U.S. patent application number 14/157967 was filed with the patent office on 2015-01-22 for anti mif antibodies.
This patent application is currently assigned to Baxter International Inc.. The applicant listed for this patent is Baxter Healthcare SA, Baxter International Inc., Dyax Corp.. Invention is credited to Rene Hoet, Randolf Kerschbaumer, Geert C. Mudde, Juergen Muellberg, Manfred Rieger, Friedrich Scheiflinger, Michael Thiele.
Application Number | 20150023978 14/157967 |
Document ID | / |
Family ID | 40535633 |
Filed Date | 2015-01-22 |
United States Patent
Application |
20150023978 |
Kind Code |
A1 |
Kerschbaumer; Randolf ; et
al. |
January 22, 2015 |
ANTI MIF ANTIBODIES
Abstract
The present invention relates to monoclonal antibodies and
antigen-binding portions thereof that specifically bind to the
C-terminal or the center region of macrophage migration inhibitory
factor (MIF). These anti-MIF antibodies and antigen-binding
portions thereof further inhibit human MIF biological function. The
invention also relates to isolated heavy and light chain
immunoglobulins derived from anti-MIF antibodies and nucleic acid
molecules encoding such immunoglobulins. The present invention also
relates to a method of identifying anti-MIF antibodies,
pharmaceutical compositions comprising these antibodies and a
method of using these antibodies and compositions for the treatment
of MIF-related conditions.
Inventors: |
Kerschbaumer; Randolf;
(Klosterneuburg, AT) ; Scheiflinger; Friedrich;
(Vienna, AT) ; Rieger; Manfred; (Korneuburg,
AT) ; Thiele; Michael; (Vienna, AT) ; Mudde;
Geert C.; (Breitenfurt, AT) ; Muellberg; Juergen;
(Lexington, MA) ; Hoet; Rene; (Zeist, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Baxter International Inc.
Dyax Corp.
Baxter Healthcare SA |
Deerfield
Cambridge
Glattpark (Opfikon) |
IL
MA |
US
US
CH |
|
|
Assignee: |
Baxter International Inc.
Deerfield
IL
Dyax Corp.
Cambridge
MA
Baxter Healthcare SA
Glattpark (Opfikon)
|
Family ID: |
40535633 |
Appl. No.: |
14/157967 |
Filed: |
January 17, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12767635 |
Apr 26, 2010 |
8668909 |
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14157967 |
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12346309 |
Dec 30, 2008 |
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12767635 |
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61094685 |
Sep 5, 2008 |
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61018988 |
Jan 4, 2008 |
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Current U.S.
Class: |
424/145.1 ;
435/320.1; 435/335; 435/69.6; 435/7.92; 530/387.3; 530/388.23;
536/23.53 |
Current CPC
Class: |
A61P 29/00 20180101;
C07K 16/24 20130101; A61P 37/00 20180101; C07K 2317/21 20130101;
C07K 2317/55 20130101; A61P 35/02 20180101; A61P 35/00 20180101;
A61P 43/00 20180101; C07K 2317/565 20130101; A61P 1/00 20180101;
A61P 1/04 20180101; A61P 11/00 20180101; A61K 2039/505 20130101;
A61P 13/12 20180101; A61P 13/00 20180101; A61P 17/06 20180101; A61P
37/08 20180101; A61P 9/14 20180101; A61P 11/06 20180101; A61P 31/04
20180101; C07K 2317/73 20130101; A61P 19/02 20180101; C07K 2317/56
20130101; A61P 9/00 20180101; C07K 2317/92 20130101; C07K 2317/52
20130101; C07K 2317/76 20130101; A61P 27/02 20180101; A61P 37/02
20180101; A61P 33/06 20180101 |
Class at
Publication: |
424/145.1 ;
435/7.92; 435/69.6; 435/335; 435/320.1; 530/387.3; 530/388.23;
536/23.53 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Claims
1. A monoclonal antibody or an antigen-binding portion thereof that
specifically binds to MIF and inhibits human MIF biological
function, wherein said monoclonal antibody or antigen binding
portion possesses at least one of the following properties: a)
inhibits glucocorticoid overriding (GCO) activity; b) inhibits
proliferation of cancer cells or fibroblasts.
2. (canceled)
3. The monoclonal antibody or antigen-binding portion according to
claim 1, wherein said antibody or antigen-binding portion binds
human MIF with a K.sub.D less than 500 nM.
4. The monoclonal antibody or antigen-binding portion according to
claim 1, wherein said antibody or said antigen-binding portion
binds to active MIF.
