U.S. patent application number 09/791551 was filed with the patent office on 2003-12-25 for method for preparing anti-mif antibodies.
Invention is credited to Hanna, Nabil, Kloetzer, William S..
Application Number | 20030235584 09/791551 |
Document ID | / |
Family ID | 26881101 |
Filed Date | 2003-12-25 |
United States Patent
Application |
20030235584 |
Kind Code |
A1 |
Kloetzer, William S. ; et
al. |
December 25, 2003 |
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.sup.-/-). 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.
Inventors: |
Kloetzer, William S.;
(Carlsbad, CA) ; Hanna, Nabil; (Rancho Santa Fe,
CA) |
Correspondence
Address: |
PILLSBURY WINTHROP LLP
1600 TYSONS BOULEVARD
MCLEAN
VA
22102
US
|
Family ID: |
26881101 |
Appl. No.: |
09/791551 |
Filed: |
February 26, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60185390 |
Feb 28, 2000 |
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60233625 |
Sep 18, 2000 |
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Current U.S.
Class: |
424/145.1 ;
530/388.23; 800/6 |
Current CPC
Class: |
A61K 39/3955 20130101;
A61K 39/3955 20130101; A01K 2227/105 20130101; C07K 14/52 20130101;
A01K 67/0276 20130101; A61P 13/12 20180101; A01K 2267/01 20130101;
A01K 2217/075 20130101; A61K 31/00 20130101; A61K 2300/00 20130101;
A61K 39/3955 20130101; A01K 2267/0331 20130101; A61P 19/02
20180101; C12N 15/8509 20130101; C07K 16/24 20130101; A61P 29/00
20180101; A61P 17/06 20180101; A61P 31/04 20180101; A61P 35/00
20180101; A61K 2039/505 20130101; A61P 27/02 20180101 |
Class at
Publication: |
424/145.1 ;
530/388.23; 800/6 |
International
Class: |
A61K 039/395; C07K
016/24; C12P 021/02; A01K 067/00 |
Claims
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 .ltoreq.50 nM; or (2)
blocking MIF-induced activity at a concentration of 10 .mu./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 .ltoreq.0.1
nM; or (2) blocking MIF-induced activity at a concentration of 10
.mu./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 .ltoreq.50 nM; or
(2) blocks MIF-induced activity in vitro at concentrations of 10
.mu./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').sub.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.sup.-8 M to about 10.sup.-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.sup.-/-), 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.sup.-/-), 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.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority from U.S. Provisional
Patent Application Serial No. 60/185,390, filed Feb. 28, 2000, and
U.S. Provisional Patent Application Serial No. 60/233,625, filed
Sep. 18, 2000, the entirety of both applications being incorporated
herein.
FIELD OF THE INVENTION
[0002] The invention relates to a method of making high-affinity
anti-macrophage migration inhibitory factor (MIF) antibodies in
animals which are homozygously deficient of a MIF gene
(MIF.sup.-/-). The invention further relates to high affinity
anti-MIF antibodies, compositions comprising said antibodies and
methods of treating diseases using said anti-MIF antibodies.
SUMMARY OF THE INVENTION
Migration Inhibitory Factor
[0003] Macrophage migration inhibitor factor (MIF) was one of the
first identified lymphokines [Bloom et al., Science 153: 80-82
(1966)] and is a pleiotropic cytokine released by macrophages,
T-cells and the pituitary gland during inflammatory responses. It
acts as a pro-inflammatory cytokine, playing a major role in
endotoxin shock and counter-regulating the anti-inflammatory
effects of dexamethasone [Bozza et al., J Exp. Med. 189: 341-6
(1999)]. MIF promotes tumor necrosis factor alpha (TNF.A-inverted.)
synthesis, T-cell activation [Leech et al., Arthritis Rheum. 42:
1601-8 (1999)], enhances interleukin-1 (IL-1) and interferon gamma
(IFN( ) production [Todo, Mol. Med. 4: 707-14 (1998)], impacts
macrophage-macrophage adherence, up-regulates HLA-DR, increases
nitric oxide synthase and nitric oxide concentrations, and inhibits
Mycoplasma avium growth (U.S. Pat. No. 5,681,724). Certain of these
features indicate that MIF also plays a role in the pathogenesis of
rheumatoid arthritis (RA) (Id.). MIF is implicated in the
activation of macrophages and counter-regulation of glucocorticoid
activity [Chesney et al., Mol. Med. 5: 181-91 (1999)]. Recombinant
forms of MIF and the DNAs encoding them have been previously
described, see for example (WO 90/11301). MIF also has a reported
role in the innate host response to staphylococcal and
streptococcal exotoxins (Calandra et al., Proc. Natl. Acad. Sci.
USA 95: 11383-8 (1998)).
[0004] MIF inhibition has been suggested for the treatment of acute
lung injury to suppress the level of neutrophil attraction to the
site of injury (Makita et al., Am J. Respir. Crit. Care Med. 158:
573-9 (1998)). MIF localizes to the cytoplasm of leukemic cells and
has been linked to a role in leukemia associated inflammatory
events (Nishihira et al., Biochem. Mol. Biol. Int. 40: 861-9
(1996)).
[0005] Several forms of MIF have been identified. The first
characterized was that of Weiser et al., Proc. Natl. Acad. Sci. USA
86: 7522-6 (1989). This MIF (MIF-1) is 115 amino acids and 12.5 kDa
(Id.). MIF-2 is a 45 kD protein identified in a human T-cell
hybridoma clone (F5) (Hirose et al., Microbiol. Immunol. 35: 235-45
(1991)). The sequence of MIF-2 is very similar to MIF-1, but
differs in that it is a more hydrophilic species than MIF-2 (Oki et
al., Lymphokine Cytokine Res. 10: 273-80 (1991)).
[0006] MIF-3 is an 119 amino acid residue sequence (ATCC No. 75712;
WO 95/31468). Antibodies and antagonists have been developed to
MIF-3, which can be used to protect against lethal endotoxemia and
septic shock or to treat ocular inflammations (WO 95/31468).
[0007] A related protein to MIF is the glycosylation-inhibiting
factor (GIF), (Galat et al., Eur. J Biochem. 224: 417-21 (1994)).
The cDNA expressing the human form of GIF is described by Mikayama
et al., Proc. Natl. Acad. Sci. U.S.A. 90: 10056-60 (1993). The
amino acid sequences for MIF-1 and GIF are now recognized to be
identical. The correct amino acid sequence is 114 amino acids and
forms a 12,345 Da protein (Swiss-Protein accession number
P14174).
Anti-MIF Antibodies
[0008] Polyclonal and monoclonal anti-human MIF antibodies have
been developed against recombinant human MIF (Shimizu et al., FEBS
Lett 381: 199-202 (1996); Japanese Patent No. 9077799A; German
Democratic Republic Patent No. 230876A; European Patent No. 162812;
and ATCC Accession Nos. 00201X0003, 1024674 and 1014477). One
monoclonal antibody against human MIF (IC5/B) has been developed
and utilized to study signals to mononuclear phagocytes in
pseudolymphomas and sarcoidosis [Gomez et al., Arch. Dermatol. Res.
(Germany) 282: 374-8 (1990); see also Weiser et al., Cell. Immunol.
90: 167-78 (1985)]. Additional human monoclonal anti-MIF antibodies
were developed by Kawaguchi et al., J Leukoc. Biol. 39: 223-232
(1986) and Weiser et al., Cell. Immunol. 90: 167-78 (1985).
Anti-murine MIF monoclonal antibodies have also been prepared [See,
e.g., Malomy et al., Clin. Exp. Immunol. 71: 164-70 (1988); and Liu
et al., J. Immunol. 137: 448-55 (1986)].
[0009] Anti-MIF antibodies have been suggested for therapeutic use
to inhibit TNF.A-inverted. release (Leech et al., 1999). As such,
anti-MIF antibodies may have wide therapeutic applications for the
treatment of inflammatory diseases. Related thereto, the
administration of anti-MIF antibodies also reportedly inhibited
adjuvant arthritis in rats (Leech et al., Arthritis Rheum. 41:
910-7 (1998)).
[0010] MIF has also been implicated in the pathogenesis of
immunologically induced kidney disease. Lan et al., J. Exp. Med.
185: 1455-65 (1997) proposed the use of agents which block MIF
activity to treat rapidly progressive glomerulonephritis in
patients, and also suggested that MIF may be important in
immune-mediated diseases generally.
[0011] Calandra et al., I. Inflamm. 47: 39-51 (1995) reportedly
used anti-MIF antibodies to protect animals from experimentally
induced gram-negative and gram-positive septic shock. Anti-MIF
antibodies were suggested as a means of therapy to modulate
cytokine production in septic shock and other inflammatory disease
states (Id.).
[0012] Anti-MIF antibodies have been proposed for use to treat
diseases where cellular/mucosal immunity should be stimulated or as
a diagnostic or prognostic marker in pathological conditions
involving the production of MIF (WO 96/09389).
[0013] MIF antagonists have been proposed to treat lymphomas and
solid tumors which require neovascularization, (WO 98/17314). WO
98/17314 by Bucala et al. reportedly describes inhibition of murine
Bull lymphoma growth in vivo by a neutralizing monoclonal antibody
against MIF administered at the time of tumor implantation (Chesney
et al., 1999). Previous studies have shown that TH2 lymphacytes
produce higher amounts of MIF upon stimulation than TH1 cells.
(Bacher et al., 1996. PNAS 93:7849.) Since MIF is functionally
involved in T-cell activation, neutralization of TH2 cell-derived
may promote the ratio of TH1 to TH2 cells, thereby also prevent
influencing host immunity against tumors (Chesney, 1999). Also, the
use of anti-MIF antibodies for inhibiting proliferation of human
endothelial cells has been reported [Chesney et al., Mol. Med. 5:
181-91 (1999); and Ogawa et al., Cytokine 12:309-314 (2000)].
Specifically, Ogawa et al. (2000) showed that certain anti-MIF
antibodies directly block VEGF stimulated endothelial cell growth,
presumably through neutralization of endogenously produced MIF.
Knock-Out Animals for Use in Preparing Antibodies to
Self-Antigens
[0014] Transgenic animals have been prepared wherein foreign
antigens are now expressed in the transgenic animal as a
self-antigen. For example, a virus protein was expressed in a
transgenic mouse model as a self-antigen in the pancreatic islets
of Langerhans, as described by Oldstone et al., Cell 65: 319-31
(1991). Typically, however, it is difficult to produce antibodies
against self-antigens or autoantigens such as MIF. Autoantigens are
normal constituents of the body, which remain typically are not
recognized by the immune system.
[0015] A knock-out (KO) mouse or animal is one in which the animal
is homozygously deficient of a functional gene (Declerck et al., J.
Biol. Chem. 270: 8397-8400 (1995)). In general, antibodies will not
be raised against self-antigens nor against highly conserved
domains of proteins that do not vary between species. However,
certain KO mice have been produced in which monoclonal
auto-antibodies against various autoantigens have been raised.
Castrop et al., Immunobiol. 193: 281-7 (1995) reported preparation
of the use of a KO mouse for the generation of monoclonal
antibodies to T-cell factor-1 (TCF-1), which had been historically
difficult to prepare antibodies to due to the extreme evolutionary
conservation of TCF-1. Reportedly, because TCF-1 is highly
expressed in thymus, intrathymic selection mechanisms will impose
tolerance for TCF-1 in the immune system, likely through clonal
deletion of TCF-1-reactive T-cells (Id.). The anti-TCF-1 antibodies
were raised against a fusion protein comprising TCF-1 fused to
maltose binding protein (MBP).
[0016] LaTemple et al., Xenotransplantation 5: 191-6 (1998) used a
KO mouse to .A-inverted.1,3galactosyltransferase (.A-inverted.1,3GT
KO) to produce a natural, anti-Gal antibody. However, the antibody
was only produced in low amounts.
[0017] Declerck et al., (1995) reported the preparation of
anti-murine tissue-type plasminogen activator (t-PA) in a KO mouse,
wherein the mouse lacked a functional t-PA gene. Declerck et al.,
also suggested that this approach could be applied to other classes
of proteins allowing the generation of monoclonal antibodies
against conserved epitopes, which could not be raised in wild-type
animals because of their "self-antigen" nature. See also Declerck
et al., Thromb. Haemost (Germany) 74: 1305-9 (1995).
[0018] To better study the biologic role of MIF, a mouse strain
lacking MIF was generated by gene targeting in embryonic stem cells
(Bozza et al., 1999). Using this mouse model, Bozza et al.
determined that MIF is involved in a host response to gram negative
bacteria induced sepsis.
[0019] Therefore, not withstanding what has been previously
reported in the literature, there exists a need for preparing
anti-MIF antibodies, especially monoclonal antibodies and fragments
thereof with improved affinity and avidity for purposes of studying
MIF function as well as regulating MIF activity. The methods of
preparing the antibodies of this invention, as well as the
antibodies themselves, can in turn be used to modulate MIF activity
in diseases and conditions mediated by MIF, such as sepsis,
rheumatoid arthritis, other autoimmune diseases, cancer, as well as
injuries which induce MIF production.
SUMMARY OF THE INVENTION
[0020] It is an object of the invention to provide novel and
improved methods for producing high-affinity anti-MIF antibodies in
animals which are homozygous deficient for a MIF gene
(MIF.sup.-/-). The gene can be MIF-1, MIF-2, MIF-3 or a MIF-like
gene, but preferably is the MIF described by Weiser et al., (1989).
