JPH04504111A - Human macrophage migration inhibitory factor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Human macrophage migration inhibitory factor The present invention generally relates to novel protein factors important in the control of diverse inflammatory responses. It is. More specifically, the present invention provides novel human macrophage migration inhibitory factors. It is related to The present invention also provides for obtaining this factor in homogeneous form and translating it into a recombinant gene. Provides a method of production using genetic engineering technology.

Background of the invention In response to antigenic or mitogenic stimuli, lymphocytes act as immune modulators of cellular immunity. called lymphokines, which play important roles in inflammation and effector mechanisms. [S.

Cohen et al., Biology of the Lymphokines, New York, USA. Kademik Press, pp. 511-576 (19V9) and A, Harmful Birds, etc., rFA SEB Journal, 38:2462-2473 (1988)]. first reported Lymphokine activity was measured in cultures of antigen-sensitized and activated guinea pig lymphocytes. observed in the culture supernatant.

This activity inhibits the migration of guinea pig macrophages from capillaries in vitro. It was named migration inhibitory factor (MIF) because of its ability to inhibit migration [B, R, Bloom et al. Science, 153:8O-82 (1966) and J. R. David, “ Proceedings of the National Academy of Sciences 'S of the United Staves of America', 153 near 2-7 7 (1966) Ko.

Since this MIF activity was first observed, numerous publications have identified single putative MIF molecules. Separation and identification are reported. For example, p, s, Babageorjou et al. Null of Immunology, 108:494-504 (1972), R,E , Loughlin et al., Cellular Immunology, 5:435 (1972), T. Yoshihichi et al., Journal of Immunology, 117:548-554 (19 76), H,G.

Rimold et al., Journal of Immunology J, 118: 2015-20 19 (1977), L. H. Block et al., Journal of Experiments. Tal Medicine J, 147:541-553 (1978) Botzants, G. A et al., Science, 205+300-3001979), P, N., Ding et al. , Journal of Interferon Research, 1:23 (1981) , W, Y, Weiser et al., Journal of Immunology, 126:195 8-1962 (1981), S., Z., Saratubin et al., Science, 223 Near 03-707 (1984), W, Y, Weiser et al., “Cellular Immunology. 88:109-122 (1984), W. Y. Weiser et al. Immunology, 93:532-540 (1985), D, T.

Lamela et al., Journal of Immunology J, 140:4211-4216. (1988) and G., Tsubadoro et al., “Clinical and Experimental See Le Immunology J, 72:510-515 (1988).

However, other lymphokines exhibiting MIF activity, in particular e.g. Feron gamma and IL-4 have only recently been identified [G, B, Tu Leman et al., Journal of Immunology, 134:305-309 (1 985) and McInnes et al., “Journal of Experimental Medicine', 167:598-611 (1988). Other known lymphokines From these observations that ``rMIF'' activity also exists in ``rMIF,'' Considerable suspicion has arisen that there may be new substances.

Detection of MIF activity is associated with delayed hypersensitivity and cellular immunity [R.E., Loughlin et al. New England Journal of Medicine, 282:1340-13 43 (1970) and J. R. David et al., Progr., in Aller. G. Immunol, 16:300-449 (1972). [S, Alu Askari et al., Nature, 205:916-917 (1965 ) and J. T. Barrington, Cellular Immunology, 30:261-2. 71 (1977) and rheumatoid polyarthritis synovial s) [Odink et al., Nature, 330:8O-82 (1987)] Correlates with various inflammatory responses including

The industry remains at a high level of expertise regarding the production of pharmaceutical compositions useful in stimulating host defenses. There is a need for bioactive proteins that affect lophage function, such as MIF. .

Summary of the invention The present invention provides - in embodiments, a novel human protein that is substantially free of other associated mammalian proteins. Clophage migration inhibitory factor (MIF) is provided. This protein is a recombinant gene Manufactured using engineering technology. The bioactive MIF protein of this invention has about 115 Consists of amino acid sequence. Its amino acid sequence is shown later.

Active MIF is derived from MIF cDNA) transfection CO81 cells. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SD It has an apparent molecular weight of about 12 kd as measured using S-PAGE).

The MIF protein of this invention has shown biological activity in various assays; Demonstrates its role as a general activator of several different macrophage functions There is. The MIF of this invention is effective in human macrophage in assays using human peripheral blood cells. Prevent the migration of arches.

MIF exhibits greater than 20% inhibition of biological activity in this assay.

Additionally, MIF is a macrophage in culture measured by several different criteria. Helps stimulate activity. MIF-treated macrophages have higher than normal levels of IL. -1βHLA-DR mRNA is expressed.

In addition, MIF alone stimulates macrophages to attack intracellular pathogens, Interferon in this system also kills Shumania Donovani. - Enhance the effect of gamma.

Another aspect of the invention is a DNA sequence encoding the expression of human MIF protein.

The DNA sequence encoding active MIF is the same as the nucleotide sequence in Table 1 or characterized by containing substantially the same sequence or a fragment thereof. M of the present invention The IF DNA sequence was derived from the same MIF-cDNA transfected CO5-1 cell. Molecules of a novel protein band revealed by 5DS-PAGE of supernatant fluid derived from cells. It encodes a polypeptide of 115 amino acids, which corresponds to approximately the same amount.

When this protein band was removed from the gel and electroeluted, it showed strong MIF activity. did. RNA fraction extracted from ConA-stimulated and unstimulated human peripheral blood lymphocytes The analysis results show that this cDNA is a hybrid with single-mRNA derived from stimulated lymphocytes. cells, but not into unstimulated lymphocytes.

The present invention also provides D Vectors containing NA sequences are provided. The invention also provides for use in the production of recombinant MIF. A host cell transformed with the vector is provided.

Vectors and transformed cells of the invention may be used in another embodiment, namely recombinant human MIF Used in novel methods for producing proteins or peptide fragments thereof.

In this method, appropriate expression control sequences capable of controlling protein expression are put into a functional form. encodes the associated expression of a MIF protein or peptide fragment thereof. Claimed in claims featuring cell lines transformed by the DNA sequence This method can use several known cells as host cells for protein expression. current The currently preferred cell lines for the production of MIF are mammalian cell lines and bacterial cells. It is.