5. The monoclonal antibody according to claim 1, wherein said
antibody is an IgG4 sub-format antibody.
6. The monoclonal antibody according to claim 5, wherein said IgG4
sub-format has a single mutation, whereby the CPSC sub-sequence in
the Fc region of IgG4 becomes CPPC.
7. The monoclonal antibody or the antigen-binding portion according
to claim 1, for use in treating an immunological disease, wherein
said immunological disease is an inflammatory disease or a
hyperproliferative disorder.
8. A pharmaceutical composition, comprising the monoclonal antibody
or the antigen-binding portion according to claim 1 and a
pharmaceutically acceptable carrier.
9. A method for treating an immunological disease selected from
inflammatory disease or a hyperproliferative disorder in a subject,
including a human, comprising the step of administering to said
subject in need thereof a therapeutically effective amount of the
monoclonal antibody or the antigen-binding portion according to
claim 1, wherein said antibody or said antigen binding portion
further inhibits human MIF biological function.
10. An isolated cell line that produces the monoclonal antibody or
the antigen-binding portion according to claim 1.
11. An isolated nucleic acid molecule comprising a nucleotide
sequence that encodes the heavy chain, or the light chain, of the
monoclonal antibody or the antigen-binding portion according to
claim 1.
12. A vector comprising the nucleic acid molecule according to
claim 11, wherein the vector optionally comprises an expression
control sequence operably linked to said nucleic acid molecule.
13. A host cell comprising the nucleic acid molecule according to
claim 11.
14. A host cell comprising a nucleic acid molecule encoding the
heavy chain and a nucleic acid molecule encoding the light chain of
the monoclonal antibody or the antigen-binding portion according to
claim 1.
15. A method of producing a monoclonal antibody or an
antigen-binding portion thereof, comprising culturing the host cell
according to claim 14 under suitable conditions and recovering said
antibody or antigen-binding portion thereof.
16. A process for the identification of anti-MIF antibodies capable
of inhibiting human MIF biological function and inducing a
beneficial effect in an animal model by carrying out the following
steps: a) selecting an antibody that binds to active MIF and does
not bind to non-active MIF b) testing said antibody in in-vitro
assays c) selecting an antibody, which inhibits GCO and/or cell
proliferation.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of co-pending U.S.
application Ser. No. 12/346,309, filed Dec. 30, 2008, which claims
benefit of U.S. provisional application No. 61/018,988, filed Jan.
4, 2008 and U.S. provisional application No. 61/094,685, filed Sep.
5, 2008. Each application is herein incorporated by reference.
FIELD OF THE INVENTION
[0002] The present invention relates to monoclonal antibodies and
antigen-binding portions thereof that specifically bind to the
C-terminal or the center region of macrophage migration inhibitory
factor (MIF). These anti-MIF antibodies and antigen-binding
portions thereof further inhibit human MIF biological function. The
invention also relates to isolated heavy and light chain
immunoglobulins derived from anti-MIF antibodies and nucleic acid
molecules encoding such immunoglobulins. The present invention also
relates to a method of identifying anti-MIF antibodies,
pharmaceutical compositions comprising these antibodies and a
method of using these antibodies and compositions for the treatment
of MIF-related conditions.
BACKGROUND
[0003] Macrophage migration inhibitory factor (MIF) is a cytokine
initially isolated based upon its ability to inhibit the in vitro
random migration of macrophages (Bloom et al. Science 1966, 153,
80-2; David et al. PNAS 1966, 56, 72-7). Although MIF has been
known since 1966 its precise function in the majority of cells is
not known, but it seems that MIF is a critical upstream regulator
of the innate and acquired immune response.
[0004] The human MIF cDNA was cloned in 1989 (Weiser et al., PNAS
1989, 86, 7522-6), and its genomic localization was mapped to
chromosome 22. The product of the MIF gene is a amino acid protein
of a molecular mass of 12.5 kDa. The protein is highly conserved
with a sequence homology between human, mouse, rat, and bovine MIF
between 90-96%. However, MIF has no significant sequence homology
to any other protein. The three-dimensional structure of MIF is
unlike any other cytokine or pituitary hormone. The protein
crystallizes as a trimer of identical subunits. Each monomer
contains two antiparallel alpha-helices that pack against a
four-stranded beta-sheet. The monomer has an additional two
beta-strands that interact with the beta-sheets of adjacent
subunits to form the interface between monomers. The three
beta-sheets are arranged to form a barrel containing a
solvent-accessible channel that runs through the center of the
protein along a molecular three-fold axis (Sun et al. PNAS 1996,
93, 5191-5196).
