WO2015071416A2 - Facteur d'inhibition de la migration des macrophages en tant que cible therapeutique - Google Patents

Facteur d'inhibition de la migration des macrophages en tant que cible therapeutique Download PDF

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Publication number
WO2015071416A2
WO2015071416A2 PCT/EP2014/074617 EP2014074617W WO2015071416A2 WO 2015071416 A2 WO2015071416 A2 WO 2015071416A2 EP 2014074617 W EP2014074617 W EP 2014074617W WO 2015071416 A2 WO2015071416 A2 WO 2015071416A2
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mif
modified
antibody
modification
compound
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WO2015071416A3 (fr
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Michael Thiele
Alexander SCHINAGL
Friedrich Scheiflinger
Randolf Kerschbaumer
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Baxter Healthcare SA
Baxter International Inc
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Baxter Healthcare SA
Baxter International Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

Definitions

  • the present invention relates to applications of specific forms of the MIF protein in medicine.
  • the invention is based upon the recognition that modification of position 81 of MIF determines the switch of MIF to its disease- associated state.
  • Macrophage migration inhibitory factor 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).
  • MIF is known as a critical upstream regulator of the innate and acquired immune response that exerts a pieiotropic spectrum of activities.
  • the human MiF cDNA was cloned in 1989 (Weiser et al., PNAS 1989, 86, 522-6), 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 antiparailel 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, 5191-5
  • MIF secretion from macrophages was induced at very low concentrations of glucocorticoids (Calandra et al. Nature 1995, 377, 68-71).
  • MIF also counter-regulates the effects of glucocorticoids and stimulates the secretion of other cytokines such as tumor necrosis factor TNF-a and interleukin IL-1 ⁇ (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.
  • MIF is a mediator of many pathologic conditions (i.e., MIF-related disorders) 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 (Ml), sepsis and cancer, though not limited thereto, and MIF also mediates many other MIF-related disorders.
  • IBD inflammatory bowel disease
  • RA rheumatoid arthritis
  • ARDS acute respiratory distress syndrome
  • asthma glomerulonephritis
  • IgA nephropathy IgA nephropathy
  • Ml myocardial infarction
  • sepsis sepsis and cancer, though not limited thereto
  • MIF also mediates many other MIF-related disorders.
  • Polyclonal and monoclonal anti-MIF antibodies have been developed against recombinant human M
  • 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.
  • 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 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
  • MIF is a molecule which is involved in a multitude of different interactions.
  • WO2013/050453 discloses that an oxidised form of MIF (oxMIF) can be detected after onset of (MIF-related) disorders, e.g. in body fluid samples, or on cells or cell surfaces and that oxMIF is correlated with a disease state and/or disease progression.
  • Antibodies that are specific to the disease-related, oxidised form of MIF (oxMIF-specific antibodies) are known.
  • oxMIF-specific antibodies are known.
  • there is a need for more information on the involvement of MIF in MIF-related disorders in order to further improve the treatment and prevention of MIF- related disorders and related medical applications.
  • the present invention achieves this objective by revealing that the switch of MIF from a non-pathogenic to a disease-related state, i.e. the redox-switch from redMIF to oxMIF is associated with and promoted by the presence of a modification of position 81.
  • the disease-related, oxidised form of MIF - oxMIF - is defined by the binding of oxMIF-specific antibodies, i.e. antibodies which bind preferentially to oxMIF as opposed to redMIF which is not particularly correlated with disease.
  • the inventors have shown that the modification of M!F at position 81 (cysteine 81) by treatment with sulfhydryi -reactive agents induces binding of such oxMIF- specific antibodies.
  • cysteine 81 was mutated to serine, which is isosteric to cysteine but is not modified by sulfhydryi -reactive agents
  • treatment with sulfhydryi -reactive agents did not induce binding of oxMIF-specific antibodies.
  • the inventors have thus established a clear link between modification of MIF position 81 and the oxMIF conformation of the protein which is involved in MIF-related disorders.
  • the sequence of human MIF is represented by SEQ ID NO: 15.
  • the present invention thus provides the use of MIF carrying a modification at 81 as a target for the treatment or prevention of a MIF-related disorder.
  • the present invention thus relates to therapeutic methods of treating or preventing a disease state or disease progression in a subject in need thereof by administering to said subject an effective amount of a compound that
  • Said preferentially-binding compound may be an is an antibody or a molecule comprising an antigen- binding portion of an antibody and/or said preferentially-binding compound may induce a form of said modified MIF that binds the antibody RAMO, RABO, RAM9 or RAB9 (or any other oxMIF-specific antibody, e.g. selected from the oxMIF-specific antibodies disclosed herein) to a lesser extent than does said modified MIF in the absence of the compound.
  • references to “modification of MIF at position 81 " or "MIF modified at position 81 " or “MIF carrying a modification at position 81”, and the like, may be abbreviated by a reference to “modified MIF”.
  • modification refers throughout this application and in ail embodiments to a modification of MIF whereby the MIF in its modified form or state binds to the antibody RAM9 (and/or to RAB9, RAMO, RABO, and/or any other oxMIF-specific antibody, e.g. selected from any of the oxMIF-specific antibodies disclosed herein below).
  • a modification in particular at MIF position 81 may be a modification compared to an unmodified cysteine residue, or compared to the free cysteine sulfhydryl group. That is, in particular, a modification at MIF position 81 may be a modification of MIF cysteine 81 , and is preferably a modification on the sulphur atom of cysteine 81.
  • MIF position 81 carrying a modification preferably MIF cysteine 81 carries a modification compared to unmodified cysteine, or more preferably the sulphur atom of cysteine 81 carries a modification compared to the free cysteine sulfhydryl group, as described herein.
  • modification of the sulfhydryl group of a cysteine residue refers to derivattzation of the sulfhydryl group, i.e. the sulfhydryl group is derivatized. That is, the sulfur atom of a cysteine residue carries a modification compared to the free sulfhydryl group of cysteine, i.e. the sulfur atom of the cysteine is attached to a moiety other than H (i.e. to a modifying moiety).
  • the invention also encompasses a method of monitoring the effectiveness of a treatment of a MIF-related disorder, comprising a step of determining in samples isolated from a subject before and after said treatment whether MIF position 81 carries a modification, wherein the treatment is identified as effective if MIF position 81 is modified to a lesser extent after treatment than before treatment.
  • the invention also provides a method of assaying a test compound for preferential binding to a form of MIF which is modified at position 81 , said method comprising the steps of
  • the compound may be selected if said compound binds to the modified MiF in the test sample preferentially or differentially compared to the control sample.
  • the compound may be selected if said compound binds to the modified MIF in the test sample with greater affinity than in the control sample.
  • a test compound may, without limitation, preferably be an antibody or a molecule comprising an antigen-binding portion of an antibody.
  • the invention also provides a method of screening for a compound that prevents the modification of MIF at cysteine 81 , said method comprising the steps of
  • the compound is selected if the sulfhydrykeactive reagent modifies cysteine 81 to a lesser extent in the test sample than in the control sample.
  • the invention also provides a method of assaying a test compound for a conformational effect upon MIF which is modified at position 81 , said method comprising the steps of
  • the compound is selected if said conformational assessment indicates a detectable degree of similarity of the conformation of the modified MIF in the test sample with conformation (ii) compared to conformation (i), or if said conformational assessment indicates that the conformation of the modified MIF in the test sample otherwise detectably deviates from conformation (i).
  • the conformation of MiF may be assessed by an immunoassay.