Another aspect of this invention provides recombinant MIF or one or more peptides thereof. A pharmaceutical composition comprising a therapeutically effective amount of a defragment is provided. These pharmaceutical compositions Treatment of disease states or wounds in which macrophage activation plays an important role It can be used in a method. For example, MIF protein or active fragment thereof is commonly used in cancer therapy, treating infections, accelerating wound healing and stimulating the immune system. can be used. MIF also stimulates the immune response to certain antigens, especially vaccines. Can be used for reinforcement.

Accordingly, yet another aspect of the invention provides that MIF or its These and and/or other pathological conditions. These treatment methods require only a few other cytokines, hematopoietin, interleukins, growth factors or tumor An effective amount of a specific antibody is administered simultaneously or with MIF or a peptide fragment thereof. may include subsequent administration.

In the following, other aspects and advantages of the invention will be described in detail. Further details are given in the description.

Detailed description of the invention The present invention provides bioactive human macrophage migration inhibitory factor (MIF) to substantially other is provided in a form without accompanying milk proteins. This protein is useful for therapeutic applications. It can be produced by recombinant techniques that allow for the production of pure, active MIF in large quantities.

The active human MIF of this invention is a protein with about 115 amino acids shown in Table 1 below. Features an array. MIF can be measured using 5DS-PAGE under reducing conditions. According to this, the DNA sequence of human MIF had an apparent molecular weight of about 12 kd. Ba G, G, Wong et al., Science, 228:810-815 (1985 ), Y.C., Young et al., Cell, 47:3-10 (1986) and A.E. Previously reported in Nature, 333:571-573 (1988). Phytohemagglutinin and PMA-stimulated Super human T cell hybridoma line, T-CEMB [W, Weiser, Briham and mRNA derived from Co., Ltd. and available upon request from Wimmins Hospital. It was cloned from a cDNA library prepared from A. this library - allows expression of cDNA inserts in mammalian cells, e.g. CO3-1 cells. was constructed in an expression vector. Library screening is performed using CDN Performed by transfecting CO3-1 cells with a pool of A clones. I was told. Express MIF activity by assaying supernatant fluid for MIF activity. A CDNA clone was identified.

mRNA from several cell sources was combined with selected MIF cDNA clones. Tested for hybridization ability. Northern plot analysis is T cell lines, CEM and T cells)\ibridoma lines, T-CEMB and Lectin-stimulated human peripheral blood lymphocytes (PBL) are hybridized with MIF clones. demonstrated that it synthesizes dizygous mRNA at easily detectable levels. . However, this message was It could not be detected in previous RNA samples. RNA translocation in activated human PBL The presence of the transcript indicates that the human MIF gene is expressed as a product of activated lymphocytes. It suggests.

The MIF cDNA sequence from this clone, shown in Table 1 below, is 115 amino acids Code the acid sequence (1 letter code).

70 90 1:L.

1:30 150 170 QQLAQATGK’PPQYIAVHVVPDQLMAFGGSSEPCAL CSLH5 Engineering GK Engineering GGAQNR5YSKLLCGLLAERLRISP DRVY engineering NYYDMNA370:190 410 The cDNA sequence in Table 1 begins with the ATG codon at nucleotides 51-53, 3 It contains a long open reading frame with 45 nucleotides.

ATG followed by 114 codons and TAA termination at nucleotides 396-398 Triplets follow. 345 nucleotides were observed by pulse-labeling experiments. 11 with a theoretical molecular weight of 12,540, which is consistent with the mass of the MIF-specific protein band. Encodes a 5 amino acid polypeptide.

MIF has been thought to be a secreted protein, but the DNA sequence internally contains the conventional secretory leader sequence [D, Pehlman et al., “Journal of ・Molecular Biology, 167:391-409 (1983)] and similar Contains no similar hydrophobic amino acid stretches. Also, the characteristics of protein signals and peptides The lack of highly hydrophobic sequences is also observed in MIF-related proteins, such as EK and Odei. Nuku et al., supra. The apparent absence of a leader sequence indicates that MIF secretion This suggests that the mechanism of secretion is different from that of typical secreted proteins. This MIF is so fragile that it cannot be secreted actively or effectively. Alternatively, the cells are MIF is exported by a mechanism similar to that of IL-1.

Additionally, the cDNA sequence of MIF consists of amino acids 73-75 (73-75 (Asn-A Two potentials at r and 110-112 (Asn-Asn-5et) encodes an asparagine-linked glycosylation site [e.g., R, J.

Wintzler, “The Chemistry of Glycoproteins in Homo "Nal Proteins and Pebutize", Volume 1, edited by C, H, Riichi, Akade. See Mitsuku Press, New York, p. 1 (1973). Betainterface Similar to Elon and IL-2, the MIF DNA sequence consists of amino acids 56.59 and It encodes three cysteine residues located at position 80. These three sisti The amino acid residues are responsible, at least in part, for the loss of MIF biological activity upon storage. can be obtained.

The nucleotide sequence of this MIF cDNA of the present invention was recorded in Diembank. compared with the nucleotide sequence. No significant similarities in nucleotide sequences were observed. It wasn't served. Notably, human MIF is a gamma interferon or IL-4, which has no sequence similarity to other cytokines with MIF activity. Not shared.

Furthermore, the MIF cDNA of this invention and the one reported by Odink et al. There is a sequence difference between the cDNAs encoding two proteins, MRP-8 and MRP-14. There is no similarity at all (i.e., the MIF of this invention It is immunologically different from proteins.

The cDNA sequences of the present invention can be used to detect functional polypeptides produced by mammalian cells. encodes biologically active human MIF. -Cl in plasmid p7-1 The sequence was published March 17, 1989 in Park Croft, Rockville, Maryland. American Type Culture Collection, 12301 Avenue Drive. Deposited under number 40582. This deposit is for the purpose of patent procedure. Necessary provisions of the Budapest Treaty on the Deposit of Goods and the provisions thereunder (Budapest Treaty) Satisfies the requirements.