[0005] It was reported that MIF secretion from macrophages was
induced at very low concentrations of glucocorticoid (Calandra et
al. Nature 1995, 377, 68-71). However, as a proinflammatory
cytokine, MIF also counter-regulates the effects of glucocorticoids
and stimulates the secretion of other cytokines such as tumor
necrosis factor TNF-.alpha. and interleukin IL-1.beta. (Baugh et
al, Crit. Care Med 2002, 30, S27-35) thus assuming a role in the
pathogenesis of inflammatory and immune diseases. MIF is also
directly associated with the growth of lymphoma, melanoma, and
colon cancer (Nishihira et al. J Interferon Cytokine Res. 2000,
20:751-62).
[0006] MIF is a mediator of many pathologic conditions and thus
associated with a variety of diseases including inflammatory bowel
disease (IBD), rheumatoid arthritis (RA), acute respiratory
distress syndrome (ARDS), asthma, glomerulonephritis, IgA
nephropathy, cancer, myocardial infarct (MI), and sepsis.
[0007] Polyclonal and monoclonal anti-MIF antibodies have been
developed against recombinant human MIF (Shimizu et al., FEBS Lett.
1996; 381, 199-202; Kawaguchi et al., J. Leukoc. Biol. 1986, 39,
223-232, and Weiser et al., Cell. Immunol. 1985, 90, 167-78).
[0008] Anti-MIF antibodies have been suggested for therapeutic use
to inhibit TNF-.alpha. release. Calandra et al., (J. Inflamm. 1995.
47, 39-51) reportedly used anti-MIF antibodies to protect animals
from experimentally induced gram-negative and gram-positive septic
shock. Anti-MIF antibodies were suggested as a means of therapy to
modulate cytokine production in septic shock and other inflammatory
disease states.
[0009] U.S. Pat. No. 6,645,493 discloses monoclonal anti-MIF
antibodies derived from hybridoma cells, which neutralize the
biological activity of MIF. It could be shown in an animal model
that these mouse derived anti-MIF antibodies had a beneficial
effect in the treatment of endotoxin induced shock. Some of the
described anti-MIF antibodies (III.D.9, XIV.14.3 and XIV.15.5) were
used in the present invention for comparative experiments.
[0010] US 2003/0235584 discloses methods of preparing high affinity
antibodies to MIF in animals in which the MIF gene has been
homozygously knocked-out.
[0011] Glycosylation-inhibiting factor (GIF) is a protein described
by Galat et al. (Eur. J. Biochem. 1994, 224, 417-21). MIF and GIF
are now recognized to be identical. Watarai et al. (PNAS 2000, 97,
13251-6) described polyclonal antibodies binding to different GIF
epitopes to identify the biochemical nature of the
posttranslational modification of GIF in Ts cells. Watarai et al
(PNAS 2000, 97, 13251-6) reported that GIF occurs in different
conformational isoforms in vitro. One type of isomer occurs by
chemical modification of a single cysteine residue. The chemical
modification leads to conformational changes within the GIF protein
and changes its biological function.
[0012] Given the complexity of involvement of MIF in various
diseases an elucidation of the function of epitope-specific
anti-MIF antibodies and its use for therapeutic approaches is
highly desirable. Therefore, there exists a need for
epitope-specific anti-MIF antibodies, which inhibit human MIF
biological function for the treatment of diseases and conditions
mediated by MIF.
SUMMARY OF THE INVENTION
[0013] The present invention relates to antibodies and
antigen-binding portions thereof that specifically bind to the
C-terminal or the center region of macrophage migration inhibitory
factor (MIF).
[0014] The invention further relates to nucleic acid molecules
encoding these antibodies or antigen-binding portions thereof, as
well as to vectors comprising such a nucleic acid and to host cells
comprising such a vector, as well as to methods for recombinant
production of polypeptides encoded by nucleic acid molecules.
[0015] The invention also relates to pharmaceutical compositions
comprising an anti-MIF antibody or an antigen-binding portion
thereof. The pharmaceutical composition may also contain
pharmaceutically acceptable carrier or other therapeutic
agents.
[0016] The invention also relates to the use of an anti-MIF
antibody or an antigen-binding portion thereof, in the manufacture
of a medicament for the treatment of immunological diseases such as
inflammatory diseases and hyperproliferative disorders.
[0017] The invention further relates to an anti-MIF antibody or
antigen-binding portion thereof, for use in treating immunological
diseases such as inflammatory diseases and hyperproliferative
disorders.
