Patent Publication Number: US-2015071942-A1

Title: Combination therapy of anti-mif antibodies and chemotherapeutics

Description:
The present invention pertains to anti-MIF antibodies, in particular their use in combination with cancer therapeutics, i.e. chemotherapeutics, in the treatment of cancer. 
     BACKGROUND 
     Macrophage migration inhibitory factor (MIF) is a cytokine initially isolated based upon its ability to inhibit the in vitro random migration of peritoneal exudate cells from tuberculin hypersensitive guinea pigs (containing macrophages) (Bloom et al. Science 1966, 153, 80-2; David et al. PNAS 1966, 56, 72-7). Today, MIF is known as a critical upstream regulator of the innate and acquired immune response that exerts a pleiotropic spectrum of activities. 
     The human MIF cDNA was cloned in 1989 (Weiser et al., PNAS 1989, 86, 7522-6), and its genomic localization was mapped to chromosome 22. The product of the human MIF gene is a protein with 114 amino acids (after cleavage of the N-terminal methionine) and an apparent molecular mass of about 12.5 kDa. MIF has no significant sequence homology to any other protein. The protein crystallizes as a trimer of identical subunits. Each monomer contains two antiparallel alpha-helices that pack against a four-stranded beta-sheet. The monomer has additional two beta-strands that interact with the beta-sheets of adjacent subunits to form the interface between monomers. The three subunits are arranged to form a barrel containing a solvent-accessible channel that runs through the center of the protein along a molecular three-fold axis (Sun et al. PNAS 1996, 93, 5191-5196). 
     It was reported that MIF secretion from macrophages was induced at very low concentrations of glucocorticoids (Calandra et al. Nature 1995, 377, 68-71). However, MIF also counter-regulates the effects of glucocorticoids and stimulates the secretion of other cytokines such as tumor necrosis factor TNF-α and interleukin IL-1 β (Baugh et al., Crit Care Med 2002, 30, S27-35). MIF was also shown e.g. to exhibit pro-angiogenic, pro-proliferative and anti-apoptotic properties, thereby promoting tumor cell growth (Mitchell, R. A., Cellular Signalling, 2004. 16(1): p. 13-19; Lue, H. et al., Oncogene 2007. 26(35): p. 5046-59). It is also e.g. directly associated with the growth of lymphoma, melanoma, and colon cancer (Nishihira et al. J Interferon Cytokine Res. 2000, 20:751-62). 
     MIF is a mediator of many pathologic conditions and thus associated with a variety of diseases including inter alia inflammatory bowel disease (IBD), rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), asthma, glomerulonephritis, IgA nephropathy, myocardial infarction (MI), sepsis and cancer, though not limited thereto. 
     Polyclonal and monoclonal anti-MIF antibodies have been developed against recombinant human MIF (Shimizu et al., FEBS Lett 1996; 381, 199-202; Kawaguchi et al, Leukoc. Biol. 1986, 39, 223-232, and Weiser et al., Cell. Immunol. 1985, 90, 167-78). 
     Anti-MIF antibodies have been suggested for therapeutic use. Calandra et al., (J. Inflamm. (1995); 47, 39-51) reportedly used anti-MIF antibodies to protect animals from experimentally induced gram-negative and gram-positive septic shock. Anti-MIF antibodies were suggested as a means of therapy to modulate cytokine production in septic shock and other inflammatory disease states. 
     U.S. Pat. No. 6,645,493 discloses monoclonal anti-MIF antibodies derived from hybridoma cells, which neutralize the biological activity of MIF. It could be shown in an animal model that these mouse-derived anti-MIF antibodies had a beneficial effect in the treatment of endotoxin-induced shock. 
     US 200310235584 discloses methods of preparing high affinity antibodies to MIF in animals in which the MIF gene has been homozygously knocked-out. 
     Glycosylation-inhibiting factor (GIF) is a protein described by Galat et al. (Eur. J. Biochem, 1994, 224, 417-21). MIF and GIF are now recognized to be identical. Watarai et al. (PNAS 2000, 97, 13251-6) described polyclonal antibodies binding to different GIF epitopes to identify the biochemical nature of the posttranslational modification of GIF in Ts cells. Watarai et al, supra, reported that GIF occurs in different conformational isoforms it vitro. One type of isomer occurs by chemical modification of a single cysteine residue. The chemical modification leads to conformational changes within the GIF protein. 
     One of those medical diseases and disorders in which MIF has been implicated for the past decades is—as pointed out above—cancer. Cancer is generally treated via various routes, one of them being the use of so-called chemotherapeutic agents (which are the basis of anticancer chemotherapy). The concept underlying chemotherapy in the general sense thereof, posits that a disease or disorder (caused by bacteria, viruses, parasites and cancer cells) can be effectively treated by way of chemical compounds. One particular indication of chemotherapy is cancer. Chemotherapeutic agents can act for example by killing cells that divide more rapidly than other cells, and thus target cancer cells which commonly divide more rapidly than non-cancerous cells. Most chemotherapeutic agents drugs work by impairing cell division, i.e., they act at one or several stages of the cell cycle and thus are able to target cells that divide more rapidly. Chemotherapeutic agents can be either cytostatic, i.e. they slow down or abrogate the growth or division of cells; other chemotherapeutic drugs can cause damage to cells and kill them; in that case they are termed cytotoxic. Most cytotoxic drugs inflict a damage that per se does not suffice to kill a cell but that generates a stimulus to initiate programmed cell death (apoptosis). 
     In general, major classes of chemotherapeutic drugs are alkylating agents, anti-metabolites, anthracycdines, plant alkaloids, topoisomerases and other anti-tumour agents. Most commonly, as mentioned above, these drugs affect cell division; they can also affect DNA synthesis or function. Other chemotherapeutics do not directly interfere with DNA. These are newer classes of chemotherapeutic agents, which are referred to as signal interceptors, which include monoclonal antibodies and tyrosine kinase inhibitors like imatinib mesylate. Examples for alkylating agents, which alkylate nucleophilic functional groups are mechlorethamine, cyclophosphamide, chlorambucil, melphalane, trofosfamide, ifosfamide, carmustine, lomustine, dacarbazine, temozolomide, mitomycine C and many others. Cisplatin, carboplatin, oxaliplatin and other platinum containing compounds form stable complexes with DNA. 
     Cytotoxic anti-metabolites are folic acid analogues (e.g., methotrexat/aminopterin, raltitrexed, pemetrexed), purines (e.g., 6-mercaptopurine, azathioprine, thioguanine, fludarabine, cladribine) or pyrimidines (cytarabine, gemcitabine, 5-fuloruracil and its prodrugs, deazacytidine). Antimetabolites either inhibit DNA-synthesis by interfering with crucial steps in the do novo synthesis of purine and pyrimidine nucleotides or they become incorporated into DNA during the S-phase of the cell cycle, where they interfere with DNA-folding, DNA-repair or methylation. Alternatively, some compounds also become incorporated into RNA. 
     Examples for alkaloids and terpenoids which are derived from plants and block cell division by preventing microtubule function are vinca-alkaloids and taxanes. Particularly well known vinca-alkaloids are vincristine, vinblastine, vinorelbine and vindesine. Podophyllotoxin is an additional example of a plant-derived compound. An example for a taxane is docetaxel or paclitaxel. Estramustin is an example of a synthetic compound that targets tubulin. 
     Examples of topoisomerase inhibitors, which are inhibitors of enzymes that maintain the topology of DNA, include camphtotecines like irinotecan and topotecan (type 1 topoisomerase inhibitors) or amsacrin, etoposide, etoposide phosphate and teniposide (topoisomerase-type 2 inhibitors). 
     Finally, examples of antineoplastic intercalating agents include dactinomycin, doxorubicin, epirunbicin, bleomycin and others. 
     A comprehensive overview is comprised in Goodman and Gilman, The Pharmacological Basis of Therapeutics, 12 th  Edition, “General Principles of Cancer Chemotherapy. Introduction” as shown below. Several tumors are susceptible to hormone therapy: glucocorticoids (e.g., prednisolone, dexamethasone and may others) promote apoptosis of lymphoma cells. They are therefore included in typical chemotherapeutic regimen. Similarly, several types of cancer are susceptible to hormonal interventions. This includes, e.g., breast cancer, ovarian cancer and prostate cancer. Hormonal ablation can be achieved by suppressing pituitary release of gonadotropins with gonadotropin-releasing hormone receptor agonists (e.g., buserelin, goserelin, leuprolide, hisrelin etc.), which induce desensitization of the receptor and hence inhibit hormone production, or with gonadotropin-releasing hormone receptor antagonists (e.g., degarelix). Alternatively, the action of estrogens and of androgens may be blocked by hormone receptor antagonists: compounds that act as partial agonists at estrogen receptors (also referred to as selective estrogen receptor modulators, SERM&#39;s) include tamoxifen, raloxifen and toremifen. Fulvestrant is an example of a pure estrogen receptor antagonist. Androgen receptors can be blocked by antagonists such as flutamide, bicalitamide and cyproterone. Finally, hormonal ablation can be achieved by blocking the pertinent enzymes, which are responsible for their synthesis. In the case of estrogens, it is the aromatase (CYP19), which is blocked by compounds such as aminoglutethimide, formestane, exemestane, anastrazole and letrozole. Androgen production can be suppressed by inhibiting the enzyme 17 α-hydroxylase/C17,20 lyase (CYP17A1) with abiraterone. Regardless of by which approach hormonal input is blocked, the growth of susceptible cancer cells is suppressed and their apoptosis is promoted. 
     Although chemotherapeutic agents have been shown to be useful and successful in the treatment of several different cancer types, chemotherapeutic regimen have a range of side effects, depending on the type of medication used. Most common side effects include depression of the immune system which can result in potentially fatal infections, fatigue, anaemia, a tendency to bleed easily, gastrointestinal distress, like nausea and vomiting, diarrhea or constipation and hair loss. Further, damage to specific organs may occur, which results e.g. in heart damage, liver damage, kidney damage, damage to the inner ear, damage to the peripheral nervous system and brain dysfunction. 
     All of these side effects increase severely if the dosage of the given chemotherapeutic drug is augmented. Decreasing the amount of the drug administered will usually also result in lower incidence and/or alleviated side effects. 
     In other cases it would be of benefit if the effects of the chemotherapeutic drugs could be enhanced or increased if high-dose treatment is necessary. 
     Thus, there remains an urgent need in the art for the provision of a therapy for cancer which allows reduction of the administration dose of a given chemotherapeutic and/or enhances the effects of a given chemotherapeutic. 
     DESCRIPTION OF THE INVENTION 
     This object has been solved by the present invention. 
     In particular it could be shown that by a combination therapy of anti-MIF antibodies and a given chemotherapeutic agent a synergistic effect could be detected which would allow treatment of cancer with a lower dosage of the chemotherapeutic and/or achieving a higher effect with a similar dosage as compared to treating this cancer with the chemotherapeutic alone. 
     In particular, a treatment by a combination therapy of anti-oxMIF antibodies and a given chemotherapeutic could be shown to be associated with a synergistic effect, as described above and exemplified in the examples of the present invention. 
     Elevated MIF levels, i.e. levels of MIF in general are detected after the onset of various diseases, inter alia after the onset of cancer. However, MIF circulates also in healthy subjects, which makes a clear differentiation difficult. oxMIF, on the contrary, is not present in healthy subjects. 
     It has been discovered after thorough research of MIF and antibodies thereto that the antibodies RAB9, RAB4 and RAB0, as well as RAM9, RAM4 and RAM0, specifically bind to oxMIF (and are incapable of binding to redMIF). 
     In earlier experiments carried out by the inventors, it could be shown that oxidative procedures like cystine-mediated oxidation, GSSG (ox. Glutathione)-mediated oxidation or incubation of MIF with Proclin300 or protein crosslinkers (e.g. BMOE) causes binding of MIF to the above mentioned antibodies. 
     The surprising conclusions reached by the present inventors are:
         Redox modulation (Cys/Glu-mediated mild oxidation) of recombinant MIF (human, murine, rat, CHO, monkey)) or treatment of recombinant MIF with Proclin300 or protein crosslinkers leads to the binding of Baxter&#39;s anti-MIF antibodies RAB9, RAB4 and RAB0   Reduction of oxMIF leads to the loss of Ab binding   Specificity for oxMIF-isoforms correlates with biological Ab efficacy (especially in vivo).   oxMIF levels can be correlated with a disease state.       