A preferred method for preparing high affinity anti-MIF antibody or
immunogenic fragment thereof comprises the steps of: (A) preparing
a transgenic animal in which the MIF gene is functionally knocked
out; (B) immunizing said transgenic animal with MIF or an
immunogenic polypeptide fragment thereof; and (C) obtaining
anti-MIF antibodies from said animal.
[0021] It is a more specific object of the invention to provide a
novel method of preparing high-affinity anti-MIF antibody
fragments, such as Fv, Fab, F(ab').sub.2, Fab' and scFV.
[0022] Another object of the invention is to provide for a method
of obtaining cells which produce high-affinity anti-MIF antibodies
from a MIF knock-out animal for purposes of preparing anti-MIF
antibody producing cell lines or cell lines which produce
recombinant forms of anti-MIF antibody fragments.
[0023] Another object of the invention is to provide a novel
nucleic acid encoding a MIF gene targeting construct comprising (A)
a selectable marker and (B) DNA sequence homologous to a MIF gene
or portion thereof, 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 prevented and wherein the animal, which is a
homozygous MIF deficient mutant, is a suitable bioreactor for
production of high affinity anti-MIF antibodies.
[0024] It is a further object of the invention to provide a
transgenic animal genome comprising a homozygous disruption of the
endogenous MIF gene, wherein said disruption comprises the
insertion of a selectable marker sequence, and wherein said
disruption results in said animal, which lacks an endogenous MIF as
compared to a wild type animal and wherein said animal is a
bioreactor for anti-MIF antibodies possessing high affinity.
[0025] Another object of the invention is to provide a method for
producing a transgenic animal lacking endogenous MIF, said method
comprising the steps of: (A) introducing a MIF targeting construct
comprising a selectable marker sequence into a embryonic stem cell;
(B) introducing said animal embryonic stem cell into a animal
embryo; (C) transplanting said embryo into a pseudopregnant animal;
(D) allowing said embryo to develop to term; and (B) 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.sup.-/-), wherein said disruptions results in an animal which
lacks at least one endogenous MIF as compared to a wild type
animal.
BRIEF DESCRIPTION OF THE DRAWING FIGURES
[0026] FIG. 1 shows the generation of high affinity, anti-MIF Mabs
in MIF gene knock out mice as assayed by ELISA of the first fusion
which was produced by human MIF S/OVA immunized mice (E1).
[0027] FIG. 2 shows the generation of high affinity, anti-MIF Mabs
in MIF gene 5 knockout mice as tested by ELISA of the second
fusion, which was produced by immunizing mice with solubler MIF.
The antigen used in the ELISA for detection was biotin-MIF.
[0028] FIG. 3 shows that MIF catalyzes keto-enol tautomerase to
tautomerize p-hydroxyphenylpyruvate.
[0029] FIG. 4 shows the MIF mediated signaling events that occur in
the protein kinase A (PKA) and MAP kinase (MAPK) signaling cascade,
as described by Mitchell et al., .J Biol. Chem. 274: 18100-6
(1999).
[0030] FIG. 5 depicts the transcription-based assay for determining
anti-MIF antibody neutralization activity using the SRE-SEAP
transcription and secretion assay.
[0031] FIG. 6 shows the percent inhibition of induced by anti-MIF
antibodies on MIF induced SRE-SEAP transcription and secretion
10:g/ml antibody and 10:g/ml rMIF were used in each reaction.
[0032] FIG. 7 shows the anti-MIF Mab effects on MIF stimulated
MMP-1 release from dermal fibroblasts.
[0033] FIG. 8 shows the anti-MIF Mab effects on VEGF-stimulated
proliferation of human umbilical vein endothelist (HOVE) cells.
[0034] FIG. 9 shows anti-MIF Mab effects on MIF+LPS induced
lethality in BALB/c mice when 10 mg LPS/kg body weight is
administered per mouse.
[0035] FIG. 10 shows anti-MIF Mab effects on MIF+LPS induced
lethality in BALB/c mice when 12.5 mg LPS/kg body weight is
administered per mouse.
[0036] FIG. 11 shows anti-MIF Mab effects on MIF+LPS induced
lethality in BALB/c mice when 15 mg LPS/kg body weight is
administered per mouse.
[0037] FIGS. 12A and 12B show the results of an assay that measured
the effect of VEGF stimulation of HUVE cell proliferation over time
in the absence of VEGF or at concentrations of 25 ng or 100 ng of
VEGF over time.
[0038] FIG. 13 shows the results of an assay that evaluated effect
of anti-MIF antibody on HOVE cells proliferation (various
antibodies tested) at a concentration of 50 mg/ml in wells
containing 625 cells/well after three (3) days.
[0039] FIG. 14 shows the results of an assay that, similar to the
assay shown in FIG. 13, compares the effect of different anti-MIF
antibodies at a concentration of 50 mg/ml on HUVE cell
proliferation in microwells containing 2500 cells/well after five
(5) days.
[0040] FIG. 15 shows the results of an assay that compares the
effect of different anti-MIF antibodies on MIF-enhanced archidonic
acid release in RAW264.7 cells transfected with the MIF gene (at
antibody concentrations of 4 mg/ml and 20 mg/ml).
[0041] FIG. 16 contains an assay that compares binding of two lead
candidate anti-MIF mabs, which were immobilized, particularly with
respect to the capture of biotin-human MIF at different antibody
concentrations.
[0042] FIGS. 17 to 30 contain amino acid and DNA sequences for lead
antibodies according to the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0043] A. Definitions
[0044] By "MIF" or "macrophage migration inhibitory factor" is
meant the protein or nucleic acid encoding the protein which is
responsible for attracting macrophages to a site. A preferred MIF
is mammalian MIF, with most preferred being a human MIF. "MIF" also
includes GIF (glycosylation-inhibiting factor), MIF-1, MIF-2,
MIF-3, MIF-like proteins, and fragments of the MIF or MIF-like
proteins. Additional forms of MIF encompassed by the term include
those listed in Table 1, and as described in Weiser et al., (1989)
and U.S. Patent Application Ser. Nos. 08/243,342; 08/462,350;
08/462,350 and 08/602,929; in PCT applications WO 96/09384; WO
90/11301; WO 94/26923; WO 95/31468 (to MIF-3); and in U.S. Pat.
Nos. 5,328,990; 5,350,687; 4,299,814; 4,708,937 and European Patent
No. 263072 (to macrophage inhibitory related peptides 8 and 14).
The "MIF" proteins can also be in the form of a fusion protein.
[0045] By "knock-out animal," "KO animal," and "transgenic animal"
is meant an animal in which a MIF gene has been functionally
disrupted or inactivated. This inactivation refers to a
modification of the gene in a manner which decreases or prevents
expression of that gene and/or its product in a cell. The
expression of the gene's product is completely suppressed. A
functionally disrupted gene includes a modified gene which
expresses a truncated polypeptide having less than the entire
coding sequence of the wild-type gene.
[0046] By "animal" is meant to include preferably such mammals as
primates, bovines, canines, felines, ovines, porcines, and rodents,
etc. Preferable rodents include mice, hamsters, rabbits and guinea
pigs. However, animals can include any eukaryote.
[0047] By "antibody" is intended to refer broadly to any
immunologic binding agent, such as IgG (including IgG.sub.1,
IgG.sub.2, IgG.sub.3, and IgG.sub.4), 1gM, IgA, IgD, 1gE, as well
as antibody fragments. As used herein, "isotype" refers to the
antibody class (e.g., IgM or IgG.sub.1) that is encoded by heavy
chain constant region genes. As used herein, "isotype switching"
refers to the phenomenon by which the class, or isotype, of an
antibody changes from one immunoglobulin (Ig) class to one of the
other Ig classes. Antibodies in the broadest sense covers intact
monoclonal antibodies, polyclonal antibodies, as well as
biologically active fragments of such antibodies and altered
antibodies.
[0048] By "monoclonal antibody" is meant an antibody obtained from
a population of substantially homogeneous antibodies, i.e., the
individual antibodies comprising the population are identical
except for possible naturally occurring mutations that may be
present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single antigenic site.
Furthermore, in contrast to conventional (polyclonal) antibody
preparations, which typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. In
addition to their specificity, the monoclonal antibodies are
advantageous in that they are synthesized by the hybridoma culture,
uncontaminated by other immunoglobulins. The modifier "monoclonal"
indicates the character of the antibody as being obtained from a
substantially homogeneous population of antibodies, and is not to
be construed as requiring production of the antibody by any
particular method. For example, the monoclonal antibodies to be
used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al., Nature 256:
495-7 (1975), or may be made by recombinant DNA methods (see, e.g.,
U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage antibody libraries using the techniques
described for example in Clackson et al., Nature 352: 624-8 (1991)
and Marks et al., J. Mol. Biol., 222: 581-97 (1991).
[0049] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with, or homologous to,
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired therapeutic activity, e.g., high affinity recognition of a
MIF protein (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl.
Acad. Sci. USA, 81: 6851-5 (1984)).
[0050] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies), which contain minimal
sequence derived from a non-human immunoglobulin. For the most
part, humanized antibodies are human immunoglobulins (recipient
antibody) in which residues from a complementarity-determining
region (CDR) of the recipient are replaced by residues from a CDR
of a non-human species (donor antibody) such as mouse, rat, rabbit
or other mammal having the desired specificity, affinity, and
capacity. In some instances, Fv framework region (FR) residues of
the human immunoglobulin are replaced by corresponding non-human
residues. Furthermore, humanized antibodies may comprise residues
which are found neither in the recipient antibody nor in the
imported CDR or framework sequences. These modifications are made
to further refine and optimize antibody performance. In general,
the humanized antibody will comprise substantially all of at least
one, and typically two, variable domains, in which all or
substantially all of the CDR regions correspond to those of a
non-human immunoglobulin and all or substantially all of the FR
regions are those of a human immunoglobulin sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc). For further details, see Jones
et al., Nature 321: 522-5 (1986); Reichmann et al., Nature 332:
323-9 (1988); and Presta Curr. Op. Struct. Biol. 2: 593-6
(1992).
[0051] By "antibody fragment" or "immunogenic fragment" is meant an
immunoglobulin, including segments of proteolytically-cleaved or
recombinantly-prepared portions of an antibody molecule that are
capable of selectively reacting with a particular antigen or
antigen family (e.g., MIF). Nonlimiting examples of such
proteolytic and/or recombinant fragments include "Fab",
"F(ab').sub.2", and "Fab'", "scFv" and "Fv" fragments. Recombinant
techniques for producing Fv fragments are set forth in WO 88/0
1649, WO 88/-06630, WO 88/07085, WO 88/07086, and WO 88/09344. By a
"V.sub.H" fragment is meant that the variable region has at least a
portion of a heavy chain variable region capable of being used as
an antigen binding functionality. The preparation and use of a
light chain variable region (VL) as an antigen binding
functionality is set forth in an article by Williams et al., Proc.
Natl. A cad. Sci. (USA) 86: 5537-41 (1989).
[0052] By "high-affinity antibody" is meant an antibody which binds
to a MIF or GIF epitope with an affinity lower than 10..sup.-8 M
(e.g., 10.sup.-9M, 10.sup.-10 M, etc.). These antibodies should be
capable of recognizing the native MIF or GIF epitopes, unlike MIF
antibodies 15.5 and 3D9, which recognize primarily denatured MIF
with only weak recognition of native, undenatured MIF. Available
antibodies against MIF include XIV 15.5 and 3D9. These all exhibit
affinities less than 10.sup.-6M against native, soluble MIF
protein. As a result, the in vivo biological potency is weak and is
achieved at 20-30 mg/kg of antibody, which is too high for medical
usage. Accordingly, the anti-MIF or anti-GIF antibodies produced by
the knock-out animal will preferably yield a therapeutic response
in a human when administered at dosages of about to about 15 mg/kg
or less.
[0053] By "nucleic acid" is meant to include DNA, genomic DNA, RNA,
mRNA and cDNA. The preferred nucleic acids of the invention include
those that encode immunoglobulins or fragments thereof which
recognize MIF. The term also may encompass a MIF targeting
construct for the purpose of making a MIF.sup.-/- mouse.
[0054] By "gene" is meant the segment of DNA involved in producing
a polypeptide chain. It includes regions preceding and following
the coding region, as well as intervening sequences (e.g., introns)
between the coding sequences (exons).
[0055] By "homologous recombination" is meant the process by which
a nucleic acid molecule with similar genetic information aligns
itself with a second nucleic acid molecule and exchanges nucleotide
strands. A nucleotide sequence of the recombinant nucleic acid
which is effective to achieve homologous recombination at a
predefined position of a target nucleic acid therefore indicates a
nucleotide sequence which facilitates the exchange of nucleotides
strands between the recombinant nucleic acid molecule at a defined
position of a target gene. The effective nucleotide sequence
generally comprises a nucleotide sequence which is complementary to
a desired target nucleic acid molecule (e.g., the gene locus to be
modified), thus promoting nucleotide base pairing. Any nucleotide
sequence can be employed as long as it facilitates homologous
recombination at a specific and selected position along the target
nucleic acid molecule (e.g., a gene encoding a MIF protein).
