TW201605886A - MIF as therapeutic target - Google Patents

Abstract

The present invention pertains to the recognition that a specific structural alteration of MIF is involved in MIF-related disorders, providing a new approach to the treatment and prevention of MIF-related disorders, as well as other specific medical applications, e.g. for example monitoring of disease progression, drug screening and diagnostic assays and respective uses of diagnostic kits.

Description

MIF as a therapeutic target

The present invention relates to the use of specific forms of MIF proteins in medicine. The present invention is based on the recognition that the modification of position 81 of MIF determines the conversion of MIF to its disease-related state.

Macrophage migration inhibitory factor (MIF) is a cytokine originally isolated based on its ability to inhibit the random migration of intraperitoneal exudate cells (containing macrophages) of tuberculin allergic guinea pigs (Bloom et al. Science 1966, 153, 80-2; David et al. PNAS 1966, 56, 72-7). Today, MIF is known to be a key upstream regulator of congenital and acquired immune responses that exert a broad spectrum of activity.

Human MIF cDNA was cloned in 1989 (Weiser et al, PNAS 1989, 86, 7522-6) and its genomic localization maps to chromosome 22. The product of the human MIF gene is a protein having 114 amino acids (after N-terminal methionine cleavage) and an apparent molecular mass of about 12.5 kDa. MIF does not have significant sequence homology to any other protein. The protein crystallizes in the same subunit of the trimer. Each monomer contained two antiparallel alpha-helices that encapsulated four beta sheets. The monomer has an additional two beta strands that interact with the beta sheets of adjacent subunits to form a linkage between the monomers. The three subunits are arranged to form a cylinder containing a solvent accessible channel through the protein center along the axis of the molecule 3 times (Sun et al. PNAS 1996, 93, 5191-5196).

It has been reported that MIF is secreted from macrophages at very low concentrations of glucocorticoids. Induction (Calandra et al. Nature 1995, 377, 68-71). However, MIF also counteracts 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). . It has also been shown, for example, that MIF exhibits angiogenic, proliferative and anti-apoptotic properties that promote tumor cell growth (Mitchell, RA, Cellular Signalling, 2004. 16(1): pp. 13-19; Lue, H. et al., Oncogene 2007.26(35): Pages 5046-59). It is also directly related, for example, to 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 pathological conditions (ie, MIF-related disorders) and This includes, inter alia, inflammatory bowel disease (IBD), rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), asthma, spheroid nephritis, IgA nephropathy It is associated with various diseases of, but not limited to, myocardial infarction (MI), sepsis, and cancer, and MIF also mediates many other MIF-related disorders.

Multi-strain and monoclonal anti-MIF antibodies against recombinant human MIF have been developed (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, 167778).

Anti-MIF antibodies have been recommended for therapeutic use. Calandra et al., (J. Inflamm. (1995); 47, 39-51) It was reported that anti-MIF antibodies were used to protect animals from experimentally induced gram-negative and gram-positive septic shock. Anti-MIF antibodies are recommended as a therapeutic means of regulating cytokine production in septic shock and other inflammatory conditions.

US 6,645,493 discloses monoclonal anti-MIF antibodies derived from fusion tumor cells, Neutralize the biological activity of MIF. It can be shown in animal models that these anti-MIF antibodies derived from mice have beneficial effects in the treatment of endotoxin-induced shock.

US 200310235584 discloses that in animals that have homozygously deleted the MIF gene A method of preparing high affinity antibodies against MIF.

Glycosylation-inhibiting factor (GIF) The protein described by Galat et al. (Eur. J. Biochem, 1994, 224, 417-21). MIF is now considered to be the same as GIF. Watarai et al. (PNAS 2000, 97, 13251-6) describe polyclonal antibodies that bind to different GIF epitopes to discern the biochemical properties of post-translational modifications of GIF in Ts cells. Watarai et al. (supra) reported that GIF appeared in test tubes in the form of isomorphic isoforms. According to Watarai et al., an isoform is produced by chemical modification of a monocysteine residue (ie, cysteine 60), which occurs in inhibiting T(Ts) cells and according to Watarai et al. , producing a biologically active form of GIF.

After decades, it has been shown that MIF is involved in many different interactions. molecule. WO 2013/050453 discloses that an oxidised form of MIF (oxMIF) and a oxMIF and disease state and/or disease can be detected after a (MIF-related) disorder, such as in a body fluid sample or on a cell or cell surface. Progress related. Antibodies specific for disease-related, oxidized forms of MIF (oxMIF-specific antibodies) are known. However, more information on MIF-related disorders in MIF-related disorders is needed to further improve the treatment and prevention of MIF-related disorders and related medical applications.

The present invention discloses the transition of MIF from non-pathogenic to disease-related state by exposing That is, the redox transition from redMIF to oxMIF is associated with the presence of a modification of position 81 and is facilitated by the presence of a modification of position 81 to achieve this goal. The disease-associated, oxidized form of MIF (oxMIF) is defined by the binding of an oxMIF-specific antibody (ie, an antibody that preferentially binds to oxMIF compared to redMIF not specifically associated with the disease). The inventors have shown that the binding of such oxMIF-specific antibodies is induced at position 81 (cysteine 81) by modification of MIF treated with a sulfhydryl reactant. In contrast, when wild-type cysteine 81 is mutated to serine (which is ordinated with cysteine but not modified by a sulfhydryl reagent), treatment with a sulfhydryl reagent does not induce Binding of oxMIF-specific antibodies. The inventors have thus established a clear link between the modification of MIF position 81 and the oxMIF conformation of the proteins involved in MIF-related disorders.

The sequence of human MIF is represented by SEQ ID NO: 15.

The invention thus provides for the use of a modified MIF at 81 as a target for the treatment or prevention of a MIF related disorder.

The invention is therefore directed to a method of treating or preventing a disease state or progression of a disease in an individual in need thereof, by administering to the individual an effective amount of the following compound

(a) preventing modification of the MIF at position 81, or (b) preferentially binding to the modified MIF carrying the position 81 as compared to the modified MIF not containing position 81.

The preferential binding compound can be an antibody or a molecule comprising an antigen binding portion of the antibody, and/or the preferential binding compound can induce binding of the antibody RAM0, RAB0 to a lesser extent than the modified MIF in the absence of the compound. The modified MIF in the form of RAM9 or RAB9 (or any other oxMIF-specific antibody, eg, selected from the oxMIF-specific antibodies disclosed herein).

In this article, mention "modification of MIF at position 81" or "at position 81" The modified MIF" or "the modified MIF at position 81" and the like can be simplified by referring to "modified MIF". Such modifications, throughout this application and in all specific examples, refer to the binding of MIF to antibody RAM9 (and/or RAB9, RAM0, RAB0, and/or any other oxMIF-specific antibody, eg, in its modified form or state, eg, Modification of MIF selected from any of the oxMIF-specific antibodies disclosed herein below.

Usually, since the position 81 of the MIF is cysteine, such pairs are modified. The reference to MIF can be used interchangeably with, for example, "modified MIF carrying cysteine 81" and the like or "MIF modified with cysteine 81" (or equivalently modified by C81). .

Preferably, herein, the modification (specifically at the MIF position 81) may be Modifications compared to unmodified cysteine residues or compared to free cysteine sulfhydryl groups. That is, specifically, the modification at the MIF position 81 may be a modification of MIF cysteine 81 and preferably a modification on the sulfur atom of cysteine 81. That is, as described herein, if a modified MIF position 81 is referred to herein, preferably MIF cysteine 81 carries a modification compared to unmodified cysteine, or more preferably The sulfur atom of cysteine 81 carries a modification compared to the free cysteine sulfhydryl group.

Modification does not cover the replacement of cysteine sulfur atoms by oxygen alone, ie cysteine only 81 is mutated to serine. Herein, the modification of the sulfhydryl group of the cysteine residue refers to the derivatization of the sulfhydryl group, that is, the sulfhydryl group derivatization. That is, the sulfur atom of the cysteine residue carries a modification compared to the free sulfhydryl group of the cysteine, that is, the sulfur atom of the cysteine is attached to a moiety other than H (ie, the modified moiety). .

The invention also encompasses a method for monitoring the effectiveness of treatment for MIF-related disorders The method comprising the step of determining whether the MIF position 81 carries a modification in a sample isolated from the individual before and after the treatment, wherein if the degree of modification of the MIF position 81 is smaller after treatment than before the treatment, the treatment is identified as Effective.

The invention also provides an analysis of whether a test compound preferentially binds to the in situ A method of setting a modified form of MIF at 81, the method comprising the following steps

(a) modifying the MIF at position 81 to obtain a MIF modified with cysteine 81, (b) combining the compound to be tested in the test sample and in a control sample in which the MIF is unmodified at position 81 and Modified MIF, (c) assessing the binding of the compound to the modified MIF in the test sample and in the control sample

Wherein the compound is selected if the compound binds to the modified MIF to a greater extent in the test sample than in the control sample.

For example, if the compound is preferentially or otherwise bound to the modified MIF in a test sample as compared to a control sample, the compound can be selected. For example, if the compound binds to the modified MIF with greater affinity in the test sample than in the control sample, the compound can be selected.

In the methods according to the invention disclosed herein, the test compound can be, but is not limited to, preferably an antibody or a molecule comprising an antigen binding portion of the antibody.

The present invention also provides a method of screening for a compound that prevents modification of MIF at cysteine 81, the method comprising the following steps

(a) combining MIF with the compound to be tested in the test sample, (b) in the test sample containing the compound, and in the control sample in which the compound is absent The MIF is treated by a sulfhydryl reaction reagent under the condition that the reagent modifies the sulfhydryl group of the cysteine 81 of MIF in at least the control sample, and (c) the cysteamine by the sulfhydryl reagent is evaluated. A modification of acid 81 wherein the compound is selected if the sulfhydryl reaction reagent is less modified in the test sample than cysteine 81 in the control sample.

The invention also provides a method of analyzing the effect of a test compound on the conformation of a modified MIF at position 81, the method comprising the steps

(a) providing a test sample containing the modified MIF at position 81, (b) combining the test compound with the modified MIF in the test sample, (c) evaluating the configuration with the modified MIF at position 81 ( i) (Control 1) and the configuration of MIF containing the unmodified cysteine residue at position 81 (ii) (Control 2) compared to the modified MIF in the test sample a form, wherein the configuration evaluation indicates that the configuration and configuration (ii) of the modified MIF in the test sample has a detectable degree of similarity compared to the configuration (i), or if the configuration evaluation indicates The configuration of the modified MIF in the test sample is otherwise detectably self-constructed (i) deviated and the compound is selected.

The configuration of MIF can be assessed by immunoassay. Advantageously, the immunoassay employs an antibody selected from the group consisting of RAM0, RAB0, RAM9 and RAB9 (and/or any other oxMIF-specific antibody, eg selected from the oxMIF-specific antibodies disclosed herein), and if the test sample (ie, is in the test) The compound is selected such that the modified MIF in the presence of the compound binds to the antibody to a lesser extent than the modified MIF in the absence of the compound (Control 1).

Preferably, the modification of the MIF position 81 in the case of "control group 1" The same in the test sample.