     This additional knowledge regarding (ox)MIF served as a basis for the further studies of the present inventors. Thus, preferred embodiments of the present invention are:
     1. An anti-MIF antibody in combination with a chemotherapeutic agent for use in the treatment of cancer, wherein the chemotherapeutic agent is preferably gemcitabine, cisplatin, and/or doxorubicin.   2. The anti-MIF antibody in combination with chemotherapeutic agent according to item 1 for use in the treatment of cancer, wherein the cancer is selected from the following group: pancreatic cancer, ovarian cancer, prostate cancer, breast cancer, lung cancer and colon cancer, more preferred pancreatic cancer, prostate cancer and ovarian cancer.   3. The combination according to item 1 or 2 wherein the anti-MIF antibody is selected from the following group: anti-MIF antibody RAM9, RAM0 and/or RAM4.   4. The combination therapy according to any of items 1 to 3 wherein the chemotherapeutic is gemcitabine.   5. The combination therapy according to any of items 1 to 4, the anti-MIF antibody is RAM9, the chemotherapeutic is doxorubicin, optionally in combination with cisplatin and the cancer is ovarian cancer.   6. The combination therapy according to any of items 1 to 4, the anti-MIF antibody is RAM9, the chemotherapeutic is gemdcitabine and the cancer is pancreas carcinoma.   7. The combination therapy according to any of items 1 to 4, the anti-MIF antibody is RAM, the chemotherapeutic is doxorubicin, optionally in combination with cisplatin and the cancer is ovarian cancer.   8. The combination therapy according to any of items 1 to 4, the anti-MIF antibody is RAM0, the chemotherapeutic is gemcitabine and the cancer is pancreas carcinoma.   9. The combination therapy according to any of items 1 to 4, the anti-MIF antibody is RAM4, the chemotherapeutic is doxorubicin, optionally in combination with cisplatin and the cancer is ovarian cancer.   10. The combination therapy according to any of items 1 to 4, the anti-MIF antibody is RAM4, the chemotherapeutic is gemcitabine, and the cancer is pancreas carcinoma.   11. The combination therapy according to any of items 1 to 4, the anti-MIF antibody is RAM0, the chemotherapeutic is mitoxantrone, and the cancer is prostate cancer.   12. A kit comprising the combination as defined in any of items 1-11 above, and instructions for use.   13. A combination as defined in any of items 1-11 above, or the kit of item 12, for use in the treatment of cancer.   

     The above-mentioned antibodies are characterized and supported by both their sequences as well as by deposits as plasmids in  E. coli , comprising either the light or the heavy chain of each of the above mentioned antibodies RAB0, RAB4 and RAB9, respectively, as well as RAM0, RAM4 and RAM9, respectively. 
     The plasmids are characterized by their DSM number which is the official number as obtained upon deposit under the Budapest Treaty with the German Collection of Microorganisms and Cell Cultures (DSMZ), Mascheroder Weg 1b, Braunschweig, Germany. The plasmids were deposited in  E. coli  strains, respectively. The plasmid with the DSM 25110 number comprises the light chain sequence of the anti-MIF antibody RAB4. The plasmid with the DSM 25112 number comprises the heavy chain (IgG4) sequence of the anti-MIF antibody RAB4. 
     The co-expression of plasmids DSM 25110 and DSM 25112 in a suitable host cell results in the production of preferred anti-MIF antibody RAB4. 
     The plasmid with the DSM 25111 number comprises the light chain sequence of the anti-MIF antibody RAB9. The plasmid with the DSM 25113 number comprises the heavy chain (IgG4) sequence of the anti-MIF antibody RAB9. 
     The co-expression of plasmids DSM 25111 and DSM 25113 in a suitable host cell results in the production of preferred anti-MIF antibody RAB9. 
     The plasmid with the DSM 25114 number comprises the light chain sequence of the anti-MIF antibody RAB0. The plasmid with the DSM 25115 number comprises the heavy chain (IgG4) sequence of the anti-MIF antibody RAB0. 
     The co-expression of plasmids DSM 25114 and DSM 25115 in a suitable host cell results in the production of preferred anti-MIF antibody RAB0. 
     Similarly, the light and heavy chains of RAM0, RAM9 and RAM4 have been similarly deposited under the Budapest Treaty on Apr. 12, 2012 with the DSMZ, Braunschweig, Germany. The following designations have been used: 
     RAM9-heavy chain:  E. coli  GA.662-01.pRAM9hc-DSM 25860. 
     RAM4-light chain:  E. coli  GA.906-04.pRAM4lc-DSM 25861. 
     RAM9-light chain:  E. coli  GA.661-01.pRAM9lc-DSM 25859. 
     RAM4-heavy chain:  E. coli  GA.657-02-pRAM4hc-DSM 25862. 
     RAM0-light chain:  E. coli  GA.906-01.pRAM0lc-DSM 25863. 
     RAM0-heavy chain:  E. coli  GA784-01.pRAM0hc-DSM 25864. 
     The term “prophylactic” or “therapeutic” treatment is art-recognized and refers to administration of a drug to a subject. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the subject) then the treatment is prophylactic, i.e., it protects the subject against developing the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate or maintain the existing unwanted condition or side effects therefrom). 
     As used herein an anti-(ox)MIF compound refers to any agent that attenuates, inhibits, opposes, counteracts, or decreases the biological activity of (ox)MIF. An anti(ox)MIF compound may be an agent that inhibits or neutralizes (ox)MIF activity, for example an antibody, particularly preferred, the antibodies as described herein, even more preferred the antibodies RAB9, RAB4 and/or RAB0. Very preferred antibodies are RAM9, RAM4 and/or RAM0. 
     The preferred MIF antagonist in accordance with the present invention is an anti-MIF antibody. Even more preferred the anti-MIF antibody is an antibody against oxMIF. In other embodiments, the anti-oxMIF antibodies, e.g., the antibodies mentioned above or an antigen-binding portion thereof bind oxMIF with a K D  of less than 100 nM, preferably a K D  of less than 50 nM, even more preferred with a K D  of less than 10 nM. Very preferred, the antibodies bind to oxMIF with a K D  of less than 5 nM. 
     The invention further relates to kits comprising an anti-MIF antibody or an antigen-binding portion thereof as well as a chemotherapeutic agent according to the invention. A kit may include in addition to the antibody and the chemotherapeutic agent, further therapeutic agents and uses thereof. A kit also can include instructions for use in a therapeutic method. 
     Earlier results have shown that an anti-MIF antibody that only binds oxMIF and does not bind redMIF and further inhibits GCO and/or cell proliferation induces a beneficial effect in an animal model. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention is further described in the figures as enclosed. 
    
