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Patent Application Titled "Method of Inhibiting Binding Or Activity of Mif by Administering a Mif Antagonist" Published Online

July 15, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- According to news reporting originating from Washington, D.C., by NewsRx journalists, a patent application by the inventors Bucala, Richard J. (Cos Cob, CT); Leng, Lin (New York, NY); Metz, Christine N. (Great Neck, NY), filed on November 26, 2013, was made available online on July 3, 2014 (see also Baxter Healthcare SA).

The assignee for this patent application is Baxter Healthcare SA.

Reporters obtained the following quote from the background information supplied by the inventors: "The present invention relates to methods and compositions for using the MHC class II invariant chain polypeptide, Ii (also known as CD74), as a receptor for macrophage migration inhibitory factor (MIF), including methods and compositions for using this receptor, as well as agonists and antagonists of MIF which bind to this receptor or which otherwise modulate the interaction of MIF with CD74 or the consequeces of such interaction, in methods for treatment of conditions characterized by locally or systemically altered MIF levels, particularly inflammatory conditions and cancer.

"Macrophage migration inhibitory factor (MIF), the first cytokine activity to be described, has emerged to be seen as a critical regulator of the innate and adaptive immune response.sup.1-3. MIF is encoded by a unique gene, and crystallization studies have shown MIF to define a new protein fold and structural superfamily.sup.4. Despite the fact that the biological activity attributed to MIF first was described almost 30 years ago, information regarding MIF's precise role in cell physiology and immunity has emerged only recently.sup.1-9,18. MIF is centrally involved in macrophage and T cell activation and in the development of septic shock, arthritis, and other inflammatory conditions.sup.2. Also, MIF has been linked to cancer.sup.32.

"MIF is critically involved in the expression of innate and acquired immunity. MIF is released by a variety of cell types and is a necessary factor for the activation or proliferative responses of macrophages.sup.18, T cells.sup.6, and fibroblasts.sup.7. MIF's mitogenic effects proceed via an autocrine/paracrine activation pathway involving the p44/p42 (ERK-1/2) mitogen-activated protein kinase cascade.sup.7. MIF -/- mice are highly resistant to endotoxic shock.sup.3, and immunoneutralization of MIF confers protection against septic shock.sup.25 and a variety of immuno-inflammatory pathologies such as delayed-type hypersensitivity.sup.26, arthritis.sup.27, and glomerulonephritis.sup.28. MIF's actions on cells also show a number of unique features. These include a global, counter-regulatory action on glucocorticoid-induced immunosuppression.sup.5,6, the induction of a sustained pattern of ERK-1/2 activation.sup.7, and functional antagonism of p53-dependent apoptosis.sup.6.

"MIF's pro-inflammatory properties have been linked to its capacity to counter-regulate the immunosuppressive effects of glucocorticoids.sup.5,6, and its interactions with cells have been presumed to require a receptor-based mechanism of action.sup.7,8 or to reflect a specialized, intracellular mode of action.sup.9. Numerous in vitro and in vivo studies have been consistent with MIF acting by engagement of a cell surface receptor, however lack of progress toward the identification of candidate receptors has prompted interest in either specialized, intracellular modes of action or the potential biological role of MIF's tautomerase activity.sup.2,21. There also is evidence that MIF may function as an isomerase.sup.4.

"The MHC class II-associated invariant chain, Ii (CD74).sup.10, has been established to play an important role in the processing and transport of MHC class II proteins from the endoplasmic reticulum to the Golgi.sup.10. Most Ii dissociates from the class II complex as antigenic peptides load onto their class II binding sites. Approximately 2-5% of total cellular Ii also is expressed on the cell surface.sup.17, where it has been shown to function as an accessory molecule for T cell activation.sup.11. Ii has been previously implicated in signaling and accessory functions for immune cell activation.sup.11-13.

"U.S. Pat. No. 5,559,028 to Humphreys, et al. discloses gene constructs for expression of wild type and mutant Ii chains in recombinant cells. U.S. Pat. No. 5,726,020 to Humphreys, et al. discloses and claims expressible reverse gene constructs and oligonucleotides that hybridize with an Ii mRNA molecule, thereby inhibiting translation of the Ii mRNA molecule."