Generally, in the method according to the invention, the configuration of the MIF can be by any pair A method of morphological change sensitivity in MIF is evaluated by the presence of a modification of MIF at position 81, in particular the presence or absence of modification of MIF cysteine 81, specifically at position 81 at MIF Depending on the sulfur atom. For example, the configuration of MIF can be assessed, for example, by immunoassay using one or more conformationally sensitive anti-MIF antibodies as described herein, ie, the binding of such antibodies depends on the modified state of MIF position 81. For example, as described herein, the presence and/or absence of a modification at position 81 can be achieved by structuring a sensitive position-81-modification-selective antibody (and/or conformation-selective cysteine 81 sulfur) The combination of base-specific antibodies) was evaluated. For example, the configuration can be assessed by ELISA or surface plasmon resonance.

In other embodiments, the configuration can be evaluated by X-ray crystallographic analysis. The configuration can be assessed, for example, by nuclear magnetic resonance (NMR) analysis or by circular dichroism.

In accordance with the above, the present invention also provides an analytical test compound pair at position 81 A method of constituting a modified MIF, the method comprising the following steps

(a) providing a test sample containing the modified MIF at position 81, (b) combining the test compound with the modified MIF in the test sample, (c) evaluating the modified MIF in the test sample and selecting Binding of RAM0, RAB0, RAM9, and RAB9 and/or any other oxMIF-specific, oxMIF conformation-sensitive antibody (eg, selected from an oxMIF-specific antibody disclosed herein) to (i) in the absence of a test compound , the sulfur atom of MIF cysteine 81 carries a modified MIF compared to the free cysteine sulfhydryl group (control group 1), Wherein the modified MIF in the test sample (ie, in the presence of the test compound) binds to the antibody to a lesser extent than the modified MIF (control 1) in the absence of the compound, then selects The compound.

The present invention also provides a method for analyzing a test compound for binding to a modified form of MIF by X-ray crystallography, the method comprising the following steps

(a) modifying the MIF at position 81 to obtain a modified MIF, (b) crystallizing the modified MIF in the presence of the test compound or by crystallizing the modified MIF and then subjecting the test compound to the The crystals of the modified MIF are contacted to provide crystals of the modified MIF, and (c) the crystals of the MIF are determined using the crystals.

Optionally, the same X-ray crystallographic method can encompass the additional crystallization and the structure determination step by the same test compound but by MIF in which position 81 is unmodified.

The invention also provides a crystal comprising a modified MIF having position 81. The crystal may optionally contain another compound, such as a test compound, for example, a combination with the modified MIF as the test compound to be evaluated.

Herein, a compound such as a compound for binding to MIF modified at position 81 (for example, a compound for therapeutic or diagnostic methods or a test compound to be analyzed) is not particularly limited, but may be, for example, a small molecule. An organic molecule, a molecule of less than 1000 Da, less than 500 Da, or any form of antibody or antibody derivative (eg, a Fab fragment or any molecule comprising an antigen binding portion of an antibody) that binds to MIF (especially to its position 81 modified form).

The invention also provides position 81 of the MIF protein as a diagnostic marker and/or for Monitor the use of MIF-related disorders. In particular, the invention provides a modified state of position 81 of the MIF protein as a diagnostic marker and/or for monitoring a condition associated with MIF.

The invention provides a method for diagnosing a disease related to MIF, which is included in The step of determining whether the MIF position 81 carries a modification in the body-separated sample, wherein if the MIF position 81 is identified as carrying such a modification, the individual is diagnosed with a MIF-related disorder. That is, the MIF location 81 carries a discovery of such a modification indicative of a MIF related disorder.

The invention provides a method for diagnosing a disease related to MIF, which is included in In the sample isolated, whether the sulfur atom of MIF cysteine 81 carries a modification step compared to the free cysteine sulfhydryl group, wherein if the sulfur atom of MIF cysteine 81 is recognized as carrying the species Modification, the individual is diagnosed with a MIF-related disorder. That is, the discovery that the sulfur atom of cysteine 81 carries this modification is indicative of a MIF related disorder.

The present invention provides a modified state of MIF position 81 for use as a diagnostic marker, A finding in which the MIF position 81 carries a modification indicates a MIF-related disorder.

The present invention provides a modified state of the sulfhydryl group of MIF cysteine 81 as a diagnosis The use of a marker in which a sulfur atom of MIF cysteine 81 carries a modification compared to a free cysteine sulfhydryl group indicates a MIF-related disorder.

The methods and uses according to the invention encompass the determination of a given condition or pathology Whether the condition involves modified MIF or methods and uses associated with modified MIF.

The invention also provides for the use of a diagnostic kit in a method or use according to the invention And wherein the diagnostic kit comprises preferentially binding to a MIF in which cysteine 81 is present or to a modified MIF at carrying position 81 (ie, specifically, on the sulfur atom of cysteine 81) Compound. The kit may additionally comprise a buffer, a control reagent (eg, recombinant cysteamine therein) MIF in the presence or absence of acid 81 or MIF with or without modification at position 81, such as a modification on the sulfur atom of cysteine 81, a compound that preferentially binds to any of these forms of MIF), multiple anti-MIF antibodies And / or combined with detection antibodies.

Herein, the expression "MIF-related disorders" covers, for example, the status of (MIF-related) diseases, (MIF-related) disease states, and (MIF-related) diseases.

In certain embodiments of methods and crystals for screening or analyzing test compounds according to the present invention, position 81 (e.g., a sulfhydryl group of MIF cysteine 81) is selectively modified. That is, the MIF is not substantially modified at other positions by the same modified moiety, or specifically, the MIF is not substantially modified by the same modified moiety at the sulfur atom of the other cysteine in the MIF sequence (ie, , does not carry the same modification in essence). For example, MIF does not substantially carry the same modification on the sulfur atom of cysteine 57. For example, MIF does not substantially carry the same modification on cysteine 60. In certain preferred embodiments, the cysteine 57 and the cysteine 60 of MIF are not substantially modified by the same modified moiety as cysteine 81. In certain preferred embodiments, either or both of cysteine 57 and cysteine 60 may be substituted with an amino acid other than cysteine (ie, mutated to, ie, by... Instead, it is replaced, for example, by serine. In other embodiments, cysteine 57 can be unmodified, that is, can comprise a free sulfhydryl group. In other embodiments, the cysteine 60 can be unmodified, that is, can contain a free sulfhydryl group. In other embodiments, both cysteine 57 and cysteine 60 are unmodified, i.e., comprise a free sulfhydryl group.

Whether the position (cysteine) 81 (specifically its sulfur atom or sulfhydryl group) can be modified within the meaning of the present invention can be carried out by mass spectrometry, for example by preparing a peptide mass fingerprint (peptide mapping and mass spectrometry). Evaluation. That is, the methods of the invention (e.g., diagnostic methods or methods of screening for compounds) can encompass mass spectrometric analysis of MIF. That is, the method of the present invention (for example Diagnostic methods or methods of screening compounds can encompass the preparation of samples comprising MIF for mass spectrometry. Methods for determining the mass fingerprinting of modified amino acid residues are well known to those skilled in the relevant art and are part of the common general knowledge of those skilled in the art. The method of the invention may thus also encompass the digestion of MIF by a proteinase (eg, trypsin) in the sample to be assessed. Subsequently, the resulting peptide (MIF fragment) forms the digestion, in particular, the peptide containing cysteine 81 (and preferably no other cysteine residues) can be analyzed by mass spectrometry.

Position can also be performed by immunoassay, ie using one or more antibodies (cysteine Whether the amino acid 81 is evaluated by modification. That is, the methods of the invention (e.g., diagnostic methods or methods of screening for compounds) can encompass contacting MIF with one or more antibodies. This can result in the formation of a complex between the MIF (eg, C81 modified MIF) and the antibody. It is thus possible to use a MIF that selectively binds to a position 81 (e.g., the sulfhydryl group of cysteine 81 is modified), i.e., binds to a position 81 (e.g., a sulfhydryl group of cysteine 81) and a cysteine The MIF of the free sulfhydryl group at the amine acid 81 is evaluated as to whether the position 81 has been modified compared to the antibody of the MIF of the preferentially modified form.

Methods for preparing specific antibodies (eg, monoclonal antibodies) are also familiar with related techniques. Well known. Such binding of the antibody can be used (or at least for) to bind the antibody to the MIF under non-reducing and/or oxidative and/or natural conditions, depending on the presence of a modification at position 81 of the MIF protein (eg, having a sulfhydryl group). protein. Binding epitopes may or may not include position 81. The antibody may be an anti-MIF antibody (eg, a conformationally sensitive antibody that binds to a modified state of position 81), such as a cysteine 81 modification-specific antibody, which is free of sulfur atoms in MIF cysteine 81 The cysteine sulfhydryl group is specifically selected when compared to its binding to MIF in the absence of such modification (ie, MIF containing a free cysteine sulfhydryl group at position 81) Integrate to MIF selectively or preferentially. For example, the binding epitope of an antibody can be an epitope (eg, a linear or conformational epitope, which may or may not include cysteine 81), and its feasibility for antibody binding is modified at position 81 and The MIF position 81 is increased compared to when it is unmodified.

For example, the binding of an antibody to MIF can be seen at position 81 of the MIF protein. Depending on the presence of the modification of the cysteine, ie, the antibody binds to the MIF when the cysteine 81 is unmodified (for example under reducing conditions) or in the absence of cysteine 81, the antibody MIF can be preferentially bound when cysteine 81 is modified (eg, under non-reducing conditions). For example, the antibody can be RAM9 or RAB9. The antibody can also be RAM0 or RAB0. The combination may be in 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 only depending on the presence of modifications on the sulfur atom of MIF cysteine 81.

The methods and uses of the invention may also employ anti-MIF antibodies (eg, conformational sensitivity) An antibody, which binds to the modified state of the cysteine 81, such as a cysteine 81 sulfhydryl selective antibody, which carries a free cysteine sulfhydryl group in the sulfur atom of MIF cysteine 81 It binds preferentially to MIF when it binds to MIF in which cysteine 81 is modified. Such antibodies can be employed, for example, as a control group.

Within the meaning of the present invention, if the assay contains cysteine 81 (but no other half) The mass of the MIF peptide of cystine) is altered compared to the corresponding peptide containing the free sulfhydryl group at cysteine 81, and position 81 (eg, the sulfhydryl group of MIF cysteine 81) can be identified as modified .

Preferably, the MIF gene of the amino acid of the MIF protein is at position 81. The evaluation or assay encoding a cysteine (eg, whether the MIF gene encodes cysteine at position 81) is performed by a method including polymerase chain reaction (PCR).

The invention also relates to an antibody as disclosed herein, which specifically recognizes the MIF position Set the presence of the modification at 81. The invention also relates to an anti-MIF antibody that specifically binds to a position 81 carrying a modification (eg, on a sulfur atom compared to a free cysteine sulfhydryl group), wherein the antibody binds undetectably or by comparison Low affinity binds to MIF that does not contain this modification. The invention also relates to a complex between a modified MIF having an epitope at position 81 and an antibody as referred to herein.

Herein, the antibody may have any type, for example, preferably having an IgG1 type or IgG4 type. Herein, the antibody is referred to as "RAM" having an IgG1 type, and an antibody called "RAB" has an IgG4 type.

As used herein, unless otherwise indicated, the reference to a combination (eg, with respect to antibodies or The test compound) is a combination under physiologically relevant conditions (for example at any temperature between 1 ° C and 45 ° C). In the case of X-ray crystallography methods, the combinations mentioned are intended to be combined under crystallization conditions well known to those skilled in the art.