Furthermore, the present invention also provides for DNA sequences that are free of concomitant DNA sequences encoding other primate proteins (, MI These novel DNA sequences encoding expression for the F polypeptide are included.

These DNA sequences are identical or substantially identical to the DNA and peptide sequences described above. Sequences containing the same sequence and stringent hybridization Conditions [T., Maniatis et al., “Molecular Cloning (A Laboratory Manual), Cold Spring Harbor Laboratory (1982) , pp. 387-389, below, hybridization with the DNA sequence of MIF reported above. Contains the array to be sized. An example of the above stringent hybridization conditions is , 65°C, hybridization in 4x SSC, followed by 0.05°C at 65°C for 1 h. Washing in 1X5SC. Alternatively, typical rigorous hybridization The reaction conditions are 50% formamide, 4XSSC at 42°C.

sequence of MIF or its active fragment under mild hybridization conditions. Regarding MIF peptides that hybridize with MIF and have MIF biological properties. The DNA sequence encoding the expression also encodes a novel MTF polypeptide.

Examples of those non-stringent hybridization conditions are 4X SSC at 50°C or Hybridization with 30-40% formamide at 42°C. example For example, regions with significant homology to the sequence of MIF, such as glycosylation sites or Proteins that share disulfide bonds and have one or more MIF biological properties DNA sequences encoding the Even without dication, it clearly encodes a MIF polypeptide.

Similarly, encoding a MIF polypeptide encoded by the sequence of MIF, DNA sequences that differ in codon sequence due to the degeneracy of the genetic code are also encompassed by this invention. It will be done. Allelic variations (which may or may not result in amino acid changes) natural base modification in some species populations), MIF proteins demonstrating MIF biological activity The DNA sequences encoding the sequences and peptide fragments thereof are also It is encompassed by the present invention together with derivatives or derivatives. point mutation or Modifications induced to increase the activity, half-life, or productivity of the polypeptide being encoded. Other variations in the DNA sequence of MIF induced by It will be done.

MIF polypeptides can also be produced by known conventional chemical synthesis. synthesis Methods of constructing polypeptides of the invention by means known to those skilled in the art. . Synthetically constructed MIF polypeptide sequences may have primary, secondary or tertiary structural and by sharing conformational features with MIF polypeptides, they may have common MIF biological properties. That is, they are used in therapeutic and immunological Biological activity or immunity against naturally purified MIF polypeptides in a biological process Can be used as a scientific substitute.

Modifications made to peptide or DNA sequences can be made by those skilled in the art using known methods. It can be performed by an expert. Interesting modifications in the MIF sequence include Substitutions, insertions or deletions of selected amino acid residues may be included. The above position Mutagenesis techniques involving substitutions, insertions or deletions are well known to those skilled in the art. It is. [See, e.g., U.S. Pat. No. 4,518,584.

Other specific mutations of the MIF polypeptide sequences described herein include one or more may involve further modification of glycosylation sites.

Absent glycosylation or only partial glycosylation The site of the molecule that is modified by the addition of a lycodylation recognition site or an 〇-linked carbohydrate. caused by amino acid substitutions or deletions in. Asparagine-linked glycosylation The recognition site is a tripeptide that is specifically recognized by the appropriate cellular glycosylation enzyme. Contains a code array.

These tripeptide sequences are asparagine-X-threonine or asparagine -X-serine, where X is usually any amino acid.

at one or both of the first or third amino acid position of the glycosylation recognition site. of various amino acid substitutions or deletions (and/or amino acid deletions in the second position). As a result, the modified tripeptide sequence becomes non-glycosylated. those modified Expression of the nucleotide sequence results in a variant that is not glycosylated at that site. produced.

MIF sequences predicted to retain MIF activity in whole or in part Other analogs and derivatives are also disclosed herein by one skilled in the art. It can be easily manufactured according to the method described above. One of the above modifications is the idiomatic Polyethylene glycol to existing glue residues in the MIF sequence by technique or the insertion of a residue into the sequence. The above modifications are encompassed by this invention. considered to be included.

The present invention also provides methods for producing MIF polypeptides. The method of the present invention of a MIF polypeptide or an active fragment thereof under the control of a known regulatory sequence. A suitable cell or cell line transformed with a DNA sequence encoding expression Including the culture of Suitable cells or cell lines include mammalian cells, e.g. can be hamster ovary cells (CHO) or 3T3 cells. Breasts are appropriate host cells and transformation, culture, amplification, screening and product production. The selection of purification methods is well known in the art. For example, Gething and Samplez Nature J, 293:620-625 (1981), or alternatively , Kaufman et al., Mo1. Ce1l.

Biol, 5(7):1750-1759 (1985) or Howley et al. See U.S. Pat. No. 4,419,446. Other suitable mammalian cell lines include monkeys These are the CO5-1 cell line and the CV-1 cell line.

Also useful as host cells suitable for the present invention are bacterial cells.

For example, various strains of Escherichia coli (e.g. HBIOI, MCl061 and Strains used in the Examples below) are used as host cell strains in the biotechnology field. It is well known as. Also, Bacillus subtilis, Pseudomonas, and other species Various strains of fungi can also be used in this method.

Additionally, many strains of yeast cells known to those skilled in the art can also be used with the polypeptides of the present invention. It can be used as a host cell for the expression of tido.

Furthermore, if desired, insect cells can also be utilized as host cells in the methods of the invention. Ru. For example, Miller et al., Genetic Engineering, 8:277- 298 (Plenum Press 1986) and references cited therein.

The present invention also provides vectors used in methods for expressing novel MIF polypeptides. do. These vectors contain novel MIF vectors encoding the MIF polypeptides of the invention. Contains FDNA sequences. Alternatively, vectors incorporating the modified sequences described above may also be used. , is an embodiment of the invention and is useful for producing MIF polypeptides. Also, this The vector used in the method operably carries the DNA coding sequence of the invention. a selected protein capable of directing its replication and expression in the selected host cell. Contains regulatory sequences.