  • the immunoassay employs an antibody selected from RAMO, RABO, RAM9 and RAB9 (and/or any other oxMIF-specific antibody, e.g. selected from oxMIF-specific antibodies disclosed herein ⁇ , and the compound is selected if the modified MIF in the test sample (i.e. in the presence of the test compound) binds the antibody to a lesser extent than does said modified MIF in the absence of the compound (control 1).
  • the modification of MIF position 81 in the case of "control 1" is the same as that in the test sample.
  • the conformation of MIF may be assessed by any methodology that is sensitive to a conformational change in M!F that depends on the presence of a modification of MIF at position 81 , in particular the presence or absence of a modification of MIF cysteine 81 , in particular on the sulphur atom of MIF at position 81.
  • the conformation of MIF may be assessed by immunoassay, e.g. using one or more conformation-sensitive anti-MIF antibodies as described herein, i.e. the binding of which is dependent on the modification state of MIF position 81.
  • the presence and/or absence of a modification of position 81 may be assessed by binding of a conformation-sensitive position-81-modification-selective antibody (and/or a conformation-selective cysteine 81 sulfhydryl-specific antibody), as described herein.
  • the conformation may be assessed by ELISA or surface plasmon resonance.
  • the conformation may be assessed by X-ray crystallographic analysis.
  • the conformation may, for example, also be assessed by nuclear magnetic resonance (NMR) analysis or by circular dichroism.
  • the invention also provides a method of assaying a test compound for a conformational effect upon MIF which is modified at position 81 , said method comprising the steps of
  • the compound is selected if the modified MIF in the test sample ⁇ i.e. in the presence of the test compound) binds the antibody to a lesser extent than does said modified MIF in the absence of the compound (control 1 ).
  • the invention also provides a method of assaying a test compound for binding to a modified form of MIF by X- ray crystallography, said method comprising the steps of
  • the same X-ray crystallographic method may encompass additionally carrying out said crystallisation and structure determination steps with same test compound but with MIF in which position 81 is not modified.
  • the invention also provides a crystal containing MIF with a modification of position 81.
  • Said crystal may optionally further contain another compound, for example a test compound, e.g. a test compound, the binding of which to said modified MIF is to be assessed.
  • a compound, e.g. for binding to MiF that is modified at position 81 e.g. a compound for use in a therapeutic or diagnostic method or a test compound to be assayed
  • a compound, e.g. for binding to MiF that is modified at position 81 is not particularly limited, but may e.g.
  • the invention also provides the use of position 81 of the MIF protein as a diagnostic marker and/or for monitoring MIF-related disorders.
  • the invention provides the use of the modification state of position 81 of the MIF protein as a diagnostic marker and/or for monitoring MIF-related disorders.
  • the present invention provides a method of diagnosing a MIF-related disorder, comprising a step of determining in a sample isolated from a subject whether MIF position 81 carries a modification, wherein, if MIF position 81 is identified as carrying such a modification, the subject is diagnosed with a MIF-re!ated disorder. That is, a finding that MIF position 81 carries such a modification is indicative of a MIF-related disorder.
  • the present invention provides a method of diagnosing a MIF-related disorder, comprising a step of determining in a sample isolated from a subject whether the sulphur atom of MIF cysteine 81 carries a modification compared to the free cysteine suifhydryl group, wherein, if the sulphur atom of MIF cysteine 81 is identified as carrying such a modification, the subject is diagnosed with a MIF-related disorder. That is, a finding that the sulphur atom of cysteine 81 carries such a modification is indicative of a MIF-related disorder.
  • the present invention provides the use of the modification state of MIF position 81 as a diagnostic marker, wherein a finding that MIF position 81 carries a modification is indicative of a MIF-related disorder.
  • the present invention provides the use of the modification state of the suifhydryl group of MIF cysteine 81 as a diagnostic marker, wherein a finding that the sulphur atom of MIF cysteine 81 carries a modification compared to the free cysteine suifhydryl group is indicative of a MIF-related disorder.
  • Such methods and uses according to the invention encompass methods and uses of determining whether modified MIF is involved in, or correlated with, a given disorder or pathologic condition
  • the invention also provides the use of a diagnostic kit in a method or use according to the invention, wherein the diagnostic kit comprises a compound that preferentially binds to MIF in which cysteine 81 is present, or to MIF carrying a modification at position 81 (i.e., in particular, on the sulfur atom of cysteine 81).
  • a kit may additionally comprise buffers, control reagents (e.g. recombinant MIF in which cysteine 81 is present or absent, or MIF with or without a modification position 81 , e.g. a modification on the sulfur atom of cysteine 81 , a compound that preferentially binds to any such forms of MIF), polyclonal anti-MIF antibody, and/or conjugated detection antibody.
  • control reagents e.g. recombinant MIF in which cysteine 81 is present or absent, or MIF with or without a modification position 81 , e.g. a modification on the sulfur atom of
  • MIF-related disorder encompasses, for example, a (MIF-related) disease, a (MIF- related) disease state, the state of progression of a (MIF-related) disease.
  • position 81 e.g. the suifhydryl group of MIF cysteine 81
  • the MIF is not substantially modified by the same modifying moiety at other positions, or, in particular, the MIF is not substantially modified by the same modifying moiety (i.e., does not substantially carry the same modification) at the sulphur atom of other cysteines in the MIF sequence.
  • MIF does not substantially carry the same modification on the sulphur atom of cysteine 57.
  • MIF does not substantially carry the same modification on cysteine 60.
  • cysteine 57 nor cysteine 60 of MIF are substantially modified by the same modifying moiety as cysteine 81.
  • cysteine 57 and cysteine 60 may be substituted by (i.e. mutated to, i.e.
  • cysteine 57 may be unmodified, i.e. may comprise a free sulfhydryl group.
  • cysteine 60 may be unmodified, i.e. may comprise a free sulfhydryl group.
  • both cysteine 57 and cysteine 60 are unmodified, i.e. comprise a free sulfhydryl group.
  • Methods of peptide mass fingerprinting to determine the modification of amino acid residues are well known to the skilled person in the relevant technical area, as part of the skilled person's common general knowledge. Methods of the invention may thus also encompass digesting MIF in a sample to be assessed with a protease (e.g. trypsin). Subsequently, peptides (MIF fragments) resulting form said digestion, in particularly a peptide containing cysteine 81 (and preferably containing no other cysteine residue) may be analysed by mass spectroscopy.
  • a protease e.g. trypsin
  • the assessment of whether position (cysteine) 81 is modified may also be performed by an immunoassay, i.e. using one or more antibodies. That is, the methods of the invention (e.g. diagnostic methods or methods of screening compounds) may encompass contacting MIF with one or more antibodies. This may lead to the formation of a complex between MIF (e.g. C81-modified MIF) and an antibody.
  • the assessment of whether position 81 is modified may thus be performed using an antibody that selectively binds to MIF in which position 81 (e.g. the sulfhydryl group of cysteine 81 is modified), i.e. binds a form of MIF in which position 81 ⁇ e.g. the sulfhydryl group of cysteine 81) is modified with preference compared to MIF with a free sulfhydryl group at cysteine 81.
  • binding of an antibody may depend upon the presence of a modification (e.g. of the sulfhydryl group) of position 81 of the MIF protein for (or at least for) the binding of the antibody to the MIF protein under non-reducing and/or oxidising and/or native conditions.
  • the binding epitope may include or may not include position 81.