Preferred binding or enhanced binding, etc. (eg for antibodies or another compound) This article may also be referred to as a differential combination. By way of example, a preferred enhanced or differential binding means that the compound, in particular, the antibody as described herein binds (by a KD value of less than 100 nM, preferably less than 50 nM, and more preferably less than 10 nM) to MIF The first form; and to a lesser extent (eg, by lower affinity, for example, characterized by a KD of 400 nM or more or a detectable binding absence (ie, the compound is not bound)) binding to the second form of MIF . The first form of MIF can be, for example, in the form of a MIF associated with a MIF-related disorder (eg, specifically a C81-modified MIF), and the second form of MIF can be in a form that is not so relevant (eg, does not contain C81) Modified sulfur atom), or vice versa.

For example, differential binding can be referred to by less than 100 nM (preferably less than 50) The KD value of nM, and more preferably less than 10 nM, binds to the modified MIF at position 81 (eg, on the sulfur atom of cysteine 81) and, for example, at a KD of 400 nM or more or a detectable binding is absent ( That is, the compound does not bind) is characterized by less binding to the MIF that does not contain the modification on the sulfur atom of cysteine 81 (or vice versa).

In this paper, such as "diagnostic markers" or "MIF-related" diseases in the diagnosis The term "marked" in the context of the present invention will cover whether MIF is a possibility to evaluate the factors involved in this (MIF related) disease. In one aspect, the modified MIF as a marker supplies information about the disease state, its progression and acts as a marker to determine the effectiveness of a given treatment; in addition, a modified MIF detect in a sample (eg, a body fluid sample or a cell sample) The test can serve as an indicator of better anti-MIF therapy. Detection of modified MIF is thus used to improve known diagnostic techniques in a given disease or condition. It assists the practitioner in assisting in the treatment of a given disease or condition and helps to improve the specificity of the diagnosis. The modified MIF is therefore a specific and suitable secondary marker. Its detection can serve as an adjunct test in the management of patients with MIF-related diseases. The disease in question is, in a preferred embodiment, a disease known or suspected to be MIF-related (see the diseases detailed below) but may also be a disease that has not been suspected to be MIF-related to date.

In a preferred embodiment, the presence of a modified MIF in the sample The test will indicate to the practitioner that the individual (who has taken the sample from the individual (or)) may benefit from treatment for MIF. Such treatment may be selected from an anti-MIF molecule, such as an anti-modified MIF antibody or a small molecule directed against a modified MIF.

Higher MIF content, usually after the onset of various diseases, especially The amount of MIF detected after the onset of cancer. However, MIF also circulates in healthy individuals, which makes It is difficult to differentiate clearly. In contrast, the oxMIFs of the invention linked to modified MIF are not present in healthy individuals and are therefore a stronger diagnostic marker for MIF related disorders. According to the present invention, an increased amount of modified MIF can be analyzed in a patient sample such as blood, serum, and urine as compared to a healthy individual.

As shown here, in particular, C81 by linking a moiety to the C81 sulfur atom The derivatization of sulfhydryl groups promotes the conversion of MIF to disease-related state oxMIF. An oxMIF-specific antibody that specifically binds to oxMIF and is unable to bind to redMIF can be useful in the context of the present invention. Non-limiting examples of oxMIF specific antibodies are Baxter antibodies RAM9, RAB9, RAM4, RAB4, RAM0 and RAB0.

Such as cystine-mediated oxidation, GSSG (ox. glutathione) mediated oxidation The oxidative procedure by MIF incubation with Proclin300 or a protein crosslinker (e.g., BMOE) can result in binding to the antibodies mentioned above.

The anti-system mentioned above by means of two of its sequences as well as by Escherichia coli (Strain TG1) is deposited as a plastid to characterize and support, comprising the light or heavy chain of each of the antibodies mentioned above. The plastid is characterized by its number of DSMs, as collected by the German Collection of Microorganisms and Cell Cultures (DSMZ), Mascheroder Weg 1b, Braunschweig, Germany, according to the Budapest Treaty. The official number obtained during deposition. These plastids were separately deposited in E. coli strains.

The plastid with the number of DSM 25110 contains the light chain of the anti-MIF antibody RAB4 sequence. The plastid with the number of DSM 25112 contains the heavy chain (IgG4) sequence of the anti-MIF antibody RAB4. Co-expression of plastid DSM 25110 and DSM 25112 in a suitable host cell results in Good anti-MIF antibody production of RAB4.

The plastid with the number of DSM 25111 contains the light chain of the anti-MIF antibody RAB9 sequence. The plastid with a number of DSM 25113 comprises the heavy chain (IgG4) sequence of the anti-MIF antibody RAB9. Co-expression of plastid DSM 25111 and DSM 25113 in a suitable host cell results in the production of a preferred anti-MIF antibody RAB9.

The plastid with the number of DSM 25114 contains the light chain of the anti-MIF antibody RAB0 sequence. The plastid with the number of DSM 25115 contains the heavy chain (IgG4) sequence of the anti-MIF antibody RAB0. Co-expression of plastid DSM 25114 and DSM 25115 in a suitable host cell results in the production of a preferred anti-MIF antibody RAB0.

The antibodies RAM0, RAM9 and RAM4 are also deposited; both have been deposited by DSZM, Braunschweig, Germany according to the Budapest Treaty on April 12, 2012, with the following names: 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: GA.784-01.pRAM0hc-DSM 25864.

The biological sample in the context of the present invention is preferably a body fluid sample of the individual to be diagnosed on which (which/whom). The body fluid sample is any body fluid sample known to those skilled in the art. Such exemplary but non-limiting samples may be blood, plasma, serum, saliva, urine, nasal fluid, ascites, ocular fluid, amniotic fluid, aqueous humor, vitreous fluid, tear fluid, Cooper Cowper's fluid, semen, interstitial fluid, lymph, breast milk, mucus (including nasal discharge and sputum), pleural fluid, pus, menstruation, vaginal lubrication, sebum, cerebrospinal fluid and synovial fluid. Additional biological samples in the context of the present application may be lavage (flush) of (hollow) body organs (eg, bronchoalveolar lavage, gastric lavage, and intestinal lavage).

A biological sample in an alternative embodiment in the context of the present application is The cell sample of the individual on which the diagnosis is made, preferably from a cell sample of circulating or diseased tissue, is more preferably a single cell suspension sample.

According to the present invention, the diagnostic methods, analysis and uses disclosed herein, specific words Methods for diagnosing MIF-related disorders as disclosed herein encompass methods for determining whether a given disorder or pathological condition involves a C81-modified MIF or a C81-modified MIF.

The invention therefore also relates to a method of assessing the progression of a disease; in the invention In this context, the term "state of disease" or "disease state" is understood to be synonymous with the term "severity of disease" and refers to the severity, extent or state (ie, stage) of a disease or condition. For example, a disease can be characterized as mild, moderate, or severe. The determination or assessment of severity or extent, i.e., the state of the disease, is well known to those skilled in the art. The actual method to be performed for this assessment will of course depend on the disease or condition being discussed. For example, the state of the disease can be determined by comparing the likelihood or length of survival of an individual with the disease to the likelihood or length of survival of other individuals with the same disease.

In other specific examples, the state of the disease can be compared by comparing the individual with the disease The symptoms of the disease are determined by the symptoms of other individuals with the same disease. In yet another embodiment, the state of the disease and its progression are reflected by changes in symptoms over the same period of time in the same patient.

In another preferred aspect, the invention may also be directed to a selection such as compliance with A method of treating an individual of a condition for treatment of a MIF compound, wherein the individual has a (MIF-related) disorder or is at risk of developing a (MIF-associated) disorder, the method comprising detecting the presence of a modified MIF in the individual and/or Or content and / or content changes. An individual having a higher level of modified MIF can be selected for prophylactic or therapeutic treatment with an anti-modified MIF compound as defined above.

The term "prophylactic" or "therapeutic" treatment is technically cognitive and points to the patient The drug is administered. The treatment is preventive if it is administered prior to the clinical manifestation of a condition (such as a disease of a host (such as a human or animal) or other state that is not pleasing to the eye) that is not pleasing to the person, that is, Protecting the host from the development of conditions that are not readily identifiable by the host, but if administered after the performance of a condition not seen by the person, the treatment is therapeutic (ie, intended to reduce, improve or maintain the existing non-my What you like to see or its side effects).

As used herein, an anti-modified MIF compound refers to any reduction, inhibition, An agent that counteracts, counteracts, or reduces the biological activity of a modified MIF. The anti-modified MIF compound can be an agent that inhibits or neutralizes the modified MIF activity, such as a small molecule or antibody. Preferred antibodies are antibodies as described herein, in particular RAM9, RAB9, RAM4, RAB4, RAM0 and RAB0; preferably RAB9 or RAB0; or RAM9 or RAM0.

The diagnostic methods or assays disclosed herein can be used to determine, for example, a patient's body fluids The presence or amount of modified MIF in a sample or cell sample. The presence or absence of oxMIF is suitable for distinguishing whether a disease is MIF-related or whether oxMIF treatment is reasonable. The OxMIF level indicates disease progression or therapeutic efficacy.

Further disclosed in the context of the present invention comprises an anti-modified MIF antibody a set of bodies or antigen binding portions thereof. The kit may include additional diagnostic or therapeutic agents other than antibodies And its use. The kit may also include instructions for use in a diagnostic or therapeutic method.

Definition and general technology

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meaning commonly understood by the ordinary skill. In general, the nomenclature and techniques described herein in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry are well known and commonly employed in the art. By. The methods and techniques of the present invention are generally carried out in accordance with the conventional methods well known in the art and as described in the various general and more specific documents cited and discussed throughout the specification, unless otherwise indicated. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (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, NY (1990), each of which is incorporated herein by reference.

"MIF" or "macrophage migration inhibitory factor" refers to a protein called a key mediator in an immune response and an inflammatory response and is called a counter-regulator 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 monomeric form is encoded as a 115 amino acid protein but is formed as a 114 amino acid protein due to cleavage of the initial methionine. "MIF" also includes "GIF" (glycosylation inhibitor) and other forms of MIF, as is well known to those skilled in the art such as, for example, by streptavidin binding peptide (SBP). Affinity markers (which are suitable for purification of proteins) are MIF fusion proteins via linkers (eg, (GGGGS) 2 linkers) as appropriate. According to SEQ ID NO: 15, the amino acid number 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 herein as The MIF isoform produced by MIF treatment is carried out with a mild oxidizing reagent such as cystine (sulfhydryl reagent). The oxMIF comprises a MIF isoform that is recombined with a oxMIF shared structure that is produced, for example, in vivo after challenge with the animal. It is specifically bound by the oxMIF-specific antibodies disclosed herein.

"Reduced MIF" or redMIF for the purposes of the present invention The MIF is restored and is not combined with RAB0, RAB9 and/or RAB4 and/or MIF to RAM0, RAM9 and/or RAM4.

The binding kinetics of the antibodies to the various forms of MIF disclosed herein can be used The Biacore 3000 system is inspected by surface plasmon resonance analysis. The antibodies were coated onto a CM5 (=carboxymethylated polydextrose) wafer and the MIF protein was pre-incubated with 0.2% Proclin 300 and the mixture injected. Proclin300 consists of an oxidative isothiazolone that stabilizes the oxMIF structure by avoiding the conversion of oxMIF to redMIF.