[0056] By "not functional" or "functionally inactive" is meant that
the MIF protein is not operational or the MIF gene cannot
synthesize a functional MIF protein. "Expression vector" is given a
functional definition, and any DNA sequence which is capable of
effecting expression of a specified DNA code in a suitable host is
included in this term. As at present, such vectors are frequently
in the form of plasmids, thus "plasmid" and "expression vector" are
often used interchangeably. However, the invention is intended to
include such other forms of expression vectors which serve
equivalent functions and which may, from time to time, become known
in the art. Typically an "expression vector" is a nucleic acid
molecule comprising (1) a promoter and other sequences (e.g.,
leader sequences) necessary to direct expression of a desired gene
or DNA sequence, and (2) the desired gene or DNA sequence.
Optionally, the nucleic acid molecule may comprise a poly A signal
sequence to enhance the stability of the gene transcript and/or to
increase gene transcription and expression.
[0057] "Transformation" refers to the introduction of DNA into a
recipient host cell that changes the genotype and consequently
results in a change in the recipient cell. "Transformation" and
"transfection" are often used interchangably.
[0058] "Host cells" refers to cells which have been recombinantly
transformed with vectors constructed using recombinant DNA
techniques. One preferred host cell, may be a MIF.sup.-/- deficient
cell. A less preferred host cell is one in which the cell is
MIF.sup.-/+. Additionally, host cells may also be those cells
transfect with a nucleic acid encoding an immunoglobulin derived
from a MIF.sup.-/- of the invention.
[0059] In descriptions of processes for isolation of antibodies
from recombinant hosts, the terms "cell," "cell culture" and "cell
line" are used interchangeably to denote the source of antibody,
unless it is clearly specified otherwise. In other words, recovery
of antibody from the "cells" may mean either from spun down whole
cells, or from the cell culture containing both the medium and the
suspended cells.
[0060] By "purified" and "isolated" is meant, when referring to a
polypeptide or nucleotide sequence, that the indicated molecule is
present in the substantial absence of other biological
macromolecules of the same type. The term "purified" as used herein
preferably means at least 75% by weight, more preferably at least
85% by weight, more preferably still at least 95% by weight, and
most preferably at least 98% by weight, of biological
macromolecules of the same type are present. An "isolated nucleic
acid molecule" which encodes a particular polypeptide refers to a
nucleic acid molecule which is substantially free of other nucleic
acid molecules that do not encode the subject polypeptide; however,
the molecule may include some additional bases or moieties which do
not deleteriously affect the basic characteristics of the
composition. Thus, for example, an isolated nucleic acid molecule
which encodes a particular CDR polypeptide consists essentially of
the nucleotide coding sequence for the subject molecular
recognition unit.
[0061] By "modulating" or "regulating" is meant the ability of an
agent to alter from the wild-type level observed in the individual
organism the wild-type activity of a MIF. MIF activity can be
regulated at transcription, translation, nucleic acid or protein
stability or protein activity.
[0062] B. Method of Preparing a Knock-Out Mouse or Other Transzenic
Animal
[0063] Transgenic animals typically can be prepared by homologous
recombination. Gene deletion or knockout can be performed as
described by Capecchi, Science 244: 1288-92 (1982); Brinster, Int.
J. Dev. Biol. 37: 89-99 (1993); and DOETSCHMAN, IN TRANSGENIC
ANIMAL TECHNOLOGY: A LABORATORY HANDBOOK 115-146 (C. A. Pinkert et
al., ed., 1994). Knock-out animals can be prepared using embryonic
stem (ES) cells or ES-like cells.
[0064] C. ES Cells
[0065] The genome of ES cells can be manipulated in vitro by
introducing a desired foreign DNA by such techniques as
electroporation, microinjection, precipitation reactions,
transfection or retroviral insertion (Bradley et al., Nature 309:
255-6 (1984); Gossler et al., Proc. Natl. Acad. Sci. U.S.A. 83:
9065-9 (1986); ROBERTSON ET AL., TERATOCARCINOMAS AND EMBRYONIC
STEM CELLS: A PRACTICAL APPROACH (1987); Kuehn et al., Nature 326:
295-8 (1987); Thompson et al., Cell 56: 313-21 (1989); Zimmer et
al., Nature 338: 150-3 (1989); and Doetschman (1994).
[0066] ES-like cell lines have been identified and can be used as
described by:
1 Hamsters Doetschman et al., Dev. Bid. 127: 224-7 (1988) Pigs
Notarianni et al., J. Reprod. Fertil. Suppl. 41: 51-6 (1990);
Piedrahita et al., Theriogenology 34: 879-90 1 (1990); and Strojek
et al., Theriogenology 33:901-13 (1990) Sheep Piedrahita et al.
(1990).
[0067] Other animals and methods of obtaining transgenic animals,
using methods other than ES cells or ES-like cells, include those
of Iannaccone et al., Dev. Biol. 163: 288-92 (1994) for rats; Stice
et al., Theriogenology 41: 301 (Abstract) (1994) for bovine
fetuses, and Wheeler, J. Reprod. Fertil. 6 (Suppl.): 1-6 (1994) and
Gerfen et al., Anim. Biotech. 6: 1-14 (1995) for pigs. The methods
by Chemey et al., Theriogenology 41: 175 (1994) can be used for
culturing bovine primordial germ cell-derived cell lines in
culture. In addition to ES or ES-like cells, inner cell mass cells
of blastocysts from animals such as bovines can be used as
described by Van-Stekelenburg-Hamers et al., Mol. Reprod. Dev. 40:
444-54 (1995) and Collas et al., Mol. Reprod. Dev. 38: 264-7
(1994).
[0068] D. Nuclear Transfer
[0069] Homologous recombination events can also be used with
nuclear transfer or transplantation. Using this technique
eliminates the need for ES or ES-like cell lines. Nuclear transfer
ca be performed using the methods described by Campbell et al.,
Nature 380: 64-8 (1996).
[0070] E. Homologous Recombination
[0071] In one aspect of the invention, a targeting vector is
employed to insert a selectable marker into a predefined position
of a gene (e.g., the gene encoding a MIF protein). The position is
selected to achieve functional disruption of the gene upon
insertion of the selectable marker. For such purposes, a preferred
embodiment is a recombinant nucleic acid molecule comprising: (1) a
5' nucleotide sequence that is effective to achieve homologous
recombination at a first predefined position of a mammalian MIF
gene operably linked to (2) the 5' terminus of a first selectable
nucleotide sequence which confers a first selection characteristic
on a cell in which it is present, and (3) a 3' nucleotide sequence
which is effective to achieve homologous recombination at a second
predefined position of the MIF gene, operably linked to the 3'
terminus of the first selectable nucleotide sequence. The
recombinant nucleic acid molecule is effective to achieve
homologous recombination in a mammalian chromosome at predefined
location, which contains a gene encoding a MIF protein. Fragments
of the targeting vector are also within the scope of the invention,
e.g., recombinant nucleic acid molecules comprising elements (1)
and (2), or comprising elements (2) and (3), etc.
[0072] Any nucleotide sequence can be employed, as long as it
facilitates homologous recombination at a specific and selected
position along the target nucleic acid molecule. Generally, there
is an exponential dependence of targeting efficiency on the extent
or length of homology between the targeting vector and the target
locus. Selection and use of sequences effective for homologous
recombination is described, e.g., in Deng et al., Mol. Cell. Bio.
12: 3365-71 (1992); Bollag et al., Annu. Rev. Genet. 23: 199-225
(1989); Waldman et al., Mol. Cell. Bio. 8: 5350-7 (1988).
[0073] An aspect of the present invention is to suppress or
functionally disrupt expression of a MIF gene. The phrases
"disruption of the gene", "gene disruption," "suppressing
expression," "gene suppression," "functional inactivation of the
gene," or "functional gene inactivation" refer to modification of
the gene in a manner which prevents expression of that gene and/or
its product (e.g., a MIF protein) in a cell. The expression of the
gene's product is completely suppressed. A functionally disrupted
gene, e.g., a functionally disrupted MIF gene, includes a modified
gene that expresses a truncated MIF polypeptide having less than
the entire coding sequence of the wild-type MIF gene. A gene can
also be functionally disrupted by affecting its mRNA structure in
such a way to create an untranslatable message, e.g., frame-shift,
decreased stability, etc.
[0074] In accordance with the present invention, a MIF gene is
modified in such a manner which is effective to disrupt expression
of the corresponding gene product. Thus, a functionally disrupted
recombinant MIF gene does not express a functional MIF polypeptide
or expresses a functional MIF polypeptide at levels which are
substantially less than wild-type levels of MIF. The gene can be
modified in any effective position, e.g., enhancers, promoters,
regulatory regions, noncoding sequences, coding sequences, introns,
exons, etc., so as to decrease or prevent expression of that gene
in a cell. Insertion into a region of a MIF gene, e.g., a MIF-1,
MIF-2 or MIF-3 gene, is usually accomplished by homologous
recombination. A recombinant nucleic acid molecule comprising
regions of gene homology and a nucleotide sequence coding for a
selectable marker gene is inserted into the promoter and/or coding
region and/or noncoding regions of a MIF gene, whereby expression
of the gene is functionally disrupted. When this knockout construct
is then inserted into a cell, the construct can integrate into the
genomic DNA. Thus, progeny of the cell will only express only one
functional copy of the gene; the other copy will no longer express
the gene product, or will express it at a decreased level, as the
endogenous nucleotide sequence of the gene is now disrupted by the
inserted nucleotide sequence. If desired, the functional gene can
be inactivated in a second analogous step.
[0075] The nucleotide sequence effective for homologous
recombination is operably linked to a nucleotide sequence,
preferably a selectable marker nucleotide sequence or gene, which
is to be inserted into the desired target nucleic acid. For
example, an aspect of the present invention is to replace all or
part of the nucleotide coding sequence for a MIF protein, with a
nucleotide sequence for a selectable marker.
[0076] The recombinant nucleic acid is preferably inserted into a
cell with chromosomal DNA that contains the endogenous gene to be
knocked out. In the cell, the recombinant nucleic acid molecule can
integrate by homologous recombination with the DNA of the cell in
such a position so as to prevent or interrupt transcription of the
gene to be knocked out. Such insertion usually occurs by homologous
recombination (i.e., regions of the targeting vector that are
homologous or complimentary to endogenous DNA sequences hybridize
to each other when the targeting vector is inserted into the cell;
these regions can then recombine so that part of the targeting
vector is incorporated into the corresponding position of the
endogenous genomic DNA).
[0077] As discussed, one or more nucleotide sequences can be
inserted into a MIF gene to suppress its expression. It is
desirable to detect the presence of the nucleotide sequence in the
gene. Such detection can be accomplished in various ways, including
by nucleic acid hybridization (e.g., Northern or Southern blot),
antibody binding to a protein epitope encoded by the inserted
nucleic acid, or by selection for a phenotype of the inserted
sequence. Accordingly, such an inserted nucleotide sequence can be
referred to as a first selectable nucleotide sequence. A first
selectable nucleotide sequence preferably confers a first selection
characteristic on a cell in which it is present. By the phrase
"selection characteristic," it is meant, e.g., a characteristic
which is expressed in a cell and which can be chosen in preference
to another or other characteristics. The selectable nucleotide
sequence, also known as selectable marker gene, can be any nucleic
acid molecule that is detectable and/or assayable after it has been
incorporated into the genomic DNA of the mammal. The selection
characteristic can be a positive characteristic, i.e., a
characteristic which is expressed or acquired by cells and whose
presence enables selection of such cells. A positive selection
characteristic can enable survival of the cell or organism, e.g.,
antibiotic resistance, ouabain-resistance (a gene for an
ouabain-resistant sodium/potassium ATPase protein). Examples of
positive selection characteristics and a corresponding selection
agent include, e.g. Neo and G4 18 or kanomycin; Hyg and hygromycin;
hisD and histidinol; Gpt and xanthine; Ble and bleomycin; and Hprt
and hypoxanthine. See, e.g., U.S. Pat. No. 5,464,764 and Capecchi,
Science 244: 1288-92 (1989). The presence of the selectable gene in
the targeted sequence can also be identified by using binding
ligands which recognize a product of the selectable gene, e.g., an
antibody can be used to identify a polypeptide product coded for by
the selectable gene, an appropriate ligand can be used to identify
expression of a receptor polypeptide encoded by the selectable
gene, or by assaying for expression of an enzyme encoded by the
selectable gene. Preferably, the selectable marker gene encodes a
polypeptide that does not naturally occur in the mammal.
[0078] The selectable marker gene can be operably linked to its own
promoter or to another promoter from any source that will be active
or can easily be activated in the cell into which it is inserted.