In addition to obtaining background information on this patent application, NewsRx editors also obtained the inventors' summary information for this patent application: "The invention is based in part upon the identification, utilizing expression cloning and functional analyses, that the Class II-associated invariant chain polypeptide, Ii (or CD74).sup.10, is a cellular receptor for MIF. Thus, MIF binds to the extracellular domain of Ii, a Type II membrane protein, and Ii is required for MIF-induced cell activation and/or phenotypic changes including, for instance, signaling via the extracellular signal-related kinase (ERK)-1/2MAP kinase cascade and cell proliferation. The inventive relationship provides a mechanism for MIPs activity as a cytokine and identify it as a natural ligand for Ii, which has been previously implicated in signaling and accessory functions for immune cell activation.

"Accordingly, one aspect of the present invention relates to methods for screening compounds to identify positive or negative modulators of MIF binding to, or activity in connection with binding to, CD74. In a first instance, such a method comprises a biochemical (i.e., acellular) binding assay, comprising: contacting an MHC class II invariant chain (Ii) polypeptide with MIF in the presence and absence of a test compound, and comparing the binding interaction of the MIF and Ii polypeptides in the presence of the test compound with their interaction in the absence of the test compound, whereby a compound that positively modulates the interaction of MIF with the Ii polypeptide is identified as an enhancer of MIF binding activity and a compound that negatively modulates the interaction of MIF with the Ii polypeptide is identified as an inhibitor of MIF binding activity. Enhancers so identified are candidate therapeutic agonists or enhancers of MIF, whereas inhibitors so identified are candidate therapeutic antagonists of MIF. For instance, a test compound may reinforce the binding of MIF to the Ii polypeptide (i.e., increase the affinity of the interaction) and thereby enhance the interaction of MIF and the Ii polypeptide. Such an enhancer is thereby identified as an agonist or enhancer of MIF, and is identified as a candidate therapeutic agent to enhance, independently or in connection with endogenous or exogenous MIF, MIF effects in subjects requiring such augmentation. Alternatively, a test compound that competes with MIF for binding to the Ii polypeptide or otherwise inhibits the interaction of the MIF with the Ii polypeptide is identified as an antagonist of MIF, and is identified as a candidate therapeutic agent to antagonize MIF effects in subjects requiring such antagonism. In this biochemical binding assay, the Ia polypeptide comprises the complete Ia sequence or an MIF-binding fragment thereof, and the assay is conveniently conducted with recombinantly prepared MIF and Ii peptides, one of which is optionally immobilized to a solid support, and one of which (or a binding partner thereto, such as an antibody) is labeled to facilitate detection and measurement of the MIF:Ii binding interaction.

"In a second aspect, the binding assay may be a cellular binding assay, comprising CD74 expressed (either normally or as a consequence of genetic engineering for Ii expression) by a cell (prokaryotic or eukaryotic), typically on the cell surface, and MIF binding thereto is detected and measured in the presence or absence of a test compound. As in the above described biochemical or acellular assay, a comparison is made of the binding interaction of the MIF and the cell-displayed Ii polypeptide in the presence of the test compound with their interaction in the absence of the test compound, whereby a compound that positively modulates the interaction of MIF with the Ii polypeptide (i.e., increases their affinity) is identified as an enhancer of MIF binding activity and a compound that negatively modulates the interaction of MIF with the Ii polypeptide (i.e., decreases their affinity) is identified as an inhibitor of MIF binding activity. Enhancers so identified are candidate therapeutic agonists or enhancers of MIF, whereas inhibitors so identified are candidate therapeutic antagonists of MIF.

"In a third aspect, the cellular assay is a signaling assay, in which the activity of an intracellular signaling cascade is measured before and after MIF is contacted to cell-displayed CD74 polypeptide, either in the presence or the absence of a test compound. Preferably, the signaling assay is an ERK-1/2 activation assay. A test compound that positively modulates the signaling activity of MIF via interaction with the Ii polypeptide is identified as an enhancer of MIF signaling activity and a compound that negatively modulates the signaling of MIF via interaction of MIF with the Ii polypeptide is identified as an inhibitor of MIF signaling activity. Enhancers so identified are candidate therapeutic agonists or enhancers of MIF, whereas inhibitors so identified are candidate therapeutic antagonists of MIF.