  • the antibody may be an anti-MIF antibody (e.g. a conformation-sensitive antibody, the binding of which is dependent on the modification state of position 81), e.g.
  • cysteine 81 modification- specific antibody which binds to MIF specifically, selectively or preferentially when the sulphur atom of MIF cysteine 81 carries a modification compared to the free cysteine sulfhydryl group, compared to its binding to MIF in the absence of such a modification (i.e. MIF containing a free cysteine sulfhydryl group at position 81 ).
  • the binding epitope of the antibody may be an epitope (e.g. a linear or conformational epitope, which may or may not include cysteine 81 ), the accessibility of which for antibody binding is enhanced when position 81 is modified, compared to when MIF position 81 is unmodified.
  • the binding of the antibody to MIF may depend on the presence of a modification of cysteine at position 81 of the MIF protein, i.e. the antibody may bind MIF preferentially (e.g. under non-reducing conditions) when cysteine 81 is modified, compared to its binding to MIF when cysteine 81 is not modified (e.g. under reducing conditions), or when cysteine 81 is absent.
  • the antibody may be RAM9 or RAB9.
  • the antibody may also be RAMO or RABO.
  • Said binding may be dependent on the presence of cysteine 57 and/or cysteine 60 (as in the case of RAM9 or RAB9) or may be independent of the presence of cysteine 57 and/or cysteine 60, and/or dependent exclusively on the presence of a modification on the sulphur atom of MIF cysteine s!
  • Methods and uses of the invention may also employ anti-MIF antibodies (e.g. conformation-sensitive antibodies, the binding of which is dependent on the modification state of cysteine 81), e.g. cysteine 81 s u If hyd ryl-selective antibodies, which bind to MIF preferentially when the sulphur atom of MIF cysteine 81 carries a free cysteine sulfhydryl group, compared to their binding to MIF in which cysteine 81 is modified. Such antibodies may be employed, e.g., as controls.
  • anti-MIF antibodies e.g. conformation-sensitive antibodies, the binding of which is dependent on the modification state of cysteine 81
  • cysteine 81 e.g. cysteine 81 s u
  • hyd ryl-selective antibodies which bind to MIF preferentially when the sulphur atom of MIF cysteine 81 carries a free cysteine sulfhydry
  • position 81 e.g. the sulfhydryl group of MIF cysteine 81
  • position 81 may be identified as being modified if the mass of a MIF peptide containing cysteine 81 (but no other cysteine) is determined to be altered compared to the corresponding peptide containing a free sulfhydryl group at cysteine 81.
  • assessment or determination of which amino acid the MIF gene encodes cysteine at position 81 is performed by a method involving a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the present invention also relates to antibodies as disclosed herein which specifically recognize the presence of a modification at MIF position 81.
  • the invention also relates an anti-MIF antibody which specifically binds to MIF in which position 81 carries a modification (e.g. on the sulfur atom, compared to the free cysteine sulfhydryl group), wherein the antibody does not detectably bind, or binds with lower affinity, to MIF which does not contain said modification.
  • the present invention also relates to complexes between MIF having a modification at position 81 and antibodies as mentioned herein.
  • an antibody may be of any type, e.g. preferably of the lgG1 type or the lgG4 type.
  • an antibody referred to as "RAM” is of the lgG1 type
  • an antibody referred to RAB" is of the lgG4 type.
  • references to binding are to binding under physiologically relevant conditions (at any temperature between, e.g. 1 and 45°C), unless otherwise indicated.
  • references to binding encompass binding under crystallization conditions, which are well known to the person skilled in the art.
  • Preferential binding, or enhanced binding, etc. may herein also be referred to as differential binding.
  • preferential, enhanced or differential binding means that a compound, in particular the antibodies as described herein, binds (e.g. with a KQ value of less than 100 nM, preferably less than 50 nM, yet more preferably less than 10nM) to a first form of MIF; and binds to a lesser extent (e.g. with lower affinity, e.g. characterized e.g. by a KQ of more than 400 nM, or by an absence of detectable binding, i.e. the compound does not bind) to a second form of MIF.
  • Said first form of MIF may, e.g., be a form of MIF that is associated with a MIF-related disorder (e.g. in particular C81-modified MIF), and said second form of MIF may be a form which is not thus associated (e.g. does not contain a modified sulphur atom of C81 ), or vice versa.
  • a MIF-related disorder e.g. in particular C81-modified MIF
  • said second form of MIF may be a form which is not thus associated (e.g. does not contain a modified sulphur atom of C81 ), or vice versa.
  • differential binding may refer to binding with a Krj value of less than 100 nM (preferably less than 50 nM, yet more preferably less than 10nM) to MIF carrying a modification at position 81 (e.g. on the sulfur atom of cysteine 81) and lesser binding, characterized e.g. by a Kp of more than 400 nM, or an absence of detectable binding (i.e., the compound does not bind) to MIF which does not contain said modification on the sulfur atom of cysteine 81 (or vice versa).
  • a Krj value of less than 100 nM (preferably less than 50 nM, yet more preferably less than 10nM) to MIF carrying a modification at position 81 (e.g. on the sulfur atom of cysteine 81) and lesser binding, characterized e.g. by a Kp of more than 400 nM, or an absence of detectable binding (i.e., the compound does not bind) to MIF which does not contain said modification on the
  • modified MIF as a marker supplies information about the disease state, its progression and serves as a marker to determine effectiveness of a given treatment; in addition, modified MIF detection in a sample, e.g. a body fluid sample or a cell sample, can serve as an indicator for a preferred anti-MIF therapy.
  • the detection of modified MIF thus serves to improve known diagnostic techniques in a given disease or disorder. It assists the practitioner in his or her decision how to treat a given disease or disorder and helps to improve specificity of the diagnosis.
  • Modified MIF is thus a specific and suitable secondary marker. Its detection can serve as an adjunctive test in the management of patients afflicted with MIF related diseases.
  • the disease in question is in a preferred embodiment a disease which is known or suspected to be MIF related (see the diseases mentioned in detail below) but can also be a disease which had so far not been suspected to be MIF related.
  • the detection of the presence of modified MIF in a sample would indicate to the practitioner that the subject, from whom (or which) the sample has been taken, might benefit from a therapy directed against MIF.
  • a therapy could be selected from anti-MIF molecules, e.g. anti-modified M!F antibodies or small molecules which are directed against modified MIF.
  • Elevated MIF levels i.e. levels of MIF in general are detected after the onset of various diseases, infer alia after the onset of cancer.
  • MIF circuiates also in healthy subjects, which makes a clear differentiation difficult.
  • oxMIF on the contrary, which the present invention links to modified MIF, is not present in healthy subjects and therefore is a much stronger diagnostic marker for MIF-related disorders.
  • patient samples such as blood, serum and urine may be assayed for increased levels of modified MIF compared to healthy subjects.
  • oxMIF-specific antibodies which specifically bind to oxMIF and are incapable of binding to redMIF
  • Non-limiting examples of oxMIF-specific antibodies are Baxter antibodies AM9, RAB9, RAM4, RAB4, RAM0 and RAB0.
  • Oxidative procedures such as cystine-mediated oxidation, GSSG (ox. Glutathione)- mediated oxidation or incubation of MIF with Proclin300 or protein crosslinkers (e.g. BMOE) may all cause binding to the above mentioned antibodies.
  • the above-mentioned antibodies are characterized and supported by both their sequences as well as by deposits as plasmids in E.coli (strain TG1 ), comprising either the light or the heavy chain of each of the above mentioned antibodies.