    
     
       DESCRIPTION OF THE FIGURES 
         FIG. 1 : Fully human anti-MIF antibody RAM9 in combination with gemcitabine in a xenograft model for pancreatic cancer. BxPC3 human pancreatic cancer cells were orthotopically implanted into nude mice; after one week, mice were treated with 60 mg/kg of RAM9 alone (Anti-MIF, right panel, solid grey line) or in combination with 100 mg/kg gemcitabine (Anti-MIF+Gemcitabine, right panel, solid black line). Control groups were left untreated (left and right panel, dotted black line) or were treated with a control human monoclonal antibody (Control IgG, left panel, solid grey line) or with gemcitabine alone (Gemcitabine, left panel, solid black line). The survival curves represent the points in time when animals had to be sacrificed in accordance with UK Home Office Guidance. 
         FIG. 2 : Combination of human anti-MIF antibody RAM0 with gemcitabine. Cob 357 human pancreatic cancer cell line was orthotopically implanted into nude mice; after one week, mice were treated with 60 mg/kg of RAM0 alone (Anti-MIF) or in combination with 100 mg/kg gemcitabine (Anti-MIF+Gem). Control groups were left untreated or were treated with gemcitabine alone (Gem) or a control human monoclonal antibody alone (Control) or with a control human monoclonal antibody in combination with gemcitabine (Control+Gem). (A) Survival curves representing the points in time when animals had to be sacrificed in accordance with UK Home Office Guidance. (B) Tumor volume was measured in as subset of animals after animals had been sacrificed on day 20 after cell implantation. (C) After treatment, mice bearing a Colo357 xenograft were injected i.v. with the FITC-conjugated tomato lectin to visualize the blood vessels on day 12. Microvessel density was quantified using ImagePro Plus software (Image-Pro plus, Media Cybernetics) and vessel density was expressed as the mean percentage of total surface area. 
         FIG. 3 : Elevation of Caspase 3 levels when A2780adr ovarian cancer cells are treated with doxorubicin in combination with RAM0 (A) and RAM9 (B). The percentage increased caspase 3 activity over non-treated cells is indicated. Cells were treated with a human isotype control IgG (Control IgG), anti-MIF antibody RAM0 or RAM9 alone, a combination of control IgG and doxorubicin (Control IgG+Dox), doxorubicin alone (Dox) or a combination of anti-MIF antibody and doxorubicin (RAM0+Dox or RAM9+Dox). 
         FIG. 4 : In vitro combination of RAM0 and cisplatin in a cisplatin-dependent cell killing assay using the human ovarian cancer cell line A2780. The EC50 of cisplatin in absence of antibody (w/o antibody) or in the presence of a human isotype control IgG or anti-MIF antibody RAM0 is depicted. Data are the means±standard deviation of 9 independent experiments. 
         FIG. 5 : In vivo combination of RAM0 and cisplatin in a mouse xenograft model using the human ovarian cancer cell line A2780. Tumor weights are indicated after treatment of inoculated mice with a human control IgG or with RAM0. Antibodies were applied either alone or in combination with cisplatin. 
         FIG. 6 : In vitro combination of RAM0 and mitoxantrone in a mitoxantrone-dependent cell killing assay using the human prostate cancer cell line LnCAP. The EC50s of mixoantrone in absence of antibody (w/o antibody) or in the presence of a human isotype control IgG or anti-MIF antibody RAM0 are depicted. The single values as well as the mean±standard deviation of 10 independent experiments are shown. 
     
    
    
     DEFINITIONS AND GENERAL TECHNIQUES 
     Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry described herein are those well known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990), which are incorporated herein by reference. “MIF” or “macrophage migration inhibitory factor” refers to the protein, which is known as a critical mediator in the immune and inflammatory response, and as a counterregulator of glucocorticoids. MIF includes mammalian MIF, specifically human MIF (Swiss-Prot primary accession number P14174), wherein the monomeric form is encoded as a 115 amino acid protein but is produced as a 114 amino acid protein due to cleavage of the initial methionine. “MIF” also includes “GIF” (glycosylation-inhibiting factor) and other forms of MIF such as fusion proteins of MIF. The numbering of the amino acids of MIF starts with the N-terminal methionine (amino acid 1) and ends with the C-terminal alanine (amino acid 115). 
     “oxidized MIF” or oxMIF is defined for the purposes of the invention as an isoform of MIF that occurs by treatment of MIF with mild oxidizing reagents, such as Cystine. As has been shown by the present inventors, recombinant oxMIF that has been treated this way comprises isoform(s) of MIF that share structural rearrangements with oxMIF that (e.g.) occurs in vivo after challenge of animals with bacteria. 
     redMIF is defined for the purposes of this invention as reduced MIF and is MIF which does not bind to RAB0, RAB9 and/or RAB4. 
     The anti-oxMIF antibodies described in this invention are able to discriminate between ox and red MIF, which are generated by mild oxidation or reduction, respectively. The anti-oxMIF antibodies are useful to specifically detect oxMIF. Discrimination between these conformers is assessed by ELISA or surface plasmon resonance. Both techniques can be performed as well known to a person skilled in the art and as described below. 
     Assessing Differential Binding of the Antibodies by Biacore. 
     Binding kinetics of oxMIF and redMIF to antibody RAB9 and RAB0 are examined by surface plasmon resonance analysis using a Biacore 3000 System. The antibodies were coated on a CM5 (=carboxymethylated dextran) chip and recombinant MIF protein, pre-incubated with 0.2% Proclin300, were injected. (Proclin300 consists of oxidative isothiazolones that stabilize the oxMIF structure). In native HBS-EP buffer (=Biacore running buffer) without addition of ProClin300, none of the recombinant MIF proteins bound to RAB9, RAB0 or to the reference antibody (irrelevant isotype control antibody) used as negative (background) binding control. 
     In a preferred embodiment, oxMIF is MIF which is differentially bound by antibody RAB9, RAB4 and/or RAB0 or an antigen-binding fragment thereof, meaning that these antibodies do bind to oxMIF while redMIF is not bound by either one of these antibodies. 
     In other embodiments, the anti-oxMIF antibodies, e.g., the antibodies mentioned above or an antigen-binding portion thereof bind oxMIF with a K D  of less than 100 nM, preferably a K D  of less than 50 nM, even more preferred with a K D  of less than 10 nM. Particularly preferred, the antibodies of the invention bind to oxMIF with a K D  of less than 5 nM. 
     The antibodies of the invention, as defined hereinabove and hereinafter, have the same specificities. They thus also show similar results in the experiments as carried out by the present inventors. (Non-)binding of an antibody, e.g. RAB9, RAB4 or RAB0 or RAM9, RAM4 or RAM0 (to oxMIF or redMIF) can be determined as generally known to a person skilled in the art, examples being any one of the following methods: ELISA with recombinant MIF in its reduced or oxidized state, or surface plasmon resonance using recombinant MIF in its reduced or oxidized state, like the well known Biacore assay, described above. 
     A preferred method for the determination of binding is surface plasmon resonance of an antibody to e.g. rec. (ox)MIF whereupon “binding” is meant to be represented by a K D  of less than 100 nM preferably less than 50 nM, even more preferred less than 10 nM whereas the non-binding to redMIF is characterized by a K D  of more than 400 nM. “Binding” and “specific binding” is used interchangeably here to denote the above. “Differential binding” in the context of this application means that a compound, in particular the antibodies as described herein, bind to oxMIF (e.g., with the K D  values mentioned above) while they do not bind to redMIF (with non-binding again being defined as above). 
     An “antibody” refers to an intact antibody or an antigen-binding portion that competes with the intact antibody for (specific) binding. See generally, Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference). The term antibody includes human antibodies, mammalian antibodies, isolated antibodies and genetically engineered forms such as chimeric, camelide/camelized or humanized antibodies, though not being limited thereto. 
     The term “antigen-binding portion” of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., (ox)MIF). Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include e.g.—though not limited thereto—the following: Fab, Fab′, F(ab′)2, Fv, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, antibodies and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific antigen binding to the polypeptide, i.e., ox or redMIF. From N-terminus to C-terminus, both the mature light and heavy chain variable domains comprise the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to each domain is in accordance with the definitions of Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia et al. J. Mol. Biol. 196:901-917 (1987), or Chothia et al., Nature 342:878-883 (1989). An antibody or antigen-binding portion thereof can be derivatized or linked to another functional molecule (e.g., another peptide or protein). For example, an antibody or antigen-binding portion thereof can be functionally linked to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or a linking molecule. 
     The term “KD” refers here, in accordance with the general knowledge of a person skilled in the art to the equilibrium dissociation constant of a particular antibody with the respective antigen. This equilibrium dissociation constant measures the affinity. The affinity determines how much complex is formed at equilibrium (steady state where association balances dissociation) (here: ox or redMIF and antibody). 
     ka=association rate constant [(M−1 s−1] 
     kd=dissociation rate constant [s−1] 
     KD=equilibrium dissociation constant=kd/ka [M] 
     The term “human antibody” refers to any antibody in which the variable and constant domains are human sequences. The term encompasses antibodies with sequences derived from human genes, but which have been changed, e.g. to decrease possible immunogenicity, increase affinity, eliminate cysteines that might cause undesirable folding, etc. The term encompasses such antibodies produced recombinantly in non-human cells, which might e.g. impart glycosylation not typical of human cells. 
     The term “humanized antibody” refers to antibodies comprising human sequences and containing also non-human sequences; in particular, a “humanized antibody” refers to a non-human antibody where human sequences have been added and/or replace the non-human sequences. 
     The term “camelized antibody” refers to antibodies wherein the antibody structure or sequences has been changed to more closely resemble antibodies from camels, also designated camelid antibodies. Methods for the design and production of camelized antibodies are part of the general knowledge of a person skilled in the art. 
     The term “chimeric antibody” refers to an antibody that comprises regions from two or more different species. 
     The term “isolated antibody” or “isolated antigen-binding portion thereof” refers to an antibody or an antigen-binding portion thereof that has been identified and selected from an antibody source such as a phage display library or a B-cell repertoire. 
     The production of the anti-(ox)MIF antibodies according to the present invention includes any method for the generation of recombinant DNA by genetic engineering, e.g. via reverse transcription of RNA and/or amplification of DNA and cloning into expression vectors. In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. In some embodiments, the vector is capable of autonomous replication in a host cell into which it is introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vector (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). 
     Anti-(ox)MIF antibodies can be produced inter alia by means of conventional expression vectors, such as bacterial vectors (e.g., pBR322 and its derivatives), or eukaryotic vectors. Those sequences that encode the antibody can be provided with regulatory sequences that regulate the replication, expression and/or secretion from the host cell. These regulatory sequences comprise, for instance, promoters (e.g., CMV or SV40) and signal sequences. The expression vectors can also comprise selection and amplification markers, such as the dihydrofolate reductase gene (DHFR), hygromycin-B-phosphotransferase, and thymidine-kinase. The components of the vectors used, such as selection markers, replicons, enhancers, can either be commercially obtained or prepared by means of conventional methods. The vectors can be constructed for the expression in various cell cultures, e.g., in mammalian cells such as CHO, COS, HEK293, NSO, fibroblasts, insect cells, yeast or bacteria such as  E. coli . In some instances, cells are used that allow for optimal glycosylation of the expressed protein. 
     The anti-(ox)MIF antibody light chain gene(s) and the anti-(ox)MIF antibody heavy chain gene(s) can be inserted into separate vectors or the genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods, e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present. 
     The production of anti-(ox)MIF antibodies or antigen-binding fragments thereof may include any method known in the art for the introduction of recombinant DNA into eukaryotic cells by transfection, e.g. via electroporation or microinjection. For example, the recombinant expression of anti-(ox)MIF antibody can be achieved by introducing an expression plasmid containing the anti-(ox)MIF antibody encoding DNA sequence under the control of one or more regulating sequences such as a strong promoter, into a suitable host cell line, by an appropriate transfection method resulting in cells having the introduced sequences stably integrated into the genome. The lipofection method is an example of a transfection method which may be used according to the present invention. 
     The production of anti-(ox)MIF antibodies may also include any method known in the art for the cultivation of said transformed cells, e.g. in a continuous or batchwise manner, and the expression of the anti-(ox)MIF antibody, e.g. constitutive or upon induction. It is referred in particular to WO 20091086920 for further reference for the production of anti-(ox)MIF antibodies. In a preferred embodiment, the anti-(ox)MIF antibodies as produced according to the present invention bind to oxMIF or an epitope thereof. Particularly preferred antibodies in accordance with the present invention are antibodies RAB9, RAB4 and/or RAB0, as well as RAM9, RAM4 and/or RAM0. 
     The sequences of these antibodies are partly also disclosed in WO 2009/086920; see in addition the sequence list of the present application and the following: 
     