That is, MIF produced by recombinant technology or synthetic methods is It may be used in pharmaceutical formulations to treat diseases that may involve diactivation. Also, above It is also possible to use active peptide fragments of MIF in pharmaceutical formulations. It is. One therapeutic use for pharmaceutical compositions containing MIF or fragments thereof is , is a cancer treatment. For example, activated macrophages can be used alone or with specific anti- When combined with tumor monoclonal antibodies, it exhibits significant tumoricidal potential. M The ability of IF to activate macrophages makes it a powerful anti-tumor agent for the treatment of cancer patients. Indicates its use alone or in combination with other therapeutic agents.

Similarly, MIF enhances macrophage-mediated killing of certain pathogens. ability to fight various infections by several pathogens, including, for example, Leishmania donovani. shows the use of this molecule in the treatment of diseases.

Furthermore, the ability of MIF to inhibit macrophage migration is an important consideration for wound treatment therapeutics. It can be used as an agent. When MIF protein is applied topically to a wound site, it becomes concentrated within the wound. The rate of wound healing is also increased by increasing the number of activated macrophages. obtain.

Additionally, MIF can be used as a general immune stimulant, particularly for specific vaccines. It can be used to increase immunity developed against. MIF is macrophage IL- 1β and HLA-DR expression indicates that this molecule This study shows that this method increases the antigen-presenting ability of T-cells. Therefore, M.I. F is useful in enhancing immune responses to various antigens. This characteristic of MIF is Demonstrates its usefulness as a general immunostimulant. For more information, see M.I.F. are useful in enhancing the immune response to certain vaccines.

This is particularly true in the case of human immunodeficiency virus, for example, where vaccine development is particularly questionable. This is very important when

Moreover, the MIF protein of this invention or its fragment can be used alone or in other sites. Tocainins, hematopoietin, interleukins, growth factors, interferos When used in combination with drugs or antibodies, it can be used to treat various infectious diseases, cancers, and and possibly treat tissue damage. In particular, MIF is IFN gamma, M-C8F or when administered with GM-CSF to enhance therapeutic benefit for the above conditions. It is expected.

Other uses for these novel polypeptides include marking them for diagnostic or therapeutic use. in the development of monoclonal and polyclonal antibodies produced by standard methods. do.

Accordingly, yet another aspect of the invention is a method and therapy for treating the conditions described above. It is a therapeutic composition. The composition comprises a therapeutically effective amount of MIF protein according to the invention or It contains a therapeutically effective fragment thereof in admixture with a pharmaceutically acceptable carrier. this The composition may be administered parenterally or systemically. Alternatively, the composition may be administered intravenously. obtain. If desired, the composition can be administered subcutaneously. For wound healing applications, this invention The MIF is formulated into a pharmaceutical formulation suitable for topical or topical administration.

For systemic administration, the therapeutic compositions used in this invention are pyrogen-free and parenteral. It takes the form of an aqueous solution that is acceptable to the public. The above-mentioned product with careful consideration of pH 1 isotonicity, stability, etc. The manufacture of pharmaceutically acceptable protein solutions is within the skill of the art.

The dosing regime associated with the treatment of the above conditions depends on various factors that modify the action of the drug, e.g. patient condition, weight, sex and diet, severity of injury or infection, time of administration. determined by the attending physician after considering other clinical factors. Generally - daily dose is 1-1000 micrograms of MIF protein per kilogram of body weight or 50 to 5000 units (i.e. 1 unit per l+1 is Half-maximal inhibition should be in the range of protein (which is the protein concentration).

The therapeutic methods and compositions of the invention may also include co-administration with other human factors.

Typical cytokinins or hematopoietins for the above uses include the known factor I L-1, IL-2, IL-3, IL-4, IL-6, G-C3F, C3F-1, GM-CSF, M-C8F1 interleukin or erythropoietin.

More specifically, MIF can be converted into IFN gamma or M-CSF or GM-CS. When combined with F, it is expected to have enhanced therapeutic activity for the treatment of the above conditions. Ru. The dosages described above are adjusted to compensate for the additional components in the therapeutic composition. . The progress of the treated patient can be monitored by conventional methods.

Hereinafter, the cloning, expression and production of human MIF and the present invention will be described in Examples. Other methods and products are detailed. These examples are for illustrative purposes only. However, it is not intended to limit the scope of the present invention.

Example 1 Isolation of mRNA and construction of cDNA library.

Significant MIF activity but barely detectable amounts of IFN-gamma methane T-CE, a human T-cell hybridoma line found to synthesize MB was chosen as the source for RNA extraction. This cell line is W, Y, W According to Zer et al., Cellular Immunology, 90:167-178 (1985). The HAT-sensitive T lymphoblastoid line CEMWH4 was transformed into a ConA-stimulated human terminal. Generated by fusion with peripheral blood T cells. Cells are RPM11640.10 % heat-inactivated fetal bovine serum (Fe2), 2 mmol Noshi glutamine and 50μ The cells are maintained in a medium consisting of g/il gentamicin.

Total RNA was stimulated with PHA (1%) and PMA (10ng/ml) for 18 hours. Charbwin et al., “Biochemistry”, 1 from stimulated T-CEMB cells. 8:5294-5299 (1979).

mRNA was purified by oligo(dT)-cellulose chromatography [H.

Aviv et al., “Proceedings of the National Academy of Sciences” Sciences of the United Stave of America, 69 :1408-1412 (1972)]. 5 microgram m Using RNA, the aforementioned wo The double-stranded cDNA reported by Ng et al. was synthesized and S! by nuclease digestion The hairpin loop was removed [T. Maniatis et al., supra]. Placing double-stranded DNA and a 5-fold excess of synthetic semi-Xho adapter [Young et al., Cell, 47 :3-10 (1986)]. Semi-xhO compatible cDNA Size fractionation was performed by agarose gel electrophoresis.

Regions of the gel containing cDNA larger than 500 base pairs were picked.