An antibody or antigen binding portion preferably by KD of less than 100nM, preferably less than the K _D 50nM, even more preferably by less than K _D 10nM binding specificity of the forms disclosed herein, the MIF (e.g. the modified MIF) . Particularly preferably, the antibodies of the invention bind to a specific form of MIF (e.g., modified MIF) disclosed herein by a KD of less than 5 nM.

The (non)binding of antibodies can be determined as commonly known to those skilled in the art to Examples of any of the following methods: differential binding ELISA to recombinant MIF, or surface plasmon resonance of recombinant MIF using any of the forms disclosed herein, similar to that described above Well known Biacore analysis.

A preferred method for assays for binding is to resonate the surface plasmon of the antibody to a MIF of the form disclosed herein (e.g., modified MIF), wherein "binding" means less than 100 nM, preferably less than 50 nM or even less preferably less than A KD of 10 nM, however, this non-binding (e.g., in the case of a modified MIF or oxMIF-specific antibody to redMIF) is characterized by a KD of 400 nM or more. "Combination" and "specific binding" are used interchangeably herein. "Differential binding" in the context of the present application encompasses a compound (specifically, an antibody as described herein) or a test compound that binds to a modified MIF (eg, by the above-referenced K _D value) and it does not bind to redMIF and/or to MIF which does not contain the modified sulfur atom of C81.

As used herein, reference to "antibody" encompasses antigen-binding antibody derivatives, their construction or Fragments are known to those of skill in the art, and specifically encompass antigen-binding molecules comprising an antigen binding portion of an antibody. "Antibody" refers to an intact antibody or a molecule consisting of or comprising an antigen binding portion of an antibody that competes (specifically) with the intact antibody. See generally, Fundamental Immunology, Ch. 7 (Paul, W. ed., 2nd edition, Raven Press, N.Y. (1989)), which is 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, but is not limited thereto.

The term "antigen-binding portion" of an antibody means One or more of the antibodies retain a fragment of the ability to specifically bind to an antigen (eg, a modified MIF). Molecules comprising an antigen binding portion of an antibody can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Such molecules include, but 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, bifunctional antibodies, antibodies and containing sufficient binding to confer specificity A polypeptide of at least a portion of an antibody of an antigen of interest, such as a MIF of the form disclosed herein, for example, to modified MIF, redMIF, oxMIF, MIF containing a free sulfhydryl group in C81, modified without C81 The MIF of the sulfur atom, or other forms of MIF as disclosed herein. From the N-terminus to the C-terminus, both the mature light chain variable domain and the heavy chain variable domain comprise the regions FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The distribution of amino acids to the various domains is based on Kabat's 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 (eg, 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 (eg, a bispecific antibody or a bifunctional antibody), a detectable agent, a cytotoxic agent, an agent And / or link molecules.

Within the context of the present invention, an antibody can be characterized, for example, as a human antibody, human Antibody, camelid antibody. Further, the antibody may be, for example, a chimeric antibody. Additionally, the antibody can be, for example, an isolated antibody.

The term "KD" refers to two mutuals based on common sense of the person familiar with the art. The equilibrium dissociation constant of the acting partner (eg, a particular antibody and its respective antigen).

The term "human antibody" refers to a variable domain and constant The domain is any antibody to a human sequence. The term encompasses having derived from human genes but has been altered to For example, reducing the potential for immunogenicity, increasing affinity, and eliminating antibodies that may result in sequences that are not desired to be folded, such as cysteine. The term encompasses such antibodies that are produced recombinantly in non-human cells, which may, for example, confer glycosylation to atypical human cells.

The term "humanized antibody" refers to a human sequence Also included are antibodies that are not human sequences.

The term "camelized antibody" refers to the structure of an antibody Or the sequence has been changed to an antibody that is closer to the antibody from the camel, also indicating the camelid antibody. The design and production of camelid antibodies are part of the common sense of those skilled in the art.

The term "chimeric antibody" is used to mean that it comes from two or Antibodies from two or more different types of regions.

The term "isolated antibody" or "isolated antigen binding" An isolated antigen-binding portion thereof refers to an antibody or antigen-binding portion thereof that has been identified and selected from an antibody source such as a phage display library or a B cell antibody library.

The antibodies disclosed herein can be borrowed by any method for producing recombinant DNA. Produced by genetic engineering, for example, by reverse transcription of RNA and/or amplification of DNA and by colonization into expression vectors. In some embodiments, the vector is a viral vector in which additional DNA fragments can 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., a bacterial vector having a bacterial origin of replication and a free mammalian vector). In other embodiments, a vector (eg, a non-episomal mammalian vector) can be integrated into the genome of the host cell when introduced into a host cell, and thereby replicated along with the host genome. In addition, certain vectors are capable of directing the performance of their operably linked genes. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

The antibodies disclosed herein can be carried out, inter alia, by means of conventional expression vectors, such as bacteria A body (such as pBR322 and its derivatives) or a eukaryotic vector is produced. The sequences encoding the antibodies can have regulatory sequences that regulate replication, expression and/or secretion from the host cell. Such regulatory sequences include, for example, a promoter (eg, CMV or SV40) and a signal sequence. The expression vector may also comprise selection and amplification markers, such as dihydrofolate reductase gene (DHFR), hygromycin-B-phosphotransferase, and thymidine-kinase. The components of the vector used, such as selection markers, replicons, enhancers, are commercially available or can be prepared by conventional methods. The expression of such vectors in various cell cultures, such as mammalian cells such as CHO, COS, HEK293, NSO, fibroblasts, insect cells, yeast or bacteria (such as E. coli) can be constructed. In some cases, cells are used to allow for optimal glycosylation of the expressed protein.

The antibody light chain gene and the antibody heavy chain gene can be inserted into the isolation vector or These genes are inserted into the same expression vector. The antibody gene is inserted into a performance vector by standard methods such as binding of the antibody gene fragment and the complementary restriction sites on the vector or blunt end engagement if unrestricted sites are present.

Production of antibodies or antigen-binding fragments thereof can include those known in the art. Any method of introducing recombinant DNA into eukaryotic cells by transfection (eg, via electroporation or microinjection). For example, recombinant expression of an antibody can be introduced into a suitable host cell strain by introducing a plastid containing the antibody-encoding DNA sequence under the control of one or more regulatory sequences (such as a strong promoter) by appropriate transfection methods, producing cells. This is achieved by stably integrating the introduced sequences into the genome. Liposomal transfection methods are examples of transfection methods that can be used in accordance with the present invention.

The production of antibodies can also include those known in the art for use in such transformed cells. Culture (eg, in a continuous or batch mode) and antibody such as constitutive or induction performance Any method. It is especially referred to WO 2009/086920 as a further reference for the production of anti-(ox) MIF antibodies.

The sequence of an oxMIF-specific antibody is also partially disclosed in WO 2009/086920. In addition, the oxMIF-specific antibody is characterized by the sequence: the amino acid sequence of the light chain of RAB9 SEQ ID NO: 1: Amino acid sequence of the light chain of RAB4 SEQ ID NO: 2: The amino acid sequence of the light chain of RAB0 SEQ ID NO: 3: The amino acid sequence of the light chain of RAB2 SEQ ID NO: 4: Amino acid sequence of the heavy chain of RAB9 SEQ ID NO: 5: Amino acid sequence of the heavy chain of RAB4 SEQ ID NO: 6: Amino acid sequence of the heavy chain of RAB0 SEQ ID NO: 7: The amino acid sequence of the heavy chain of RAB2 SEQ ID NO: 8: Amino acid sequence of RAM0hc SEQ ID NO: 9: Amino acid sequence of RAM0lc SEQ ID NO: 10: Amino acid sequence of RAM9hc SEQ ID NO: 11: Amino acid sequence of RAM9lc SEQ ID NO: 12: Amino acid sequence of RAM4hc SEQ ID NO: 13: Amino acid sequence of RAM4lc SEQ ID NO: 14: Sequence of human MIF SEQ ID NO: 15

The antibody according to the invention is preferably an isolated monoclonal antibody. Anti-MIF antibodies can be IgG, IgM, IgE, IgA or IgD molecules. In other embodiments, the anti-MIF antibody is an IgGl, IgG2, IgG3 or IgG4 subclass. In other embodiments, the antibody is a subclass of IgGl or IgG4. In other embodiments, the antibody is a subclass IgG4. In some embodiments, the IgG4 antibody has a single mutation that changes serine (serine 228, according to the Kabat numbering scheme) to proline. Thus, the CPSC subsequence in the Fc region of IgG4 becomes CPPC, which is a subsequence in IgGl (Angal et al. Mol Immunol. 1993, 30, 105-108).

The antibody can be recovered from the culture medium using standard protein purification methods, for example, via anion exchange chromatography or affinity chromatography. In one embodiment, the anti-(ox) MIF antibody can be purified from the cell culture supernatant by size exclusion chromatography.

The terms MIF "central region" and "C-terminal region" refer to human MIF comprising amino acid 35-68 and amino acid 86-115, respectively, preferably comprising amino acid 50-68 and amino acid 86 to 102, respectively. The area of human MIF.

It is especially preferred that the anti-oxMIF antibody binds to the amino acid 50-68 or the regional amino acid 86-102 of the human MIF. This is also reflected by the combination of the preferred antibodies RAB0, RAB4, RAB2 and RAB9 and the following combinations of RAM4, RAM9 and RAM0:

RAB4 and RAM4: Amino Acid 86-102

RAB9 and RAM9: Amino acid 50-68

RAB0 and RAM0: Amino acid 86-102

RAB2: amino acid 86-102.

The term "antigenic determinant" includes any protein determinant capable of specifically binding to an immunoglobulin protein or antibody fragment. An epitope determinant usually consists of a chemically active surface group of molecules such as an exposed amino acid, an amino sugar or other carbohydrate side chain and is usually It has specific three-dimensional structural features and charge-to-mass ratio characteristics.

The term "vector" means capable of transporting another nucleic acid to its connected A nucleic acid molecule of a nucleic acid. In some embodiments, the vector is a plastid, that is, an additional double-stranded DNA loop into which additional DNA fragments can be ligated.

The term "host cell" refers to a cell line that is capable of introducing a table. The recombinant protein is then produced following the 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 strain" means a specific individual cell strain and progeny of such a cell strain. Because certain modifications may occur in progeny due to either mutation or environmental influences, the progeny may actually be different from 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 invention is, for example, COS cells, CHO cells or For example, HEK293 cells or any other host cell known to those skilled in the art, and thus, for example, include bacterial cells, such as, for example, E. coli cells. In one embodiment, the antibody is expressed in a DHFR deficient CHO cell line (eg, DXB11) and with the addition of G418 as a selection marker. When a recombinant expression vector encoding antibody gene is introduced into a CHO host cell, it is produced by culturing the host cell for a period of time sufficient to permit expression of the antibody in the host cell or secretion of the antibody into the medium in which the host cell is grown. antibody.