However, the selectable marker gene need not have its own promoter
attached, as it may be transcribed using the promoter of the gene
into which it is inserted. The selectable marker gene can comprise
one or more sequences to drive and/or assist in its expression,
including, e.g., ribosome-recognition sequences, enhancer
sequences, sequences that confer stability to the polypeptide or
RNA, and/or a polyA sequence attached to its 3' end to terminate
transcription of the gene. A positive selectable marker facilitates
selection for recombinants in which the positive selectable marker
has integrated into the target nucleic acid by homologous
recombination. A gene targeting vector in accordance with the
present invention can also further comprise a second selection
characteristic encoded by a second selectable gene to further
assist in the selection of correctly targeted recombinants. A
negative selection marker permits selection against cells in which
only non-homologous recombination has occurred. In one preferred
embodiment, the second selectable marker gene confers a negative
selection characteristic upon a cell in which it has been
introduced. Such negative selection characteristics can be arranged
in the targeting vector in such a way to facilitate discrimination
between random integration events and homologous recombination. By
the term "negative selection", it is meant a selection
characteristic which, when acquired by the cell, results in its
loss of viability (i.e., it is lethal to the cell). A nucleoside
analog, gancyclovir, which is preferentially toxic to cells
expressing HSV tk (herpes simplex virus thymidine kinase), can be
used as a negative selection agent, as it selects for cells which
do not have an integrated HSV tk selectable marker. FIAU
(1,2-deoxy-2-fluoro-.A-inverted.-d-arabino- furansyl-5-iodouracil)
can also be used as a negative selection agent to select for cells
lacking HSV tk. Other negative selectable markers can be used
analogously. Examples of negative selection characteristics and a
corresponding enzyme include thymidine kinase (HSV tk) and
acyclovir, gancyclovir, or FIAU; Hprt and 6-thioguanine or
6-thioxanthine; diphtheria toxin; ricin toxin; cytosine deaminase
and fluorocytosine.
[0079] The negative selectable marker is typically arranged on the
gene targeting vector 5' or 3' to the recombinogenic homology
regions so that double-crossover replacement recombination of the
homology regions transfers the positive selectable marker to a
predefined location on the target nucleic acid, but does not
transfer the negative selectable marker. For example, a tk cassette
can be located at the 3' end of a murine MIF gene, about 150 base
pairs from the 3' stop codon. More than one negative selectable
marker can also be utilized in a targeting vector. The positioning
of, for example, two negative selection vectors at the 5' and 3'
ends of a targeting vector further enhances selection against
target cells which have randomly integrated the vector. Random
integration sometimes results in the rearrangement of the vector,
resulting in excision of all or part of the negative selectable
marker prior to the random integration event. When this occurs,
negative selection cannot be used to eliminate those cells which
have incorporated the targeting vector by random integration rather
than homologous recombination. The use of more than one negative
selectable marker substantially enhances the likelihood that random
integration will result in the insertion of at least one of the
negative selectable markers. For such purposes, the negative
selectable markers can be the same or different.
[0080] The use of a positive-negative selection scheme reduces the
background of cells having incorrectly integrated, targeted
construct sequences. Positive-negative selection typically involves
the use of two active selectable markers: (1) a positive selectable
marker (e.g., neo) that can be stably expressed following random
integration or homologous targeting, and (2) a negative selectable
marker (e.g., tk) that can only be stably expressed following
random integration. By combining both positive and negative
selection, host cells having the correctly targeted homologous
recombination event can be efficiently obtained. Positive-negative
selection schemes can be performed as described in, e.g., U.S. Pat.
No. 5,464,764; and WO 94/06908. It is recognized, however, that one
or more negative selectable markers are not required to carry out
the present invention, e.g., produce a transgenic animal in which a
MIF gene is functionally inactivated or disrupted.
[0081] A recombinant nucleic acid molecule according to the present
invention can also comprise all or part of a vector. A vector is,
e.g., a nucleic acid molecule which can replicate autonomously in a
host cell, e.g., containing an origin of replication. Vectors can
be useful to perform manipulations, to propagate, and/or obtain
large quantities of the recombinant molecule in a desired host. A
skilled worker can select a vector depending on the purpose
desired, e.g., to propagate the recombinant molecule in bacteria,
yeast, insect, or mammalian cells. The following vectors are
provided by way of example. Bacterial: pQE70, pQE60, pQE-9
(Qiagen), pBS, pD10, Phagescript, phiX174, pBK Phagemid, pNH8A,
pNH16a, pNH18Z, pNH46A (Stratagene); Bluescript KS+II (Stratagene);
ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia). Eukaryotic:
PWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene), pSVK3, PBPV, PMSG,
pSVL (Pharmacia). However, any other vector, e.g., plasmids,
viruses, or parts thereof, may be used as long as they are
replicable and viable in the desired host. The vector can also
comprise sequences that enable it to replicate in the host whose
genome is to be modified. The use of such vector can expand the
interaction period during which recombination can occur, increasing
the targeting efficiency.
[0082] In accordance with an aspect of the present invention, the
function of a MIF gene, such as MIF-1, is disrupted or knocked out
by the insertion of an exogenous or heterologous sequence into it,
interrupting its function. For example, the exogenous or
heterologous sequence can be inserted into a region of the gene,
such as MIF-1 before its first start codon. The nucleotide sequence
coding for a selectable characteristic can be inserted into the MIF
gene in such a manner by homologous recombination so that it is
operably linked to an endogenous MIF gene promoter. Upon
integration of the selectable marker gene into the desired
predefined position of the MIF gene, expression of the selectable
characteristic is driven by the endogenous MIF gene promoter,
permitting its detection in those cells in which the construct has
integrated.
[0083] The selectable marker gene can also be integrated at
positions downstream of (3' to) the first start codon of the MIF
gene. The MIF gene can be integrated out-of-reading frame or
in-reading frame with the MIF polypeptide so that a fusion
polypeptide is made, where the fusion polypeptide is less active
than the normal product. By detecting only those cells which
express the characteristic, cells can be selected which contain the
integrated sequence at the desired location. A convenient way of
carrying out such selection is using antibiotic resistance. As
described herein, neomycin resistance is utilized as the selectable
characteristic. Cells grown in the presence of a toxic
concentration of G418 will normally die. Acquisition of the
neomycin resistance gene (neo) by homologous recombination rescues
cells from the lethal effect, thereby facilitating their
selection.
[0084] The MIF gene is knocked-out or functionally interrupted by
the integration event. The insertion of the selectable gene ahead
of the MIF coding sequence effectively isolates it from a promoter
sequence, disabling its expression. If the selectable gene contains
a transcription terminator, then transcription of the gene using
the MIF promoter will terminate immediately after it and will
rarely result in the transcription of a MIF coding sequence. The
MIF gene can also be knocked out by a deletion without a
replacement, such as a site-directed deletion of a part of the
gene. Deleted regions can be coding regions or regulatory regions
of the gene.
[0085] A MIF gene can be modified at any desired position. It can
be modified so that a truncated MIF polypeptide is produced having
one or more activities of the complete MIF polypeptide. As already
discussed, such a modified gene is a functionally disrupted
gene.
[0086] If desired, the insertion(s) can be removed from the
recombinant gene. For example, a neomycin cassette can replace
exons of a mouse MIF gene to functionally inactivate it. The
neomycin cassette can be subsequently removed from the MIF gene,
e.g., using a recombinase system. The Cre-lox site specific
recombination system is especially useful for removing sequences
from a recombinant gene. To utilize the Cre-lox system, recombinase
recognition sites are integrated into the chromosome along with the
selectable gene to facilitate its removal at a subsequent time. For
guidance on recombinase excision systems, see, e.g., U.S. Pat. Nos.
5,626,159, 5,527,695, and 5,434,066. See also, Orban et al., Proc.
Natl. Acad. Sci. USA 89: 6861-5 (1992); O'Gorman et al., Science
251: 1351-5 (1991); and Sauer et al., Nuc. Acids Res. 17: 147-61
(1989).
[0087] A nucleic acid comprising a nucleotide sequence coding
without interruption means that the nucleotide sequence contains an
amino acid coding sequence for a polypeptide, with no non-coding
nucleotides interrupting or intervening in the coding sequence,
e.g., absent intron(s) or the noncoding sequence, as in a cDNA.
[0088] Another aspect of the present invention relates to host
cells comprising a recombinant nucleic acid of the invention. A
cell into which a nucleic acid is introduced is a transformed cell.
Preferred nucleic acids include the knock-out cassettes described
above, as well as nucleic acids encoding a high affinity antibody
or fragment thereof which is produced by a MIF.sup.-/- knockout
animal. Host cells include, mammalian cells, e.g., murine Ltk-,
murine embryonic stem cells, COS-7, CHO, HeLa, insect cells, such
as Sf9 and Drosophila, bacteria, such as E. coli, Streptococcus,
bacillus, yeast, fungal cells, plants, embryonic stem (ES) cells
(e.g., mammalian, such as mouse), neuronal cells (primary or
immortalized), e.g., NT-2, NT-2N, PC-12, SY-5Y, neuroblastoma. See,
also Goeddel, Methods in Enzymology 185: 3-7 (1990) A nucleic acid
can be introduced into the cell by any effective method including,
e.g., calcium phosphate precipitation, electroporation, injection,
pressure, DEAE-Dextran mediated transfection, fusion with
liposomes, and viral transfection. When the recombinant nucleic
acid is present in a mouse cell, it is preferably integrated by
homologous recombination into the mouse cell gene locus. Additional
methods are as described in SAMBROOK ET AL., MOLECULAR CLONING: A
LABORATORY MANUAL (1989).
[0089] A transformed cell can contain a recombinant gene integrated
into its chromosome at the targeted gene locus. A targeting vector
which comprises sequences effective for homologous recombination at
a particular gene locus, when introduced into a cell under
appropriate conditions, will recombine with the homologous
sequences at the gene locus, introducing a desired selectable gene
into it. When recombination occurs such that insertion results, the
nucleic acid is integrated into the gene locus. The gene locus can
be the chromosomal locus which is characteristic of the species, or
it can be a different locus, e.g., translocated to a different
chromosomal position, on a supernumerary chromosome, on an
engineered "chromosome," etc.
[0090] As discussed below, the present invention also relates to
transgenic animals containing one or more modified MIF genes. The
transgenic animals produced in accordance with the present
invention can be used as a source to establish primary or
established, e.g., immortalized, cell lines according to various
methods as the skilled worker would know. Since the animals (either
homozygotes or heterozygotes) contain a modified MIF gene, the
corresponding cell lines would be expected to have the same
genotype. The cell lines can be derived from any desired tissue or
cell-type, including, e.g., liver, epithelia, neuron, fibroblast,
mammary, lung, kidney, pancreas, stomach, thyroid, prostate,
osteoblasts, osteoclasts, osteocytes, osteoprogenitor cells, muscle
(e.g., smooth), etc.
[0091] Cell lines produced in accordance with the present invention
are useful for a variety of purposes. In one aspect of the
invention, it is desirable to create panels of cell lines which
differ in the expression of one or more genes. For example, the
present invention describes and enables the production of cell
lines which lack a MIF gene, such as the MIF-1 gene. A
MIF-functionally-disrupted cell line differs from the parental
(i.e., starting) cell line by the expression of the MIF gene. The
availability of such pairs of cell lines, i.e., plus or minus for
MIF expression (or any other desired gene, e.g., MIF-2), is useful
to distinguish the effects of MIF from those of other MIF genes
products. A cell line functionally-disrupted in one or more desired
proteases (e.g., MIF-1, MIF-2, etc.), in combination with the
parental cell line intact for other MIF or MIF-like proteins, can
be employed to specifically distinguish its activity (e.g., MIF-1)
from all other MIF proteins. Such genetic dissection can be used to
develop, e.g., drugs and therapeutics which target a specific gene
product.
[0092] Gene functionally-disrupted cell lines can also be utilized
to produce transgenic, either chimeric, heterozygous, or
homozygous, animals, e.g., non-human mammals. Such transgenic
animals are useful as models to study the physiological role of a
desired gene and to identify agents which specifically target the
desired gene or a biological pathway in which it acts. Thus, an
aspect of the invention is method of administering to a mammal
functionally-disrupted for a MIF or MIF-like gene, e.g., MIF, an
amount of an agent effective to restore MIF activity.
[0093] The present invention also relates to a non-human transgenic
animal, preferably a mammal, more preferably a mouse, which
comprises a macrophage MIF gene, which has been engineered
employing a recombinant nucleic acid according to the present
invention. Generally, a transformed host cell, preferably a
totipotent cell, whose endogenous gene has been modified using a
recombinant nucleic acid as described above is employed as a
starting material for a transgenic embryo. The preferred
methodology for constructing such a transgenic embryo involves
transformed embryonic stem (ES) cells prepared as described herein
employing a targeting vector comprising a recombinant nucleic acid
according to the invention. A particular gene locus, e.g., MIF-1,
is modified by targeted homologous recombination in cultured ES or
ES-like cells employing a targeting vector comprising a recombinant
nucleic acid according to the invention. The ES or ES-like cells
are cultured under conditions effective for homologous
recombination. Effective conditions include any culture conditions
which are suitable for achieving homologous recombination with the
host cell chromosome, including effective temperatures, pH, medias,
additives to the media in which the host cell is cultured (e.g.,
for selection, such as G418 and/or FIAU), cell densities, amounts
of DNA, culture dishes, etc. Cells having integrated the targeting
vector are selected by the appropriate marker gene present in the
vector. After homologous recombination has been accomplished, the
cells contain a chromosome having a recombinant gene. In a
preferred embodiment, this recombinant gene contains a positive
selectable marker gene fused to endogenous MIF gene sequences. The
transformed or genetically modified ES or ES-like cells can be used
to generate transgenic non-human mammals, e.g., mice, by injection
into blastocysts and allowing the chimeric blastocysts to mature,
following transfer into a pseudopregnant mother. See, e.g.,
TERATOMACARCINOMA AND EMBRYONIC STEM CELLS: A PRACTICAL APPROACH
(E. J. Robertson, ed., IRL Press). Various stem cells can be used,
as known in the art, e.g., AB-1, HM-1 D3, CCl 1.2, E-14T62a, or
RW4. Offspring born to foster mothers may be screened initially for
mosaic coat color, where a coat color selection strategy has been
employed. Alternatively, DNA from tail or other suitable tissue of
the offspring can be used to screen for the presence of the DNA
targeting vector. Offspring that appear to be mosaics are then
crossed to each other, if it believed they carry the modified gene
in their germ line, in order to generate MIF deficient homozygotes.