These semi-Xho compatible cDNA fragments were attached to glass powder [B, Vogelstein et al., “Proceedings of the National Academy of Sciences” Demy of Sciences of the United Stave of the United States of America 76:615-619 (1979)] and subsequent low salt buffers [5 ml Limole of Tris, 0.5 mmol of ethylenediaminetetraacetic acid (EDTA)] It was separated by elution using

The CO5-1 cell expression vector pXM [Y, C. Young et al., supra] was transformed into a unique Xho Linearized at the I site and ligated to equimolar amounts of semi-Xho-compatible cDNA. . Using a ligation reaction, a qualified Escherichia coli strain HB101[Y,C , Young et al. transformed the above-mentioned cocoon and obtained about 60,000 ampicillin-resistant colonies. Created a library.

Example 2 DNA production and C08-1 cell transfection.

The expression cloning system previously reported by G, G, Wong et al. cDNA encoding MIF activity was isolated using the method described below.

Bacterial colonies from the cDNA library above were filtered onto a nitrocellulose filter. -Reproduced above. Scrape colonies from each filter into L-broth and add Mid-DNA was isolated by the method described above [J. A. Meyers et al., "Journal. of Bacteriology, 127:1529-1536 (1976)]. 1 each A second DNA sample was prepared from a pool of 200-500 colonies.

Using 5 micrograms of each plasmid DNA, 0.1 mmol of chloroquine treatment with DEAE-dextran-mediated DNA transfection C08-1 cells were transfected [L, M, Sompairak et al. Rothesidinges of the National Academy of Sciences ``Of the United Stave of America'', 78 Near 575-7 578 (1981) and H. Rusman et al. Chi”, 11:1295-1308 (1983)]. transfected Culture medium fluid from CO3-1 cells was collected 72 hours after transfection. , MIF activity was assayed according to the assay method described in Example 7 below.

Plasmid DNA from the positive pool was retransfected into CO3-1 cells. and rescreen transfected supernatants for MIF activity. did. using cells from different blood donors to minimize the chance of false positives. Each sample was tested in small numbers (each 5 times) and then tested until individual clones were isolated. Each sample was further subdivided to contain fewer clones. primary pool Of the 100 supernatants from the first C08-1 cell transfection, two The sample showed the best overall MIF activity.

Select the pool with the highest MIF activity and further refine it to contain a smaller number of clones. The DNA of the sea urchins was prepared, transfected, and the MIF activity was determined. MIF the transfected supernatant until a single expressing clone is obtained. The activity was investigated.

The one clone that consistently showed the best MIF activity was retested in the MIF assay of Example 7. did. We also investigated the biological activity of this clone in guinea pig and mouse peritoneal macrophages. Tested by phage. Crude supernatant of transfected C08-1 cells A 5-fold dilution of 38% and 31% inhibition, respectively, was found. Also, this MIF activity from clones was compared with other cytokines Q (IL-2, TNF-α, T NF-β, IL-3 and IFN-gamma). The MIF supernatant was biopsied. showed greater MIF activity than all of these cytokines in the assay. but However, considerably stronger MIF activity was exhibited by TNF-α and TNF-gamma. It was.

Example 3 Protein analysis.

The polypeptide encoded by the p7-1 cDNA was isolated using a pulse-labeling experiment. Identified it. C08-1 cells transfected with p7-I DNA 5DS-PAGE of proteins secreted by the mock transfection control showed the presence of a 12kd polypeptide that is not present in . Polyacrylate this new belt Removed from the lylamide gel, 50 mmol of NH4HCOs and 200 Electroelution at 6 watts for 2 hours in elution buffer containing ng/ml human serum albumin. did. Addition of the latter prevented non-specific attachment. As a contrast, the same Remove the gel with the same molecular weight from the mock transfection supernatant and electrolyte Elution was performed. The eluate was reconstituted with medium and tested for MIF activity.

Strong MIF activity was found in the eluate. However, No activity was detected from the simulated gel. active MIF polypep When a band with 30 kd was removed from the same gel containing tide and subjected to electroelution, 30 Eluates from the kd band enhanced migration. This 30 kd protein was found in the crude supernatant at M It may be an inhibitor of MIF that inhibits and/or reduces IF activity.

Forty-eight hours after chloroquine treatment, recombinant DNA from MIF-positive clones was transfected. The crude supernatant from the transfected CO5-1 cells was removed and the cells were incubated at 37°C for 4 hours. Pulse standard with 0.5 mC1[35S]methionine in 5 ml DMEM for 0° time. I understood. The radiolabeled supernatant was collected and subjected to 15% 5DS-PAGE [U, , Laemmle et al., Nature, 227:680-685 (1970)]. electrophoresis After mobilization, the gel is washed with a fluorography-enhancing solution (enhance, two-knee England). Nuclear, Boston, MD), dried and exposed to X-ray film. did.

Example 4 RNA analysis.

PHA/PMA-stimulated or unstimulated T-CEMB cells, ConA-stimulated or unstimulated Twenty micrograms of total cellular RNA from stimulated human PBL or CEM cells was ．． Electrophoresed on a 1.2% agarose gel containing 2 molar formaldehyde. [H., Roerach et al., Biochemistry, 16:4743 (1977 )]. E. M. Southern, Journal of Molecular Biology J. , 98:503-517 (1975). NA with a nylon filter (Zetabind, Cuno, Meridan, Connecticut) It was moved to

cD by inserting cDNA DNA from the vector with XhoI restriction enzyme. Create a NA probe and incubate it in the presence of a large fragment of DNA polymerase I. Target the insert with 31p using a random oligonucleotide as a primer. [A., P., Finebar, Gu et al., “Analytical Biochemistry] J, 132:6-13 (1983)]. Nylon filter at 43℃ for 4 hours Prehybridize and incubate at 43°C for 16 hours in 5x SSC, 0.5% SDS. .

Hive consisting of 5x Denhardt's solution and 100 μg/ml denatured salmon semen DNA Hybridization with azp-labeled cDNA probe in redization solution I turned on.

After hybridization, wash the filter for 30 min at room temperature with post-wash solution (10 mmol sodium phosphate, 0.1% SDS, 1 mmol EDTA and 1xS SC) twice and for 15 min at 68°C with wash solution II (10 mmol sodium phosphate). Thorium, 0.1% SDS, 1 mmol EDTA and 0.2×SSC ), dried and applied to X-ray film.