       
         
           
               
            
               
                 SEQ ID NO: 1 for the amino acid sequence of the 
               
               
                 light chain of RAB9: 
               
               
                 DIQMTQSPSS LSASVGDRVT ITCRSSQRIM TYLNWYQQKP 
               
               
                   
               
               
                 GKAPKLLIFV ASHSQSGVPS RFRGSGSETD FTLTISGLQP 
               
               
                   
               
               
                 EDSATYYCQQ SFWTPLTFGG GTKVEIKRTV AAPSVFIFPP 
               
               
                   
               
               
                 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ 
               
               
                   
               
               
                 ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 
               
               
                   
               
               
                 LSSPVTKSFN RGEC, 
               
               
                   
               
               
                 SEQ ID NO: 2 for the amino acid sequence of the 
               
               
                 light chain of RAB4: 
               
               
                 DIQMTQSPVT LSLSPGERAT LSCRASQGVS SSSLAWYQQK 
               
               
                   
               
               
                 PGQAPRLLIY GTSSRATGIP DRFSGSASGT DFTLTISRLQ 
               
               
                   
               
               
                 PEDFAVYYCQ QYGRSLTFGG GTKVEIKRTV AAPSVFIFPP 
               
               
                   
               
               
                 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ 
               
               
                   
               
               
                 ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 
               
               
                   
               
               
                 LSSPVTKSFN RGEC, 
               
               
                   
               
               
                 SEQ ID NO: 3 for the amino acid sequence of the 
               
               
                 light chain of RAB0: 
               
               
                 DIQMTQSPGT LSLSPGERAT LSCRASQGVS SSSLAWYQQK 
               
               
                   
               
               
                 PGQAPRLLIY GTSSRATGIP DRFSGSASGT DFTLTISRLQ 
               
               
                   
               
               
                 PEDFAVYYCQ QYGRSLTFGG GTKVEIKRTV AAPSVFIFPP 
               
               
                   
               
               
                 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ 
               
               
                   
               
               
                 ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 
               
               
                   
               
               
                 LSSPVTKSFN RGEC, 
               
               
                   
               
               
                 SEQ ID NO: 4 for the amino acid sequence of the 
               
               
                 light chain of RAB2: 
               
               
                 DIQMTQSPVT LSLSPGERAT LSCRASQSVR SSYLAWYQQK 
               
               
                   
               
               
                 PGQTPRLLIY GASNRATGIP DRFSGSGSGT DFTLTISRLE 
               
               
                   
               
               
                 PEDFAVYYCQ QYGNSLTFGG GTKVEIKRTV AAPSVFIFPP 
               
               
                   
               
               
                 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ 
               
               
                   
               
               
                 ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 
               
               
                   
               
               
                 LSSPVTKSFN RGEC, 
               
               
                   
               
               
                 SEQ ID NO: 5 for the amino acid sequence of the 
               
               
                 heavy chain of RAB9: 
               
               
                 EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYSMNWVRQA 
               
               
                   
               
               
                 PGKGLEWVSS IGSSGGTTYY ADSVKGRFTI SRDNSKNTLY 
               
               
                   
               
               
                 LQMNSLRAED TAVYYCAGSQ WLYGMDVWGQ GTTVTVSSAS 
               
               
                   
               
               
                 TKGPSVFPLA PCSRSTSEST AALGCLVKDY FPEPVTVSWN 
               
               
                   
               
               
                 SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTKTYT 
               
               
                   
               
               
                 CNVDHKPSNT KVDKRVESKY GPPCPPCPAP EFLGGPSVFL 
               
               
                   
               
               
                 FPPKPKDTLM ISRTPEVTCV VVDVSQEDPE VQFNWYVDGV 
               
               
                   
               
               
                 EVHNAKTKPR EEQFNSTYRV VSVLTVLHQD WLNGKEYKCK 
               
               
                   
               
               
                 VSNKGLPSSI EKTISKAKGQ PREPQVYTLP PSQEEMTKNQ 
               
               
                   
               
               
                 VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG 
               
               
                   
               
               
                 SFFLYSRLTV DKSRWQEGNV FSCSVMHEAL HNHYTQKSLS 
               
               
                   
               
               
                 LSLGK, 
               
               
                   
               
               
                 SEQ ID NO: 6 for the amino acid sequence of the 
               
               
                 heavy chain of RAB4: 
               
               
                 EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYAMDWVRQA 
               
               
                   
               
               
                 PGKGLEWVSG IVPSGGFTKY ADSVKGRFTI SRDNSKNTLY 
               
               
                   
               
               
                 LQMNSLRAED TAVYYCARVN VIAVAGTGYY YYGMDVWGQG 
               
               
                   
               
               
                 TTVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF 
               
               
                   
               
               
                 PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS 
               
               
                   
               
               
                 SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE 
               
               
                   
               
               
                 FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV 
               
               
                   
               
               
                 QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW 
               
               
                   
               
               
                 LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP 
               
               
                   
               
               
                 SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT 
               
               
                   
               
               
                 TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH 
               
               
                   
               
               
                 NHYTQKSLSL SLGK, 
               
               
                   
               
               
                 SEQ ID NO: 7 for the amino acid sequence of the 
               
               
                 heavy chain of RAB0: 
               
               
                 EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYAMDWVRQA 
               
               
                   
               
               
                 PGKGLEWVSG IYPSGGRTKY ADSVKGRFTI SRDNSKNTLY 
               
               
                   
               
               
                 LQMNSLRAED TAVYYCARVN VIAVAGTGYY YYGMDVWGQG 
               
               
                   
               
               
                 TTVTVSSAST KGPSVFPLAP CSRSTSESTA ALGCLVKDYF 
               
               
                   
               
               
                 PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS 
               
               
                   
               
               
                 SSLGTKTYTC NVDHKPSNTK VDKRVESKYG PPCPPCPAPE 
               
               
                   
               
               
                 FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSQEDPEV 
               
               
                   
               
               
                 QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV SVLTVLHQDW 
               
               
                   
               
               
                 LNGKEYKCKV SNKGLPSSIE KTISKAKGQP REPQVYTLPP 
               
               
                   
               
               
                 SQEEMTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT 
               
               
                   
               
               
                 TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF SCSVMHEALH 
               
               
                   
               
               
                 NHYTQKSLSL SLGK, 
               
               
                   
               
               
                 SEQ ID NO: 8 for the amino acid sequence of the 
               
               
                 heavy chain of RAB2: 
               
               
                 EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYAMDWVRQA 
               
               
                   
               
               
                 PGKGLEWVSG IVPSGGFTKY ADSVKGRFTI SRDNSKNTLY 
               
               
                   
               
               
                 LQMNSLRAED TAVYYCARVN VIAVAGTGYY YYGMDVWGQG 
               
               
                   
               
               
                 TTVTVSSAST KGPSVFPLAP CSRSTSESTA 
               
               
                   
               
               
                 ALGCLVKDYF PEPVTVSWNS GALTSGVHTF PAVLQSSGLY 
               
               
                   
               
               
                 SLSSVVTVPS SSLGTKTYTC NVDHKPSNTK VDKRVESKYG 
               
               
                   
               
               
                 PPCPPCPAPE FLGGPSVFLF PPKPKDTLMI SRTPEVTCVV 
               
               
                   
               
               
                 VDVSQEDPEV QFNWYVDGVE VHNAKTKPRE EQFNSTYRVV 
               
               
                   
               
               
                 SVLTVLHQDW LNGKEYKCKV SNKGLPSSIE KTISKAKGQP 
               
               
                   
               
               