See, e.g., U.S. Pat. Nos. 5,557,032 and 5,532,158.
[0094] In addition to the ES or ES-like cell methods described
herein, transgenic animals can be created by other methods, e.g.,
by pronuclear injection of recombinant genes into pronuclei of
one-cell embryos, incorporating an artificial yeast chromosome into
embryonic stem cells, gene targeting methods and embryonic stem
cell methodology. See, e.g., U.S. Pat. Nos. 4,736,866; 4,873,191;
4,873,316; 5,082,779; 5,304, 489; 5,174, 986; 5,175, 384;
5,175,385; and 5,221,778; and Gordon et al., Proc. Natl. Acad. Sci.
U.S.A., 77: 7380-4 (1980); Palmiter et al., Cell 41: 343-5 (1985);
Palmiter et al., Ann. Rev. Genet. 20: 465-99 (1986); Askew et al.,
Mol. Cell. Bio. 13: 4115-24 (1993); Games et al., Nature 373: 523-7
(1995); Valancius et al., Mol Cell. Bio. 11: 1402-8 (1991); Stacey
et al., Mol. Cell. Bio. 14:1009-16 (1994); Hasty et al., Nature
350: 243-6 (1995); and Rubinstein et al., Nucl. Acid Res. 21:
2613-7 (1993).
[0095] As discussed, one aspect of the invention relates to a
knock-out mammal, such as a mouse, comprising cells which contain
at least one functionally disrupted, recombinant MIF gene (e.g.,
heterozygous or homozygous) at a chromosomal MIF gene locus. The
cells and animals can be created in accordance with the examples
below by inserting an exogenous nucleotide sequence into the MIF
gene. However, other methods can be used to create a functionally
interrupted gene. For example, a termination codon can be inserted
into a MIF gene, using, e.g., a replacement type vector as
described in Rubinstein et al., Nucleic Acid Res. 21: 2613-7 (1993)
or a tag-and-exchange strategy as described in Askew et al., Mol.
Cell. Bio. 13: 4115-24 (1993), etc. Functional interruption of a
MIF gene can also be achieved classically by mutagenesis, such as
chemical or radiation mutagenesis.
[0096] A recombinant nucleic acid molecule according to the present
invention can be introduced into any non-human mammal, including a
mouse (HOGAN ET AL., MANIPULATING THE MOUSE EMBRYO: A LABORATORY
MANUAL (Cold Spring Harbor Laboratory, Cold Spring Harbor, New
York, 1986)), pig (Hammer et al., Nature 315: 343-5 (1985)), sheep
(Hammer et al., Nature 315: 343-345 (1985)), cattle, rat, or
primate. See also, e.g., Church, Trends in Biotech. 5: 13-9 (1987);
Clark et al., Trends in Biotech. 5: 20-4 (1987); DePamphilis et
al., BioTechniques 6: 662-80 (1988); and STRATEGIES IN TRANSGENIC
ANIMAL SCIENCE (Glenn M. Monastersky and James M. Robl, eds.
1995).
[0097] In the examples below, a murine MIF gene is modified by
homologous recombination utilizing a gene targeting vector
comprising regions of the murine MIF gene. To carry out genetic
modification of another mammalian MIF gene, e.g., a rat or a
primate, it may be desirable to obtain analogous regions of the
target MIF gene. A MIF gene from another species, using a murine or
human MIF gene, can be accomplished by various methods known in the
art, e.g., PCR using a mixture of oligonucleotides based on a
consensus sequence or MIF (e.g., Leytus et al., Biochemistry 27:
1067-74 (1988)), nucleic acid hybridization using oligonucleotides,
cDNA, etc., at a desired stringency (e.g., SAMBROOK ET AL.,
MOLECULAR CLONING, 1989).
[0098] A transgenic animal according to the present invention can
comprise one or more MIF genes which have been modified by genetic
engineering. For example, a transgenic animal comprising a MIF gene
which has been modified by targeted homologous recombination in
accordance with the present invention can comprise other mutations,
including modifications at other gene loci and/or transgenes.
Modifications to these gene loci and/or introduction of transgenes
can be accomplished in accordance with the methods of the present
invention, or other methods as the skilled worker would know. For
instance, double-mutants can be made by conventional breeding,
i.e., crossing animals and selecting for a desired phenotype and/or
genotype. In one embodiment of the invention, a transgenic animal
can be constructed having at least a defective MIF-1 gene (e.g., a
knock-out) and one or more other MIF or MIF-like genes coding for a
MIF or MIF-like protein. In a preferred embodiment, the latter
genes are null or functionally-disrupted. Such an animal can be
homozygous (-/-) or heterozygous (-/+) for the desired loci, or a
combination thereof.
[0099] For other aspects of the nucleic acids, polypeptides,
antibodies, etc., reference is made to standard textbooks of
molecular biology, protein science, and immunology. See, e.g.,
Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (Elsevir Sciences
Publishing, Inc., New York 1986); Hames et al., NUCLEIC ACID
HYBRIDIZATION (IL Press 1985), SAMBROOK ET AL., (1989); CURRENT
PROTOCOLS IN PROTEIN SCIENCE (F. M. Ausubel et al., eds. John Wiley
& Sons, Inc.), CURRENT PROTOCOLS IN HUMAN GENETICS (Nicholas C.
Dracopoli et al., eds. John Wiley & Sons, Inc. 1994); CURRENT
PROTOCOLS IN PROTEIN SCIENCE (John E. Coligan et al., eds. John
Wiley & Sons, Inc. 1995); and CURRENT PROTOCOLS IN IMMUNOLOGY
(John E. Coligan et al., ed. John Wiley & Sons, Inc. 1991).
[0100] F. Method of Raising Antibodies in a Knock-Out Animal
[0101] Antibodies can be obtained from the blood serum of a
MIF.sup.-/- animal immunized with a MIF antigen.
[0102] i. MIF Antigen
[0103] The MIF antigen used to raise antibodies can be from a
complete MIF protein from any species, fragments thereof and fusion
proteins containing all or a portion of a MIF protein. MIF
sequences include, but at not limited to, any of the following:
2 TABLE 1 GenPept Name Accession No. Publication or Deposit L
dopachrome-methyl ester P81748 tautomerase (macrophage MIF homolog)
of Trichuris trichiura D-dopachrome tautomerase O35215 Esumi et
al., Mamm. Genome (murine) 9: 753-7 (1998). Macrophage MW homolog
P91850 Pastrana et al., Infect. Immun. (BMMIF) (Brugia malayi) 66:
5955-63 (1998). L-Dopachrome-methyl ester P81529 Pennock et al.,
Biochem. J. tautomerase (macrophage MIF 335: 495-8 (1998). homolog)
Trichinella spiralis L-Dopachrome-methyl ester P81530 Pennock et
al., (1998) tautomerase (macrophage MIF homolog) Trichuris muris
MIF-Like Protein C52E4.2. Q18785 Caenorhabditis elegans Macrophage
MIF P80928 (Glycosylation-inhibiting factor) (GIF). Sus scrofa
Macrophage MIF P30904 Sakai et al., Biochem. Mol.
(Glutathione-binding 13 kD Biol. Int. 33: 439-46 (1994) Protein).
Rattus norvegicus Macrophage MIF P14174 Weiser et al., Proc. Natl.
(Glycosylation-inhibiting Acad. Sci. U.S.A. 86: 7522-6 factor,
GIF).; Homo sapiens (1989); Mikayama et al., Proc. Natl. Acad. Sci.
U.S.A. 90: 10056-60 (1993); Kato et al., Proc. Natl. Acad. Sci.
U.S.A. 93: 3007-10 (1996) Macrophage MIF(P12A). P80177 Galat et
al., Eur. J. Biochem. Bovine 224: 417-21 (1994). Macrophage MIF
(delayed P34884 Bernhagen et al., Nature 365: early response
protein 6, 756-9 (1993); Mikayama et DER6) (Glycosylation- al.,
(1993). inhibiting factor). Murine Macrophage MIF. Gallus Q02960
Wistow et al., Proc. Natl. gallus Acad. Sci. U.S.A. 90: 1272-5
(1993). Chain B, Macrophage MIF 5822094 Y95f Mutant. Mus musculus
Chain A, Macrophage MIF 5822093 Y95f Mutant. Mus musculus Chain C,
Macrophage MIF 5822092 Y95f Mutant. Mus musculus Macrophage MIF.
Sus scrofa AAD50507 Abraham et al., Domest. Anim. Endocrinol. 15:
389-6 (1998). Macrophage MIF 4505185 Mikayama et al., (1993);
(glycosylation-inhibiting Paralkar et al., Genomics 19: factor).
Homo sapiens 48-51 (1994); Kozak et al., Genomics 27: 405-11
(1995); Budarfet al., Genomics 39: 235-6 (1997). Chain C,
Macrophage MIF 5542327 Lubetsky et al., Biochemistry with Pro-1
Mutated To Gly-1. 38: 7346-54 (1999). Homo sapiens. Chain B,
Macrophage MIF 5542326 Lubetsky et al., (1999). with Pro-1 Mutated
To Gly-1. Homo sapiens. Chain A, Macrophage MIF 5542325 Lubetsky et
al., (1999). with Pro-1 Mutated To Gly-1. Homo sapiens Chain C,
Macrophage MIF 5542179 Lubetsky et al., (1999). with Alanine
Inserted Between Pro-1 And Met-2. Homo sapiens Chain B, Macrophage
MIF 5542178 Lubetsky et al., (1999). With Alanine Inserted Between
Pro-1 And Met- 2.Met-2. Homo sapiens Chain A, Macrophage MIF
5542177 Lubetsky et al., (1999). with Alanine Inserted Between
Pro-1 and Met-2. Homo sapiens Macrophage migration CAB46355
inhibitory factor-like protein. Trichuris trichiura Macrophage MIF.
Bos taurus AAD38354 Macrophage MIF. AAC82615 Wuchereria bancrofti
Chain C, Macrophage MIF. 1942979 Sun et al., Proc. Natl. Acad. Homo
sapiens Sci. U.S.A. 93: 5191-6 (1996) Chain B, Macrophage MIF.
1942978 Sun et al., (1996). Homo sapiens Chain A, Macrophage MIF.
1942977 Sun et al., (1996). Homo sapiens Macrophage MIF. Meriones
AAC02629 unguiculatus Macrophage MIF. Brugia AAB60943 malayl
Macrophage MIF. Bovine S32394 Galat et al., FEBS Lett. 319: 233-6
(1993). Macrophage migration A44499 Lanahan et al., Mol. Cell.
Biol. inhibitory factor DER6- 12: 3919-29 (1992); Wistow mouse. et
al., (1993); Bernhagen et al., (1993); Mikayama et al., (1993); and
Mitchell et al., J. Immunol. 154: 3863-70 (1995). Macrophage
inhibitory factor A61386 Oki et al., Lymphokine (F5 cells)-human
(fragment). Cytokine Res. 10: 273-80 (1991). Macrophage migration
CAA80598 Bernhagen et al., (1993); and inhibitory factor. Homo
Wistow et al., (1993). sapiens MIF (rat liver), 115 aa AAB32392
Sakai et al., Biochem. Mol. Biol. Int. 33: 439-46 (1994). p12a
isoform = macrophage AAB32021 Galat et al., (1994).
migration-inhibitory factor [cattle, Peptide, 114 aa]. Macrophage
MIF {N-terminal AAB26003 Galat et al., (1993). partial peptide, 39
aa} Bos taurus. Macrophage MIF. Rattus AAB04024 norvegicus
Macrophage MIF. Mus CAA80583 Bernhagen et al., (1993). musculus
Macrophage MIF. Mus AAA91638 Kozak et al., (1995). musculus
Macrophage migration AAA91637 Bozza et al., Genomics 27:
inihibitory factor. Mus 412-19 (1995). musculus MIF. Mus musculus
AAA74321 Mitchell et al., J. Immunol. 154: 3863-7 (1995).
Macrophage MIF. Gallus AAA48939 Wistow et al., (1993). gallus
Macrophage MIF. Homo AAA36179 Wistow et al., (1993). sapiens
Macrophage MIF. Homo AAA21814 Paralkar et al., (1994) sapiens
Macrophage MIF.-3 (human) U.S. Pat. Nos. 5,986,060; 5,650,295; ATCC
No. 75712 Macrophage MIF.-2 Hirose et al., Microbiol. Immunol.
35:235-45 (1991). Sequence 8 from U.S. Pat. g5960276 U.S. Pat. No.
5,897,714 No. 5,807,714 (antigen- specific glycosylation inhibiting
factor (AgGIF)) Sequence 4 from U.S. Pat. g5960275 U.S. Pat. No.
5,807,714 No. 5,807,714 (antigen- specific glycosylation inhibiting
factor (AgGIF)) Sequence 4 from U.S. Pat. g5960274 U.S. Pat. No.