This Northern blot analysis analyzes T cell lines, OEM and T cell hybrids. human line, T-CEMB and lectin stimulated human PBL with MIF clone. to readily synthesize detectable levels of hybridizing mRNA. Indicated. However, despite long exposure to film, Metsenger It could not be detected in RNA samples obtained from unstimulated PBL. activated human The presence of RNA transcripts in PBL indicates that the human MIF gene is expressed and and suggest that MIF is a product of activated lymphocytes.

Example 5 DNA sequence analysis.

The nucleotide sequence of the p7-1 cDNA clone is G, G, Wong and y, c, by Ba131 nuclease digestion as described by Young et al. (supra). Generate an ordered set of partially overlapping fragments and subtract them into the M13 vector. [M., Pontz et al., “Proceedinges et al. of the national academy of sciences of the united Ted Stave of America, 79:4298-4302 (1982) and J. Messing et al., Gene, 19:269-276 (1982)] . A single chain DNA was produced and a dideoxynucleotide chain termination method [F, Sanger et al. , ``Proceedings of the National Academy of Sciences Seeds of the United Stave of America, 74:546 The nucleotide sequence was determined according to No. 3-5467 (1977) 1.

Example 6 Generation of mutant cDNA fragments.

Despite the lack of a clear signal peptide in the coding region, p7-1 Comparison of 12kd protein with MIF activity from transfected CoS cells Although the molecularly cloned protein is not MIF, as it is effectively secreted by CO The possibility arose that it was an inducer of endogenous MIF expression by 8 cells. This is possible To test the potential, two insertional mutants of the coding region of the p7-1 cDNA was constructed as follows.

The MIF cDNA clone, p7-1, contains a single PstI site but close to the insert. Due to the PstI site in the adjacent adapter, this site is isolated from the MIF cDNA. This made it unsuitable for mutational analysis. PstI by T4 DNA polymerase The p7-1 insert isolated after partial digestion of the plasmid with By ligating an EcoRI adapter to the flash end of the Two adjacent Pst1 sites were removed. This matched fragment was designated as Pst The only EcoRI site in a derivative of pXM, named pXMT4, which does not have one site. subcloned into position. Clone with cDNA in correct orientation, p7-1- 24 were selected for mutational analysis.

Two insertional mutants of MIF were constructed. The only Pst1 of p7-1-24 14-base oligodeoxynucleotide at the site, 5' TGTAATTACATG By inserting CA 3', the first mutant (p7-1-24B2) was generated. accomplished. This sequence indicates that the MIF coding region has a stop codon ( TAA).

Insert a 99 base oligodeoxynucleotide into the PstI site of p7-1-24. A second insertional mutant (p7-1-24232) was constructed by. child When inserted in either direction into the PstI site of p7-1-24, the sequence , was designed to add 33 amino acids to the MIF coding region.

Clones containing 14 base oligonucleotides or 99 base oligonucleotides used $2p-labeled 14-base oligomer or 99-base oligomer as a probe. It was identified by hybridization using Each of these plasmids secretion of the 12 kd protein and C Testing for ability to induce MIF activity when transfected into O8 cells did.

C transfected with the truncated form of MIF (p7-1-24B2) Neither 12kd protein nor MIF activity was detected in the supernatant obtained from O8 cells. It was. Additionally, a mutant with an extended coding region (p7-1-24232) Transfection of COS cells with detectable levels of MIF activity Or the 12kd protein was not obtained. However, CO8 cell supernatants Predicted size of the extended coding region of the incoming mutant p7-1-24232 may include a new species with an apparent molecular weight of about 15,500, consistent with discovered.

These experiments were carried out using p7-1 derived from p7-1-transfected CO8 cells. A group of 2 kd proteins is encoded directly by the cDNA insert of the p7-1 plasmid. and that this protein has MIF activity.

Example 7 MIF biological activity.

A. MIF test Methods previously described [W, Y., Weiser et al., supra, and J. T., Barrington et al. According to "Journal of Immunology J, 110 Near 52 (1973)" , human peripheral blood monomers as indicator cells in an agarose droplet assay system. MIF assay was performed using nuclear leukocytes.

Human peripheral blood mononuclear leukocytes obtained by Fico-High Bark centrifugation were collected using buffered salts. Wash twice with solution (HBSS) and 0.2% agarose in minimal essential medium (MEM). mixed with. Place 1 microliter of cells in the agarose mixture in 50 μm replicates. microtie using a dispenser (Hamilton Company, Reno, NV). distributed to the center of the tar well. The sample to be tested was 100 μm per well. were added to the wells in proportions. The size of each agarose droplet was measured. Leave overnight at 37℃ After incubation, the entire migration area including the original agarose droplet was measured again.

Migration area = (diameter of total area/diameter of agarose droplet) 2-1 was calculated. The percentage of inhibition (1%) for each sample was derived as follows. I %=100-(average migration of test sample/average migration of control sample) x 100°20% or greater inhibition was considered significant [W, Y, Weiser et al. Previously mentioned.

B, MIF promotion of macrophage IL-1β and HLA-DR gene expression.

Mononuclear leukocyte-induced macrophages obtained by 7-day culture of isolated mononuclear leukocytes for 6 or 24 hours after recombinant MIF of the invention or mock transfection. Incubation with supernatant from cells. Total RNA obtained from these cells Cherbwin et al., Biochemistry, 18:5294 (1974) 1.2% agarose containing 2.2 mol formaldehyde according to the method of ・Size fractionation was performed by gel electrophoresis. RNA to nylon filter Moved. IL-1-beta and HLA-DR cDNA inserts were inserted into their respective vectors. cleavage, isolation, and random brining [Finebarb et al. 'Naltical Biochemistry', 132:6 (1983) 3! P and hybridized with the filter.

Mock supernatant (rMIF supernatant) induced IL-1-beta RNA expression.

The rMIF of this invention induces a rapid and long-lasting increase in HLA-DR mRNA. However, macrophages incubated with mock supernatant showed HLA-DR It did not show any increase in the level of NA.

Activation of human macrophages on C0 hydrogen peroxide release.