"In a fourth aspect, the cellular assay is a cellular activity or cell phenotype assay, in which the activity or phenotype of a target cell is measured before and after MIF is contacted to cell-displayed CD74 polypeptide, either in the presence or the absence of a test compound. Preferably, the activity or phenotype assay is a proliferation assay or an assay for functional antagonism of p53-dependent apoptosis. A test compound that positively modulates the chosen cellular activity or phenotypic change mediated by MIF via interaction with the Ii polypeptide is identified as an enhancer of MIF cellular activity and a compound that negatively modulates the chosen cellular activity or phenotypic change mediated by MIF via interaction with the Ii polypeptide is identified as an inhibitor of MIF cellular activity. Enhancers so identified are candidate therapeutic agonists or enhancers of MIF, whereas inhibitors so identified are candidate therapeutic antagonists of MIF.

"The invention also provides an enhancer of MIF, including an agonist, or an inhibitor, including an antagonist of MIF, identified by any of the methods above. One form of such an agonist or antagonist would be an antibody or antigen-binding fragment thereof, such as an anti-CD74 antibody. Anti-CD74 antibodies and CD74-binding fragments thereof are known in the art. For instance, the anti-CD74 antibody may be a monoclonal antibody and also may be a human, humanized or chimeric antibody, made by any conventional method.

"Another aspect of the invention relates to a method of inhibiting an effect of MIF on a cell comprising on its surface an MHC class II invariant chain (Ii) polypeptide which binds MIF and thereby mediates the effect of MIF. This method comprises: contacting the cell with an antagonist or other inhibitor of MIF, where the antagonist or inhibitor inhibits, in a first instance, binding of MIF to the Ii polypeptide; in a second instance, signaling initiated by MIF:Ii interaction; and in a third instance, a change in cellular activity, metabolism or phenotype effected by MIF:Ii interaction. In any of these methods the antagonist or inhibitor may be an antibody or fragment thereof which binds to the Ii polypeptide. Alternatively, the inhibitor may be soluble Ii polypeptide or a soluble MIF-binding fragment thereof which inhibits the interaction of MIF and Ia polypeptide (or the cellular consequences of such interaction) by binding to MIF or by interacting with Ii polypeptide on the surface of a cell. In some cases, the cell comprising Ii polypeptide is present in a mammal and the antagonist or other inhibitor is administered to the mammal in a pharmaceutical composition. A mammal that would benefit from this method is a mammal suffering from a condition or disorder characterized by MIF levels locally or systemically elevated above the normal range found in mammals not suffering from such a condition. In such a case, the antagonist or inhibitor is administered in an amount effective to treat the condition or disorder. For instance, the mammal may be suffering from cancer or an inflammatory disorder, and the antagonist or inhibitor is administered in an amount effective to treat the cancer or inflammatory disorder. The inflammatory disorder may be, for instance, septic shock or arthritis.

"More particularly, one aspect of the invention is a method of inhibiting an activity of MIF, which method comprises: contacting MIF with an MHC class II invariant chain (Ii) polypeptide or a fragment thereof which binds to MIF. The fragment of the MHC class II invariant chain (Ii) polypeptide which binds to MIF may be a soluble form of the polypeptide, particularly a soluble form that comprises the extracellular binding domain of this type II transmembrane polypeptide. In some cases, the MIF to be inhibited is in a mammal and the Ii polypeptide or a fragment thereof is administered to the mammal in a pharmaceutical composition. Where the mammal suffers from cancer or an inflammatory disorder, such as septic shock or arthritis, the Ii polypeptide or fragment thereof is administered in an amount effective to treat the disorder. In a further instance, the MIF antagonist or inhibitor is administered in an amount effective to treat an infectious disease, in which disease MIF or a polypeptide evolutionarily related to MIF (as evidenced by sequence homology) deriving from the infecting pathogen (whether a virus, bacterial, fungus, or especially, a parasite) is present locally, systemically, or at the host:pathogen interface.

"Yet another aspect of the invention relates to a method of purifying MIF comprising: contacting a sample comprising MIF with an MHC class II invariant chain (Ii) polypeptide or a fragment thereof which binds to MIF, under conditions that promote the specific binding of MIF to the Ii polypeptide or fragment thereof, and separating the MIF:Ii polypeptide complex thereby formed from materials which do not bind to the Ii polypeptide or fragment thereof. In this method, the Ii polypeptide may be immobilized on a solid support matrix. The invention also provides a method of assaying for the presence of MIF comprising: contacting a sample with an MHC class II invariant chain (Ii) polypeptide or a fragment thereof which binds to MIF under conditions that promote the specific binding of MIF to the Ii polypeptide or fragment thereof, and detecting any MIF:Ii polypeptide or MIF:Ii polypeptide fragment complex thereby formed.