"MIF-related disorders" in the context of the present invention include, but are not limited to, infectious diseases, inflammation, autoimmune, cancer, cell differentiation, atherogenesis, and angiogenesis-related diseases. MIF-related disorders are, for example, type I and type II diabetes, acute lung injury, asthma, allograft rejection, graft versus host disease, wound healing disorders, and inflammatory bowel disease. Cancer is another MIF-related condition. In particular, MIF-related cancers include lymphomas, sarcomas, Prostate cancer and colon cancer, bladder cancer, pancreatic cancer, ovarian cancer, melanoma, hepatocellular carcinoma, ovarian cancer, breast cancer and pancreatic cancer.

In addition, atherosclerosis is a MIF-related disorder.

In addition, malignant ascites is a MIF-related disorder.

In addition, MIF-related disorders include sarcoma, scleroderma, psoriasis, (ulcerative) colitis, and atopic dermatitis, as well as septic shock, delayed allergic reactions, acute respiratory distress syndrome (ARDS), multiple sclerosis Symptoms, pancreatitis and ischemic heart damage.

MIF-related immune and inflammatory conditions include Gram-negative and Gram-positive sepsis, such as Pseudomonas aeruginosa infection or sepsis, DTH, spheroid nephritis, arthritis, adjuvant arthritis, juvenile arthritis, (autologous Immune) encephalomyelitis/encephalitis, (autoimmune) myocarditis, allergic encephalitis, gastritis, colitis; (immune) spheroid nephritis; pneumonia, toxic shock syndrome, viral infection, tuberculosis, hepatitis B, dengue fever Dengue fever), parasites associated with helminth MIF infection, specifically malaria, leishmaniasis, trypanosomiasis, toxoplasmosis, amoebiasis, schistosomiasis, cysticercosis, rot Insect and filariasis; kidney diseases such as leukocyte-mediated kidney injury, non-proliferative nephropathy, proliferative nephropathy, renal allograft rejection, and Finnish-type congenital renal syndrome, nephritis, such as uric acid nephropathy Nephropathy, ureteral obstruction and diabetic nephropathy of hypertensive nephropathy.

Neuralgia is another MIF-related disorder.

The best diseases to be diagnosed according to the present invention are: spheroid nephritis, sepsis, lymphoma, lupus, nephritis, psoriasis, ulcerative colitis and ophthalmic conditions, and Burkitt's lymphoma, leukemia, malignant ascites , prostate adenocarcinoma, pancreatic cancer, ovarian cancer, colorectal cancer, head and neck cancer, renal cell carcinoma, hepatocellular carcinoma, breast cancer and lung cancer. K-ras wild type cancer Both k-ras mutant cancers can be treated in accordance with the present invention.

In particular, the present invention also covers all third-line treatments of the above mentioned diseases. Treatment.

One aspect of the invention relates to a modified MIF in a sample of an individual Detection. This detection allows a skilled practitioner to determine, for example, whether MIF is a therapeutically important component of a disease or condition that afflicts an individual. This assay will assist the practitioner in determining whether the (additional) anti-(ox) MIF treatment may be beneficial to the individual in question.

Modified MIFs are also suitable for use as markers to determine the health of a given individual or Disease condition; a higher level of modified MIF will allow the discovery of an individual with MIF-related disease; the modified MIF can therefore also be used as a general marker for the health/disease condition of the individual, for example with current and extensive The determination of the C-reactive protein (CRP) used as the (secondary) marker was similar.

According to the present invention, the presence of cysteine 81 (C81) of MIF is directed against MIF The conversion of the disease-related state, that is, the redox transition to redox to oxMIF, is critical, and the derivatization of the C81 sulfhydryl group promotes (or is responsible for) this conversion to the oxMIF form. Accordingly, the present invention provides a modified 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 covers disease detection, disease status Assessment of status and monitoring of disease progression also allows monitoring of the efficacy of therapeutic treatment.

In a preferred embodiment, the MIF-related disorders according to the present invention The diagnosis can encompass the use of a compound that binds to the modified MIF or another use for detection of the modified MIF. These compounds that differentially bind to the modified MIF can be differentially knotted An antibody or small molecule that binds to the modified MIF. Diagnostic assays useful in the present invention can be any diagnostic assay known to those skilled in the art. In particular, for example, ELISA format, sandwich (ELISA) format, by using any of FACS, immunofluorescence, immunohistochemistry, and various other suitable methods (all of which are well known in the art) Perform a diagnostic analysis.

When assessing the presence of a modified C81 according to the invention of a sample, it is possible A false positive result from an oxidized form of MIF, such as CIF modified MIF, occurs. For example, oxidation of a cysteine residue in a MIF protein (and thus C81) can be induced by redox active iron and heme in a biological sample (eg, a hemolytic blood sample), or if an oxidizing agent is added to the sample, This oxidation is then induced. Such pseudo-positive results can be avoided, for example, by taking measures as described herein below, for example by deactivating redox active iron and heme, and by avoiding the addition of, for example, an oxidizing agent.

For the analysis of circulating MIF in blood, a special sample procedure is preferably employed, which includes the following measures and steps. Citrate plasma is preferred. Citrate plasma from fresh blood (not stored for more than 12 h at +4 ° C) was centrifuged at 40 g for 5 min. The supernatant was transferred to a new tube and centrifuged again for 2 min at 2000 g. The cell-free supernatant was again transferred to a new tube and centrifuged at 16000 g for 3 min. After three centrifugation steps, the cell-free supernatant can be stored at -80 °C or used directly for MIF analysis. If the MIF is analyzed in serum , the cells and insoluble fragments are preferably removed by the same three centrifugation steps prior to storage by freezing or prior to running the MIF ELISA.

The urine sample prior to MIF ELISA deposits should also preferably be removed by a centrifugation step (16000g, 5min). Typically, cells and other common particles present in biological fluids (eg, tears, saliva) must be removed in advance by a centrifugation step and then stored for testing MIF.

In addition, since denatured MIF can also be specifically bound to oxMIF or to repair The antibody to MIF is cognized and the MIF protein to be tested should retain its natural conformation during sample preparation (eg during separation and preparation of body fluids). Thus, denaturation conditions/steps such as boiling, fixing (on membranes, plastics (plates) or wafers) and chemotherapeutics (for example by reducing agents, oxidizing agents and organic solvents) are avoided.

For the analysis of MIF on the cell surface, flow cytometry is preferred. Analysis. It is especially important that the samples do not undergo hemolysis during sample preparation. Thus, all of the samples used in the flow cytometry analysis of the present invention have been prepared without any steps that would result in hemolysis of cells within the sample.

Summary of preferred embodiments of the invention

A method for treating or preventing a MIF-related disorder in an individual in need thereof, by administering to the individual an effective amount of the following compound

(a) preventing modification of the MIF at position 81, or (b) preferentially binding to the modified MIF at position 81, as compared to the modified MIF not containing cysteine 81.

2. The method of embodiment 1, wherein the preferential binding compound is an antibody or a molecule comprising an antigen binding portion of the antibody, and/or the preferential binding compound induces a modification of the modified MIF in the absence of the compound. The modified MIF in the form of binding of antibody RAM0, RAB0, RAM9 or RAB9 or another oxMIF-specific antibody to a small extent.

3. The use of a modified MIF at position 81 for use as a target for the treatment or prevention of MIF related conditions.

In a preferred embodiment, a modified MIF line is carried at position 81 (SEQ ID NO: 15) for use as a target for drug discovery analysis. Drug discovery analysis is an analysis well known to those skilled in the art. It is an analysis of the (potential) usefulness of a given compound by which a given compound is used for the treatment or prevention of a disease or condition ( "as a drug" ).

4. A method of monitoring the effectiveness of a treatment for a condition associated with a MIF comprising determining whether a MIF position 81 carries a modification step in a sample isolated from the individual before and after the treatment, wherein the MIF position is compared to the pre-treatment period after treatment The degree of modification of 81 is small and the treatment is identified as being effective.

Both "effectiveness" and "efficacy" are well-known terms and are used interchangeably herein.

5. An anti-MIF antibody or a molecule comprising an antigen binding portion of an antibody that binds to a modified MIF at position 81 but does not bind to an unmodified MIF at position 81.

6. A method of analyzing whether a test compound preferentially binds to a modified form of MIF at position 81, the method comprising the steps

(a) modifying the MIF at position 81 to obtain a modified MIF, (b) combining the compound to be tested and the modified MIF in a test sample and in a control sample in which the MIF is unmodified at position 81, (c) evaluating the combination of the compound and the modified MIF in the test sample and in the control sample

Wherein the compound is selected if the compound binds to the modified MIF to a greater extent in the test sample than in the control sample.

7. A method of analyzing the effect of a test compound on the conformation of a modified MIF at position 81, the method comprising the steps

(a) providing a test sample containing the modified molybdenum MIF at position 81, (b) combining the compound to be tested with the modified MIF in the test sample, and (c) evaluating the position in the test sample 81 configuration of modified MIF (i) (control 1) and/or configuration of MIF containing unmodified cysteine residues at position 81 (ii) compared to control 2 The configuration of the modified MIF, wherein if the conformational assessment indicates that the configuration of the modified MIF in the test sample has a detectable degree of similarity to configuration (ii) compared to configuration (i), or The compound is selected if the conformational assessment indicates that the configuration of the modified MIF in the test sample is detectably self-constructed (i) from the deviation.

8. The method of embodiment 7, wherein the configuration of the MIF is assessed in step (c) by immunoassay, preferably by ELISA.

9. The method of embodiment 7, wherein the configuration of the MIF is evaluated by X-ray crystallography in step (c).

10. A method of analyzing the effect of a test compound on the conformation of a modified MIF at position 81, the method comprising the steps

(a) providing a test sample containing the modified MIF at position 81, (b) combining the test compound with the modified MIF in the test sample, (c) evaluating the modified MIF in the test sample and Test compound in the absence of (i) the modified MIF (Control 1) compared to an antibody selected from RAM0, RAB0, RAM9 and RAB9 and/or any other oxMIF specific, oxMIF conformation sensitive antibody (eg selected from a combination of the disclosed oxMIF-specific antibodies), wherein the modified MIF in the test sample (ie, in the presence of the test compound) The modified MIF (Control 1) in the absence of the compound binds to the antibody to a lesser extent, and the compound is selected.

11. A method of screening for a compound that prevents modification of MIF at cysteine 81, the method comprising the steps

(a) combining MIF with the compound to be tested in the test sample, (b) in the test sample containing the compound and in the control sample in the absence of the compound, the reagent is at least in the reagent by the sulfhydryl reagent The MIF is treated under the condition that the thiol group of the cysteine 81 of the MIF is modified in the control sample, and (c) the modification of the cysteine 81 is evaluated by the sulfhydryl reagent, wherein the sulfhydryl reagent is The compound was selected in this test sample to a lesser extent than the modification of cysteine 81 in the control sample.

12. A method of testing a compound for binding to a modified form of MIF by X-ray crystallography analysis, the method comprising the steps

(a) modifying the MIF at position 81 to obtain a modified MIF, (b) crystallizing the modified MIF in the presence of the test compound or crystallizing the modified MIF in the absence of the test compound and The resulting crystals are then contacted with the test compound to provide crystals of the modified MIF, and (c) the crystals of the MIF are determined using the crystals.

13. A crystal comprising a modified MIF having a position at position 81.

14. The crystal of Concrete Example 13 further contains another compound.

15. Use of position 81 of the MIF protein for use as a diagnostic marker for MIF-related disorders and/or for monitoring MIF-related disorders.