[0018] The invention also relates to methods for treating a variety
of immunological diseases and conditions, such as inflammatory
diseases and hyperproliferative disorders with an effective amount
of an anti-MIF antibody, or an antigen binding portion thereof.
[0019] The invention also relates to diagnostic methods. The
anti-MIF antibody or antigen-binding portion thereof can be used to
detect MIF in a biological sample.
[0020] The invention further relates to a process for the
identification of an anti-MIF antibody capable of inhibiting active
MIF and inducing a beneficial effect in an animal model.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1: shows the amino acid sequence of the light chain
variable region of the human anti-MIF antibody of the invention
[0022] FIG. 2: shows the amino acid sequence of the heavy chain
variable region of the human anti-MIF antibody of the invention
[0023] FIG. 3: shows the DNA sequences (SEQ ID NOs:13-18) and
translations of the light chain variable region of human anti-MIF
antibodies of the invention.
[0024] FIG. 4: shows the DNA sequences (SEQ ID NOs:19-24) and
translation of the heavy chain variable region of human anti-MIF
antibodies of the invention
[0025] FIG. 5: Competition experiment of murine III.D.9 against a
control antibody (C3) and anti-MIF antibody Bax94. A clear
competition by increasing amounts of antibody Bax94 can be
observed.
[0026] FIG. 6: Antibody Bax94 (dotted line) and antibody Bax152
(dashed line) showed increased survival and delayed time to death
in the peritonitis animal model compared with a control antibody
(C3).
[0027] FIG. 7: Differential binding of antibody Bax94 to active MIF
and non-active MIF. Antibody Bax94 binds active MIF in a direct
ELISA format, whereas non-active-MIF does not bind.
[0028] FIG. 8: Table summarizing in-vitro properties of human
anti-MIF antibodies.
[0029] FIG. 9: Pro-apoptotic effects of anti-MIF antibodies in a
cell based assay. Cellular caspase-3 (effector caspase) activities
are shown after antibody treatment of PC-3 cells, Assays are done
in triplicate and data are presented as mean.+-.SD.
[0030] FIG. 10: Anti-invasive effects of anti-MIF antibodies. The
invasion of PC-3 prostate cancer cells through pores of
matrigel-coated Transwell.TM. inserts is examined. The number of
invaded cells per visual field are counted (microscopy at 400 fold
magnification). Data are presented as mean.+-.SD from 3-10 visual
field counts and significant differences are shown.
DETAILED DESCRIPTION OF THE INVENTION
Definitions and General Techniques
[0031] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Generally, nomenclatures used in connection with, and
techniques of, cell and tissue culture, molecular biology,
immunology, microbiology, genetics and protein and nucleic acid
chemistry described herein are those well known and commonly used
in the art. The methods and techniques of the present invention are
generally performed according to conventional methods well known in
the art and as described in various general and more specific
references that are cited and discussed throughout the present
specification unless otherwise indicated. See, e.g., Sambrook et
al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and
Ausubel et al., Current Protocols in Molecular Biology, Greene
Publishing Associates (1992), and Harlow and Lane Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. (1990), which are incorporated herein by
reference.
[0032] "MIF" or "macrophage migration inhibitory factor" refers to
the protein, which is known as a critical mediator in the immune
and inflammatory response, especially as a counterregulator of
glucocorticoids. MIF includes mammalian MIF, specifically human MIF
(Swiss-Prot primary accession number: P14174), wherein the
monomeric form is encoded as a 115 amino acid protein but is
produced as a 114 amino acid protein due to cleavage of the initial
Methionine. "MIF" also includes "GIF" (glycosylation-inhibiting
factor) and other forms of MIF such as fusion proteins of MIF. The
numbering of the aminoacids of MIF starts with the N-terminal
Methionine (amino acid 1) and ends with the C-terminal Alanine
(amino acid 115).
[0033] The term "active MIF" refers to naturally occurring
conformational isoforms of MIF, which are relevant for its
biological function. Active MIF includes isoforms that can be
observed on the surface of cells (such as THP1 or the like). Active
MIF also includes MIF isoforms that occur in serum of mammals after
challenge with bacteria.
[0034] An "antibody" refers to an intact antibody or an
antigen-binding portion that competes with the intact antibody for
specific binding. See generally, Fundamental Immunology, Ch. 7
(Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by
reference). The term antibody includes genetically engineered forms
such as chimeric or humanized antibodies.
[0035] The term "antigen-binding portion" of an antibody refers to
one or more fragments of an antibody that retain the ability to
specifically bind to an antigen (e.g., MIF). Antigen-binding
portions may be produced by recombinant DNA techniques or by
enzymatic or chemical cleavage of intact antibodies.