Hydrogen peroxide release ability suggests that reactive oxygen intermediates are deeply involved in their antibacterial function. This is a closely biochemically correlated phenomenon of macrophage activation [e.g. -san, Trans, R, Soc, Trop, Med, Hyg,, 77 :620 (1983) reference. Macrophage activity of rMIF regarding hydrogen peroxide Sexualization ability was measured as follows.

Human mononuclear leukocyte-induced macrophages were prepared using rMIF diluted 2 to 1000 times according to the present invention. Incubation was for approximately 48 hours in medium containing supernatant or mock supernatant. PMA The release of hydrogen peroxide from these cells induced by horseradish peroxide Measured by oxidation of scopoletin in the presence of sidase.

Incubation with rMIF supernatant diluted 2, 20 and 50 times (not with mock supernatant) Injected macrophages showed 2-3.6 times higher release of hydrogen peroxide.

D, Leishmania donoveri lethal MIF activation by macrophages.

It is known that the rMIF of this invention is sensitive to oxygen-dependent lethal mechanisms. [Muray et al., “Journal of Clinical Investigation ', 72:32 (1983)] Mortality of the intracellular parasite Leishmania donovani In order to determine whether or not it was possible to induce the following test, the following test was conducted.

Mononuclear leukocyte-derived macrophages were incubated with rMIF supernatant for approximately 48 hours. I turned on. Promast of Leishmania donovani with a 10:1 parasite to cell ratio Promastigote was added to the incubated cells. . Number of intracellular parasites/100 mice at 2.24.48 and 72 hours after infection Clophages were counted on stained preparations of coverslips.

22 in the anti-Leishmanial ability of cells treated with rMIF of the present invention. An increase of more than % was observed. rMIF combined with interferon gamma Parallel experiments are expected to increase the lethality of this parasite.

Example 8 Expression of recombinant human MIF.

To produce MIF, introduce the cDNA encoding it into an appropriate expression vector. did. Their diverse types can be found in mammals, insects, yeasts, Well known in the art regarding fungal and bacterial expression. is the above for mammalian cells. One vector is pXM[Y.

C. Young et al., Cell, 47:3-10 (1983)]. this vector is suitable for directing high-level expression of desired cDNAs in mammalian cells. In relation to the SV40 replication origin and enhancer, the adenovirus main late promoter adenovirus tripartite leader sequence cDNA copy, small hybrid intervening sequence, SV40 polyadenylation signal and adenovirus VA I genes [e.g., Kaufman, “Procedure Inges of the National Academy of Sciences The United Stave of America, 82:689-693 (19 85)]. Linearize pXM vector with endonuclease enzyme XhoI followed by synthetic oligonucleotides [collaborative oligonucleotides] that generate Xhol complementary ends. pre-modified by the addition of tive research, lexinsin, and marylantco. by separately ligating in equimolar amounts to the cDNA encoding MIF. and generate constructs for expression. These constructs can vary depending on the appropriate vector. can be expressed in any host.

a, Expression by mammalian cells.

To achieve expression of the MIF protein used in the assay described below, the cD for MIF was The pXM construct containing NA was transfected into CoS cells as described in Example 5. action. Conditioned medium from transfected CO8 cells was Contains MIF bioactivity as determined by F-test.

The mammalian cell expression vectors described here are well known to those skilled in the industry. can be synthesized using advanced techniques. Components of the vector, e.g. replicon, selection gene , enhancers, promoters, etc., can be obtained from natural sources or synthetically by known techniques. can be obtained. Kaufman et al., “Journal of Molecular Biology J, 159:511-521 (1982), and Kaufman, “Procedure. National Academy of Sciences of the National Academy of Sciences ・United Stave of America J, 82:689-693 (198 5) See. Typical mammalian host cells include transformed cell lines, especially primates. There are murine cell lines and murine cell lines. Normal diploid cells, - next group Cell lines and some explants derived from marginal in vitro cultures are also suitable. It is. If the selection gene is significantly active, the candidate cell will It does not have to be genotypically defective. Vector DNA was extracted using conventional methods. To stably integrate and subsequently amplify the integrated vector DNA , CHO cells may be used. Alternatively, the vector DNA is derived from bovine papilloma. Mavirus Genome [Lasky et al., Cell, 36:391-401 (1984 ) containing all or part of a cell line as a stable episomal element, e.g. For example, it can be carried by C127 mouse cells. Other suitable mammalian cell lines include Heela , CO3-1 monkey cells, mouse L-929 cells, Swiss Ba1b-c or NI 3T3 line derived from H mice, BHK or HaK hamster cellular Including, but not limited to:

Stable transformants are then isolated using standard immunological, biological or enzymatic assays. Screen for product expression by:

The presence of DNA and mRNA encoding MIF polypeptides can be determined by standard methods, For example, it can be detected by Southern plotting and RNA blotting. Ru. The expression vector DNA is introduced into a suitable host cell, such as a CO3-1 monkey cell. After that, the transient expression of the polypeptide-encoding DNA was determined during several days in culture. The activity of the protein in the culture medium or immunoassay is measured without selection.

In addition, a person skilled in the art can, for example, extract MIF from a plasmid using an appropriate enzyme. DNA sequence (using known recombinant genetic engineering techniques and other known vectors) ter, such as pJL3 and pJL4 [Gough et al., rEMB○ Journal, 4: 645-653 (1985) and pMT2 (pMT2-VWFI: , ATCC $67122, PCT Application PCT/US87100033). Other mammalian expression vectors comparable to pXM vectors can be constructed by using can be done. When a suitable host cell is transformed with a vector accompanied by MIF, , a MIF polypeptide can be expressed.

b. Bacterial expression system Similarly, those skilled in the art will be able to exclude mammalian regulatory sequences in close proximity to coding sequences. and engineered the sequence encoding MIF by inserting bacterial regulatory sequences, Creating bacterial vectors for intracellular or extracellular expression of the MrF of the invention by bacterial cells can be achieved. Additionally, the DNA encoding MIF is known in the art. As described above, they can be modified to include different codons to optimize bacterial expression. Like Alternatively, mature MIF-encoding sequences can be produced by methods also well known in the art. A secretory inducer that allows bacterial expression, secretion and processing of mature MIF polypeptides. operably linked in frame to a nucleotide sequence encoding a guiding polypeptide. It will be done. Results of MIF expression in Escherichia coli using the above secretion system As a result, secretion of active polypeptide is expected.