16. Use according to embodiment 15, wherein the modified state of position 81 of the MIF protein is used as a diagnostic marker for MIF-related disorders and/or for monitoring MIF-related disorders.

17. The modified state of MIF position 81 is used as a diagnostic marker, wherein the MIF position 81 carries a modified finding indicative of a MIF related disorder.

18. A use according to embodiment 16, wherein the modified state of the sulfhydryl group of MIF cysteine 81 is used as a diagnostic marker, wherein the sulfur atom of MIF cysteine 81 is compared to the free cysteine sulfhydryl group. The findings of carrying the modification indicate a MIF related condition.

19. A method of diagnosing a MIF-related disorder, comprising the step of determining whether a MIF location 81 carries a modification in a sample isolated from an individual, wherein the MIF location 81 is identified as being modified, the individual is diagnosed with a MIF-related disorder or susceptible Infected with MIF related disorders.

20. A method of diagnosing a condition associated with MIF comprising the step of determining whether a sulfur atom of MIF cysteine 81 carries a modification compared to a free cysteine sulfhydryl group in a sample isolated from an individual, wherein if MIF is half The sulfur atom of cystine 81 is identified as carrying the modification, and the individual is diagnosed with a MIF-related disorder or is susceptible to a MIF-related disorder.

21. The use of any one of the specific examples 15 to 18, or a method of the specific example 19 or 20, which is a method for determining whether a given condition or pathological condition involves a modified MIF at position 81 Or a method associated with a modified MIF at position 81.

The use of the diagnostic kit of any one of the embodiments of the present invention, wherein the diagnostic kit comprises a preferential binding to the method of any one of the embodiments The MIF in which cysteine 81 is present or the compound that carries the modified MIF at position 81.

23. The use of embodiment 22, wherein the kit further comprises a buffer, a control reagent (eg, MIF in which cysteine 81 is present or absent, or MIF with or without modification at position 81, eg, on the sulfur atom of cysteine 81 compared to free cysteine sulfhydryl Modifications, preferential binding to any of these forms of MIF compounds), multiple anti-MIF antibodies and/or marker detection antibodies.

24. The method of any one of the specific examples 1, 2 or any of the specific examples 6 to 12, or the use of the specific example 22 or 23, wherein the compound is an antibody or a molecule comprising an antigen binding portion of the antibody.

The method of any one of the embodiments 1, 2, 4, 19, 20 or 24, or the use of any one of the specific examples 3, 15 to 18, 21 to 23, wherein the MIF-related disorder is The status of MIF-related diseases, MIF-related disease states, or progression of MIF-related diseases.

26. The use or method of embodiment 25, wherein the MIF-related disorder is an inflammatory or neoplastic disorder.

27. The use or method of embodiment 25, wherein the MIF-related disorder is selected from the group consisting of colon cancer, prostate cancer, bladder cancer, pancreatic cancer, ovarian cancer, melanoma, lymphoma, hepatocytes Cancer, asthma, ARDS, rheumatoid arthritis, sepsis, IgA nephropathy, glomerulonephritis, lupus nephritis (LN), hepatitis, pancreatitis (+/- acute lung injury), Crohn's disease ( Crohn's disease), ulcerative colitis, gastric ulcer, Alzheimer's disease, multiple sclerosis, Guillain-Barre syndrome, cardiac dysfunction, angioplasty, atherosclerosis Sclerosing, myocarditis, type 1 diabetes, diabetic retinopathy, age-related macular degeneration (AMD), atopic dermatitis, psoriasis, endometriosis, neuralgia, and uveitis.

28. The use or method of claim 25, wherein the MIF-related disorder is malignant ascites, preferably in a third line of treatment.

29. A MIF molecule modified at position 81 of SEQ ID NO: 15.

The following examples and the schemes described below illustrate the invention and methods that may be useful in performing or evaluating the invention. The examples and drawings are not intended to limit the invention.

Figure 1 shows a schematic representation of wild-type and mutant recombinant fusion constructs of MIF compared to wild-type MIF as used in the examples of the present invention.

Figure 2 shows a Western blot analysis of the MIF construct of Figure 1 using antibody RAM9.

Figure 3 shows the western blot analysis of the MIF construct of Figure 1 using antibody RAM0.

Figure 4 shows an example of a sulfhydryl reaction reagent for the derivatization of a sulfhydryl group on a compound R or MIF, the resulting reaction product, and an expected increase in molecular weight in the Da of R or MIF after derivatization.

Figure 5 shows the general settings for an ELISA assay for assessing MIF and its mutant and/or derived forms.

Figure 6 shows wild-type human MIF in its ELISA assay in its reduced form (human MIF in its reduced form; huMIFred), in cysteineized form (Cys-MIF) and in the use of 5,5'-dithiol double -(2-Nitrobenzoic acid) / DTNB (DTNB-MIF) when bound to antibody RAM9.

Figure 7 shows the fusion of MIF with wild type (MIF) and wild type by ELISA. (MIF (wt) SBP) and mutant fusion (MIF (C57S) SBP, etc.) forms are unmodified, are derived from DNTB and evaluated under non-reducing conditions or in the presence of DTT in DNTB and under reducing conditions Evaluation of the combination of antibody RAM9 (Figure A) and RAM0 (Figure B) in the next evaluation.

Figure 8 (panels A and B) show mass spectrometry data obtained by MIF in the form of its wild type (MIF), wild type fusion (MIFwt-SBP) and mutant fusion (MIF (C57S) SBP, etc.), using cysteine Amino acid or DTNB (+NTB) confirms the derivatization. The peak that reflects the corresponding increase in molecular weight is produced by cysteine acidation or derivatization of DTNB.

Figure 9 shows the mass spectrum obtained from peptide mass fingerprinting of cysteine-acidified MIF.

Figure 10 shows the results of analysis of cysteine MIF by peptide mass fingerprinting.

Figure 11 shows the mass spectra obtained from peptide mass fingerprinting of free DTNB-derived MIF.

Figure 12 shows the results of an analysis of MIF derived from DTNB by peptide mass fingerprinting.

Several assays or methods that are useful in the evaluation of antibodies and/or MIF are described below.

A) GCO analysis of antibody screening: The THP1 suspension culture was centrifuged and the cells were resuspended in fresh whole medium to achieve a cell density of 106 cells per ml. This culture was transferred to wells of a 96-well microplate (90 μl per well) and potential anti-MIF antibodies were added to give a final concentration of 75 μg/ml. Each antibody was tested in triplicate. Dexamethasone was added after o/n incubation at 37 ° C to obtain a concentration of 2 nM and LPS (3 ng / ml final concentration) was added after incubation at 37 ° C for 1 hour. Supernatants were harvested after an additional 6 hours incubation at 37 °C and IL-6 concentrations were determined in a commercial ELISA. The results in triplicate were averaged and the percentage of IL-6 secretion was determined compared to the control antibody. Antibodies that cause IL-6 secretion less than 75% are evaluated as positive.

B) Analysis of the determination of IC50 values

The experimental procedure was performed as described for the screening assay except that an increased amount of antibody (typically 1-125 nM) was used. The resulting dose response curve is expressed as % inhibition compared to the negative control antibody. This curve is used for the maximum inhibitory effect of the antibody (% Inh max) and the antibody concentration (IC50) showing the maximum inhibitory effect of 50%.

C) Inhibition of cell proliferation Serum stimulates secretion of MIF in resting NIH/3T3 and MIF in turn stimulates cell proliferation. The antibody inhibits this endogenous MIF and, therefore, reduces the proliferation of resting NIH/3T3 cells. The reduction in proliferation was determined by the incorporation of 3H-thymidine.

1000 NIH/3T3 cells per well were cultured in 96-well plates at 37 ° C over the weekend in medium containing 10% serum. The cells were then starved overnight at 37 ° C by incubation in medium containing 0.5% serum. The 0.5% medium was removed and replaced with fresh medium containing 10% serum, 75 μg/ml antibody and 5 μCi/ml of 3H-thymidine. The cells were washed twice with 150 μl of cold PBS per well after 16 hours of incubation in a CO 2 incubator at 37 °C. 150 μl of a 5% (w/v) TCA solution per well was added using a multi-channel pipette and incubated at 4 ° C for 30 minutes. The plate was washed with 150 μl of PBS. 75 μl of a 0.5 M NaOH solution with 0.5% SDS per well was added, mixed and stored at room temperature. Samples were measured in a ß-counter by mixing 5 ml of Ultima Gold (Packard) with 75 μl of sample solution. Each assay was performed in triplicate and the values were compared to the values of the control antibody by t test. Antibodies that significantly reduced proliferation (P < 0.05) were evaluated as positive.

D) Binding study: Determination of epitopes of anti-MIF antibodies

Each peptide was diluted in coupling buffer to give a peptide concentration of typically 1 μg/ml, which was added to a microplate (NUNC ImmobilizerTM Amino Plate F96 Clear) and incubated overnight (100 μl per well) at 4 °C. Recombinant full length MIF and PBS were used as a control group. The plate was washed 3 times with 200 μl of PBST and (100 μl per well) antibody (2-4 μg/ml in PBS) was added and incubated for 2 hours at room temperature with gentle shaking. The plate was washed 3 times with 200 μl PBST and added (100 μl per well) to detect antibodies (eg Fc-specific anti-human IgG/HRP tag, Sigma). After incubating for 1 hour at room temperature by gentle shaking, the plate was washed 3 times with 200 μl of PBST. Each well was incubated with 100 μl of TMB (3,3',5,5'-tetramethylbenzidine) solution (T-0440, Sigma) for 30 minutes in the dark. The staining reaction was stopped by increasing 100 μl of 1.8 M H 2 SO 4 solution per well. The sample was measured at 450 nm.

E) Affinity determination of anti-MIF antibodies by Biacore

Typically, the 40 RU unit of human recombinant MIF is immobilized on a sensor wafer by a CM5 (=carboxymethylated polydextrose) matrix (Biacore). Fab fragments were injected at a concentration ranging from 6-100 nM typical diluted in HBS-EP. The wafer was renewed with 50 mM NaOH + 1 M NaCl after each cycle. Affinity was calculated according to the 1:1 Langmuir model.

F) Measurement of C81-modified MIF in serum samples by ELISA

The microtiter plate is coated by, for example, human anti-C81 modified MIF monoclonal antibody. Human serum samples were diluted 1:25 in 0.5% fish gel/PBS (pH 7.2) and applied to the plate. After washing the plate, detection of C81-modified MIF captured by the coated antibody is performed, for example, by affinity purification of a plurality of rabbit antibodies against human MIF. For example, a goat anti-rabbit antibody labeled with HRP (BioRad, Cat.: 171-6516; any other known in the art may also be used herein) Goat anti-rabbit) and TMB (3,3',5,5'-tetramethylbenzidine; T-0440, Sigma) as the chromogenic substrate (may also use any other suitable matrix known to those skilled in the art) After further incubation, the ELISA was read. The color reaction of TMB was stopped with H2SO4 and the ELISA plate was measured at 450 nm.

ELISA calibration can be achieved by recombinant humans newly derived from cysteine 81 MIF is performed, for example, using DTNB or MIF diluted in ProClin containing a buffer that results in an artificial oxMIF configuration. Standards were diluted in 0.5% fish gelatin/PBS including 0.2% ProClin300 and 4% human control plasma (i.e., pool of serum samples from 50 healthy donors). The calibration curve can range, for example, from 10 ng/ml to 0.156 ng/ml.