                 REPQVYTLPP SQEEMTKNQV SLTCLVKGFY PSDIAVEWES 
               
               
                   
               
               
                 NGQPENNYKT TPPVLDSDGS FFLYSRLTVD KSRWQEGNVF 
               
               
                   
               
               
                 SCSVMHEALH NHYTQKSLSL SLGK, 
               
               
                   
               
               
                 SEQ ID NO: 9 for the amino acid sequence of 
               
               
                 RAM0hc: 
               
               
                 EVQLLESGGG LVQPGGSLRL SCAASGFTFS WYAMDWVRQA 
               
               
                   
               
               
                 PGKGLEWVSG IYPSGGRTKY ADSVKGRFTI SRDNSKNTLY 
               
               
                   
               
               
                 LQMNSLRAED TAVYYCARVN VIAVAGTGYY YYGMDVWGQG 
               
               
                   
               
               
                 TTVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF 
               
               
                   
               
               
                 PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS 
               
               
                   
               
               
                 SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP 
               
               
                   
               
               
                 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED 
               
               
                   
               
               
                 PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH 
               
               
                   
               
               
                 QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT 
               
               
                   
               
               
                 LPPSREEMTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN 
               
               
                   
               
               
                 YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE 
               
               
                   
               
               
                 ALHNHYTQKS LSLSPGK, 
               
               
                   
               
               
                 SEQ ID NO: 10 for the amino acid sequence of 
               
               
                 RAM0lc: 
               
               
                 DIQMTQSPGT LSLSPGERAT LSCRASQGVS SSSLAWYQQK 
               
               
                   
               
               
                 PGQAPRLLIY GTSSRATGIP DRFSGSASGT DFTLTISRLQ 
               
               
                   
               
               
                 PEDFAVYYCQ QYGRSLTFGG GTKVEIKRTV AAPSVFIFPP 
               
               
                   
               
               
                 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ 
               
               
                   
               
               
                 ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 
               
               
                   
               
               
                 LSSPVTKSFN RGEC, 
               
               
                   
               
               
                 SEQ ID NO: 11 for the amino acid sequence of 
               
               
                 RAM9hc: 
               
               
                 EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYSMNWVRQA 
               
               
                   
               
               
                 PGKGLEWVSS IGSSGGTTYY ADSVKGRFTI SRDNSKNTLY 
               
               
                   
               
               
                 LQMNSLRAED TAVYYCAGSQ WLYGMDVWGQ GTTVTVSSAS 
               
               
                   
               
               
                 TKGPSVFPLA PSSKSTSGGT AALGCLVKDY FPEPVTVSWN 
               
               
                   
               
               
                 SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI 
               
               
                   
               
               
                 CNVNHKPSNT KVDKRVEPKS CDKTHTCPPC PAPELLGGPS 
               
               
                   
               
               
                 VFLFPPKPKD TLMISRTPEV ICVVVDVSHE DPEVKFNWYV 
               
               
                   
               
               
                 DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 
               
               
                   
               
               
                 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSREEMT 
               
               
                   
               
               
                 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD 
               
               
                   
               
               
                 SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK 
               
               
                   
               
               
                 SLSLSPGK, 
               
               
                   
               
               
                 SEQ ID NO: 12 for the amino acid sequence of 
               
               
                 RAM9lc: 
               
               
                 DIQMTQSPSS LSASVGDRVT ITCRSSQRIM TYLNWYQQKP 
               
               
                   
               
               
                 GKAPKLLIFV ASHSQSGVPS RFRGSGSETD FTLTISGLQP 
               
               
                   
               
               
                 EDSATYYCQQ SFWTPLTFGG GTKVEIKRTV AAPSVFIFPP 
               
               
                   
               
               
                 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ 
               
               
                   
               
               
                 ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 
               
               
                   
               
               
                 LSSPVTKSFN RGEC, 
               
               
                   
               
               
                 SEQ ID NO: 13 for the amino acid sequence of 
               
               
                 RAM4hc: 
               
               
                 EVQLLESGGG LVQPGGSLRL SCAASGFTFS IYAMDWVRQA 
               
               
                   
               
               
                 PGKGLEWVSG IVPSGGFTKY ADSVKGRFTI SRDNSKNTLY 
               
               
                   
               
               
                 LQMNSLRAED TAVYYCARVN VIAVAGTGYY YYGMDVWGQG 
               
               
                   
               
               
                 TTVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF 
               
               
                   
               
               
                 PEPVTVSWNS GALTSGVHTF PAVLQSSGLY SLSSVVTVPS 
               
               
                   
               
               
                 SSLGTQTYIC NVNHKPSNTK VDKRVEPKSC DKTHTCPPCP 
               
               
                   
               
               
                 APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED 
               
               
                   
               
               
                 PEVKFNWYVD GVEVHNAKTK 
               
               
                   
               
               
                 PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA 
               
               
                   
               
               
                 PIEKTISKAK GQPREPQVYT LPPSREEMTK NQVSLTCLVK 
               
               
                   
               
               
                 GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL 
               
               
                   
               
               
                 TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK, 
               
               
                   
               
               
                 SEQ ID NO: 14 for the amino acid sequence of 
               
               
                 RAM4lc: 
               
               
                 DIQMTQSPGT LSLSPGERAT LSCRASQGVS SSSLAWYQQK 
               
               
                   
               
               
                 PGQAPRLLIY GTSSRATGIP DRFSGSASGT DFTLTISRLQ 
               
               
                   
               
               
                 PEDFAVYYCQ QYGRSLTFGG GTKVEIKRTV AAPSVFIFPP 
               
               
                   
               
               
                 SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ 
               
               
                   
               
               
                 ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG 
               
               
                   
               
               
                 LSSPVTKSFN RGEC. 
               
            
           
         
       