Antigen-binding portions include Fab, Fab', F(ab').sub.2, Fv, and
complementarity determining region (CDR) fragments, single-chain
antibodies (scFv), chimeric antibodies, diabodies and polypeptides
that contain at least a portion of an antibody that is sufficient
to confer specific antigen binding to the polypeptide. From
N-terminus to C-terminus, both the mature light and heavy chain
variable domains comprise the regions FR1, CDR1, FR2, CDR2, FR3,
CDR3 and FR4. The assignment of amino acids to each domain is in
accordance with the definitions of Kabat, Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md. (1987 and 1991)), Chothia et. al. J. Mol. Biol. 196:901-917
(1987), or Chothia et al., Nature 342:878-883 (1989). An antibody
or antigen-binding portion thereof can be derivatized or linked to
another functional molecule (e.g., another peptide or protein). For
example, an antibody or antigen-binding portion thereof can be
functionally linked to one or more other molecular entities, such
as another antibody (e.g., a bispecific antibody or a diabody), a
detectable agent, a cytotoxic agent, a pharmaceutical agent, and/or
a linking molecule.
[0036] The term "human antibody" refers to any antibody in which
the variable and constant domain sequences are human sequences. The
term encompasses antibodies with sequences derived from human
genes, but which have been changed, e.g. to decrease possible
immunogenicity, increase affinity, eliminate cysteines that might
cause undesirable folding, etc. The term encompasses such
antibodies produced recombinantly in non-human cells, which might
impart glycosylation not typical of human cells.
[0037] The term "humanized antibody" refers to immunoglobulins,
immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab',
F(ab').sub.2, fragments, or other antigen-binding portions of
antibodies), which contain sequences derived from a non-human
immunoglobulin.
[0038] The term "chimeric antibody" refers to an antibody that
comprises regions from two or more different species.
[0039] The term "isolated antibody" or "isolated antigen-binding
portion thereof" refers to an antibody or an antigen-binding
portion thereof that has been identified and selected from an
antibody source such as a phage display library or a B-cell
repertoire.
[0040] The term "K.sub.D" refers to the equilibrium dissociation
constant of a Fab portion of a particular antibody with the
respective antigen.
[0041] The terms "center region" and "C-terminal region" of MIF
refer to the region of human MIF comprising amino acids 35-68 and
86-115, respectively.
[0042] The term "epitope" includes any protein determinant capable
of specific binding to an immunoglobulin or an antibody fragment.
Epitopic determinants usually consist of chemically active surface
groupings of molecules such as exposed amino acids, aminosugars, or
other carbohydrate side chains and usually have specific
three-dimensional structural characteristics, as well as specific
charge characteristics.
[0043] The term "vector" refers to a nucleic acid molecule capable
of transporting another nucleic acid to which it has been linked.
In some embodiments, the vector is a plasmid, i.e., a circular
double stranded DNA loop into which additional DNA segments may be
ligated.
[0044] The term "host cell" refers to a cell line, which is capable
to produce a recombinant protein after introducing an expression
vector. The term "recombinant cell line", refers to a cell line
into which a recombinant expression vector has been introduced. It
should be understood that "recombinant cell line" means not only
the particular subject cell line but also the progeny of such a
cell line. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term "recombinant cell
line" as used herein.
[0045] The term "pharmaceutically acceptable carrier" refers to any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like that are physiologically compatible.
Anti-MIF Antibodies
[0046] In one embodiment, the invention relates to isolated
monoclonal antibodies or antigen-binding portions thereof, which
specifically bind to the C-terminal or the center region of human
MIF and further inhibit human MIF biological function. In some
embodiments the monoclonal antibodies, are human monoclonal
antibodies. In other embodiments the monoclonal antibodies, are
humanized monoclonal antibodies.
[0047] In some embodiments, the light chain of the anti-MIF
antibody comprises the amino acid sequence that is the same as the
amino acid sequence of the V.sub.L of antibody Bax8 (SEQ ID NO: 1),
antibody Bax69 (SEQ ID NO: 2), antibody Bax74 (SEQ ID NO: 3),
antibody Bax94 (SEQ ID NO: 4), antibody Bax152 (SEQ ID NO: 5),
antibody BaxA10 (SEQ ID NO: 6), or an amino acid sequence which has
85%, preferably 90% sequence homology to said amino acid sequences.
In some embodiments, the light chain comprises the amino acid
sequence from the beginning of the CDR1 to the end of the CDR3 of
any one of said antibodies. In some embodiments, the light chain of
the anti-MIF antibody comprises at least the light chain CDR1, CDR2
or CDR3 of the amino acid sequences shown in FIG. 1.