G) Measurement of C81-modified MIF on the cell surface in patients with sepsis by flow cytometry

Anti-CD3 epsilon (for T cells) labeled with Alexa700 and anti-Ly6G (for granulocytes) labeled with PerCP-Cy5.5 or APC-labeled anti-CD14 (for monocytes) and PE-Cy7-labeled anti-CD19 (For B cells) Blood was simultaneously stained in cell staining buffer (Biologend) by 300 nM against C81-modified MIF antibody or control IgG (eg, from patients with sepsis). After washing, human antibodies were detected using goat R-PE labeled anti-human IgG antibodies. After washing, red blood cells were lysed with BD FACSTM lysis solution (Becton Dickinson, Franklin Lakes, USA). Data acquisition was performed by DIVA ^(TM) software (software version 6; Becton Dickinson) using FACS ^(TM) Canto II (Becton Dickinson) and data was analyzed using FlowJo ^(TM) software (Treestar, Ashland, OR, USA).

H) Preparation of multiple strains and affinity purified rabbit anti-human MIF antibodies

Immunization schedule of multiple rabbit anti-huMIF antibodies in New Zealand white rabbits

For the initial immunization, the weight of 25 μg obtained as described in Example 1 Groups of human MIF were diluted in 100 μl of PBS and mixed with 100 μl of Complete Freunds Adjuvant (CFA). 200 μl (4 x 50 μl) of the mixture was applied subcutaneously to different body parts of each rabbit. Two to three weeks after the initial immunization, a first booster immunization with 25 μg of recombinant human MIF (suspended in 100 μl of PBS) was mixed with 100 μl of Incomplete Freunds Adjuvant (IFA). Again, 200 μl (4 x 50 μl) of the mixture was applied subcutaneously to the different body parts of each rabbit. A second booster immunization was performed 2-3 weeks after the first booster immunization, and 25 μg of recombinant human MIF (suspended in 100 μl of PBS) was mixed with 100 μl of Incomplete Freunds Adjuvant (IFA). Again, 200 μl (4 x 50 μl) of the mixture was applied subcutaneously to the different body parts of each rabbit. The immunization schedule was terminated 2 weeks after the second booster immunization. Typically, plasma from multiple rabbits is combined and used for isolation of anti-MIF antibodies.

Purification of protein A and huMIF affinity purification program of multiple rabbit anti-huMIF antibodies

Isolation of rabbit anti-huMIF antibodies from immune plasma is typically performed by two affinity chromatography steps. First, the plasma was purified by Protein A affinity column (MabSelect Sure, GE Healthcare). The effect was that the rabbit plasma was diluted 1:3 with 20 mM Na2HPO4 buffer (pH 7.0) and applied to the affinity column. After the washing step (5 column volumes with 20 mM Na2HPO4 buffer, pH 7.0), the entire rabbit IgG was eluted by 100 mM glycine (pH 2.8). The eluate was combined and neutralized to pH 7.0 using 1 M Tris/HCl. For hu-MIF affinity purification, whole rabbit IgG was again diluted 1:3 with 20 mM Na2HPO4 buffer (pH 7.0) and applied to a 5 ml NHS-affinity column (GE Healthcare) as suggested by the supplier with 25 mg rhuMIF. In the washing step (5 column volumes with 20 mM Na2HPO4 buffer, pH 7.0) Thereafter, the elution of the specific rabbit anti-huMIF antibody was achieved by 100 mM glycine (pH 2.8). The eluate was combined and neutralized to pH 7.0 using 1 M Tris/HCl. Finally, hu-MIF affinity-purified specific rabbit anti-human MIF antibody (hereinafter referred to as "anti-human MIF affinity purified polyclonal antibody") was dialyzed against PBS and stored at -20 °C.

Example

In the following examples, the presence of cysteine 81 (C81) of MIF specifically illustrates the conversion of MIF to a disease-associated state, that is, the redox transition from redMIF to oxMIF is critical and the derivation of C81 sulfhydryl is promoted. (or responsible for) this conversion.

Example 1 Preparation of recombinant wild-type and mutant forms of MIF

As shown in Figure 1, the wild type of human MIF (huMIF) via the (GGGGS)2-linker (see SEQ ID NO: 16) was labeled by streptavidin binding peptide (SPB). The cysteine mutant recombinant fusion construct is prepared, colonized and expressed by standard methods. Briefly, synthetic DNA (Invitrogen, GeneArt) was selected in pet25b and expressed in E. coli Shuffle T7 Express (NEB). Soluble proteins were extracted using BugBuster® Protein Extraction Master Mix (Novagen) and purified by affinity chromatography via streptavidin column and biotin elution. Further purification was carried out using size exclusion chromatography. The sequence of the streptavidin-binding peptide (SPB) tag is: MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP (SEQ ID NO: 17). Thus, a wild-type human MIF fusion with SBP (SEQ ID NO: 18) and three cysteine-mutated SBP fusions with a single mutation of cysteine to serine at positions 57, 60 and 81 of MIF were prepared. Construction (ie, single mutations are C57S, C60S, and C81S-SEQ ID NO: 19-21, respectively).

Recombinant wild-type unlabeled huMIF (SEQ ID NO: 15) Human MIF sequences are expressed in the system of E. coli cells. Freshly thawed cells were cultured overnight at +37 °C in Luria Bertani medium (LB/Amp) supplemented with Ampicillin. On the next day, bacterial cell cultures were diluted with an equal volume of fresh LB/Amp medium and induced by adding IPTG (final concentration: 1.0 mM) for 4 hours at 30 °C. Bacterial centrifugation blocks were harvested by centrifugation and stored at < -15 °C.

Further purification of intracellular human MIF proteins to allow frozen bacteria to leave The heart pieces were resuspended in 20 mM Tris/HCl buffer (pH 7.8) and the cells were mechanically disrupted by glass beads. Cell debris was removed by centrifugation and filtered using a common 0.2 [mu]m filter. The supernatant was applied directly to an anion exchange chromatography column (HiTrap 26/16 DEAE FF, GE Healthcare, Waukesha, USA) and MIF was purified by passive binding mode. The overflow was re-buffered in 20 mM Bis/Tris (pH 6.3) and further purified by cation exchange chromatography (source 30S, GE). The purified human MIF was washed by a salt gradient of 50 mM NaCl in 20 mM Bis/Tris buffer (pH 6.3). Finally, purified human MIF was re-buffered against PBS concentrated by ultrafiltration and characterized by purity and functionality.

Example 2 Western blot detection of MIF cysteine mutation by anti-oxMIF antibody

RAM9 is a monoclonal antibody as described herein above and in WO2013/050453, which preferentially binds to the oxidative, disease-related form of MIF (oxMIF) compared to the reduced form of MIF (redMIF). RAM0 is the monoclonal antibody described above and in WO 2013/050453, which preferentially binds to the oxidative, disease-related form of MIF (oxMIF) compared to the reduced form of MIF (redMIF). Under physiological conditions, binding of the antibody to MIF occurs only after a redox-dependent (and reversible) transition of MIF to oxMIF. However, under non-physiological or degenerative conditions, MIF The structure is irreversibly altered, which also results in antibody binding. Thus, for example, MIF is immobilized on a plastic or treated with a strong detergent (SDS) to effect binding of such antibodies.

Antibody RAM 9 and RAM 0 were therefore used in Western blot analysis according to standard procedures to detect the MIF construct of Example 1 under denaturing conditions. The results are shown in Figure 2. SDS-PAGE on which Western blots are carried out is carried out under reducing conditions. The results show that C57 and C60 (but not C81) are of fundamental importance for the combination of RAM9 and MIF in a linear molecular form. Residual binding of RAM9 at the highest MIF concentration was not significant compared to its binding to wild type or in the case of C81S mutation. In contrast, the combination of RAM0 and MIF as a linear molecule is not substantially affected by either of the three cysteine mutations and detects wild-type constructs and all mutations are substantially equal.

Example 3 Mutagenic effect of different cysteine residues, effect of sulfhydryl derivatization and lack of derivatization

Antibodies RAM9 and RAM0 are used as immobilized capture antibodies in ELISA assays to detect cysteine to serine mutations, thiol-derived derivatization and derivatization of different cysteine residues in oxidative, disease-related forms The effect of the formation of MIF (oxMIF). An example of a sulfhydryl reaction reagent for the derivatization of a sulfhydryl group in MIF is shown in FIG. The general setup for the ELISA assay is shown in Figure 5.

Wild type human MIF is shown in Figure 6 in an ELISA assay in its reduced form (MIF), in the form of cysteine (Cys-MIF) or in combination with antibody RAM9 when derivatized with DTNB (DTNB-MIF). According to the disclosure in WO 2013/050453, RAM 9 is not bonded to the reduced form of MIF. However, RAM9 demonstrated significant binding to both cysteine and DTNB-derived MIF. Since RAM 9 is specific for example as described in WO 2013/050453 A disease-related form of oxMIF, which displays MIF with cysteine (by treatment with cysteine) or 2-nitro-5-thiobenzoic acid (NTB). A protein derived from a disease-associated oxMIF form is induced by the derivatization of "DTNB-derived MIF" or MIF+NTB by treatment with DTNB. The equivalent result is obtained by RAM0.

MIF by ELISA in its wild-type and mutant form, with reduced/unrepaired The form of decoration ("MIF", "MIF(wt)SBP", "MIF(C57S)SBP", etc.), derived from DNTB and evaluated under non-reducing conditions ("DTNB:+") or derived from DNTB The evaluations in combination with antibody RAM9 and RAM0 when evaluated under reducing conditions ("DTNB +, DTT +") are shown in Figures 7 and 8, respectively.

These data reconfirm the sulfhydryl group of the compound DTNB by the sulfhydryl reaction. The modification promotes the formation of disease-associated oxMIF with which antibody RAM9 and RAM0 preferentially bind. For RAM 9, this effect was not observed when C57 or C60 was replaced by serine. However, it appears that this is necessary for the binding of RAM9 by C60 and C57 itself as part of the RAM9 linear epitope (described in Kerschbaumer et al. J Biol Chem. 2012 Mar 2; 287(10): 7446- 55)). Not due to the lack of oxMIF formation in MIF (C57S) SBP-DTNB and MIF (C60S) SBP-DTNB. The data show that oxMIF is still formed when C57 or C60 is replaced by serine, since these mutants still strongly promote the binding of RAM0 by the derivatization of DTNB. The latter oxMIF-specific antibody preferentially binds to MIF (C57S) SBP-DTNB and MIF (C60S) SBP- to the extent that it binds to wtMIF that has been converted to oxMIF (see MIF-DTNB and MIF(wt)SBP-DTNB). DTNB. In contrast, the data demonstrate that the derivatization of C81 and its sulfhydryl groups plays a more fundamental role in the formation of disease-associated oxMIF forms of protein than C57 or C60: when C81 is replaced by serine, by DTNB Processing mutation MIF does not enhance binding of either of the two oxMIF-specific antibodies compared to the non-derivatized form (see MIF (C81S) SBP and MIF (C81S) SBP-DTNB). Mass spectrometry data (see Example 4 below) shows that C57 and C60 were unmodified under such conditions.

The data also shows all oxMIF species induced by DTNB in wild type and the C57S or C60S mutant can be reduced by DTT.