     
     The anti-MIF antibody of the invention is preferably an isolated monoclonal antibody. The anti-MIF antibody can be an IgG, an IgM, an IgE, an IgA, or an IgD molecule. In other embodiments, the anti-MIF antibody is an IgG1, IgG2, IgG3 or IgG4 subclass. In other embodiments, the antibody is either subclass IgG1 or IgG4. In other embodiments, the antibody is subclass IgG4. In some embodiments, the IgG4 antibody has a single mutation changing the serine (serine228, according to the Kabat numbering scheme) to proline. Accordingly, the CPSC sub-sequence in the Fc region of IgG4 becomes CPPC, which is a sub-sequence in IgG1 (Angal et al. Mol Immunol. 1993, 30, 105-108). 
     Additionally, the production of anti-(ox)MIF antibodies may include any method known in the art for the purification of an antibody, e.g., via anion exchange chromatography or affinity chromatography. In one embodiment the anti-(ox)MIF antibody can be purified from cell culture supernatants by size exclusion chromatography. 
     The terms “center region” and “C-terminal region” of MIF refer to the region of human MIF comprising amino acids 35-68 and aa 86-115, respectively, preferably as 50-68 and aa 86 to 102 of human MIF, respectively. Particularly preferred antibodies of the present invention bind to either region aa 50-68 or region aa 86-102 of human MIF. This is also reflected by the preferred antibodies of the invention, like RAB0, RAB4 RAB2 and RAB9 as well as RAM4, RAM9 and RAM0 which bind as follows: 
     RAB4 and RAM4: aa 86-102 
     RAB9 and RAM9: aa 50-68 
     RAB0 and RAM0: aa 86-102 
     RAB2: aa 86-102 
     The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or an antibody fragment. Epitopic determinants usually consist of chemically active surface groupings of molecules such as exposed amino acids, amino sugars, or other carbohydrate side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. 
     The term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In some embodiments, the vector is a plasmid, i.e., a circular double stranded DNA loop into which additional DNA segments may be ligated. 
     The term “host cell” refers to a cell line, which is able to produce a recombinant protein after introducing an expression vector. The term “recombinant cell line”, refers to a cell line into which a recombinant expression vector has been introduced. It should be understood that “recombinant cell line” means not only the particular subject cell line but also the progeny of such a cell line. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “recombinant cell line” as used herein. 
     The host cell type according to the present invention is e.g. a COS cell, CHO cell or, e.g., an HEK293 cell, or any other host cell known to a person skilled in the art, thus also for example including bacterial cells, like e.g.  E. coli  cells. In one embodiment, the anti-MIF antibody is expressed in a DHFR-deficient CHO cell line, e.g., DXB11, and with the addition of G418 as a selection marker. When recombinant expression vectors encoding antibody genes are introduced into CHO host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. 
     Anti-(ox)MIF antibodies can be recovered from the culture medium using standard protein purification methods. 
     The second active ingredient of the combination therapy as provided by the present invention is a chemotherapeutic. 
     Chemotherapeutic agents in the general sense thereof, are compounds, which can be used for the treatment of a disease or disorder that arises from bacterial, viral or parasitic infection or that is due to transformation of normal cells (cancer). One particular indication of chemotherapy is cancer. Chemotherapeutic agents can act for example by killing cells that divide more rapidly than other cells, and thus target cancer cells which commonly divide more rapidly than non-cancerous cells. Most chemotherapeutic agents work by impairing cell division at one of several stages of the cell cycle. Thus, they are able to target those cells that divide more rapidly. Chemotherapeutic agents can be either cytostatic, i.e., they slow down or abrogate the growth or division of cells; other chemotherapeutic agents can cause damage to cells and kill them; in that case they are termed cytotoxic. Most cytotoxic drugs inflict a damage that per se does not suffice to kill a cell but that generates a stimulus to initiate programmed cell death (apoptosis). 
     In general, major classes of chemotherapeutic drugs are alkylating agents, anti-metabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors and other anti-tumour agents. Most commonly, as mentioned above, these drugs affect one or several stages of the cell cycle; they can also affect DNA synthesis or DNA integrity. Other chemotherapeutics do not directly interfere with DNA. These are newer classes of chemotherapeutics and can include monoclonal antibodies and tyrosine kinase inhibitors like imatinib mesylate. Other examples are chemotherapeutic hormones and hormone antagonists, e.g. glucocorticosteroids. 
     Examples for alkylating agents, which alkylate nucleophilic functional groups are mechlorethamine, cyclophosphamide, chlorambucil, melphalane, trofosfamide, ifosfamide, carmustine, lomustine, dacarbazine, temozolomide, mitomycine C and many others. Cisplatin, carboplatin, oxaliplatin and other platinum containing compounds form stable complexes with DNA. 
     Cytotoxic anti-metabolites are folic acid analogues (e.g., methotrexat/aminopterin, raltitrexed, pemetrexed), purine analogs (e.g., 6-mercaptopurine, azathioprine, thioguanine, fludarabine, cladribine) or pyrimidine analogs (cytarabine, gemcitabine, 5-fuloruracil and its prodrugs, deazacytidine). Antimetabolites either inhibit DNA-synthesis by interfering with crucial steps in the de novo synthesis of purine and pyrimidine nucleotides or they become incorporated into DNA during the S-phase of the cell cycle, where they interfere with DNA-folding, DNA-repair or methylation. Alternatively, some compounds also become incorporated into RNA. Examples for alkaloids and terpenoids which are derived from plants and block cell division by preventing microtubule function are vinca-alkaloids and taxanes. Particularly well known vinca-alkaloids are vincristine, vinblastine, vinorelbine and vindesine. Podophyllotoxin is an additional example of a plant-derived compound. An example for a taxane is docetaxel or paclitaxel. Another example is abraxane, an albumin bound paclitaxel. Estramustin is an example of a synthetic compound that targets tubulin. 
     Examples of topoisomerase inhibitors, which are inhibitors of enzymes that maintain the topology of DNA, include camphtotecines like irinotecan and topotecan (type 1 topoisomerase inhibitors) or amsacrin, etoposide, etoposide phosphate and teniposide (topoisomerase-type 2 inhibitors). 
     Finally, examples of antineoplastic intercalating agents include dactinomycin, doxorubicin, epirubicin, bleomycin and others. 
     A comprehensive overview is comprised in Goodman and Gilman, The Pharmacological Basis of Therapeutics, 12 th  Edition, “General Principles of Cancer Chemotherapy”. 
     The following are examples for alkylating agents: 
     Mechlorethamine 
     Cyclophosphamide Ifosfamide 
     Melphalan 
     Chlorambucil 
     Procarbazine (N-methylhydrazine, MIH) 
     Busulfan 
     Camustine (BCNU) 
     Streptozocin 
     (streptozotocin) 
     Bendamustine 
     Dacarbazine (DTIC; dimethyltriazenol midazole carboxamide) 
     Temozolomide 
     Cisplatin, carboplatin, oxaliplatin 
     Antimetabolites are exemplary represented by 
     Methotrexate (Amethopterin) 
     Pemetrexed 
     Fluorouracil (5-fluorouracil; 5-FU), capecitabine 
     Cytarabine (cytosine arabinoside) 
     Gemcitabine 
     5-aza-cytidine 
     Deoxy-5-aza-cytidine 
     Mercaptopurine (6-mercaptopurine; 6-MP) 
     Pentostatin (2-deoxycoformycin) 
     Fludarabine 
     Clofarabine 
     Nelarabine 
     while Natural Products can be selected from: 
     Vinblastine 
     Vinorelbine 
     Vincristine 
     Paclitaxel, docetaxel 
     Etoposide 
     Teniposide 
     Topotecan 
     Irinotecan 
     Dactinomycin 
     (actinomycin D) 
     Daunorubicin 
     (daunomycin, rubidomycin) 
     Doxorubicin 
     Yondelis 
     Mitoxantrone 
     Bleomycin 
     Mitomycin C 
     L-Asparaginase 
     Examples for Hormones and Antagonists are: 
     Mitotane (o.p′DDD) 
     Prednisone 
     Hydroxyprogesterone caproate, 
     medroxyprogesterone acetate, megestrol acetate 
     Dietyhlstilbestrol, ethinyl estradiol 
     Tamoxifen, toremifene 
     Anastrozole, letrozole, exemestane 
     Testosterone propionate, fluoxymesterone 
     Flutamide, casodex 
     Leuprolide 
     while examples for further agents are: 
     Hydroxyurea 
     Tretinoin, arsenic trioxide 
     Histone deacetylase inhibitor (vorinostat) 
     Imatinib 
     Dasatinib, nilotinib 
     Gefitinib, erlotinib 
     Sorafenib 
     Sunitinib 
     Lapatinib 
     Bortezomib 
     Interferon-alfa, 
     Interleukin-2 
     Thalidomide 
     Lenalidomide 
     Temsirolimus, 
     Everolimus 
     Chemotherapeutics have been shown to be successful in alleviation and treatment of cancer. However, most chemotherapeutics are associated with a range of side effects which are in some cases extreme, to the extent that the treatment has to be abrogated. In any case, the side effects place a further burden on the physical and mental health of a patient and should thus be avoided as far as possible. 
     With the present invention, by combining a chemotherapeutic with an anti-MIF antibody, it is now possible to reduce the amount of the chemotherapeutic agent which is necessary for a given treatment compared to a situation where the chemotherapeutic agent is given as the sole active ingredient. A further possibility enabled by the present invention is to maintain the dose of the chemotherapeutic as compared to the chemotherapeutic given alone and have a much higher treatment response in the patient. 
     This increase of the treatment response in the patient also indicates the possibility to achieve a treatment response as with a chemotherapeutic alone, with a combination of anti-MIF antibody with a lower dose of chemotherapeutic agent, e.g. in cases where the side effects of the chemotherapeutic do not allow continuous treatment with the higher dose. 
     It was shown quite surprisingly by the present inventors that the effect obtained by combining a chemotherapeutic with an anti-MIF antibody showed a much higher treatment response than with either the anti-MIF antibody or the chemotherapeutic agent alone. 
     A treatment response can easily be determined by a person skilled in the art and refers to diminishing or ameliorating or alleviating a given condition. Methods to determine such a treatment response are well known and can be for example determination of the likelihood or length of survival of a subject having a disease and being treated with a combination of MIF antagonist and chemotherapeutic agent with the likelihood or length of survival in other subjects having the same disease and being treated with either agent alone, or by determining the change of symptoms within one and the same patient over a period of time. An example well known to a person skilled in the art is the Kaplan-Meier-Plot. 
     Other methods/assays are well known and can be derived for example from general textbooks, like The Pharmacological Basis of Therapeutics, 12 th  Edition, “General Principles of Cancer Chemotherapy. Introduction”. This reference is incorporated hereby in its entirety by reference; additional methods/assays are those as described in the present examples. 
     Preferred chemotherapeutics according to the present invention are doxorubicin and gemcitabine. Doxorubicin can be used in a preferred embodiment in combination with cisplatin. 
     Also preferred is e.g. abraxane. 
     Particularly preferred combinations are
         a treatment of ovarian cancer with doxorubicin, preferably in combination with cisplatin, together with an anti-MIF antibody, or   a treatment of pancreatic carcinoma with gemcitabine and/or abraxane, preferably together with an anti-MIF antibody.       