  • the plasmids are characterized by their DS 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 £. co!i 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 251 12 number comprises the heavy chain (lgG4) 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 (lgG4) 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 251 14 number comprises the light chain sequence of the anti-MIF antibody RABO.
  • the plasmid with the DSM 251 15 number comprises the heavy chain (lgG4) sequence of the anti-MIF antibody RABO.
  • the co-expression of plasmids DSM 25114 and DSM 25115 in a suitable host cell results in the production of preferred anti-MIF antibody RABO.
  • 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.
  • RAMO - light chain E.coli GA.906-01.pRAMOIc - DSM 25863.
  • RAMO - heavy chain E.coli GA.784-01.pRAMOhc - DSM 25864.
  • a biological sample in the context of the present disclosure is preferably a body fluid sample of the subject on which/whom the diagnosis shall be performed.
  • a body fluid sample is any sample of a body fluid as known to a person skilled in the art. Exemplary, but not limiting, such a sample can be blood, plasma, serum, saliva, urine, nasal fluid, ascites, ocular fluid, amniotic fluid, aqueous humour, vitreous humour, tear fluid, Cowper's fluid, semen, interstitial fluid, lymph, breast milk, mucus (incl. snot and phlegm), pleural fluid, pus, menses, vaginal lubrication, sebum, cerebrospinal fluid and synovial fluid.
  • a biological sample in the context of this application can be lavages (washing outs) of a (hollow) body organ (e.g. bronchoalveolar lavage, stomach lavage and bowel lavage).
  • a biological sample in the context of this application in an alternative embodiment is a cell sample, most preferably a cell sample from the circulation or the diseased tissue, more preferably as a single cell suspension sample, of the subject on which the diagnosis shall be performed.
  • the diagnostic methods, assays and uses disclosed herein in particular methods of diagnosing a MIF-related disorder as disclosed herein, encompass methods of determining whether C81- modified MIF is involved in, or correlated with, a given disorder or pathologic condition.
  • the present invention thus also pertains to a method for evaluating the progression of a disease; in the present context the term "state of a disease” or “disease state” is to be understood as synonymous with the term “severity of a disease” and refers to the seriousness, degree or state (i.e. stage) of a disease or condition.
  • a disease may be characterised as mild, moderate or severe.
  • the determination or assessment of the degree of severity or the degree, i.e. state of the disease is well known to a person skilled in the art.
  • the actual method which will be carried out for this assessment of course depends on the disease or condition in question.
  • the state of a disease may be determined by comparing the likelihood or length of survival of a subject having a disease with the likelihood or length of survival in other subjects having the same disease.
  • the state of the disease may be determined by comparing the symptoms of a disease in a subject having a disease with the symptoms in other subjects having the same disease.
  • the state of the disease and its progression is reflected by the change of symptoms within one and the same patient over a period of time,
  • the present invention can also be directed to a method of selecting a subject as being eligible for a treatment with an anti-modified MIF compound, wherein the subject has a (MIF-related) disorder, or is at risk of developing a (MIF-related) disorder, comprising detecting the existence and/or level and/or change of level of modified MIF in said subject.
  • a subject having an elevated level of modified MIF can be selected for a prophylactic or therapeutic treatment with an anti modified MIF compound as defined above.
  • prophylactic or therapeutic treatment is art-recognized and refers to administration of a drug to a patient. If it is administered prior to clinical manifestation of the unwanted condition (e.g.
  • the treatment is prophylactic, i.e., it protects the host 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 thereof).
  • an anti-modified MIF compound refers to any agent that attenuates, inhibits, opposes, counteracts, or decreases the biological activity of modified MIF.
  • An anti-modified MIF compound may be an agent that inhibits or neutralizes modified MIF activity, for example a small molecule or an antibody.
  • Preferred antibodies are the antibodies as described herein, particularly, RAM9, RAB9, RAM4, RAB4, RAMO and RABO; preferably RAB9 or RABO; or RAM9 or RAMO.
  • Diagnostic methods or assays disclosed herein can be used to determine the presence or level of modified MIF in e.g. body fluid samples or cellular samples of patients.
  • the presence or absence of oxMIF is suitable to distinguish, if the disease if MIF relevant or to decide of oxMIF treatment is reasonable.
  • OxMIF levels indicate disease progression or treatment efficacy.
  • kits comprising an anti- modified MIF antibody or an antigen-binding portion thereof.
  • a kit may include in addition to the antibody, further diagnostic or therapeutic agents and uses thereof.
  • a kit also can include instructions for use in a diagnostic or therapeutic method.
  • 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), the sequence of which is represented by SEQ ID NO: 15. The monomelic 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, e.g.
  • an affinity tag which is useful for purification of the protein ⁇ , such as SBP (streptavidin binding peptide), optionally via a linker (e.g. a (GGGGS)2 linker), as is well known to the person skilled in the art.
  • a linker e.g. a (GGGGS)2 linker
  • 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), in accordance with SEQ ID NO: 15.
  • Oxidized MIF or oxMIF is defined herein as an isoform of MIF that occurs by treatment of MIF with mild oxidizing reagents (sulfhydryl-reactive reagents), such as cystine.
  • oxidizing reagents such as cystine.
  • Such oxMIF comprises isoform(s) of MIF that share structural rearrangements with oxMIF that (e.g.) occurs in vivo after challenge of animals with bacteria, it is bound specifically by the oxMIF-specific antibodies disclosed herein.
  • Reduced MIF or redMIF is defined for the purposes of this invention as reduced MIF and is MIF which does not bind to RABO, RAB9 and/or RAB4 and/or to RAMO, RAM9 and/or RAM4.
  • Binding kinetics of antibodies to the various forms of MIF disclosed herein may be examined by surface plasmon resonance analysis using a Biacore 3000 System.
  • Proclin300 consists of oxidative isothiazolones that stabilizes oxMIF structure by avoiding a conversion of oxMIF to redMlF.
  • Antibodies or antigen-binding portions thereof preferably bind a specific form of MIF disclosed herein (e.g. modified MIF) with a rj of less than 100 nM, preferably a of less than 50 n , even more preferred with a K D of less than 10 nM.
  • the antibodies of this invention bind to a specific form of MIF disclosed herein (e.g. modified MIF) with a KQ of less than 5 nM.
  • Non-binding of an antibody can be determined as generally known to a person skilled in the art, examples being any one of the following methods: Differential Binding ELISA with recombinant MIF, or surface plasmon resonance using recombinant MIF in any of the forms disclosed herein, like the well-known Biacore assay, described above.
  • a preferred method for the determination of binding is surface plasmon resonance of an antibody to the forms of MIF disclosed herein (e.g. modified MIF) wherein "binding" refers to a KQ of less than 100 nM, preferably less than 50 nM, even more preferred less than 10 nM whereas the non-binding (e.g. in the case of modified MIF or ox;!F-specific antibodies to redMlF) is characterized by a Krj of more than 400 nM.
  • Binding and “specific binding” is used interchangeably herein.
  • “Differential binding” in the context of this application encompasses that a compound, in particular the antibodies as described herein, or test compounds, bind to modified MIF (e.g. with the KD values mentioned above) while they do not bind to redMlF and/or to MIF which does not contain a modified sulphur atom of C81.
  • references to “antibodies” encompass antigen-binding antibody derivatives, constructs or fragments which are known to a person skilled in the relevant art, in particular antigen-binding molecules comprising antigen-binding portions of antibodies.