5,897,714 No. 5,807,714 (antigen- specific glycosylation inhibiting
factor (AgGIF)) Sequence 4 from U.S. Pat. g5960273 U.S. Pat. No.
5,897,714 No. 5,807,714 (antigen- specific glycosylation inhibiting
factor (AgGIF)) Chain A, Human g1942169 Kato et al., (1996).
glycosylation-inhibiting factor Chain B, Human g1942170 Kato et
al., (1996). glycosylation-inhibiting factor Chain C, Human
g1942171 Kato et al., (1996). glycosylation-inhibiting factor
Glycosylation-inhibiting g2135300 Weiser et al., (1989); and
factor-human Paralkar et al., (1994). Glycosylation-inhibiting
g1085446 Galat et al., (1994). factor-bovine
Glycosylation-inhibiting g402717 Mikayama et al., (1993). factor
Glycosylation-inhibiting g402702 Mikayama et al., (1993).
factor
[0104] G. Method of Preparing Cell Lines Which Express Anti-MIF
Antibodies
[0105] Once antibody secreting cells, which produce antibodies of a
desired anti-MIF affinity, are isolated, these cells can be
utilized using standard procedures to produce cell lines which
produce the desired antibodies.
[0106] i. Hybridoma Preparation
[0107] Hybridomas secreting monoclonal antibodies can be prepared
as described by Kohler and Milstein, Nature 256: 495-7 (1975) or by
Galfr et al., Methods Enzymol. 73 (Pt. B): 3-46 (1981). Briefly,
homozygous deficient MIF mice (MIF.sup.-/-) are immunized by
subcutaneous injection of about 0.1 to 100:g (preferably 10:g) of
MIF protein in complete Freund's adjuvant, followed approximately 2
weeks later by intraperitoneal injection of about 10:g of MIF in
incomplete Freund's adjuvant. Antisera is collected about 1 week
later and is analyzed in a micro-ELISA using microtiter plates
coated with MIF protein (about 1 .mu./ml) and detection of bound
immunoglobulins with horseradish peroxidase-conjugated rabbit
anti-mouse IgG. The specific antibody concentration in these
antisera is retrospectively calculated by ELISA on microtiter
plates coated with the respective antigen using purified monoclonal
antibodies for calibration. After an interval of at least 4 weeks,
the mice are boosted intraperitoneally with 10:g of MIF protein in
saline on days 4 and 2 before the cell fusion. Spleen cells are
isolated and fused with either P3x63.Ag.8-6.5.3 or Sp2/O-AG14
myeloma cells. After selection in
hypoxanthine-aminopterine-thyinidine medium, the supernatants are
screened for specific antibody production with an one-site,
non-competitive, micro-ELISA using microtiter plates coated with
MIF and detection of bound immunoglobulins as described above.
Positive clones are used for the production of ascites in
pristane-primed mice. The IgG fraction of the monoclonal antibodies
can be purified from ascites by affinity-chromatography on protein
A-Sepharaose.
[0108] It should be noted that injection schedules, the animal
immunized, and the amount and type of MIF antigen used (e.g., MIF
fusion protein, MIF peptides or porteins) can be varied as would be
known to the skilled artisan. See, e.g., ED HARLOW ET AL.,
ANTIBODIES: A LABORATORY MANUAL (1988).
[0109] ii. Antigen
[0110] The MIF antigen used to immunize the knock-out mice or other
knock-out animal can be derived from various sources. MIF can be
purified from biological samples by chromatography or other
purification procedure. Alternatively, MIF can be prepared
recombinantly in eukaryotes or prokaryotes as previously described.
Whole MIF proteins can be injected into the animal, as well as MIF
peptides. MIF peptides for use in raising anti-peptide anti-MIF
antibodies are preferably greater than 6 consecutive MIF amino
acids in length. Peptide antigens can be 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 and 50 amino
acids in length. Peptides can be prepared synthetically,
recombinantly or by proteolytic cleavage of the MIF protein to
produce proteolytic MIF fragments. Recombinant forms of MIF or MIF
peptides can be in the form of a fusion protein, wherein MIF is
fused to another protein or polypeptide such as maltose binding
protein (MBP), -galactosidase or other suitable protein. MIF
peptides can also be expressed recombinantly.
[0111] H. Diseases to be Treated Using Anti-MIF Antibodies
[0112] Diseases mediated by MIF include inflammatory diseases,
retinopathy, e.g. diabetic or SLE-associated retinopathy, delayed
type hypersensitivity (DTH), conditions mediated by DTH, cancer,
pathological conditions induced by viruses and other pathogens,
adult respiratory distress syndrome (ARDS), autoimmune diseases,
endotoxic shock, pathological conditions involving
neovascularization and trauma.
[0113] In the instance of septic shock, MIF has been reported to be
a major secreted protein released by anterior pituitary cells in
response to lipopolysaccharide (LPS) and may be a critical mediator
of septic shock (Calandra et al., Nature 377: 68-71 (1995); and
Bernhagen et al., Nature 365: 756-9 (1993). Some have suggested
that the counteraction or neutralization of MIF may serve as an
adjunct therapy in sepsis (Bozza et al., J. Exp. Med. 189: 341-6
(1999)).
[0114] In cancer, MIF has been reported to be spontaneous expressed
by human cancer cells (Shimizu et al., Biochem. Biophys. Res.
Commun. 264: 751-8 (1999); and Bini et al., Electrophoresis 18:
2832-41 (1997)). MIF reportedly also mediates or is produced in
elevated quantities in colonic adenomas (Shkolnik et al., Am. J.
Gastroenterol. 82: 1275-8 (1987)), human T-cell leukemia virus
(HTLV) induced T-cell leukemia (Koeffler et al., Blood 64: 482-90
(1984)), prostatic adenocarcinoma (Meyer-Siegler et al., Urology
48: 448-52 (1996)), pseudolymphoma, sacroidosis, and acute
myeloblastic leukemia (AML). Hypoxia can also induce transcription
of MIF and MIF found, in the serum of head and neck cancer
patients, has been correlated with the degree of hypoxia occurring
in these patients (Koong et al., Cancer Res. 60: 883-7 (2000)). MIF
has been reported to suppress p53 activity and has been suggested
as a link between inflammation and tumorigenesis (Hudson et al., J.
Exp. Med. 190:1375-82(1999)). Anti-MIF antibodies have been shown
to inhibit growth and visualization of colon tumors in mice (Ogawa,
1999).
[0115] Delayed type hypersensitivity (DTH) related diseases include
atopic dermatitis (Shimizu et al., Biochem. Biophys. Res. Commun.
240: 173-8 (1997)). Autoimmune diseases with potential MIF
involvement include Gaucher's Disease, rheumatoid arthritis (see
Leech et al., Arthritis Rheum. 42: 1601-8 (1999); Onodera et al.,
J. Biol. Chem. 275: 444-50 (2000); and Onodera et al., Cytokine 11:
163-7 (1999)), asthma, immunologically induced kidney disease and
systemic lupus erythematosus. In rheumatoid arthritis, MIF seems to
act by inducing expression of matrix metalloproteinases (MMPs),
such as MMP-1 and MMP-3, by synoviocyte fibroblasts (Onodera et
al., 2000). MIF also has been indicated to play a role in psoriasis
(Steinhoffet al., Br. .J. Dermatol. 141: 1061-6 (1999)). Moreover,
although it was known that MIF played a role in experimental
glomerulonephritis (GN), only recently have researchers reported
that MIF is markedly up-regulated in proliferative forms of human
GN and that this up-regulation correlated with leukocyte
infiltration, histologic damage and renal function impairment (Lan
et al., Kidney Int. 57: 499-509 (2000)).
[0116] In one aspect, the anti-MIF antibodies or the immunogenic
fragments thereof are contemplated for use in modulating the
diseases and conditions described above. Preferably, the antibodies
or their immunogenic fragments would inhibit the activity of MIF in
a subject, wherein the subject is preferably human. More
specifically, the anti-MIF antibodies contemplated are proposed for
use alone or as an adjunct therapy to prevent disease progression.
Some anti-MIF antibodies, prepared by methods other than those
disclosed herein and with different specificities and affinities,
have been shown to, for example, protect mice against (1)
LPS-induced septic shock related death (Bernhagen et al., 1993));
(2) lethal peritonitis induced by cecal ligation and puncture (CLP)
(Calandra et al., Nature Med. 6: 164-70 (2000)), anti-glomerular
basement membrane (GBM) induced glomerulonephritis (Lan et al., J.
Exp. Med. 185: 1455-65 (1997)), collagen type II induced rheumatoid
arthritis in mice (Mikulowska et al., J. Immunol. 158: 5514-7
(1997)) and adjuvant induced arthritis in rats (Leech et al.,
Arthritis Rheum. 41: 910-7 (1998)), and has slowed 38C13 B cell
lymphoma growth and vascularization in mice (Chesney et al., Mol.
Med. 5: 1181-91(1999)), and carcinoma growth and neovascularization
(Ogawa et al., Cytokine 12: 309-14 (2000)).
[0117] I. Anti-MIF Antibody or Antibody Fragment Compositions and
Administration
[0118] An antibody or fragment thereof of the invention is
administered to subjects in a biologically compatible form suitable
for pharmaceutical administration in vivo. By "biologically
compatible form suitable for administration in vivo" is meant a
form of the antibody or fragment to be administered in which any
toxic effects are outweighed by the therapeutic effects of the
antibody or fragment. An antibody or fragment can be administered
in any pharmacological form, optionally in a pharmaceutically
acceptable carrier. Administration of a therapeutically effective
amount of the antibody or fragment thereof is defined as an amount
effective, at dosages and for periods of time necessary to achieve
the desired result (e.g., inhibition of the progression or
proliferation of the disease being treated). For example, a
therapeutically active amount of an antibody or fragment thereof
may vary according to such factors as the disease stage (e.g.,
stage I versus stage IV), age, sex, medical complications, and
weight of the individual, and the ability of the antibody or
fragment thereof to elicit a desired response in the individual.
The dosage regimen may be adjusted to provide the optimum
therapeutic response. For example, several divided doses may be
administered daily, or the dose may be proportionally reduced as
indicated by the exigencies of the therapeutic situation.
[0119] The active compound, an antibody or fragment thereof, by
itself or in combination with other active agents, such as
conventional anti-cancer drugs, steroids (e.g., glucocorticoids and
cortico steroids) and additional antibodies or fragments thereof.
Examples of steroids for use in combination with anti-MIF
antibodies include dexamethasone and cortisol. Examples of
glucocorticoids include: 21-Acetoxypregnenolone, Alclometasone,
Algestone, Anicinonide, 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, Prednisolone Sodium Phosphate, Prednisone,
Prednival, Prednylidene, Rimexolone, Tixocortol, Triamcinolone,
Triamcino lone, Acetonide, Triamcino lone B enetonide,
Triamcinolone Hexacetonide. The immunoconjugate, alone or in
combination with other agents, may be administered in a convenient
manner such as by injection (subcutaneous, intramuscularly,
intravenous, etc.), inhalation, transdermal application or rectal
administration. Depending on the route of administration, the
active compound may be coated with a material to protect the active
compound from the action of enzymes, acids and other natural
conditions, which may inactivate the compound. A preferred route of
administration is by intravenous (I.V.) injection. Examples of
conventional anti-cancer drugs include, but are not limited to
methotrexate, taxol, cisplatin, tamoxifen, et seq.
[0120] To administer an antibody or fragment thereof by other than
parenteral administration, it may be necessary to coat the antibody
or fragment thereof with, or co-administer the antibody or fragment
thereof with, a material to prevent its inactivation. For example,
an antibody or fragment thereof can be administered to an
individual in an appropriate carrier or diluent, co-administered
with enzyme inhibitors or in an appropriate carrier or vector, such
as a liposome. Pharmaceutically acceptable diluents include saline
and aqueous buffer solutions. Liposomes include
water-in-oil-in-water emulsions, as well as conventional liposomes
(Strejan et al., J. Neuroimmunol. 7: 27 (1984)). Additional
pharmaceutically acceptable carriers and excipients are known in
the art or as described in REMINGTOM'S PHARMACEUTICAL SCIENCES
(18th ed. 1990).
[0121] The active compound may also be administered parenterally or
intraperitoneally. Dispersions of the active compound also can be
prepared in glycerol, liquid polyethylene glycols, and mixtures
thereof and in oils. Under ordinary conditions of storage and use,
these preparations may contain one or more preservatives to prevent
the growth of microorganisms.
[0122] Pharmaceutical compositions suitable for injectable use
include sterile, aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersions. In all cases, the
composition must be sterile and must be fluid to the extent that
easy syringability exists. It must be stable under the conditions
of manufacture and storage and must be preserved against the
contaminating action of microorganisms, such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyethylene glycol, and the like), and suitable
mixtures thereof. The proper fluidity can be maintained, for
example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of
dispersion, and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorBio acid, thimerosal and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols, such as manitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0123] Sterile injectable solutions can be prepared by
incorporating an active compound (e.g., an anti-MIF antibody or
fragment thereof) in the required amount in an appropriate solvent
with one or a combination of ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the active compound into
a sterile vehicle, which contains a basic dispersion medium and the
required other ingredients from those enumerated above. In the case
of sterile powders for the preparation of sterile injectable
solutions, the preferred methods of preparation are vacuum drying
and freeze-drying, which yields a powder of an active ingredient
plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0124] When the active compound is suitably protected, as described
above, the protein may be orally administered, for example, with an
inert diluent or an assimilable edible carrier. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like. The
use of such media and agents for pharmaceutically active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active compound, use thereof in
the therapeutic compositions is contemplated. All compositions
discussed above for use with an anti-MIF antibody or fragment
thereof may also comprise supplementary active compounds in the
composition.