Example 4 Mass Spectrometry Analysis of Derived MIF and Cysteine Mutants

Derivatization of MIF and its cysteine mutant by cysteine or DTNB was confirmed by mass spectrometry.

Information on full length proteins obtained by MALDI-TOF (such as those shown in Figure 8) demonstrates the generation of peaks reflecting a corresponding increase in molecular weight by cysteine acidation or derivatization of DTNB.

The strongest signal from the sample of cysteylated human MIF is the monoprotonated molecular ion at m/z 12470, followed by the 2 times protonated ion at mass m/z 6235 corresponding to the mass of 12468 Da. This corresponds well to the theoretical quality of a human MIF sequence that does not have an N-terminal methionine and has a monocysteine (+119 Da). Non-cysteined human MIF is visible with a weaker signal at m/z 12352. The additional peaks at m/z 12565 and m/z 12684 are attributed to the adduct of the sinapic acid with the matrix.

The strongest signal from a sample of human MIF derived from DTNB is a monoprotonated molecular ion at m/z 12351 (unmodified) and m/z 12549 (with NTB moiety, +197 Da) and at m/z 6177 And m/z 6275 correspond to 2 times protonated ions of the mass of 12352Da (unmodified) and 12549Da (with NTB part), respectively. The signal at m/z 12760 is attributed to the adduct with sinapic acid, m/z 24693 is a dimer and is not explained at m/z 11868 And two additional signals at m/z 18083.

Human wild-type MIF (MIF) from SBP fused by DTNB The strongest signal for the sample of wt-SBP+NTB) is the monoprotonated molecular ion at m/z 17269 (unmodified) and m/z 17467 (with DTNB, +197Da) and at m/z 8636 and m/ z 8732 corresponds to 2 times protonated ions of 17270 Da (unmodified) and 17462 Da (with DTNB), respectively. The signal at m/z 17675 is attributed to an adduct with sinapic acid.

The strongest letter from the sample of human MIF (C57S) SBP created by DTNB No. is a single protonated molecular ion at m/z 17251 (unmodified), m/z 17453 (with DTNB) and m/z 17651 (with 2 DTNB). The double protonated ions at m/z 8627, m/z 8726, and m/z 8824 correspond to the masses of 17252 Da, 17450 Da, and 17646 Da.

The strongest signal from the sample of human MIF (C60S) SBP-DTNB is at m/z Single protonated molecular ion at 17246. This corresponds well to the theoretical quality of the sequence 17247. The signal at m/z 17460 is assigned to the protein modified by DTNB and additional oxidation. The corresponding 2 times protonated ions are visible at m/z 8624 (17246 Da) and m/z 8731 (17460 Da).

The sample of human MIF (C81S) SBP created by DTNB did not produce an indication Any signal that is derived from the protein. The main peak at m/z 17249 corresponds to a single protonated unmodified molecular ion.

Mass fingerprinting of thiol-modified MIF peptides (MIF pancreatic protein) Mass spectrometry after enzymatic digestion) allows the derivatization to be localized to a specific cysteine residue, specifically cysteine 81. Peptides expected from trypsin digestion of human MIF are shown in Table 1 below:

The trypsin is digested with cysteine to digest MIF and used 4800 The peptide consisting of residues 79-87 (LLCGLLAER) was identified as cysteamine by Proteomics Analyzer (AB Sciex) after mass spectrometry of the resulting peptide by the MALDI-TOF method. In addition to the expected expression of this peptide in the unmodified form at MH+ 987.57, an additional mass of +119.00 Da, ie an additional mass corresponding to the cysteine, was observed at MH+ 1106.57. (See Figures 9 and 10). No additional mass, i.e., no cysteine acidification, was observed in the peptides with residues 13-67, which contained cysteine 57 and 60.

The pancreatic proteinase digestion of MIF has been treated with DTNB and used 4800 After proteomics Analyzer (AB Sciex) mass spectrometric analysis of the resulting peptide by the MALDI-TOF method, the peptide consisting of residues 79-87 (LLCGLLAER) was identified as being modified by DTNB treatment. In addition to the expected unmodified form of this peptide at MH+ 987.57, an additional mass of +196.97 Da, also corresponding to the 2-nitro-5-thiobenzoic acid moiety, was also observed at MH+ 1184.54. The quality corresponds to the signal of this peptide (see Figure 11 and Figure 12). No additional mass was observed in the peptide with residues 13-67, which contained cysteine 57 and 60, ie no 2- Modified with nitro-5-thiobenzoic acid.

Therefore, peptide mass fingerprinting data confirmed the derivatization of cysteine 81 in both cases.

Example 5 Production of MIF-specific antibodies modified by C81

The antibody is produced in mammalian cells, preferably in CHO cells, preferentially in CHO cells encoded by genes that have been genetically knocked out of MIF (endogenous CHO-MIF). Contamination of antibodies with endogenous CHO-MIF can be eliminated in gene knockout cells, which is desirable because of the sensitivity of the assay.

Typically, C81-modified MIF-specific antibodies are produced during batch fermentation using a disposal bioreactor (wave system) of up to 25 L volume. Stable CHO cell strains encoding the heavy and light chain genes of the produced antibodies, respectively, were inoculated into PowerCHO medium (Invitrogen) and incubated at 37 ° C and 5% CO 2 .

During the incubation period, each of the other human antibodies was continuously expressed into the cell culture medium. At the end of incubation (<50% survival), the cells were isolated by common centrifugation and filtration steps. The clarified cell culture supernatant (ccs) was concentrated by ultrafiltration and used for purification of the antibody.

Human antibodies (MabSelect Sure, GE Healthcare) were purified from protein ccs by protein A affinity chromatography. After equilibration of the protein A material with 5 column volume (cv) of 20 mM sodium phosphate processing buffer (pH 7), the concentrated supernatant of the isotype control group was completely applied to the affinity column. Wash out impurities or undesired proteins with the operating buffer. The antibody was eluted by pH shift using 100 mM glycine (pH 3) and dialyzed against 250 mM glycine buffer.

Alternatively, the concentrated cell culture supernatant can be applied to the Protein A tube prior to equilibration with 5 cv of 20 mM Tris/HCl buffer (pH 7) including 150 mM sodium chloride buffer and 0.1% Tween 80. column. Impurities can be washed out by two washing steps: 1.) adding 1 M NaCl in the equilibration buffer and 2.) 100 mM sodium phosphate (pH 5) including 0.1% Tween 80. The antibody can be eluted by 100 mM glycine buffer (pH 3) including 0.1% Tween 80 and then dialyzed against 250 mM glycine buffer (pH 5).

All references cited herein are hereby incorporated by reference in their entirety.

<110> Baxter Health Company

Baxter International

<120> MIF as a therapeutic target

<130> 176986

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Claims (21)

Use of a compound for the manufacture of a medicament for the treatment or prevention of a MIF-related disorder in an individual in need thereof, wherein the compound (a) prevents modification of the MIF at position 81, or (b) preferentially binds to The modified MIF is carried at position 81 as compared to a modified MIF that does not contain the position 81. The use of the first aspect of the patent application, wherein the MIF-related disorder is a state of progression of a MIF-related disease, a MIF-related disease state, or a MIF-related disease. A use of a modified MIF at position 81 (SEQ ID NO: 15) for use as a target for the treatment or prevention of a MIF-related disorder or as a target for drug discovery analysis. The use of claim 1 or 3, wherein the compound is an antibody or a molecule comprising an antigen binding portion of the antibody. The use of the third aspect of the patent application, wherein the MIF-related disorder is a state of progression of a MIF-related disease, a MIF-related disease state, or a MIF-related disease. The use of the second or fifth aspect of the patent application, wherein the MIF-related disorder is an inflammatory disease or a neoplastic disease. The use of the second or fifth aspect of the patent application, wherein the MIF-related disorder is selected from the group consisting of colon cancer, prostate cancer, bladder cancer, pancreatic 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 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, neuralgia and uveitis. For example, the use of the second or fifth aspect of the patent application, wherein the MIF-related disorder is malignant ascites, preferably in the third-line treatment. A method of monitoring the efficacy of a treatment for a condition associated with a MIF comprising determining whether the MIF position 81 carries a modification in a sample isolated from the individual before and after the treatment, wherein the modification of the MIF position 81 after treatment is compared to before treatment The degree is small and the treatment is identified as being effective. The method of claim 9, wherein the MIF-related disorder is a state of progression of a MIF-related disease, a MIF-related disease state, or a MIF-related disease. An anti-MIF antibody or a molecule comprising an antigen binding portion of an antibody that binds to a modified MIF at position 81 but does not bind to an unmodified MIF at position 81. A method of analyzing whether a test compound preferentially binds to a MIF in a modified form at position 81, the method comprising the steps of (a) modifying MIF at position 81 to obtain a modified MIF, (b) testing Combining the compound to be tested with the modified MIF in the sample and in a control sample in which the MIF is unmodified at position 81, (c) evaluating the compound in the test sample and the control sample and the modified a combination of MIF, if the compound is in the test sample to a greater extent than in the control sample Upon binding to the modified MIF, the compound is selected. A method of screening for a compound that prevents modification of MIF at cysteine 81, the method comprising the steps of (a) combining MIF with a compound to be tested in a test sample, and (b) testing the test sample containing the compound And treating the MIF by a sulfhydryl reagent in a control sample in which the compound is absent, wherein the reagent modifies the sulfhydryl group of cysteine 81 of MIF in at least the control sample, (c) The sulfhydryl reaction reagent evaluates the modification of cysteine 81, wherein if the sulfhydryl reagent is less modified in the test sample than the cysteine 81 in the control sample, then the selection is The compound. The method of claim 13, wherein the compound is an antibody or a molecule comprising an antigen-binding portion of the antibody. A modified state of MIF position 81 is used as a diagnostic marker, wherein the MIF position 81 carries a modified finding indicative of a MIF related disorder. A method of diagnosing a MIF-related disorder, comprising the step of determining whether a MIF location 81 carries a modification in a sample isolated from an individual, wherein the MIF location 81 is identified as being modified, the individual is diagnosed with a MIF-related disorder or Infected with MIF related disorders. The method of claim 16, wherein the MIF-related disorder is a state of progression of a MIF-related disease, a MIF-related disease state, or a MIF-related disease. The method of claim 10 or 17, wherein the MIF-related disorder is an inflammatory disease or a neoplastic disease. For example, the method of claim 10 or 17 of the patent scope, wherein the MIF related disease system is selected Free group consisting of colon cancer, prostate cancer, bladder cancer, pancreatic cancer, ovarian cancer, melanoma, lymphoma, hepatocellular carcinoma, asthma, ARDS, rheumatoid arthritis, sepsis, IgA nephropathy, spheroid Nephritis, lupus nephritis (LN), hepatitis, pancreatitis (+/- acute lung injury), Crohn's disease, ulcerative colitis, gastric ulcer, Alzheimer's disease, multiple sclerosis, Green-Bar Syndrome, cardiac dysfunction, angioplasty, atherosclerosis, myocarditis, type 1 diabetes, diabetic retinopathy, age-related macular degeneration (AMD), atopic dermatitis, psoriasis, endometriosis , neuralgia and uveitis. The method of claim 10 or 17, wherein the MIF-related disorder is malignant ascites, preferably in a third-line treatment. A MIF molecule modified at position 81 of SEQ ID NO: 15.