethylbenzothiacoline-6-sulfonic acid) diammonium salt (ABTS, Boehringer Mannheim, 55 mg in 100 ml citrate / phosphate buffer, 0.05 M citrate, 0.1 M Na 2 HPO 4, pH 4.0 containing 16 and a photometric measurement at 405 nm.

10 instead of the biotin antibody 7D10 conjugate, one analogously prepared biotin antibody 1C5 conjugate may also be used, but an ELISA conducted in this way shows a lower sensitivity to human MIF.

Example 11 Test equipment for MIF-ELISA.

A test kit for the enzyme immunoassay described in Example 10 contains the following: polypropylene microtiter plates 20 ml solution of the monoclonal antibody 1C5 (10 µg / ml) 20 10 ml solution of a biotin conjugate with the monoclonal antibody 7D10 (molar ratio biotin to antibody 7D10 = 5: 1, 10 µg / ml) 1 ml solution of an avidin horseradish peroxidase conjugate (0.3 µg / ml) 25 l mg 2,2-acino-bis- (3-ethylbenzothiazoline-6-sulfonic acid) - diammonium salt 20 ml citrate / phosphate buffer (0.05M citrate / 0.1M Na2HPO4)

1 ml 30% H 2 O 2 100 ml PBS

30 200 ml of PTG-20 buffer (PBS containing 0.2% gelatin and 0.05% Tween® 20).

Claims (22)

1. Purified human MIF, characterized in that it contains only proteins of human origin which exhibit epitopes that are recognized and bound by the monoclonal antibody 1C5 to human MIF, and are active in standard test methods where measurement is made of the migration of macrophages, and its single proteins. Purified human MIF according to claim 1, characterized in that it consists of at least four single proteins having a molecular weight of approx. 8, ca. 14, ca. 28 and approx. 45 kg / mol and optionally other single proteins or oligomeric protein aggregates having a molecular weight of more than approx. 45 kg / mol. Protein according to claim 1, characterized in that it has the N-terminal amino acid sequence X 1 -leu-thr-glu-leu-glu-lys-ala-leu-asn-ser-ile-ile-asp-val-tyr. -is-light-bull in which the significance of amino acid X1 is not determined. 20 4. Protein according to claim 1 or 3, characterized in that it has the N-terminal amino acid sequence met-leu-thr-glu-leu-glu-lys-ala-leu-α-sn-ser-ile-ile-asp-val- tyr-his-lys-tyr-s2er-leu-ile-lys-gly-a2sn-phe-his-ala-val-tyr-arg-asp-asp-40 45 45 leu-lys-lys-leu-leu-glu -tnr-glu-X ^ -pro-gln-tyr-ile-arg-lys-lys-g ^ Py-ala-asp-val-trp-p ^ e-lys-glu-leu-asp-iTe-asn -X62-X62 ~ Xg ^ -a ^ a-val, wherein the meaning of amino acids X ^ 2, X62 'X63 and Xg ^ is not determined, though X ^ 2 can only mean ser or cys. Protein according to claim 1, 3 or 4, characterized in that it has an approximate molecular weight of 8 kg / Mol. Protein according to claim 1 or 3, characterized in that it has an approximate molecular weight of 14 kg / Mol. 35 DK 171646 Bl Process for the preparation of purified human MIF and its single proteins, characterized in that a human MIF-containing solution, optionally after purification steps known per se, 5 a) is contacted with carrier material on human-MIF specific monoclonal antibodies, unbound proteins and other foreign substances are removed and the human MIF bound to the antibodies is selectively cleaved and isolated and b) if desired, the purified human MIF is separated into its single proteins. 8. Monoclonal antibodies against human macrophage migration inhibition factor (human MIF). Monoclonal antibody according to claim 8, characterized in that it is the monoclonal antibody 1C5. Monoclonal antibody according to claim 8, characterized in that it is the monoclonal antibody 7D10. 20 Method for producing monoclonal antibodies according to claim 8, characterized in that hybridoma cells producing these monoclonal antibodies are a) cultured in vitro and the monoclonal antibodies isolated from the culture supernatants or b) propagated in vivo in a suitable mammal , and the monoclonal antibodies are isolated from the body fluids of this mammal. Hybridoma cell lines, characterized in that they produce monoclonal antibodies against human MIF. Hybridoma cell lines according to claim 12, characterized in that they are hybrids of mouse myeloma cells and mouse lymphocytes. DK 171646 B1 Hybridoma cell lines according to claim 12, characterized in that they are hybrids of mouse myeloma cells and rat lyphocytes. Hybridoma cell line according to claim 12, characterized in that it is the hybridoma cell line 1C5, which is deposited in the "Collection Nationale de Cultures de Microorganisms" of the Pasteur Institute in Paris under number 1-316. The hybridoma cell line of claim 12, characterized in that it is the hybridoma cell line 7D10 deposited in the "Collection Nationale de Cultures de Microorganisms" of the Pasteur Institute in Paris under number 1-418. A method of producing hybridoma cells according to claim 12, characterized by immunizing suitable mammals with MIF or MIF conjugates, fusing antibody producing cells taken from the mammal with myeloma cells, cloning the hybridoma cells formed and selecting the cell clones producing the desired antibodies. Use of monoclonal antibodies according to claim 8 for qualitative and quantitative determination of human MIF. Test equipment for the qualitative and quantitative determination of human MIF, characterized in that it contains monoclonal antibodies according to claim 8 and, where appropriate, aids. Use of monoclonal antibodies and derivatives according to claim 8 for the purification of human MIF. Pharmaceutical compositions, characterized in that they contain a therapeutically effective amount of purified 35 human MIF or its single proteins according to claim 1 and a significant amount of pharmaceutical aid. DK 171646 B1 Pharmaceutical composition, characterized in that it contains a therapeutically effective amount of human MIF monoclonal antibody according to claim 8 and a significant amount of pharmaceutical aid. 5