[0125] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of a dosage. "Dosage unit form," as used herein, refers
to physically discrete units suited as unitary dosages for the
mammalian subjects to be treated; each unit containing a
predetermined quantity of active compound is calculated to produce
the desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on: (A) the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved; and (B) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0126] In Vitro Functional Assays for Testing MIF-Neutralizing
Antibodies
[0127] Several assays are available for testing whether a
particular anti-MIF antibody produced from a MIF knockout animal,
or a humanized antibody a portion of which is derived from an
anti-MIF antibody produced from a knock out animal neutralize
MIF-induced activity.
[0128] For example, one assay is the phenylpyruvate tautomerase
(PPT) assay. This assay is based on the fact that MIF interconverts
the enol- and keto- forms of phenylpyruvate and
(p-hydroxyphenyl)pyruvate (Hermanowski-Vostka et al., Biochem. 38:
12841-9 (1999)). As shown in FIG. 3, MIF catalyzes the
tautomerization of p-hydroxyphenylpyruvate. MIF also has been shown
to possess D-dopachrome tautomerase and thiol protein
oxidoreductase activities (Matsunaga et al., Cell. Mol. Biol. 45:
1035-40 (1999)). Accordingly, similar assays could be developed for
the D-dopachrome and thiol protein oxidoreductase activities, as is
described for the PPT assay.
[0129] Another in vitro assay can be performed in, for example
NIH-3T3 cells, to determine the MIF activity inhibition based on
the role of MIF (see FIG. 4) in the activation of the p44/p42
extracellular signal-regulated (ERK) mitogen-activated protein
kinases (MAP) pathways, as discussed by Mitchell et al., J. Biol.
Chem. 274: 18100-6 (1999). The activation of ELC by ERK 1/2 is
discussed by Mitchell et al., (1999). The assay is a
transcription-based assay for testing the efficacy of MIF
neutralization by anti-MIF antibodies. A construct comprising a
serum response element (SRE), promoter and Secreted alkaline
phosphatase (SEAP) is created and transiently transfected into an
appropriate cell line, such as NIH-3T3 cells. The expression of the
SEAP gene is proportional to the transcriptional activation of ELK1
(e.g., EL1-pELK1.sup.+). The impact of anti-MIF antibodies on MIF
stimulated SRE-mediated transcription ascedrtained by measuring the
alkaline phosphatase concentrations secreted (see FIG. 5). The
alkaline phosphatase can be assessed using, for example a
chemiluminescence detection system. Similar studies can be
performed on MIF signaling events involving other phosphorylation
of pathways involving transcription activation of AP-1, NF-KB and
other factors.
[0130] Other in vitro studies for examining the activity of MIF
signaling includes growth arrest and apoptosis studies. The
potential target interactions include a MIF-mediated cascade
involving override of 53 effects, tumor necrosis factor
.A-inverted. (TNF), sodium nitroprusside and glucocorticoids. In
vitro assay systems, such as those described above, could be
suitably altered to study each of these interactions, and thereby
study the anti-MIF activity of the antibodies or fragments thereof
in inhibiting said MIF activity.
[0131] Another assay would be based on MIF induction of MMP-1
release from cells. As discussed, MIF can up-regulate the matrix
metalloproteinases (MMPs), such as MMP-1 (interstitial collagenase)
and MMP-3 (stromelysin) (Onodera et al., 2000). Anti-MIF antibodies
can then be tested for their ability to inhibit MIF induced MMP-1
release, for example, from human adult dermal fibroblasts. Other
cells which produce MMPs would also be suitable for such assays,
such as MMP-1 release from RA synovial fibroblasts.
[0132] Still another bioassay includes anti-MIF antibody inhibition
of VEGF-stimulated endothelial cells. These assays include changes
in proliferation and regulation of cell cycle and apoptosis.
[0133] K In Vivo Models for Testing MIF-Neutralizing Antibodies
[0134] There are several in vivo models for testing the efficacy of
a particular anti-MIF monoclonal antibody in an animal model.
Lipopolysaccharide (LPS) induced disease is an animal model in
which to examine septic shock (see, e.g., Bernhagen et al., Nature
365: 756-9 (1993)). Spontaneous mouse glomerulonephritis (GN) in
mice strains such as female NZB/W F1 and NZM2410; GN can also be
rapidly induced by injection of rabbit anti-GBM (glomerular
basement membrane) serum (Lan et al., J. Exp. Med. 185:1455-65
(1997)). The animal models of adjuvant-induced arthritis (see Leech
et al., Arthritis Rheum. 41: 910-7 (1998)) in rats, and collagen
type II induced arthritis in mice (see Mikulowska et al., J.
Immunol. 158: 5514-7 (1997)) are appropriate animal models for
studying methods of treating human rheumatoid arthritis.
[0135] These animal models would be used to determine the
inhibitory activity of anti-MIF monoclonal antibodies on
MIF-induced activity in each of these diseases. For example, in the
MIF/LPS lethality animal model, mice would be preinjected with an
anti-MIF monoclonal antibody or negative control antibody. Two
hours later the mice would receive an injection of MIF and LPS.
Seven hours after the injection of MIF and LPS, the mice would
receive an injection of MIF alone. The number of mice which survive
this regimen of LPS-induced lethality would then be examined as
compared to the control mice receiving an antibody other than an
anti-MIF antibody (control) or mice not receiving any LPS. Survival
would be plotted, typically at 24 hr, 48 hr, 72 hr and 96 hr after
the MIF and LPS injection.
Uses
[0136] The present invention further is directed to use of anti-MIF
antibodies for treatment and prophylaxsis of diseases, wherein
suppression or modulation of MIF is therapeutically beneficial.
Examples thereof include diseases involving cytokine-mediated
toxicity. More specific examples are inflammatory diseases and
autoimmune diseases, such as rheumatoid arthritis and other
autoimmune diseases, graft-vs-host disease, TNF induced toxicity,
endotoxin associated toxicity, septic shock, infections such as
malarial, bacterial and viral infections, allergy, etc. Also,
anti-MIF antibodies can be used to suppress undesirable immune
responses. Such antibodies may be administered alone or in
combination with other active agents, as described above.
[0137] The examples and methods provided below serve merely to
illustrate particular embodiments of the invention and are not
meant to limit the invention.
EXAMPLE 1
[0138] Preparation of a MIF Knock-Out Mouse
[0139] Targeting vector construction and generation of MIF.sup.-/-
Mice. A mouse MIF genomic fragment is isolated from a 129SV/J.
genomic library (Bozza et al., Genomics 27: 412-19 (1995)), and a
6.1 kb XbaI fragment containing the 5' upstream region, exons 1-3,
and the 3' region is subcloned in pBluescript.RTM.. The vector is
digested with EcoRV (sites present in the 3' region of the gene and
in the polylinker of the plasmid), releasing a 0.7 kb fragment. The
vector is religated and digested with AgeI, which disrupts part of
exon 2, the second intron, and exon 3. The neo cassette is inserted
by blunt ligation after end-filling the vector and the neo
cassette. The disrupted genomic vector is digested with XbaI/XhoI
and ligated into the HSV-TK vector. The targeting vector is
linearized with XhoI, and 30 .mu.g is transfected by
electroporation into 10.sup.7 J1 embryonic stem (ES) cells that are
maintained on a feeder layer of neo embryonic fibroblasts in the
presence of 500 U/ml of leukemia inhibitory factor. After 8 days of
selection with G418 (200 .mu.g/ml) and FIAU (2 .mu.g), 30 clones
are analyzed by Southern blot hybridization using the 0.7 kb
EcoRV/XbaI 3' fragment as a probe. An ES cell line clone displaying
a novel 7 kb XbaI allele predicted to occur after homologous
recombination is injected into day 3.5 C57BL/6 blastocysts. The
blastocysts are transferred into pseudopregnant females. Chimeric
mice are bred with C57BL/6 mice and agouti offspring can be
analyzed for the MIF disrupted allele by Southern blot
hybridization.
[0140] Results. The mouse MIF gene can be disrupted by replacing
part of exons 2 and 3 with a neo cassette. The targeting vector is
electroporated in J1 ES cells and G418-FIAU-resistant colonies are
isolated. Correctly targeted ES cells are used to generate chimeric
animals by injection into C57BL/6 blastocysts. Highly chimeric
animals transmitted the mutated allele through the germline.
Homozygous mice are generated by intercrosses of heterozygous mice.
Northern blot analysis from liver RNA of lipopolysaccharide
(LPS)-treated animals can be used to confirm that the gene
disruption creates a null mutation (Bozza et al., J. Exp. Med. 189:
341-6 (1999)). ELISA of serum from LPS-treated animals can be used
to further confirm the absence of MIF protein in the MIF.sup.-/-
mice (see Bozza et al., 1999). As described by Bozza et al., of the
218 animals obtained from heterozygous matings described above, 16%
were homozygous for the null allele. The newborn MIF.sup.-/- mice
developed normally in size and behavior and were fertile. The
litter size of heterozygous and homozygous matings were normal.
Both gross examination and histopathological analysis of several
organs (kidney, liver, spleen, adrenal, thymus, lungs, heart, brain
and intestine) of MIF.sup.-/- mice revealed no abnormalities. Flow
cytometric analysis of splenocytes and thymocytes of MIF.sup.-/-
mice demonstrated normal lymphocyte populations (Bozza et al.,
1999).
EXAMPLE 2
[0141] Preparation of Anti-MIF Antibodies in a MIF Knock Out
Mouse
[0142] Six week old mice, which are MIF knock out mice, are
immunized by subcutaneous injection of 100 .mu.g of MIF protein,
MIF peptides fragment in Freund's Complete Adjuvant on day one,
followed by a similar injection in Freund's Incomplete Adjuvant at
day 10. Intraperitoneal injections are then performed at weekly
intervals of 100 .mu.g of MIF (or a MIF peptide fragment) in
phosphate buffered saline (PBS). Blood is collected by supraorbital
functions.
EXAMPLE 3
Preparation of Hybridomas
[0143] For hybridoma fuision, the spleen of the mice immunized in
Example 2 are isolated and 1.times.10.sup.8 splenocytes are fused
to an equal number of Ag8 myeloma cells using the standard
polyethylene glycol protocol. Selection in
hypoxanthine/aminopterine/thymidine is initiated directly after
replating the cell suspension into fifteen 96-well flat bottom
plates. Supernatants are screened 10-14 days after the hybridoma
fusion. Positive hybridomas can then be repeatedly subcloned.
[0144] Analysis of antibody affinity can be assayed by ELISA. For
example, one .mu.g of protein/ml PBS is coated in a 96-well
polyvinylplate for 3 hours at 37.degree. C. After three washes with
PBS/0.05% Tween-20, the plates are blocked with PBS/0.1% bovine
serum albumin (BSA) for 1 hour at 37.degree. C. Again three washes
are performed before the first antibody is incubated. Sera or
antibodies are diluted in PBS/0.05% Tween-20/1% fetal calf serum
(FCS). The serum incubation is performed for 1 hour at 37.degree.
C., followed by 3 washes. The enzyme conjugate RAMPO (Dakopats), is
diluted 1000-fold and incubated for 1 h at 37.degree. C. Tetra
methyl benzidine (TMB) is used as the substrate for the peroxidase
reaction. This reaction is stopped after 15 minutes, at room
temperature by adding equal volume of 1 N H.sub.2SO.sub.4, at which
time the optical density can be measured at 450 nm. As noted in
Table 2 below, no high affinity anti-MIF generating hybridomas were
produced from BALB/c mice, whereas using the MIF knockout mouse,
numerous anti-MIF producing hybridomas were generated.
3TABLE 2 Generation of Mabs that Bind MIF with High Affinity #
fusion # hybridomas # anti-MIF mouse immunogen fusions date
generated hybridomas BALB/c MIF 2 Dec. 22, 1998 573 0 BALB/c MIF 2
Mar. 11, 1999 344 0 BALB/c MIF 2 May 10, 1999 384 0 BALB/c MIF 3
Aug. 05, 1999 500 0 BALB/c MIF/OVA 1 Dec. 21, 1999 ? 0 MIF KO
MIF/OVA 4 Dec. 21, 1999 3242 671 MIF KO MIF 3 Feb. 14, 2000 2304 12
KO = MIF knockout mouse
EXAMPLE 4
Phenylpyruvate Tautomerase Assay
[0145] The assay for relative phenylpyruvate tautomerase activity
of MIF was modified from Lubetsdy et al., Biochemistry, 38:
7346-7354 (1999). We used p-hydroxyphenylpyruvate (HPP) (Aldrich)
as substrate. HPP was dissolved in 50 mM ammonium acetate (pH 6.0)
at room temperature for overnight and stored in refrigerator until
use. For catalytical activity measurement, 20 .mu.l of HPP was
added to 1.96 ml of 0.435 M boric acid (pH 6.2) and allowed to
equilibrate in 1 ml quartz cuvette at room temperature for five
minutes. To initiate the catalytic activity, 20 .mu.l of 0.01 mg/ml
rhMIF was added to above solution and thoroughly mixed. Activity
was measured by following the increase in absorbance at 330 nm for
five minutes. To study the effect of mouse anti-MIF anticlonal
antibodies on the phenylpyruvate tautomerase activity of rhMIF, 0.2
.mu.g of rhMIF is pre-incubation with 12.5 .mu.g of antibody at
25.degree. C. for one hour, then the 30 .mu.l protein mixture was
added to 1.97 ml of assay solution that contains HPP. For each
antibody clone, the mean activity (slope of absorbance increase)
was calculated from triplicate measurements. The relative activity
was calculated by taking the percentage for the slope of the
antibody-rhMIF samples to that of the rhMIF alone.