"Still another method provided by the invention is a method for reducing an effect of MIF on a cell comprising on its surface an MHC class II invariant chain (Ii) polypeptide or fragment thereof which binds MIF and thereby mediates the effect of MIF. This method comprises: providing to the cell an antisense nucleic acid molecule in an amount effective to reduce the amount of Ii polypeptide produced by the cell. The antisense nucleic acid molecule specifically binds to a portion of mRNA expressed from a gene encoding the MHC class II invariant chain (Ii) polypeptide and thereby decreases translation of the mRNA in the cell and, ultimately, the level of Ii polypeptide on the surface of the cell. In this method the cell comprising the Ii polypeptide may be in a mammal, for instance, a mammal suffering from a condition or disorder characterized by MIF levels locally or systemically elevated above the normal range in mammals not suffering from such a condition or disorder. For instance, the mammal may be suffering from a cancer or an inflammatory disorder, such as septic shock or arthritis. In such a case, the antisense nucleic acid is administered in a pharmaceutical composition, in an amount effective to treat the condition or disorder.

BRIEF DESCRIPTION OF THE FIGURES

"FIG. 1 illustrates high affinity binding of MIF to THP-1 monocytes. a, Alexa-MIF shows full retention of dose-dependent MIF biological activity as assessed by activation of the p44/p42 (ERK-1/2) MAP kinase cascade, visualized by western blotting of cell lysates using antibodies specific for phospho-p44/p42 or total p44/p42; and b, suppression of p53-dependent apoptosis induced by serum starvatuin (CM: complete medium, SFM: serum-free medium). MIF or Alexa-MIF were added at 50 ng/ml. Data shown are Mean.+-.SD of triplicate wells and are representative of 3 independent experiments. Further evidence for the retention of native structure by Alexa-conjugation was provided by the measurement of MIF tautomerase activity using L-dopachrome methyl ester as substrate.sup.25. No difference in the tautomerase activity of Alexa-MIF versus unconjugated MIF was observed (Alexa-MIF: .DELTA.OD.sub.475=0.275 sec.sup.-1 .mu.g.sup.-1 protein, versus rMIF: .DELTA.OD.sub.475=0.290 sec.sup.-1 .mu.g.sup.-1; P.dbd.NS) c, Flow cytometric analysis shows the binding of Alexa-MIF to THP-1 monocytes is markedly enhanced by IFN-.delta. treatment. Competition for Alexa-MIF binding was performed in the presence of 1 .mu.g/ml unlabeled, rMIF d, Direct visualization of Alexa-MIF binding to THP-1 monocytes by confocal microscopy THP-1 cells were grown on cover slips, incubated with INF.gamma. (1 ng/ml) for 72 hrs and stained with Alexa-MIF (left panel) or Alexa-MIF plus excess, unlabeled rMIF (right panel). Cell bound Alexa-MIF was rapidly internalized upon shifting cells from 4.degree. to 37.degree. for 15 mins (right panel). Magnification: 630.times.e, Binding characteristics of Alexa-MIF to IFN.gamma.-activated, THP-1 monocytes. The inset shows the binding data transformed by Scatchard analysis, indicating two distinct binding activities; one with K.sub.d=3.7.times.10.sup.-8 m the other with K.sub.d=3.5.times.10.sup.5, data are representative of 3 independent experiments.