  • An “antibody” refers to an intact antibody or a molecule consisting of or comprising an antigen-binding portion of an antibody 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 herein by reference in its entirety ⁇ .
  • the term antibody includes human antibodies, mammalian antibodies, isolated antibodies and genetically engineered forms such as chimeric, camelized or humanized antibodies, though not being limited thereto.
  • 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. modified MIF).
  • Molecules comprising antigen-binding portions of antibodies may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Such molecules include, though are not limited to, the following: Fab, Fab', F(ab')2, Fv, and complementarity determining region (CDR) fragments, single-domain antibodies, and single-chain Fv antibodies (scFv), chimeric antibodies, diabodies, antibodies and polypeptides that contain at least a portion of an antibody that is sufficient to confer specific binding to an antigen of interest, e.g. a form of MIF disclosed herein, e.g. to modified MIF, redMlF, oxMIF, MIF containing a free sulfhydryl group in C81 , MIF which does not contain a modified sulphur atom of C81 , or other forms of MIF disclosed herein.
  • 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 a!. J. Mol. Biol. 196:901-917 (1987), or Chothia et a!., Nature 342:878-883 (1989).
  • An antibody or antigen-binding portion thereof can be derivatized or linked to another functional moiecule (e.g., another peptide or protein).
  • 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.
  • another antibody e.g., a bispecific antibody or a diabody
  • detectable agent e.g., a detectable agent, cytotoxic agent, a pharmaceutical agent, and/or a linking molecule.
  • an antibody may e.g. characterised as a human antibody, a humanized antibody, a camelized antibody. Further, an antibody may be, e.g. a chimeric antibody, Further, an antibody may be, e.g. an isolated antibody.
  • KQ refers, in accordance with the common general knowledge of a person skilled in the art to the equilibrium dissociation constant of two interaction partners, e.g. a particular antibody and its respective antigen.
  • 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 ceils, which might e.g. impart glycosylation not typical of human cells.
  • humanized antibody refers to antibodies comprising human sequences and containing also non- human sequences.
  • 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.
  • chimeric antibody refers to an antibody that comprises regions from two or more different species.
  • 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-ceSi repertoire.
  • Antibodies disclosed herein may be produced by 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.
  • the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • 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).
  • the vector e.g. non-episomal mammalian vectors
  • 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”).
  • Antibodies disclosed herein 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.
  • promoters e.g., CMV or SV40
  • the expression vectors can also comprise selection and amplification markers, such as the dihydrofolate reductase gene (DHFR), hygromycin-B-phosphotransferase, and thymidine-kinase.
  • selection markers such as the dihydrofolate reductase gene (DHFR)
  • hygromycin-B-phosphotransferase hygromycin-B-phosphotransferase
  • thymidine-kinase thymidine-kinase.
  • the components of the vectors used 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 ceils 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.
  • Antibody light chain gene(s) and 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 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.
  • the recombinant expression of an antibody can be achieved by introducing an expression plasmid containing the antibody encoding DNA sequence under the control of one or more regulating sequences such as a strong promoter, into a suitable host ceil 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 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 antibody, e.g.
  • oxMIF-specific antibodies are partly also disclosed in WO 2009/086920. Further oxMIF- specific antibodies are characeterised by the following sequences:
  • SEQ ID NO: 8 for the amino acid sequence of the heavy chain of RAB2:
  • DIQMTQSPSS LSASVGDRVT ITCRSSQRIM TYLNWYQQKP GKAPKLLIFV ASHSQSGVPS RFRGSGSETD FTLTISGLQP EDSATYYCQQ SFWTPLTFGG GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG LSSPVTKSFN RGEC.
  • He Gly Lys lie Giy Gly Ala Gin Asn Arg Ser Tyr Ser Lys Leu Leu Cys Gly Leu Leu Ala Giu Arg Leu Arg He Ser Pro Asp Arg Val Tyr
  • an antibody according to the present disclosure is preferably an isolated monoclonal antibody.
  • the anti-MIF antibody can be an !gG, an IgM, an IgE, an IgA, or an IgD molecule.
  • the anti-MIF antibody is an SgG1 , lgG2, lgG3 or lgG4 subclass.
  • the antibody is either subclass lgG1 or lgG4.
  • the antibody is subclass lgG4.
  • the lgG4 antibody has a single mutation changing the serine (serine228, according to the Kabat numbering scheme) to proline.
  • CPSC CPSC sub-sequence in the Fc region of lgG4 becomes CPPC, which is a subsequence in lgG1 (Angal et al. Mol Immunol. 1993, 30, 105-108).
  • Antibodies can be recovered from the culture medium using standard protein purification methods, e.g. via anion exchange chromatography or affinity chromatography.
  • the anti-(ox)M!F antibody can be purified from cell culture supematants by size exclusion chromatography.
  • center region and C-terminai region of MIF refer to the region of human MIF comprising amino acids 35-68 and aa 86-115, respectively, preferably aa 50-68 and aa 86 to 102 of human MIF, respectively.
  • Particularly preferred anti-oxMIF antibodies bind to either region aa 50-68 or region aa 86-102 of human MIF. This is also reflected by the binding of the preferred antibodies RAB0, RAB4 RAB2 and RAB9 as well as RA 4, RAM9 and RAM0 which bind as follows:
  • RAB4 and RAM4 aa 86- 102
  • RAB9 and RAM9 aa 50-68
  • RAB2 aa 86 - 102
  • epitopic determinants 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.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • the vector is a plasmid, i.e., a circular double stranded DNA loop into which additional DNA segments may be !igated.
  • the term "host cell” refers to a cell line, which is capable of producing 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, a 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.
  • the antibody is expressed in a DHFR-deficient CHO cell line, e.g., DXB1 1 , and with the addition of G418 as a selection marker.
  • the antibodies are produced by cuituring the host cells for a period of time sufficient to allow for expression of the antibody in the host ceils or secretion of the antibody into the culture medium in which the host cells are grown.
  • MIF-related disorder in the present context includes but is not limited to infectious diseases, inflammation, autoimmunity, cancer, cell differentiation, atherogenesis and angiogenesss-related diseases.
  • MIF-related disorders are e.g., type i and It-diabetes, acute lung injury, asthma, ailog raft-rejection, graft-versus-host- disease, wound healing disturbances and inflammatory bowel disease.
  • Cancer is a further MIF-related disorder.
  • MIF-related cancers include lymphoma, sarcoma, prostatic cancer and colon cancer, bladder cancer, pancreas cancer, ovarian cancer, melanoma, hepatocellular carcinoma, ovarian cancer, breast cancer and pancreatic cancer.
  • Atherosclerosis is a MIF-related disorder.
  • malignant ascites is a MIF-related disorder.
  • MIF-related disorders include sarcoidosis, scleroderma, psoriasis, (ulcerative) colitis, as well atopic dermatitis, as well as septic shock, delayed hypersensitivity, acute respiratory distress syndrome (ARDS), multiple sclerosis, pancreatitis and ischemic cardiac injury.