[0048] In some embodiments, the heavy chain comprises an amino acid
sequence of the variable domain (V.sub.H) of antibody Bax8 (SEQ ID
NO: 7), antibody Bax69 (SEQ ID NO: 8), antibody Bax74 (SEQ ID NO:
9), antibody Bax94 (SEQ ID NO: 10), antibody Bax152 SEQ ID NO: 12),
antibody BaxA10 (SEQ ID NO: 12), or an amino acid sequence which
has 85%, preferably 90% sequence homology to said amino acid
sequences. In some embodiments, the heavy chain comprises the amino
acid sequence from the beginning of the CDR1 to the end of the CDR3
of any one of said antibodies. In some embodiments, the heavy chain
of the anti-MIF antibody comprises at least the heavy chain CDR1,
CDR2 or CDR3 of the amino acid sequences shown in FIG. 2.
Class and Subclass of Anti-MIF Antibodies
[0049] The anti-MIF antibody of the invention is an isolated
monoclonal antibody. The anti-MIF antibody can be an IgG, an IgM,
an IgE, an IgA, or an IgD molecule. In other embodiments, the
anti-MIF antibody is an IgG and is an IgG1, IgG2, IgG3 or IgG4
subclass. In other embodiments, the antibody is either subclass
IgG1 or IgG4. In other embodiments, the antibody is subclass IgG4.
In some embodiments the IgG4 antibody has a single mutation
changing the serine (serine-228, according to the Kabat numbering
scheme) to proline. Accordingly, the CPSC sub-sequence in the Fc
region of IgG4 becomes CPPC, which is a sub-sequence in IgG1 (Angal
et al. Mol. Immunol. 1993, 30, 105-108).
MIF Epitopes Recognized by Anti-MIF Antibodies
[0050] In some embodiments, the invention relates to anti-MIF
antibodies or antigen-binding portions thereof that specifically
bind to the regions spanning from amino acids 35-68 or 86-115 of
human MIF, respectively, preferably the anti-MIF antibodies
specifically bind to the regions spanning from amino acids 50 to
68, or 86 to 102, respectively, and inhibit human MIF biological
function.
[0051] In other embodiments, the invention relates to anti-MIF
antibodies, which specifically bind to active MIF and further
inhibit human MIF biological function. In some embodiments, active
MIF is membrane-bound.
[0052] It was surprisingly found that anti-MIF antibodies of the
invention had the surprising property of competing anti-MIF
antibody 111.0.9 in binding studies with human MIF. Competition of
III.D.9 can be determined as described in Example 5.
Binding Affinity of Anti-MIF Antibodies to Human MIF
[0053] The invention relates to anti-MIF antibodies or
antigen-binding portions thereof, which bind to human MIF with a
K.sub.D of 5.times.10.sup.-7M or less. In other embodiments, the
antibodies bind to human MIF with a K.sub.D of 5.times.10.sup.-8M
or less, 5.times.10.sup.-9M or less, or 5.times.10.sup.-10 M or
less.
[0054] The binding affinity of anti-MIF antibodies or
antigen-binding portions thereof to human MIF can be determined by
methods known in the art. The binding affinity for example can be
measured by surface plasmon resonance (BIACORE). Example 10
exemplifies a method for determining affinity constants of anti-MIF
antibodies by BIACORE technology.
[0055] In some embodiments, the invention further relates to
anti-MIF antibodies or antigen-binding portions thereof, which bind
to active MIF with a K.sub.D of less than 500 nM and further
inhibit human MIF function biological function. In some
embodiments, the anti-MIF antibodies or antigen-binding portions
thereof bind active MIF with a K.sub.D of less than 50 nM.
Production of Anti-MIF Antibodies
[0056] Anti-MIF antibodies or antigen-binding portions thereof
according to the present invention may be prepared by many methods
known to the person skilled in the art, such as screening of phage
display libraries of antibody fragments. Different formats of phage
display libraries may be utilized, e.g. scFv or Fab fragments
libraries or the like. A phage display library is screened for
antibody fragments with desired affinities for certain MIF epitopes
and the genetic material is recovered from the appropriate clone.
In consecutive rounds of generating and screening libraries,
antibody fragment can be isolated with an increased affinity
compared to the affinity of the original antibody fragment
isolated. The affinity of an identified anti-MIF fragment can be
further enhanced by affinity maturation.