[0146] As shown in FIG. 3, when anti-MIF monoclonal antibodies
(12.5 .mu.g monoclonal antibody) are added to the reaction mixture
containing MIF (0.2 .mu.g MIF), the antibodies inhibits PPT
activity. These results are summarized in Table 3 below. The
outcomes are presented in the percent MIF-induced PPT activity
remaining after the addition of each anti-MIF antibody.
4TABLE 3 Anti-MIF Mabs Effects on MIF Phenylpyruvate tautomerase
activity (12.5 Mg Mab + 0.2 .mu.g MIF) Antibody Subelone Off-Rate %
MIF PPT Activity 30B7-11 <1.0E-06 -12 19B11-7 1.0E-05 0 22F11-6
2.0E-05 65 34D11-1 5.0E-05 54 2D8-3 8.0E-06 57 33G7-9 <1.0E-06
67 6B5-5 <1.0E-06 82 2G2-5 6.0E-05 92 9G10-12 3.6E-05 98 2B8
(murine anti- -- 105 CD2O Mab) 1OB11-3 9.0E-06 136 IA9-7 2.0E-05
136 29B12-1 <1.0E-06 146 IIA9-8 <1.0E-06 159 6E2-12
<1.0E-06 188
EXAMPLE 5
Anti-MIF Mab Inhibition of MIF Induced SRE-SEAP
Transcription/Secretion
[0147] The results depicted in FIG. 6 demonstrate that the addition
of anti-MIF antibodies to a reaction containing MIF inhibited the
stimulation of MIF induced SRE-SEAP transcription and secretion.
The most inhibitory of the antibodies tested were the 6B5-5, 2G2-5
and 22F11-6 antibodies.
EXAMPLE 6
MIF Stimulated MMP-1 Release Assay
[0148] MIF is known to stimulate MMP-1 release from normal synovial
fibroblasts or rheumatoid arthritis [Onodera, et al. (2000)]. When
anti-MIF antibodies are added along with MIF, MIF stimulated MMP-1
release from the fibroblasts is inhibited (FIG. 7). The antibodies
10B11-3, 2D8-3, 19B11-7 and 33G7-9 all inhibit MIF-induced MMP-1
release (FIG. 7, upper left panel). Additionally, 22F11-6, 6B5-5,
34D11-1, 9G10-12 and 2G2-5 also inhibit MIF induced MMP-1 release
(FIG. 7, upper right panel). Of these, the antibodies which prevent
MIF-induced MMP-1 release, according to FIG. 7, lower panel, were
10B11-3, 6B5-5 and 22F1 1-6. Additional antibodies were tested for
MIF inhibitory activity of MMP-1 release as seen in FIG. 8. In all
instances, the concentration of antibody administered was 10
.mu.g/ml. The results from this Example and Example 5 above can be
summarized in Table 4 below:
5TABLE 4 Anti-MIF Monoclonal Antibody Effects on In Vitro Bioassays
10 .mu.g mab/ml MIF Stimulated 10 .mu.g mab/ml % MIF SRE MIF
Stimulated Binds Binds Subclone PPT Transcription MMP-1 Release
Human Murine MAB Off-Rate Activity (% MIF Activity) (% MIF
Activity) MIF MIF None -- -- 100% 100% N/A N/A 10B11-3 9.0E-06 136
0 (-83%) 0 (-14.2%) <0.1E-09 - Kd 22F11-6 2.0E-05 65 42% 0
(-4.3%) <0.1E-09 + Kd 6B5-5 <1.0E-06 82 36% 0 (-2.5%) ++ -
2D8-3 8.0E-06 57 0 (-21%) + 34D11-1 5.0E-05 54 0% + 3367-9
<1.0E-06 67 5% + ++ ++ 29B12-1 <1.0E-06 146 25% + ++ -
19B11-7 1.0E-05 0 29% + <0.1E-09 +++ Kd 2G2-5 6.0E-05 92 32% +
++ - 6E2-12 <1.0E-06 188 0 (-250%) - 30B7-11 <1.0E-06 -12 0
(-53%) - IA9-7 2.0E-05 136 0 (-46%) - 9G10-12 3.6E-05 98 0 (-22%) -
IIA9-8 1.0E-06 159 122% - Mab Kd 24-31 (murine anti- -- -- CD154
mab) IDEC-114 (anti-CD80 2.2E-09 IDEC-152 (anti-BD23 1.2E-09
mab)
[0149] Also supplied in Table 4 is the human MIF and murine MIF
binding capabilities of each of the listed antibodies.
EXAMPLE 7
[0150] MIF/LPS Lethality Model For Assessing Anti-MIF
Antibodies
[0151] BALB/c mice were injected (all injections i.p. in this
experiment) with lipopolysaccharide (LPS strain: E cell 0111:BY,
Sigma Catalog #L2630) at 10 mg LPS/kg body weight. Some of the
LPS-treated mice were then injected with 5 mg/kg monoclonal
antibody (negative control) or an anti-MIF antibody (specific to
human MIF). Additionally, MIF (R&R MIF Lot #US1600MBCO) was
administered to said mice at a concentration of 2.5 mg/kg at the
time of LPS injection (T=0) and seven hours later (T=7 hours). Mice
pre-treated with anti-MIF monoclonal antibody at T=-2 hours had a
greater percent survival than animals which received LPS and MIF or
LPS and MIF and the negative-control antibody. These results are in
FIG. 9.
[0152] A similar experiment was conducted wherein BALB/c mice were
treated as described above, except that 12.5 mg/kg of LPS was
administered instead of 10 mg/kg. As shown in FIG. 10, mice
pre-treated with anti-MIF again had greater survival percentage
than animals which did not receive antibody or which received the
negative-control antibody.
[0153] Further, another similar experiment was effected except that
15.0 mg/kg LPS body weight was administered (rather than the
previous 10.0 or 12.5 mg/kg body weight). Again, the animals which
received anti-MIF had better survival percentages than animalsl
which did not receive antibody or received the negative-control
antibody.
[0154] These results are summarized in Table 5 as well as other
activities of the tested antibodies specific to MIF.
6TABLE 5 Anti-MIF Mab Effects on MIF + LPS Lethality in BALB/c Mice
10 .mu.g mab/ml MIF Stimulated 10 .mu.g mab/ml % MIF SRE MIF
Stimulated Binds Binds Blocks PPt Transcription MMP-1 Release Human
Murine LPS MAB Activity (% MIF Activity) (% MIF Activity) MIF MIF
Lethality 10B11-3 136 0 (-83%) 0 (14.2%) <0.1E-09 - Kd 22F11-6
65 42% 0 (-4.3%) <0.1E-09 + + Kd 6B5-5 82 36% 0 (-2.5%) ++ -
2D8-3 57 0 (-21%) + 34D11-1 54 0% + 33G7-9 67 5% + ++ ++ + 29B12-1
146 25% + ++ - + 19B11-7 0 29% + <0.1E-09 +++ + Kd 2G2-5 92 32%
+ ++ - 6E2-12 188 0 (-250%) - 30B7-11 -12 0 (-53%) - IA9-7 136 0
(-46%) - 9G10-12 98 0 (-22%) - IIA9-8 159 122% - 24-31 - (murine
anti- CD154 mab)
[0155] The characteristics of two of the lead candidate antibodies
are summarized 5 below in Table 6:
7 TABLE 6 Mab 10B11-3 Mab 22F11-6 human MIF Kd <50 nm <0.1 nM
<0.1 nM Neutralizes MIF in vitro 10 100% 58% .mu.g/ml MIF
stimulated transcription SRE: SEAP Neutralizes MIF in vitro 10
.mu.g/ml 100% 100% MIF stimulated MMP-1 release
[0156] The following Table 7 lists the antibodies generated from
MIF knockout (KO) mice as well as anti-CD80 and anti-CD23
antibodies.
8TABLE 7 Summary of Anti-MIF mabs generated from MIF gene knockout
mice Parent Parent CGM CGM Subclone Off-Rate Off-Rate CGM HYBRIDOMA
Hybridoma Fusion #1 #2 Off-Rate STATUS 29B12-1 1 3.5E-05
<1.0E-06 <1.0E-06 purified from ascites fluid 30B7-11 1
9.4E-06 <6.0E-06 <1.0E-06 purified from ascites fluid IIA9-8
1 3.6E-05 <6.0E-06 <1.0E-06 purified from ascites fluid 6B5-5
1 1.7E-05 3.0E-05 <1.0E-06 purified from ascites fluid 33G7-9 1
1.8E-05 -- <1.0E-06 purified from ascites fluid 6E2-12 1 2.9E-05
6.0E-05 <1.0E-06 purified from ascites fluid 2D8-3 1 8.8E-05
4.5E-05 8.0E-06 purified from ascites fluid 10B11-3 1 1.7E-05
<1.0E-06 9.0-E06 purified from ascites fluid 19B11-7 1 3.4E-06
<1.0E-06 1.0E-06 purified from ascites fluid L2E1-9 1 1.0E-05
<1.0E-05 1.0E-06 expanded for CGM IA9-7 1 4.5E-05 <1.0E-06
2.0E-05 purified from ascites fluid 22F11-6 1 6.6E-05 2.0E-05
2.0E-05 purified from ascites fluid 7E10-11 1 9.4E-05 8.0E-05
2.0E-05 expanded for CGM 25D11 1 3.0E-05 purified from CGM 25D-11
2.8E-05 subclones frozen 9G10-12 1 2.8E-05 <1.0E-06 3.6E-05
purified from ascites fluid 22A5-12 1 9.2E-05 5.0E-05 4.0E-05
expanded for CGM 14H5 1 4.0E-05 subclones frozen 34D11-1 1 7.1E-05
1.0E-05 5.0E-05 purified from ascites fluid IDEC-114 5.4E-05
(anti-CD80 mab) 2G2-5 1 9.4E-05 4.0E-5 6.0E-05 purified from
ascites fluid Summary of Anti-MIF mabs generated from MIF gene
knockout mice Parent Parent CGM CGM Subclone Off-Rate Off-Rate CGM
HYBRIDOMA Hybridoma Fusion #1 #2 Off-Rate STATUS L3A11-5 2 1.0E-05
8.0E-05 7.0E-05 expanded for CGM L4A10-8 2 1.0E-05 5.0E-05 8.0E-05
expanded for CGM K8H8-9 2 1.0E-05 <1.0E-05 9.0-E-05 expanded for
CGM 33C4 1 9.4E-05 subclones frozen L4C9-4 2 2.0E-04 8.0E-05
1.0E-05 purified from CGM K8C9-8 2 2.0E-04 1.0E-05 1.0E-05 expanded
for CGM L1A6-7 2 3.0E-04 N/D 1.0E-05 purified from CGM 22C11-8 1
2.0E-04 1.6E-04 1.6E-04 ready to purify from CGM IIB1-4 1 2.2E-05
1.0E-05 2.0E-04 expanded for CGM 11H2-9 1 8.1E-05 1.5E-04 2.0E-04
expanded for CGM 33F6-10 1 7.8E-05 2.0E-04 2.0E-04 expanded for CGM
19D3-9 1 6.3E-05 3.0E-04 2.0E-04 purified from CGM L4G3 2 2.0E-04
subclones frozen 5A11-10 1 8.9E-05 3.0E-05 4.0E-05 expanded for CGM
IDEC-152 4.8E-04 (anti-CD23 mab) Total monoclonal hybridomas 34
Total mabs purified from ascites fluid 14 Total mabs to be purified
from CGM 20
EXAMPLE 8
Identification of Sequences
[0157] The DNA and amino acid sequences of several lead candidate
antibodies were identified, particularly for 6B5, 10B11, 19B11,
22F11, 29B12 and 33G7 and are contained in FIGS. 17-30. These
sequences may be further mutated in order to enhance binding
affinity.
EXAMPLE 9
Administration of an Anti-MIF Antibody for Therapy
[0158] Anti-MIF antibody is administered at doses that may range
from 1-5 mg/kg to patients with an inflammatory disease who are not
being treated with other drugs, or to those who are being treated
with steroids such as Dexamethasone or other anti-inflammatory
drugs. In certain cases of chronic inflammatory conditions such as
asthma, RA or nephritis, the combination treatment with anti-MIF
antibody and, for example, steroids may lead to the reduction of
the steroid maintenance dose. Under such conditions the antibody
may be used as a steroid salvage therapy which will bring the
steroid dose down to avoid the side effects of steroid high dose
therapy. The anti-MIF antibody may be administered i.v., i.m. or
s.c. at intervals that may vary from weekly to monthly dosing
regimens.
[0159] Although the present invention has been described in detail
with reference to examples above, it is understood that various
modifications can be made without departing from the spirit of the
invention. All references discussed above are hereby incorporated
by reference in their entirety.
* * * * *