Method for preparing anti-MIF antibodies

Abstract

The specification provides methods of preparing high-affinity antibodies to a macrophage migration inhibitory factor (MIF) in animals in which the MIF gene has been homozygously knocked-out (MIF −/− ). Also provided are methods of preparing hybridomas which produce the anti-MIF antibodies, methods of administering the antibodies to treat inflammatory or cancerous conditions and/or diseases modulated by MIF, as well as compositions comprising said high-affinity anti-MIF antibodies.

What is claimed is:  
     
         1 . A method of preparing a high-affinity anti-MIF antibody or fragment thereof comprising the steps of: 
 (a) preparing a transgenic animal in which the MIF gene is functionally knocked out;    (b) immunizing said transgenic animal with a MIF protein or a polypeptide fragment thereof, and    (c) obtaining a high-affinity anti-MIF antibodies or fragment thereof from said animal.    
     
     
         2 . The method of  claim 1 , wherein the high-affinity anti-MIF antibody or fragment thereof recognizes and binds to MIF-1 or fragment thereof, MIF-2 or fragment thereof, MIF-3 or fragment thereof, or a MIF-like protein or fragment thereof.  
     
     
         3 . The method of  claim 1 , wherein the animal is selected from the groups consisting of rodent, canine, porcine, feline, equine, ovine and bovine.  
     
     
         4 . The method of  claim 3 , wherein the rodent is selected from the groups consisting of rat, mouse, hamster and guinea pig.  
     
     
         5 . The method of  claim 1 , wherein the MIF or immunogenic polypeptide thereof is selected from human MIF-1, human MIF-2, human MIF-3, a MIF fusion protein, or a MIF peptide of at least about 7 consecutive amino acids.  
     
     
         6 . The method of  claim 1 , wherein the high-affinity anti-MIF antibody or fragment thereof is an anti-peptide antibody specific to a MIF epitope, a humanized antibody, a human antibody or a chimeric antibody.  
     
     
         7 . A high-affinity anti-MIF antibody produced by the method of  claim 1 .  
     
     
         8 . A high-affinity anti-MIF antibody produced from a MIF knockout mouse, wherein said antibody is characterized by: (1) binding soluble human MIF with an affinity of ≦50 nM; or (2) blocking MIF-induced activity at a concentration of 10 μ/ml or less.  
     
     
         9 . The high affinity antibody of  claim 8 , wherein the MIF-induced activity which is blocked is one or more of the following: MIF stimulated MMP release, PPT activity, LPS lethality, or MIF stimulated SRE transcription.  
     
     
         10 . The high affinity antibody of  claim 9 , wherein the MMP stimulated by MIF is selected from MMP-1 and MMP-3.  
     
     
         11 . The high-affinity anti-MIF antibody of  claim 8 , wherein said antibody binds soluble human MIF with an affinity of ≦0.1 nM; or (2) blocking MIF-induced activity at a concentration of 10 μ/ml or less.  
     
     
         12 . A high affinity anti-MIF monoclonal antibody produced from a knock-out mouse, wherein the monoclonal antibody is 10B11-3, 22F11-6, 6B5-5, 29B12-1, 19B11-7 or 33G7-9.  
     
     
         13 . A nucleic acid encoding an anti-MIF monoclonal antibody of  claim 12 .  
     
     
         14 . A vector comprising the nucleic acid of  claim 13 .  
     
     
         15 . An isolated cell transfected with the vector of  claim 14 .  
     
     
         16 . A high affinity anti-MIF monoclonal antibody produced from a knock-out mouse, wherein the monoclonal antibody is 1OB11-3, 22F11-6, 29B12-1, 30B7-11, 11 A9-8, 6B5-5, 33G7-9, 6E2-12, 2D8-3, 10B11-3, 19B11-7, L2E1-9, 1A9-7, 22F11-6, 7E10-11, 25D-11, 9G10-12, 22A5-12, 14H5 and 34D11-1.  
     
     
         17 . A nucleic acid encoding an anti-MIF monoclonal antibody of  claim 16 .  
     