Nucleic Acids, Vectors, Host Cells, and Recombinant Methods of
Making Anti-MIF Antibodies
[0057] The invention further relates to nucleic acid molecules
encoding anti-MIF antibodies or antigen-binding portions thereof
according to the present invention, as well as to vectors
comprising such nucleic acid and to host cells comprising such a
vector, as well as to methods of recombinantly producing a
polypeptide encoded by the nucleic acid molecule.
[0058] In some embodiments, the DNA sequence encoding the V.sub.L
region of the anti-MIF antibody comprises the nucleotide sequence
that is the same as the sequence of the V.sub.L of antibody Bax8
(SEQ ID NO: 13), antibody Bax69 (SEQ ID NO: 14), antibody Bax74
(SEQ ID NO: 15), antibody Bax94 (SEQ ID NO:16), antibody Bax152
(SEQ ID NO: 17), antibody BaxA10 (SEQ ID NO: 18) as shown in FIG.
3, or a sequence, which has 85%, preferably 90% sequence homology
to any of said nucleotide sequences.
[0059] In some embodiments, the DNA sequence encoding the V.sub.H
region of the anti-MIF antibody comprises the nucleotide sequence
that is the same as the sequence of the V.sub.H of antibody Bax8
(SEQ ID NO: 19), antibody Bax69 (SEQ ID NO: 20), antibody Bax74
(SEQ ID NO: 21), antibody Bax94 (SEQ ID NO: 22), antibody Bax152
(SEQ ID NO: 23), antibody BaxA10 (SEQ ID NO: 24) as shown in FIG.
4, or a sequence, which has 85%, preferably 90% sequence homology
to any of said nucleotide sequences.
[0060] The production of the anti-MIF antibodies according to the
present invention include any method for the generation of
recombinant DNA by genetic engineering, e.g. via reverse
transcription of RNA and/or amplification of DNA and cloning into
expression vectors.
[0061] In some embodiments, the vector is a viral vector, wherein
additional DNA segments may be ligated into the viral genome. In
some embodiments, the vector capable of autonomous replication in a
host cell into which introduced (e.g., bacterial vectors having a
bacterial origin of replication and episomal mammalian vectors). In
other embodiments, the vector (e.g., non-episomal mammalian
vectors) can be integrated into the genome of a host cell upon
introduction into the host cell, and thereby replicated along with
the host genome. Moreover, certain vectors are capable of directing
the expression of genes to which they are operatively linked. Such
vectors are referred to herein as "recombinant expression vectors"
(or simply, "expression vectors").
[0062] Anti-MIF antibodies can be produced by means of conventional
expression vectors, such as bacterial vectors (e.g., pBR322 and its
derivatives), or eukaryotic vectors. Those sequences that encode
the antibody can be provided with regulatory sequences that
regulate the replication, expression and/or secretion from the host
cell. These regulatory sequences comprise, for instance, promoters
(e.g., CMV or SV40) and signal sequences. The expression vectors
can also comprise selection and amplification markers, such as the
dihydrofolate reductase gene (DHFR),
hygromycin-B-phosphotransferase, and thymidine-kinase. The
components of the vectors used, such as selection markers,
replicons, enhancers, can either be commercially obtained or
prepared by means of conventional methods. The vectors can be
constructed for the expression in various cell cultures, e.g., in
mammalian cells such as CHO, COS, HEK293, NS0, fibroblasts, insect
cells, yeast or bacteria such as E. coli. In some instances, cells
are used that allow for optimal glycosylation of the expressed
protein.
[0063] The anti-MIF antibody light chain gene and the anti-MIF
antibody heavy chain gene can be inserted into separate vectors or
both genes are inserted into the same expression vector. The
antibody genes are inserted into the expression vector by standard
methods, e.g., ligation of complementary restriction sites on the
antibody gene fragment and vector, or blunt end ligation if no
restriction sites are present.
[0064] The production of anti-MIF antibodies or antigen-binding
portions thereof may include any method known in the art for the
introduction of recombinant DNA into eukaryotic cells by
transfection, e.g. via electroporation or microinjection. For
example, the recombinant expression of anti-MIF antibody can be
achieved by introducing an expression plasmid containing the
anti-MIF antibody encoding DNA sequence under the control of one or
more regulating sequences such as a strong promoter, into a
suitable host cell line by an appropriate transfection method
resulting in cells having the introduced sequences stably
integrated into the genome. The lipofection method is an example of
a transfection method which may be used according to the present
invention.
[0065] The production of anti-MIF antibodies may also include any
method known in the art for the cultivation of said transformed
cells, e.g. in a continuous or batchwise manner, and the expression
of the anti-MIF antibody, e.g. constitutive or upon induction.