  • Immune and inflammatory disorders include gram negative and gram positive sepsis, e.g. P. aeruginosa infections or sepsis, DTH, glomerulonephritis, arthritis, adjuvant arthritis, juvenile arthritis, (autoimmune) encephalomyelitis/encephalitis, (autoimmune) myocarditis, allergic encephalitis, gastritis, coiitis; (immune)giomerulonephritis; pneumonia, toxic shock syndrome, viral infections, tuberculosis, hepatitis 8, dengue fever, parasitic and helminthic MIF-related infections, in particular malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, amoebiasis, schistosomiasis, cysticercosis, trichenellosis and filariasis; kidney diseases, like leukocyte-mediated renal injury, non-proliferative renal disease, proliferative renal disease, renal allografta, and othersograf
  • Most preferred diseases to be diagnosed according to the present invention are: glomerulonephritis, sepsis, lymphoma, lupus nephritis, psoriasis, ulcerative colitits and ophthalmological conditions, as well as Burkitt's lymphoma, leukemia, malignant ascites, prostate adenocarcinoma, pancreatic adenocarcinoma, ovarian carcinoma, colorectal carcinoma, head and neck cancer, renal cell carcinoma, hepatocellular carcinoma, breast cancer and lung cancer. Both k-ras wild-type as well as k-ras mutated cancers can be treated in accordance with the present invention.
  • the present invention also covers third line treatments of all above mentioned disorders.
  • One aspect of the present invention is directed to detection of modified M!F in a sample of a subject.
  • detection allows a skilled practitioner, e.g., to determine whether or not MIF is a therapeutically important component of a disease or disorder afflicting the subject. This determination will aid the practitioner's decision whether or not an (additional) anti-(ox)MIF treatment could be beneficial for the subject in question.
  • Modified MIF is also useful as a marker to determine a health or disease condition of a given subject in general; an elevated level of modified MIF will allow the finding that the subject is afflicted with a MIF related disease; modified MIF can thus also be used as a (secondary) general marker for a health/disease condition of a subject, similar e.g. to the determination of C-reactive protein (CRP) which is currently and widely used as such a (secondary) marker.
  • CRP C-reactive protein
  • cysteine 81 (C81) of MIF is essentia! for the switch of MIF to a disease-related state, i.e. the redox-switch from redMIF to oxMIF, and it is the derivatisation of the C81 sulfhydryl group which promotes (or is responsible for) this switch to the oxMIF form.
  • the invention provides the use of the modification state of the sulfhydryl group of MIF cysteine 81 as a diagnostic marker, for the diagnosis of MIF-related disorders.
  • Diagnosis in the context of this specification encompasses detection of a disease, evaluation of a disease state and monitoring of a disease progression, which also allows monitoring efficacy of a therapeutic treatment.
  • diagnosis of said MIF-reiated disorders according to the invention may encompass the use, or the further use, of compounds binding to modified MIF for the detection of modified MIF.
  • These compounds, which differentially bind modified MIF can be antibodies or small molecules, which differentially bind to modified MIF.
  • the diagnostic assay which can be used in the present invention can be any diagnostic assay which is well-known to a person skilled in the art.
  • the diagnostic assay can be carried out e.g. in an ELISA format, a sandwich (ELISA) format with use of FACS, immunofluorescence, immunohistochemistry, and any of various other suitable methods, all of which are well-known in the art.
  • oxidised forms of MIF such as C81-modifed MIF
  • oxidation of cysteine residues in the MIF protein ⁇ and thus C81 can be induced by redox-active iron and heme in biosamples (e.g. hemolytic blood samples) or if oxidizing agents are added to the sample.
  • Such false positive results may be avoided, e.g., by taking measures as described herein below, e.g. by de-activating redox-active iron and heme, and by avoiding the addition of e.g. oxidizing agents.
  • Citrated plasma is preferred. Citrated plasma from fresh blood (stored at +4°C not longer than 12 h) is centrifuged at 40 g for 5 min. The supernatant is transferred into a new tube and centrifuged again at 2000 g for 3 min. The cell free supernatant is transferred again into a new tube and centrifuged at 16000 g for 3 min.
  • the cell free supernatant can be stored at - 80°C or directly used for the analysis of MIF, If MIF is to be analysed in sera, cells and insoluble fragments are preferably removed by the same three centrifugation steps prior storage by freezing or prior running the MIF ELISA.
  • Sediments in urine samples should also preferably be removed by a centrifugation step (16000 g for 5 min) prior to use in the MIF ELISAs.
  • a centrifugation step (16000 g for 5 min) prior to use in the MIF ELISAs.
  • cells and other common particles occurring in biological fluids e.g. tear fluid, saliva
  • the MIF protein to be tested is to be kept in its native conformation during sample preparation (e.g. during the isolation and preparation of body fluids). Therefore denaturating conditions/steps such as for example boiling, immobilization (on membranes, plastic (plate) or chips) and chemical treatments (e.g. with reducing agents, oxidizing agents and organic solvents), are avoided.
  • a flow cytometry assay For the analysis of MIF on cellular surfaces, preferably a flow cytometry assay is used. It is particularly important that the samples do not undergo hemolysis during sample preparation. Therefore, all samples for the present flow cytometry analysis have been prepared without any step which would lead to a hemolysis of the cells within the sample.
  • the MIF carrying a modification at position 81 is used as a target for a drug discovery assay.
  • a drug discovery assay is an assay well known to a person skilled in the art. It is an assay whereby the (potential) usefulness of given compounds/a given compound for the treatment or prevention of a disease or disorder ("as a drug") is assessed,
  • a method of monitoring the effectiveness of a treatment of a MIF-related disorder comprising a step of determining in samples isolated from a subject before and after said treatment whether MiF position 81 carries a modification, wherein the treatment is identified as effective if MIF position 81 is modified to a lesser extent after treatment than before treatment.
  • antibody or molecule binds a MIF which is modified at position 81 but does not bind MIF which is not modified at position 81.
  • a method of assaying a test compound for preferential binding to a form of MIF which is modified at position 81 comprising the steps of
  • the compound is selected if said compound binds to the modified MIF in the test sample to a greater extent than in the control sample.
  • a method of assaying a test compound for a conformational effect upon MIF which is modified at position 81 comprising the steps of
  • the compound is selected if said conformational assessment indicates a detectable degree of similarity of the conformation of the modified MIF in the test sample with conformation (ii) compared to conformation (i), or if said conformational assessment indicates that the conformation of the modified MIF in the test sample otherwise detectab!y deviates from conformation (i).
  • step (c) the conformation of MIF is assessed by an immunoassay, preferably by ELISA.
  • step (c) the conformation of MIF is assessed by X-ray crystallography.
  • a method of assaying a test compound for a conformational effect upon MIF which is modified at position 81 comprising the steps of
  • a method of screening for a compound that prevents the modification of MIF at cysteine 81 comprising the steps of
  • the compound is selected if the sulfhydryl-reactive reagent modifies cysteine 81 to a lesser extent in the test sample than in the control sample.
  • a crystal containing MIF with a modification at position 81 is a crystal containing MIF with a modification at position 81.
  • position 81 of the MIF protein as a diagnostic marker for a MIF-related disorder and/or for monitoring a MIF-related disorder.
  • a method of diagnosing a MIF-related disorder comprising a step of determining in a sample isolated from a subject whether MIF position 81 carries a modification, wherein, MIF position 81 is identified as being modified, the subject is diagnosed with a MIF-related disorder, or as being susceptible thereto.
  • a method of diagnosing a MIF-related disorder comprising a step of determining in a sample isolated from a subject whether the sulphur atom of MIF cysteine 81 carries a modification compared to the free cysteine sulfhydryl group, wherein, if the sulphur atom of MIF cysteine 81 is identified as carrying such a modification, the subject is diagnosed with a MIF-related disorder, or as being susceptible thereto.
  • a diagnostic kit in a use according to any one of embodiments 15-18, or a method according to any one of embodiments 19-21 , wherein the diagnostic kit comprises a compound that preferentially binds to MIF in which cysteine 81 is present, or to MIF carrying a modification at position 81.