     
         18 . A high affinity, humanized anti-MIF antibody, wherein said antibody is characterized by having at least one of the following: (1) binds soluble human MIF with an affinity of ≦50 nM; or (2) blocks MIF-induced activity in vitro at concentrations of 10 μ/ml or less.  
     
     
         19 . The high affinity, humanized anti-MIF antibody of  claim 18 , wherein the antibody is also cross reactive with murine MIF.  
     
     
         20 . The high-affinity anti-MIF antibody of  claim 7 , wherein the antibody recognizes and binds to MIF-1 or fragment thereof, MIF-2 or fragment thereof, MIF-3 or fragment thereof or a MIF-like protein or fragment thereof.  
     
     
         21 . A method of preparing a cell line producing a high-affinity monoclonal anti-MIF antibody or fragment thereof by preparing hybridomas using the cells producing the anti-MIF antibodies of  claim 1 .  
     
     
         22 . The method of  claim 21;  wherein the high-affinity anti-MIF antibody or fragment thereof recognizes and binds to MIF-1 or fragment thereof, MIF-2 or fragment thereof, MIF-3 or fragment thereof, or a MIF-like protein or fragment thereof.  
     
     
         23 . A high-affinity anti-MIF monoclonal antibody or fragment thereof produced by the cell line of  claim 21 .  
     
     
         24 . The high affinity anti-MIF antibody fragment of  claim 23 , wherein the fragment is selected from the group consisting of: FV, scFV, Fab, Fab′ and F(ab′) 2 .  
     
     
         25 . The high-affinity anti-MIF monoclonal antibody or fragment thereof of  claim 6 , wherein the antibody has a dissociation constant (KD) of about 10 −8  M to about 10 −9  M or less for a MIF epitope.  
     
     
         26 . An isolated nucleic acid comprising a MIF targeting construct comprising (A) a selectable marker and (B) DNA sequence homologous to a MIF gene, wherein said isolated nucleic acid is introduced into an animal at an embryonic stage, and wherein said nucleic acid disrupts endogenous MIF gene activity wherein MIF protein production is blocked and wherein said animal is suitable for production of high-affinity anti-MIF antibodies.  
     
     
         27 . The isolated nucleic acid of  claim 26 , wherein the MIF targeting construct targets a MIF-1 gene, a MIF-2 gene, a MIF-3 gene or a gene encoding a MIF-like protein.  
     
     
         28 . The isolated nucleic acid of  claim 26 , wherein the selectable marker sequence confers a positive selection characteristic.  
     
     
         29 . The isolated nucleic acid of  claim 26 , wherein the selectable marker is a neomycin resistance (neo) gene.  
     
     
         30 . A transgenic animal genome comprising a homozygous disruption of the endogenous MIF gene (MIF −/− ), wherein said disruption comprises the insertion of a selectable marker sequence, and wherein said disruption results in said animal with negligible or no expression of MIF as compared to a wild type animal and wherein said animal is capable of producing high affinity anti-MIF antibodies.  
     
     
         31 . The transgenic animal of  claim 30 , wherein the MIF gene which is homozygously disrupted is selected from the group consisting of a MIF-1 gene, a MIF-2 gene, a MIF-3 gene or gene encoding a MIF-like protein.  
     
     
         32 . The transgenic animal of  claim 30 , wherein the selectable marker sequence is a neomycin cassette.  
     
     
         33 . The transgenic animal of  claim 30 , wherein the anti-MIF antibodies produced by said animal have a dissociation constant (KD) for a MIF epitope of about 10 M to about 10−9 M or less.  
     
     
         34 . A method for producing a transgenic animal lacking an endogenous MIF gene, said method comprising: 
 (a) introducing a MIF targeting construct comprising a selectable marker sequence into an embryonic stem (ES) cell or ES-like cell;    (b) introducing said animal ES cell or ES-like cell into an animal embryo;    (c) transplanting said embryo into a pseudopregnant animal;    (d) allowing said embryo to develop to term; and    (e) identifying a transgenic animal whose genome comprises a disruption of the endogenous MIF gene at least one allele;    (f) breeding the transgenic animal of step E to obtain a transgenic animal whose genome comprises a homozygous disruption of the endogenous MIF gene (MIF −/− ), wherein said disruption results in an animal which lacks endogenous MIF as compared to a wild type animal.    
     