     In a preferred embodiment of the above combinations the anti-MIF antibody is selected from the group of RAB9, RAB4 and RAB0. 
     “Cancer” in the present context encompasses all disorders or diseases in which a cell or a group of cells displays uncontrolled growth, invasion (Intrusion and destruction of adjacent tissues) and sometimes metastasis. 
     Further, in a preferred embodiment, the cancer can be MIF-related. MIF-related cancers are e.g. lymphoma, sarcoma, prostatic cancer and colon cancer, bladder cancer, pancreas cancer, ovarian cancer, melanoma, hepatocellular carcinoma, ovarian cancer, breast cancer and pancreatic cancer, as well as endometriosis. Possible dosage forms which are envisaged by the present application are tablets, capsules, sachets or pills. The granules can be used as such as a preferred dosage form, can be filled into capsules or sachets or can be further compressed into tablets or pills. 
     Further dosage forms which are also encompassed by the present application are drinks or syrups, elixirs, tinctures, suspensions, solutions, hydrogels, films, lozenges, chewing gums, orally disintegrating tablets, mouth-washes, toothpaste, lip balms, medicated shampoos, nanosphere suspensions and microsphere tablets, as well as aerosols, inhalers, nebulisers, smoking or freebase powder forms and dosage forms for topical application like creams, gels, liniments or balms, lotions, ointments, ear drops, eye drops and skin patches. 
     Further encompassed are suppositories which can be used e.g. rectally or vaginally. All these dosage forms are well-known to a person skilled in the art. 
     Preferred dosage forms according to the present invention are oral forms like granules, coated granules, tablets, enteric coated tablets, pellets, suppositories and emulsions. Even more preferred are granules and tablets. Other preferred dosage forms are parenteral or topical dosage forms. A particular preferred administration route for the anti MIF antibody is a subcutaneous or intravenous application. A preferred administration route for the chemotherapeutic agent is oral application (e.g., a granule, liquid, sachet or tablet). A further preferred application form for the chemotherapeutic is topical application, wherein a topical application can encompass an application to the skin and/or a spray, like a nasal spray or inhaler. A further preferred administration route for a chemotherapeutic is an intravenous application or an application via a subcutaneous injection (including slow release formulations). 
     The administration can however principally be by all known routes. 
     The term “combination” or “combination therapy” are used interchangeably here. They refer to a dosing regimen where the anti-MIF antibody is administered together with or sequentially to the chemotherapeutic or vice versa. The dosing regimen would be typically daily for chemotherapeutics and every 2 weeks for the anti-MIF antibody. Preferred dosing regimens are: 
     As explained above, it is possible to administer the anti-MIF antibody together with the chemotherapeutic agent(s) or sequentially. “Together with” in this context means that not more than 10 minutes have passed between the administration of the anti-MIF antibody and the administration of the chemotherapeutic. “Sequentially” means that more than 10 minutes have passed between the administration of the anti-MIF antibody and the administration of the chemotherapeutic agent. The time period can then be more than 10 min., more than 30 minutes, more than 1 hour, more than 3 hours, more than 6 hours or more than 12 hours. Anti-MIF antibody and chemotherapeutic agents are principally dosed in a way to ensure that both compounds are present within the body during the same time period (for a certain time span). An anti-MIF antibody has a half-life of typically 2-4 weeks, chemotherapeutic agents a half-life of 2-48 hours. 
     Therefore, the above combination therapy also explicitly encompasses a sequential dosing regime where the skilled person takes into account the well known half life of the respective chemotherapeutic drug in question and the antibody in question. In view of the fact that antibodies generally have a half-life of 2-4 weeks, administration of the antibody in question could be only every 2 weeks, every 3 weeks or once a month. The chemotherapeutic drug to be administered in the inventive combination therapy with such an antibody has in a typical embodiment a half-life of 2-48 h; therefore, administration of the chemotherapeutic could be every 5 hours, every 6 hours, three times a day, twice a day, once daily, once a week or once per three week cycle in a typical embodiment. 
     Dosing of chemotherapeutics agent, as well as the combined dosing with antibodies, according to the present invention, however, will need to be determined by the practitioner on a case-by-case basis according to the specific disorder to be treated and the particulars of the afflicted subject. The person of skill in the art is aware of the respective guidelines for a given chemotherapeutic agent. 
     As a general understanding in curative chemotherapy, one would wish to apply the highest tolerated dose to achieve the desired dose intensity. The dose is reduced only if there is toxicity (i.e., neutrophil counts &lt;4000 (but &gt;2500)=administer half the dose, see cisplatin or cyclphosphamide). Most chemotherapeutic agents are administered on the basis of (m)g/m 2  body surface. Differences in tolerance and efficacy between mouse, rat and man are typically accounted for by basing the dose on body surface. 
     In a particularly preferred embodiment, the active ingredient would be an ingredient which should be delivered with a controlled, e.g. a delayed release. That is, the orally administrable dosage forms of the present invention comprising such an active ingredient might be provided with a coating. Thus, in a preferred embodiment the present invention is directed to granules with coatings and in particular to granules comprising active ingredients which shall be released in a controlled manner, whereby these granules have a coating. 
     More preferred, this coating is pharmacologically acceptable coating and particularly preferred is an enteric coating, a prolonged release coating or a delayed release coating; all such coatings are well known to a person skilled in the art. 
     A subset of in vivo protective anti-MIF mAbs (e.g. RAB9, RAB4, RAB0), which are directed against the pro-inflammatory cytokine MIF (Macrophage Migration Inhibitory Factor) do not bind to unmodified MIF in its reduced state. By contrast, these mAbs were shown to be highly selective for a redox dependent MIF isoform. 
     A particularly preferred antibody is antibody RAB9. 
     Another particularly preferred antibody is antibody RAB4. 
     Yet another particularly preferred antibody is antibody RAB0. 
     A very preferred antibody is antibody RAM9. 
     As is shown by the present invention, the combination therapy proposed here is advantageous in that it results in a synergistic effect of both components. 
     The present invention will be in the following described by way of the examples, whereby the examples shall be considered by no means as limiting the present invention. 
     REFERENCE EXAMPLES 
     A) GCO-Assay for Antibody Screening 
     A THP1 suspension culture is centrifuged and cells are resuspended in fresh full medium to a cell density of 10 6  cells per m. This culture is transferred into wells of a 96-well microplate (90 μl/well) and a potential anti-MIF antibody is added to give a final concentration of 75 μg/ml. Each antibody is tested in triplicate. After o/n incubation at 37° C. dexamethasone is added to give a concentration of 2 nM and after one hour incubation at 37° C. LPS is added (3 ng/ml final concentration). After further six hours incubation at 37° C. the supernatant is harvested and the IL-6 concentrations are determined in a commercially available ELISA. The results of the triplicates are averaged and the percentage of IL-6 secretion is determined in comparison to the control antibodies. Antibodies that result in an IL-6 secretion of less than 75% are evaluated as positive. 
     B) Assay for Determination of IC Values 
     The experimental procedure is carried out as described for the screening assay with the exception that increasing amounts of antibody are used (typically from 1-125 nM). The resultant dose response curve is expressed as % inhibition in comparison to a negative control antibody. This curve is used for calculation of the maximum inhibitory effect of the antibody (% Inh max) and the antibody concentration that shows 50% of the maximum inhibitory effect (IC50). 
     C) Inhibition of Cell Proliferation 
     Serum stimulates secretion of MIF in quiescent NIH/3T3 and MIF in turn stimulates cell proliferation. Antibodies inhibiting this endogenous MIF, therefore, decrease the proliferation of quiescent NIH3T3 cells. The reduction of proliferation is determined by the incorporation of  3 H-thymidine. 
     1000 NIH/3T3 cells per well are incubated in a 96 well plate over the weekend at 37° C. in medium containing 10% serum. Cells are then starved over night at 37° C. by incubation in medium containing 0.5% serum. The 0.5% medium is removed and replaced by fresh medium containing 10% serum, 75 μg/ml antibody and 5 μCi/ml of 3H-thymidine. After 16 hours incubation in a CO 2  incubator at 37° C. cells are washed twice with 150 μl of cold PBS per well. Using a multi-channel pipette 150 μl of a 5% (w/v) TCA solution per well are added and incubated for 30 minutes at 4° C. Plates are washed with 150 μl PBS. Per well 75 μl of a 0.5M NaOH solution with 0.5% SDS are added, mixed and stored at room temperature. Samples are measured in a β-counter by mixing 5 ml of Ultima Gold (Packard) and 75 μl sample solution. Each determination is done in triplicate and the values are compared with the values of the control antibody by a t-test. Antibodies that significantly reduce proliferation (P&lt;0.05) are evaluated as positive. 
     D) Binding Studies: Epitope Determination of Anti-MIF Antibodies 
     Each peptide is diluted in coupling buffer to give a peptide concentration of typically 1 μg/ml added to microplates (NUNC Immobilizer™ Amino Plate F96 Clear) and incubated over night at 4° C. (100 μl/well). As controls recombinant full length MIF and PBS are used. The plate is washed 3 times with 200 μl PBST and antibodies (2-4 μg/ml in PBS) are added (100 μl/well) and incubated for 2 hours at room temperature with gentle shaking. The plate is washed 3 times with 200 μl PBST and detection antibody (e.g. Fc specific anti-human IgG/HRP labelled, Sigma) is added (100 μl/well). After incubation for 1 hour at room temperature with gentle shaking, the plate is washed 3 times with 200 μl PBST. Each well is incubated with 100 μl TMB (3,3′,5,5′-tetramethylbenzidine) solution (T-0440, Sigma) for 30 minutes in the dark. Staining reaction is stopped by adding 100 μl of 1.8 M H 2 SO 4 -solution per well. Samples are measured at 450 nm. 
     E) Affinity Determination of Fab Fragments of Anti-MIF Antibodies by Biacore 
     Typically, 40 RU units of human recombinant MIF are immobilized on a sensor chip with a CM5 (=carboxymethylated dextran) matrix (Biacore). Fab fragments are injected at a concentration range of typically 6-100 nM diluted in HBS-EP. After each cycle the chip is regenerated with 50 mM NaOH+1 M NaCl. Affinities are calculated according to the 1:1 Langmuir model. 
     EXAMPLES 
     Introduction 
     Therapy of cancer and hyperproliferative disorders using chemotherapeutic drugs is often hampered by severe side effects or a required increase of dosage. In addition, many tumors have proven to be resistant to chemotherapeutic intervention. In this invention we describe that the effectiveness of chemotherapeutic compounds (e.g., gemcitabine, doxorubicin, or cisplatin) can be increased by combination with a MIF antagonist (e.g. an anti-oxMIF antibody). Such a combination has the potential to result in an improved therapy of cancer and hyperproliferative disorders in comparison to the respective monotherapy and to prolong significantly the life expectancy of patients. 
     Overview 
     
       
         
           
               
               
               
               
               
             
               
                   
               
               
                 Tumor 
                 Chemotherapy 
                 Antibody 
                 In vivo/in vitro 
                 Effect 
               
               
                   
               
             
            
               
                 Pancreatic 
                 Gemcitabine 
                 RAM9 
                 In vivo 
                 Synergy 
               
               
                 cancer 
                   
                 RAM0 
                 In vivo 
                 Synergy 
               
               
                 Ovarian 
                 Doxorubicin 
                 RAM0 
                 In vitro 
                 Synery 
               
               
                 cancer 
                   
                 RAM9 
                 In vitro 
                 Synergy 
               
               
                   
                 Cisplatin 
                 RAM0 
                 In vitro 
                 Synergy 
               
               
                   
                   
                 RAM0 
                 In vivo 
                 Synergy 
               
               
                 Prostate 
                 Mitoxantrone 
                 RAM0 
                 In vitro 
                 Synergy 
               
               
                 cancer 
               
               
                   
               
            
           
         
       
     