  • kits additionally comprises buffers, control reagents (e.g. recombinant MIF in which cysteine 81 is present or absent, or MIF with or without a modification at position 81 , e.g. a modification on the sulphur atom of cysteine 81 compared to the free cysteine sulfhydryl group, a compound that preferentially binds to any such forms of MIF), polyclonal anti-MIF antibody, and/or a labelled detection antibody.
  • control reagents e.g. recombinant MIF in which cysteine 81 is present or absent, or MIF with or without a modification at position 81 , e.g. a modification on the sulphur atom of cysteine 81 compared to the free cysteine sulfhydryl group, a compound that preferentially binds to any such forms of MIF
  • polyclonal anti-MIF antibody e.g. a labelled detection antibody.
  • MIF-related disorder is an inflammatory disease or a neoplastic disease.
  • MIF-related disorder is selected from the group consisting of colon cancer, prostate cancer, bladder cancer, pancreas cancer, ovarian cancer, melanoma, lymphoma, hepatocellular carcinoma, asthma, ARDS, rheumatoid arthritis, sepsis, IgA nephropathy, glomerulonephritis, Lupus Nephritis (LN), hepatitis, pancreatitis (+/- acute lung injury), Crohn's disease, ulcerative colitis, gastric ulcer, Alzheimer's disease, multiple sclerosis, Guillain- Barre syndrome, cardiac dysfunction, angioplasty, atherosclerosis, myocarditis, type 1 diabetes, diabetic retinopathy, age-related macular degeneration (AMD), atopic dermatitis, psoriasis, endometriosis, neuropathic pain and uveitis.
  • MIF-related disorder is selected from the group consisting of colon cancer, prostate cancer, bladder cancer, pan
  • Figure 1 shows schematic representations of wild-type and mutant recombinant fusion constructs of
  • FIG. 1 shows Western blot assays of the MIF constructs of Figure 1 using the antibody RAM9.
  • Figure 3 shows Western blot assays of the MIF constructs of Figure 1 using the antibody RAMO.
  • Figure 4 shows examples of sulfhydrykeactive reagents for derivatisation of sulfhydryl groups on a compound R or MIF, the resulting reaction products, and the expected increases in molecular weight in Da for R or MIF after derivatisation.
  • Figure 5 shows the general setup of an ELISA assay for assessing MIF and its mutant and/or derivatised forms.
  • Figure 6 shows the binding of wild-type human MIF in its reduced form (huMIFred), in cysteinylated form (Cys-MIF), and when derivatised using 5,5 ' -dithiobis-(2-nitrobenzoic acid/DTNB (DTNB-MIF), to the antibody RAM9 in an ELISA assay.
  • Figure 7 shows the assessment by ELISA of the binding of MIF in its wild-type (MIF), wild-type fusion
  • Figure 8 shows mass-spectroscopic data obtained with MIF in its wild-type (MIF), wild-type fusion (MIFwt-SBP), and mutant fusion (MIF(C57S)SBP, etc), confirming derivatisation with cysteine or DTNB (+NTB). Cysteinylation or derivatisation with DTNB give rise to peaks reflecting correspondingly increased molecular weights.
  • Figure 9 shows a mass spectrum obtained from peptide mass fingerprinting of cysteinylated MIF
  • Figure 10 shows results of an analysis of cysteinylated MIF by peptide mass fingerprinting
  • Figure 1 1 shows a mass spectrum obtained from peptide mass fingerprinting of MIF derivatised by
  • Figure 12 shows results of an analysis of MIF derivatised by DTNB by peptide mass fingerprinting REFERENCE EXAMPLES AND GENERALLY APPLICABLE METHODS
  • a THP1 suspension culture is centrifuged and cells are resuspended in fresh full medium to a cell density of 106 cells per ml. This culture is transferred into wells of a 96-well microplate (90 ⁇ /well) and a potential anti- MIF antibody is added to give a final concentration of 75 g/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.
  • 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).
  • Serum stimulates secretion of IF in quiescent NIH/3T3 and 1F in turn stimulates cell proliferation.
  • Antibodies inhibiting this endogenous MIF therefore, decrease the proliferation of quiescent NIH/3T3 cells.
  • the reduction of proliferation is determined by the incorporation of ⁇ -thymidine.
  • 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 pg/ml antibody and 5 ⁇ Ci/ml of 3H-thymidine. After 16 hours incubation in a CO2 incubator at 37"C cells are washed twice with 150 ⁇ of cold PBS per well. Using a multi-channel pipette 150 ⁇ of a 5% (w/v) TCA solution per well are added and incubated for 30 minutes at 4°C. Plates are washed with 150 ⁇ PBS.
  • Each peptide is diluted in coupling buffer to give a peptide concentration of typically 1 pg/ml added to microplates (NUNC ImmobilizerTM Amino Plate F96 Clear) and incubated over night at 4°C (100 ⁇ /well).
  • MIF and PBS are used as controls recombinant full length MIF and PBS.
  • the plate is washed 3 times with 200 ⁇ PBST and antibodies (2-4 g/ml in PBS) are added (100 ⁇ /well) and incubated for 2 hours at room temperature with gentle shaking.
  • the plate is washed 3 times with 200 ⁇ PBST and detection antibody (e.g. Fc specific anti- human IgG/HRP labeled, Sigma) is added (100 ⁇ /well).
  • 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 NaCI. Affinities are calculated according to the 1 :1 Langmuir model.
  • Microliter plates are coated e.g. with a human anti-C81 -modified MIF monoclonal antibody.
  • Human serum samples are diluted 1 :25 in 0.5% fish gelatin/PBS, pH 7.2 and applied to the plate.
  • detection of C81 -modified MIF captured by the coating antibody is performed e.g. with an affinity purified polyclonal rabbit antibody anti-human MIF.
  • the read-out of the ELISA is done after further incubation e.g.
  • the standards are diluted in 0.5% fish gelatin/PBS including 0.2% ProClin300 and 4% human control plasma (i.e. a pool of serum samples from 50 healthy donors).
  • the range of the calibration curve may be, e.g., 10 ng/ml to 0.156 ng/ml.
  • Blood (e.g. from a patient with sepsis) is stained in Ceil Staining Buffer (Biologend) with either Alexa700- labeled anti-CD3s (for T cells) and PerCP-Cy5.5-labeled anti-Ly6G (for granulocytes) or APC-labeled anti- CD14 (for monocytes) and PE-Cy7-labeled anti-CD19 (for B cells) in parallel with 300 nM anti-C81 -modified MIF antibody or control IgG. After washing, the human antibodies are detected using the goat R-PE-labeled anti-humanlgG antibodies.
  • the red blood cells are lysed with the BD FACSTM Lysing solution (Becton Dickinson, Franklin Lakes, USA). Data acquisition is performed using a FACSTM Canto II (Becton Dickinson) with the DIVATM software (software version 6; Becton Dickinson) and the data are analyzed using the FlowJoTM software (Treestar, Ashland, OR, USA).
  • human MIF (suspended in 100 ⁇ PBS) was mixed with 100 ⁇ IFA (Incomplete Freunds Adjuvants). Again, 200 ⁇ (4 x 50 ⁇ ) of the mixture was appiied s.c. to different body portions of each rabbit. The immunization procedure was terminated 2 weeks after the second boost. Typically, plasma from multiple rabbits was pooled and used for the isolation of the anti MIF antibodies.