     
         35 . The method of  claim 34 , wherein the embryonic stem cell is a mouse embryonic stem cell and the animal is a mouse.  
     
     
         36 . A nucleic acid encoding a high-affinity anti-MIF monoclonal antibody or fragment thereof of  claim 23 .  
     
     
         37 . A therapeutic composition comprising an anti-MIF antibody or fragment thereof of  claim 23  and a pharmaceutically acceptable carrier.  
     
     
         38 . The therapeutic composition of  claim 37  further comprising a steroid.  
     
     
         39 . The therapeutic composition of  claim 37  further comprising an immunosuppressive, cytotoxic or other anti-cancer agent.  
     
     
         40 . The therapeutic composition of  claim 38 , wherein the steroid is a glucocorticoid or a corticosteroid.  
     
     
         41 . The therapeutic composition of  claim 40 , wherein the glucocorticoid is selected from the group consisting of: 21-Acetoxypregnenolone, Alclometasone, Algestone, Aincinonide, Beclomethasone, Betamethasone, Budesonide, Chloroprednisone, Clobetasol, Clobetasone, Clocortolone, Cloprednol, Corticosterone, Cortisone, Cortivazol, Deflazacort, Desonide, Desoximetasone, Dexamethasone, Diflorasone, Diflucortolone, Difluprednate, Enoxolone, Fluazacort, Flucloronide, Flumethasone, Flunisolide, Flucinolone Acetonide, Fluocinonide, Fluocortin Butyl, Fluocortolone, Fluorometholone, Fluperolone Acetate, Fluprednidene Acetate, Fluprednisolone, Flurandrenolide, Fluticasone Propionate, Formocortal, Halcinonide, Halobetasol Propionate, Halometasone, Halopredone Acetate, Hydrocortamate, Hydrocortisone, Loteprednol Etabonate, Mazipredone, Medrysone, Meprednisone, Methylprednisolone, Mometasone Furoate, Paramethasone, Prednicarbate, Prednisolone, Prednisolone 25-Diethylaminoacetate, Predisolone Sodium Phosphate, Prednisone, Prednival, Prednylidene, Rimexolone, Tixocortol, Triamcinolone, Triameinolone, Acetonide, Triamcinolone Benetonide, Triamcinolone Hexacetonide.  
     
     
         42 . A method of treating a MIF-mediated disease comprising the step of administering a pharmaceutically acceptable amount of the composition of  claim 37 .  
     
     
         43 . A method of treating an inflammatory disease comprising administering a therapeutically effective amount of an anti-MIF antibody according to  claim 7 .  
     
     
         44 . The method of  claim 43  wherein said disease is selected from the group consisting of arthritis, psoriasis, glomerulonephritis, septic shock, and atopic dermatitis.  
     
     
         45 . A method of inhibiting angiogenesis comprising administering a therapeutically effective amount of an anti-MIF antibody according to  claim 7 .  
     
     
         46 . A method of treating cancer comprising administering a therapeutically effective amount of an anti-MIF antibody according to  claim 7 .  
     
     
         47 . The method of  claim 46  wherein said antibody inhibits angiogenesis.  
     
     
         48 . The method of  claim 43  wherein the treated disease is septic shock.  
     
     
         49 . The method of  claim 43  wherein the treated disease is glomerulonephritis.  
     
     
         50 . The method of  claim 43  wherein the treated disease is rheumatoid arthritis.  
     
     
         51 . The method of  claim 43  wherein the treated disease is atopic dermatitis.  
     
     
         52 . The method of  claim 42  wherein the treated disease is retinopathy.  
     
     
         53 . The method of  claim 52  wherein retinopathy is associated with diabetes or lupus.