Compounds expressed by either route in bacterial host cells are then all known Depending on the method, harvesting, purification and/or physicochemical, biochemical and/or clinical It can be characterized with respect to floor parameters.

Expression by CO insect or yeast cells By performing the same operation, constructs for expressing MIF polypeptide in insect cells can be obtained. Insect vectors can be constructed [e.g. in published European patent application 155476] See reported methods]. MIF cDNA is expressed in insect cells.

by constructing similar yeast vectors using yeast regulatory sequences. cDNA encoding MIF is expressed, and extracellular active MIF is secreted.

[For example, published PCT application WO3610O639 and European patent application see the method described in application EP 123289].

Example 9 Construction of a CHO cell line expressing high levels of MIF.

In one method for producing high levels of the MIF protein of the present invention from mammalian cells, MIF is Cells containing multiple copies of the encoding cDNA are constructed.

Kaufman and Sharp, “Journal of Molecular Biology J (1982) (supra), the cDNA is labeled with an amplifiable marker, e.g. DHFR gene and cells containing increasing concentrations of methotrexate (MTX). (simultaneous) transfection.

This method can be used with several different cell types.

For example, MIF operably associated with other plasmid sequences to enable expression. The pXM vector containing the gene was subjected to calcium phosphate co-precipitation and transfection. DHFR expression plasmids, such as pAdD26SVpA3 [Kauf Mann, “Proceedings of the National Academy of Sci. 82:689-693 (1985) Ooni Koto, DHFR deletion C Introduce into HO cells, DUKX-BI1. DHFRHF transformants are dialysis selected for growth in alpha medium containing fetal bovine serum.

Transformants were analyzed for MI by bioassay, immunoassay, or RNA plotting. We checked for expression of F, followed by Kaufman et al., Mo1. Ce1l B Increasing concentrations of MTX (0,0 in successive steps at 2,0, 2,1,0 and 5 micromolar MTX). Select positive pools for amplification by growth. Clone the amplified line and MIF protein expression is monitored by the MIF assay. MIF expression is MT It is expected to increase as the level of X resistance increases.

In any of the above expression systems, the cell lines generated are treated with appropriate drug selection. The resulting cell line was further amplified by selection, and the resulting cell line was re-cloned and described in this specification. Expression levels can be assessed using the MIF assay described in .

Various modifications and variations of the present invention are also encompassed by the present specification and will be appreciated by those skilled in the art. It is expected that this will be obvious. The above modifications to the compositions and methods of the invention and modifications are considered to be included within the scope of the following claims.

international search report Ichikko-^-””””” PCT/lls 90101355S^35384

Claims (1)

[Claims] (1) Human macrophage migration inhibitory factor that is substantially free of other protein-like substances protein. (2) The following amino acid sequence: [There is an array] or the entire or substantially identical amino acid sequence to a fragment thereof; 2. The protein of claim 1, comprising a portion. (3) The following DNA sequence: [There is an array] DNA that is the same or substantially the same as, a fragment thereof or a hybrid thereto. 2. Encoded by all or a portion of the DNA capable of being oxidized. protein on the plate. (4) The following features: (1) Apparent molecular weight under reducing conditions in SDS-PAGE is approximately 12 kd , (2) biological activity in the MIF bioassay showing a inhibition rate higher than 20%; (3) IL-1beta and HLA-DR gene expression in macrophages biological activity in potentiation, (4) In activation of macrophage lethal effect of Leishmania donovani biological activity 2. The protein of claim 1, having one or more of the following. (5) DNA encoding the expression of MIF accompanied by an expression control sequence in a functional manner The claim is made by culturing a cell line transformed by the sequence. 1. Protein according to 1. (6) DNA encoding the expression of MIF accompanied by an expression control sequence in a functional manner culturing cell lines transformed with the sequence or fragment thereof. A method for producing MIF or a fragment thereof, comprising: (7) The following array: [There is an array] a sequence of nucleotide bases identical or substantially identical to, a fragment thereof or A D encoding MIF containing a DNA sequence capable of hybridizing thereto. NA sequence. (8) The DNA sequence according to claim 7, which is accompanied by an expression control sequence in a functional manner. transformed cells. (9) The cell according to claim 8, comprising a mammalian or bacterial cell. (10) Homogeneous MIF with biological activity in MIF assay with inhibition rate greater than 20% . (11) The therapeutic use of MIF or a biologically active fragment thereof in a pharmaceutically effective excipient. A pharmaceutical composition comprising a therapeutically effective amount. (12) Additionally, additional cytokines, hematopoietin, growth factors or tumor 12. The composition of claim 11, comprising a therapeutically effective amount of activating antibody. (13) Cytokines are IL-1, IL-2, IL-3, IL-4, IL-6 , GM-CSF, G-CSF, M-CSF, interferon, erythropoietin 13. The composition of claim 12, wherein the composition is selected from the group consisting of: (14) The additional cytokine is IFN-gamma, M-CSF or GM-CSF. 14. The composition according to claim 13. (15) A plasmid vector comprising the DNA sequence according to claim 7. (16) administering to the patient an effective amount of MIF or a fragment thereof; How to treat cancer. (17) comprising administering to the patient an effective amount of MIF or a fragment thereof; How to treat infections. (18) administering to the patient an effective amount of MIF or a fragment thereof; Methods for promoting wound healing. (19) comprising administering to the patient an effective amount of MIF or a fragment thereof; How to stimulate the immune system. (20) administering the vaccine after or simultaneously with a therapeutic composition comprising MIF; methods to increase vaccine effectiveness, including; (21) Furthermore, at least one hematopoietin, cytokine, growth factor or or the antibody simultaneously or subsequently with MIF. 9. The method described in 9. (22) The cytokine is M-CSF, GM-CSF or IFN-gamma 22. The method of claim 21.