  • the eluate was pooled and neutralized to pH 7,0 using 1 M Tris/HCI.
  • hu-MIF affinity purification the total rabbit IgG was again diluted 1 :3 with 20 mM Na2HPC ⁇ 4 buffer, pH 7.0 and applied to the 5 ml NHS-affsnity column (GE Healthcare) coupled with 25 mg rhuMIF as recommended by the supplier, After a washing step (5 column volumes with 20 mM Na2HP04 buffer, pH 7.0) the elution of the specific rabbit anti huMIF antibodies was effected with 100 mM glycine, pH 2.8. The eluate was pooled and neutralized to pH 7.0 using 1 M Tris/HCI. Finally, the hu-MIF affinity purified specific rabbit anti human MIF antibodies (in the following "anti-human MiF affinity purified polyclonal antibody”) were dialyzed against PBS and stored at -20°C.
  • Wild-type and cysteine-mutant recombinant fusion constructs of human MIF (huMIF) with streptavidin binding peptide (SPB) tag via a (GGGGS)2-linker (cf. SEQ ID NO: 16), as shown in Figure 1 were prepared, cloned and expressed by standard methods. Briefly, synthetic DNA (Invitrogen, GeneArt) was cloned in pet25b and expressed in £ coli Shuffle T7 Express (NEB). Soluble protein was extracted using BugBuster® Protein Extraction Master Mix (Novagen) and purified by affinity chromatography via streptavidin column and biotin elution. Further purification was done using size exclusion chromatography.
  • streptavidin binding peptide (SPB) tag is: MDEKTTGWRGGHWEGLAGELEGL ARLEHHPQGQREP (SEQ ID NO: 17).
  • SBP streptavidin binding peptide
  • Recombinant wiid-type untagged huMIF (SEQ ID NO: 15) was produced in E.coli cells including an expression system with the human M!F sequence.
  • Fresh thaw cells were cultivated in Luria Bertani medium supplemented with Ampicillin (LB/Amp) over night at +37°C.
  • the bacterial cell culture was diluted with an equal volume of fresh LB/Amp medium and the expression induced by addition of IPTG (final concentration: 1.0 mM) at 3G°C for 4 hours.
  • the bacterial pellet was harvested by centrifugation and stored at ⁇ -15°C.
  • the frozen bacterial pellet was resuspended in 20 mM Tris/HCi buffer, pH 7.8 and cells were disrupted mechanicaliy by glass beads. Cell debris was removed by centrifugation and filtration using a common 0.2 m filter. The supernatant was directly applied to an anion exchange chromatography column (HiTrap 26/16 DEAE FF, GE Healthcare, Waukesha, USA) and MIF was purified by a passive binding mode. The flow through was rebuffered in 20 mM Bis/Tris pH 6.3 and further purified by a cation exchange chromatography (Source 30S, GE).
  • RAM9 is a monoclonal antibody described herein above and also in WO20 3/050453, which binds preferentially to the oxidized, disease-associated form of MIF (oxMIF), compared to the reduced form of MIF (redMIF).
  • RAM0 is a monoclonal antibody described herein above and also in WO2013/050453, which binds preferentially to the oxidized, disease-associated form of MIF (oxMIF), compared to the reduced form of MIF (redMIF).
  • oxMIF oxidized, disease-associated form of MIF
  • redMIF reduced form of MIF
  • the antibodies RAM9 and RAMO were used as immobilised capture antibodies in an ELISA assay to probe the effects of cysteine-to-serine mutations, sulfhydryl derivatisation and the lack of derivatisation of different cysteine residues on the formation of the oxidized, disease-associated form of MIF (oxMIF).
  • oxMIF oxidized, disease-associated form of MIF
  • sulfhydryl-reactive reagents for derivatisation of sulfhydryl groups in MIF are shown in Figure 4.
  • the general setup of an ELISA assay is shown in Figure 5.
  • RAM9 shows significant binding to both cysteinylated and DTNB-derivatised MIF.
  • RAM9 is specific for the disease-associated form described as oxMIF e.g. in W02013/050453, this shows that derivatisation of MIF with either cysteine (by treatment with cystine) or a 2-nitro-5-thiobenzoic acid (NTB) moiety (by treatment with DTNB - leading to "DTNB-derivatised MIF" or MIF+NTB) induces the disease- associated oxMIF form of the protein. Equivalent results were obtained with RAMO.
  • oxMIF is still formed when C57 or C60 are replaced by serine, because derivatisation of these mutants by DTNB still strongly promotes the binding of RAMO.
  • the latter oxMlF-specific antibody preferentially binds MIF(C57S)SBP -DTNB and IF(C60S ⁇ SBP -DTNB to a degree that is comparable to its binding to wtMIF that has been converted into oxMIF (cf. MIF-DTNB and MIF(wt)SBP-DTNB).
  • Antibodies are produced in mammalian cells, preferentially in CHO cells, preferentially in CHO cells where the gene encoding for IF (endogenous CHO-MIF) has been knocked out genetically, in the knock-out cells the contamination of the antibody with endogenous CHO-M!F can be abolished, which is desirable as sensitivity of the assays can be enhanced.
  • IF endogenous CHO-MIF
  • C81-modified MIF specific antibodies are produced in a batch fermentation process using a disposal bioreactor (wave system) up to 25 L volume.
  • Stable CHO cell lines harboring the genes encoding for the heavy and light chain of the produced antibody, respectively, are seeded into an PowerCHO medium (Invitrogen Inc.) and incubated at 37°C and 5% CO2.
  • the respective human antibodies are continuously expressed into the cell culture medium.
  • the cells are separated by common centrifugation and filtration steps.
  • the clarified cell culture supernatant (ccs) is concentrated by ultrafiltration and used for the purification of antibodies.
  • the human antibodies are purified from the concentrated ccs by Protein A affinity chromatography (MabSelect Sure, GE Healthcare). After equilibration of the Protein A material with 5 column-volumes (cv) of 20 mM sodium phosphate running buffer, pH 7 the concentrated supernatant of the isotype control is completely applied to the affinity column. Impurities or undesirable proteins are washed out with the running buffer.
  • the antibodies are eluted by a pH shift using 100 mM glycine, pH 3 and dialyzed against 250 mM glycine buffer, pH 5.
  • the concentrated cell culture supernatant may be applied to the Protein A column prior equilibrated with 5 cv of 20 mM Tris/HCt buffer including 150 mM sodium chloride buffer and 0.1% Tween 80, pH 7. Impurities may be washed out by two washing steps: 1.) addition of 1 M NaCI in the equilibration buffer and 2.) 100 mM sodium phosphate including 0.1% Tween 80, pH 5. Antibodies may be eluted by 100 mM glycine buffer, pH 3 including 0.1% Tween 80 and then dialyzed against 250 mM glycine buffer, pH 5.

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Abstract

La présente invention concerne la découverte qu'une altération structurelle spécifique du facteur d'inhibition de migration est impliquée dans des troubles liés au facteur d'inhibition de migration, fournissant une nouvelle approche au traitement de troubles liés au facteur d'inhibition de migration, ainsi que d'autres applications médicales spécifiques, par exemple, pour le suivi de la progression de maladies, le criblage de médicaments et des méthodes diagnostiques et des utilisations respectives de trousses de diagnostic.
PCT/EP2014/074617 2013-11-14 2014-11-14 Facteur d'inhibition de la migration des macrophages en tant que cible therapeutique Ceased WO2015071416A2 (fr)

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