     Anti-MIF in Combination with Antimetabolites 
     (Exemplified by a Combinations of Anti-MIF Antibodies with Gemcitabine) 
     Example 1 
     Fully Human Anti-MIF Antibody RAM9 in Combination with Gemcitabine in a Xenograft Model for Pancreatic Cancer Using the BxPC3 Cell Line 
     BxPC3 cells are derived from a human pancreatic adenocarcinoma and grow inside the pancreas once they have been injected orthotopically in nude mice and present as a solid tumor. CD1-Foxn1/nu nude mice are orthotopically inoculated with 1×10 6  BxPC3 cells (injection of 30 μl matrigel:tumor cell suspension into the pancreas) and the tumor was allowed to establish for 7 days. Mice were treated with 100 mg/kg of gemcitabine (administered every three days for four injections in total) in combination with 60 mg/kg of RAM9 (administered every other day for the duration of the experiment). Mice of control groups were treated with 100 mg/kg gemcitabine alone or with 60 mg/kg of RAM9 alone as monotherapy. Additional control groups were left untreated or received a control human IgG1 monoclonal antibody. The growth of internal tumors was judged by external signs (e.g. abdominal distension causing more than 20% increase in normal body weight, or a 20% loss in normal body weight) or clinical signs (e.g. dyspnoea, jaundice, neurological signs, digestive disturbances). Animals reaching the home office limit according to the UK home office guidance were sacrificed (for animal welfare reasons). The resulting survival curve was used as primary outcome of the study. 
     Gemcitabine as a monotherapy only marginally prolonged the survival of the xenografted mice and had, in fact, a similar effect as RAM9 monotherapy. However, anti-MIF antibody RAM9 in combination with gemcitabine conferred a substantial survival advantage as compared to either agent alone and led to an overall survival of 20% of the mice, suggesting a synergistic effect of the two agents ( FIG. 1 ). 
     Example 2 
     Fully Human Anti-MIF Antibody RAM0 in Combination with Gemcitabine in a Xenograft Model for Pancreatic Cancer Using the COLO357 Cell Line 
     CD1-Foxn1/nu nude mice are orthotopically inoculated with 1×10 5  Colo357 cells (injection of 30 μl matrigel:tumor cell suspension into the pancreas) and the tumor was allowed to establish for 7 days. Mice were treated i.p. with 100 mg/kg of gemcitabine (administered every three days for four injections in total) and 60 mg/kg of RAM0 (administered every other day for the duration of the experiment). Mice of control groups were treated with 100 mg/kg gemcitabine alone or with 60 mg/kg of RAM0 alone as monotherapy. Additional control groups were left untreated or received a control human IgG1 monoclonal antibody. The growth of internal tumors was judged by external signs (e.g. abdominal distension causing more than 20% increase in normal body weight, or a 20% loss in normal body weight) or clinical signs (e.g. dyspnoea, jaundice, neurological signs, digestive disturbances). Animals reaching the home office limit according to the UK home office guidance were sacrificed (for animal welfare reasons). The resulting survival curve was used as primary outcome of the study. 
     Gemcitabine as a monotherapy only prolonged survival to a marginal and statistically insignificant extent RAM0 as a monotherapy already has a significant effect on survival. However, the beneficial effect of the combination “gemcitabine plus anti-MIF antibody” is greater than the effect of the two agents alone, thus indicating a synergistic effect between gemcitabine and the anti-MIF antibody ( FIG. 2A ). The prolongation of survival time correlates with reduced tumor volume and the combination treatment resulted in a strongly reduced tumor volume ( FIG. 2B ). 
     We further analyzed Wood vessel density in a small cohort of animals. All mice were orthotopically injected with Colo357 cells, tumours were established for 1 week and the different groups were treated as described above. On day 12, mice were anesthetized and injected with FITC-conjugated tomato lectin to visualize the blood vessel density within the tumor. The lectins were allowed to circulate for 3 min before animals were perfused with 4% paraformaldehyde. Tumors were cut into 50 μm sections. Cell nuclei were stained using propidium iodide, and vessels were visualized using confocal microscopy (Zeiss LSM 510 META), and microvessel density was quantified using ImagePro Plus software (Image-Pro plus, Media Cybernetics). Vessel density was expressed as the mean 
     percentage of total surface area Our results indicate that the synergistic effect of the combination of RAM0 with gemcitabine normalises blood vessel density in the tumour microenvironment. Neither gemcitabine nor RAM0 have a significant effect as monotherapy ( FIG. 2C ). 
     Anti-MIF in Combination with Intercalating Agents 
     (Exemplified by a Combinations of an Anti-MIF Antibody with Doxorubicin) 
     Example 3 
     Induction of Apoptosis in Doxorubicin-Resistant Human Ovarian Cancer Cells by Combined Application of Doxorubicin and Anti-MIF Antibodies 
     In an in vitro assay, doxorubicin resistant A2780adr ovarian cancer cells were treated either with anti-MIF antibodies RAM9 or RAM0 alone or in combination with doxorubicin. The induction of apoptosis was assessed by detection of caspase 3 activity after 72 hours of treatment. Non-treated cells or cells treated with antibody alone did not show any enhanced caspase 3 activity compared to isotype control antibody treated or untreated cells. Doxorubicin induced a significant but minor enhanced caspase 3 activity. The combination of anti-MIF antibodies and doxorubicin further enhanced caspase 3 activity and therefore the induction of apoptosis. The results show, that a previously doxorubicin resistant cell line becomes sensitive to this chemotherapy drug by combined application with MIF neutralizing antibodies. Combination of Doxorubicin with RAM0 ( FIG. 3A ) or RAM9 ( FIG. 3B ) gave comparable results. These results confirm the synergistically increased caspase activity observed in vivo when anti-MIF is combined with an antimetabolite. 
     Anti-MIF in Combination with Alkylating Agents 
     (Exemplified by a Combinations of an Anti-MIF Antibody with Cisplatin) 
     Example 4 
     Anti-MIF Antibody Sensitizes A2780 Ovarian Cancer Cells to the Action of Cisplatin In Vitro 
     The synergistic effect of cisplatin and an anti-MIF antibody was demonstrated in a cisplatin-dependent cell killing assay. A2780 cells were incubated with increasing concentrations of cisplatin either in the absence or in presence of 50 nM RAM0 or a human isotype control antibody. After 48 hours of incubation cells were detached with Accutase™, labeled with calcein-AM and the level of calcein fluorescence was determined by flow cytometry. The mean fluorescence intensity in the absence of any drug or antibody was set to 1 to normalize for interassay variations. The mean fluorescent intensities were blotted against the cisplatin concentration and the half maximum active concentrations of cisplatin (EC50-values) were calculated. The EC50-value was reduced significantly (p&lt;0.01) when cisplatin was combined with RAM0 ( FIG. 4 ). The anti-MIF antibody as a monotherapy (in absence of cisplatin) has no effect on cell killing in comparison to the isotype control antibody (data not shown). 
     Example 5 
     Anti-MIF Antibody Sensitizes A2780 Ovarian Cancer Cells to the Action of Cisplatin In Vivo 
     The data summarized above showed that, in cell culture, RAM0 rendered ovarian cancer cells more susceptible to cell killing by cisplatin. This effect is of high relevance, in particular if it can also be observed in vivo. This was explored in female Mf1 nude mice inoculated with A2780 xenografts ( FIG. 5 ). Mice (n=10/group) were inoculated with 1*10 6  A2780 cells resuspended in matrigel. RAM0 (15 mg/kg) or a human isotype control antibody (15 mg/kg) was injected every other day starting one day after inoculation of the xenograft. Cisplatin was administered once a week starting on day 2 after xenograft inoculation. Cisplatin did not have any effect on tumor growth as a monotherapy. RAM0 had a minor but not significant effect. However, RAM0 sensitizes the tumor to cisplatin treatment and the combination of cisplatin and RAM0 shows a significantly reduced tumor growth (p=0.04). 
     Anti-MIF in Combination with Intercalating Agents/Natural Agents 
     (Exemplified by a Combinations of an Anti-MIF Antibody with Mitoxantrone) 
     Example 6 
     LnCAP Prostate Cancer Cells are Sensitized to the Cytotoxic Action of Mitoxantrone by Anti-MIF Antibody RAM0 
     The synergistic effect of mitoxantrone and an anti-AF antibody was demonstrated in a mitoxantrone-dependent cell killing assay. LnCAP cells were incubated with increasing concentrations of mitoxantrone either in the absence or in presence of 100 nM RAM0 or a human isotype control antibody. After 48 hours of incubation cells were detached with Accutase™, labeled with calcein-AM and the level of calcein fluorescence was determined by flow cytometry. The mean fluorescence intensity in the absence of any drug or antibody was set to 1 to normalize for interassay variations. The mean fluorescent intensities were plotted against the mitoxantrone concentration and the half maximum active concentrations of mitoxantrone (EC 50 -values) were calculated. 10 independent EC 50  values were determined for each mitoxantrone/antibody combination (n=10) and the results are depicted in  FIG. 6 . The mean ECs-value for mitoxantrone alone was determined to be 1.6 nM. The combination of mitoxantrone and a control antibody gave the identical mean EC 50 . However, the mean ECs was significantly reduced to 0.97 nM when mitoxantrone was combined with RAM0. The anti-MIF antibody as a monotherapy (in absence of mitoxantrone) has no effect on cell killing in comparison to the isotype control antibody (data not shown). 
     CONCLUSIONS 
     The strong enhanced efficacy of combining a MIF antibody with standard of care chemotherapeutic drugs has been shown in the above different experiments. In a spontaneous pancreas model an anti-MIF antibody was tested in combination with an antimetabolite (gemcitabine). Compared to the expected additive outcome of anti-MIF antibody and chemotherapeutic drug, the combination resulted in a significantly prolonged survival of the mice. Secondly, in an in vitro assay, a doxorubicin-resistant cell line was rendered doxorubicin-sensitive by co-incubation with anti-MIF antibodies as demonstrated by the increase of caspase activity. In addition, anti-MIF antibody cisplatin resistant cancer cells were rendered cisplatin sensitive in an in vivo model. Furthermore, in an in vitro assay, a significant reduction of mean ECs could be shown for a combination of mitoxantrone and an anti-MIF antibody. This is the first description of a synergistic effect of chemotherapeutic drugs with MIF-inhibitors. A combination therapy using chemotherapeutic agents and MIF antibodies can thus overcome current limitations of chemotherapeutic intervention and improve the therapeutic result in patients with hyperproliferative disorders.