"FIG. 2 show that Ii is a cell surface binding protein for MIF a, Sequential cycles of fluorescence-activated cell-sorting of COS-7 cell transfectants shows enrichment for MIF binding activity b, Diagrams indicating structure of Ii (35 kDa isoform), and three of ten representative cDNA clones with MIF binding activity. IC, TM, and EC are the intracellular, transmembrane, and extracellular domains. M1 and M17 refer to two sites of alternative translation initiation. c, Flow cytometry analysis of MIF binding to Ii-expressing cells. Enhanced binding of Alexa-MIF to Ii-transfected versus control vector-transfected COS-7 cells (left panel), inhibited binding of Alexa-MIF to Ii-transfected COS-7 cells incubated with anti-Ii mAb (clone LN2) versus an isotypic mAb control (con mAb) (middle panel), and enhanced binding of Alexa-MIF to IFN.gamma.-stimulated, THP-1 monocytes incubated with anti-Ii mAb (clone LN2) versus an isotypic mAb control (right panel). The data shown are representative of at least three independent experiments. The anti-Ii mAb, LN2 (PharMingen), is reactive with an epitope residing within 60 amino acids of the extracytoplasmic, C-terminus of the protein d, MIF binds to the extracellular domain of Ii in vitro. [.sup.35S]-Ii protein was prepared in a coupled transcription and translation reaction utilizing plasmids encoding Ii fragments of different lengths. Protein-protein interaction was assessed by measuring bound radioactivity in 96-well plates that were pre-coated with MIF (n=6 wells per experiment). The data shown are representative of three experiments, showing that MIF binding is severely compromised with vectors expressing Ii fragments of amino acids 1-72 or 1-109 versus robust binding with vectors expressing Ii fragments of amino acids 1-149 or 1-232 (full-length).

"FIG. 3 illustraes Ii mediation of MIF stimulation of ERK-1/2 (p44/p42) phosphorylation in COS-7 cells a, ERK-1/2 phosphorylation is induced by MIF in COS-7 cells transfected with Ii vector (COS-7/Ii) or control vector (COS-7/V). Cells were treated without or with various doses of rMIF for 2.5 hrs and analyzed for phospho-p44/p42 and total p44/p42 by western blotting b, There is dose-dependent inhibition of MIF-induced ERK-1/2 phosphorylation by anti-Ii mAb. COS-7 cells were transfected with an Ii vector and stimulated with 50 ng/ml MIF for 2.5 hrs in the presence of an isotypic control mAb or an anti-Ii mAb (clone LN2) at different doses. In control experiments, anti-Ii showed no effect on ERK-1/2 phosphorylation in the absence of added MIF (data not shown).

"FIG. 4 illustrates western blots of MIF-induced phosphorylation a, MIF dose dependently stimulates ERK-1/2 (p44/p42) phosphorylation in human Raji B cells, as visualized by western blotting for phospho-p44/p42 b, There is inhibition of MIF-induced ERK-1/2 phosphorylation in Raji cells by anti-Ii mAb also. Raji cells were stimulated with 50 ng/ml of MIF for 2.5 hrs in the presence of an isotype control antibody (Con Ab) or the two anti-Ii mAbs, --B741 or LN.sup.2, each added at 50 .mu.g/ml. c, Anti-Ii inhibits MIF-induced Raji cell proliferation quantified by .sup.3H-thymidine incorporation d, Anti-Ii inhibits MIF-induced proliferation of human fibroblasts also. Antibodies were added to a final concentration of 50 .mu.g/ml. The results shown are the Mean.+-.SD of triplicate assays and are representative of at least three separate experiments. Anti-Ii antibodies showed no effect on cell proliferation in the absence of added MIF (data not shown).

"FIG. 5 shows the complete nucleotide sequence (SEQ ID NO: 1) and longest translated amino acid sequence (beginning at nt 8; SEQ ID NO:2) of the human mRNA for the Ii polypeptide (HLA-DR antigens associated invariant chain p33 [GenBank Accession Nr. X00497 M14765]), as reported in Strubin, M. et al., The complete sequence of the mRNA for the HLA-DR-associated invariant chain reveals a polypeptide with an unusual transmembrane polarity. EMBO J., 3, 869-872 (1984)."

For more information, see this patent application: Bucala, Richard J.; Leng, Lin; Metz, Christine N. Method of Inhibiting Binding Or Activity of Mif by Administering a Mif Antagonist. Filed November 26, 2013 and posted July 3, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=%2Fnetahtml%2FPTO%2Fsearch-adv.html&r=3635&p=73&f=G&l=50&d=PG01&S1=20140626.PD.&OS=PD/20140626&RS=PD/20140626

Keywords for this news article include: Antibodies, Kinase, Sepsis, Peptides, Chemistry, Monocytes, Immunology, Phagocytes, Amino Acids, Biochemical, Septic Shock, Myeloid Cells, Blood Proteins, Immunoglobulins, Baxter Healthcare SA, Enzymes and Coenzymes, Mononuclear Leukocytes, Leukocyte Migration-Inhibitory Factors, Macrophage Migration-Inhibitory Factors.

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