[0066] The host cell type according to the present invention may be
any eukaryotic cell. In one embodiment the cell is a mammalian cell
with the ability to perform posttranslational modifications of
anti-MIF antibodies. For example said mammalian cell is derived
from a mammalian cell line, like for example a cell line selected
from the group consisting of
SkHep-, CHO-, HEK293-, and BHK-cells. In one embodiment, the
anti-MIF antibody is expressed in a DHFR-deficient CHO cell line,
e.g., DXB11, and the addition of G418 as a selection marker. When
recombinant expression vectors encoding antibody genes are
introduced into mammalian host cells, the antibodies are produced
by culturing the host cells for a period of time sufficient to
allow for expression of the antibody in the host cells or secretion
of the antibody into the culture medium in which the host cells are
grown. Anti-MIF antibodies can be recovered from the culture medium
using standard protein purification methods.
[0067] Additionally, the production of anti-MIF antibodies may
include any method known in the art for the purification of an
antibody, e.g. via anion exchange chromatography or affinity
chromatography. In one embodiment the anti-MIF antibody can be
purified from cell culture supernatants by size exclusion
chromatography.
Properties of Anti-MIF Antibodies
[0068] The invention relates to anti-MIF antibodies or
antigen-binding portion thereof, which possess at least one of the
following properties: [0069] a) bind to the C-terminal or the
center region of human MIF [0070] b) inhibit glucocorticoid
overriding (GCO) activity, [0071] c) inhibit proliferation of cells
lines such as fibroblasts or cancer cells (e.g. NIH/3T3 or PC-3)
[0072] d) bind to active MIF [0073] e) does not bind to non-active
MIF [0074] f) compete mouse anti-MIF antibody III.D.9.
[0075] In some embodiments, active MIF is an isoform of active MIF
that occurs by treatment of human MIF with mild oxidizing reagents,
such as Cystine or by immobilizing human MIF on a support such as
an ELISA-plate or beads. In other embodiments, active MIF is an
isoform of active MIF that occurs in vivo after challenge of
animals with bacteria. In other embodiments, active MIF is an
isoform of active MIF that occurs in vivo on the surface of cells
(e.g. THP1, CFB).
[0076] In some embodiments, non-active MIF is reduced MIF (e.g. as
described in Example 7) or, intracellular stored MIF.
[0077] In other embodiments, the anti-MIF antibodies or
antigen-binding portion thereof bind active MIF with a K.sub.D less
than 500 nM.
Pharmaceutical Compositions of Anti-MIF Antibodies and Methods of
Treatment
[0078] The invention also relates to compositions comprising an
anti-MIF antibody or an antigen-binding portion thereof, for the
treatment of a subject in need of treatment for MIF-related
conditions, specifically immunological diseases such as
inflammatory diseases and hyperproliferative disorders.
[0079] In some embodiments, the subject in need of treatment is a
human. Hyperproliferative disorders, such as cancerous diseases,
that may be treated by anti-MIF antibodies of the invention can
involve any tissue or organ and include but are not limited to
brain, lung, squamous cell, bladder, gastric, pancreatic, breast,
head, neck, liver, renal, ovarian, prostate, colorectal,
esophageal, gynecological, nasopharynx, or thyroid cancers,
melanomas, lymphomas, leukemias or multiple myelomas. In
particular, anti-MIF antibodies of the invention are useful to
treat carcinomas of the breast, prostate, colon and lung.
[0080] The invention also encompasses methods for the treatment of
inflammatory diseases such as vasculitis, arthritis, sepsis, septic
shock, endotoxic shock, toxic shock syndrome, acquired respiratory
distress syndrome, glomerulonephritis, inflammatory bowel disease,
Crohn's disease, ulcerative colitis, peritonitis, nephritis, atopic
dermatitis, asthma, conjunctivitis, fever, Malaria or psoriasis in
a subject, including a human, comprising the step of administering
to said subject in need thereof a therapeutically effective amount
of an anti-MIF antibody or antigen-binding portion thereof.
[0081] In other embodiments the composition comprising said
anti-MIF antibody of the invention is used for the treatment of an
inflammatory disease selected from the group consisting of
glomerulonephritis, inflammatory bowel disease, nephritis and
peritonitis.
[0082] The treatment may also involve administration of one or more
anti-MIF antibody of the invention, or an antigen-binding fragment
thereof, alone or with a pharmaceutically acceptable carrier. Some
examples of pharmaceutically acceptable carriers are water, saline,
phosphate buffered saline, dextrose, glycerol, ethanol and the
like, as well as combinations thereof. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, or sodium chloride in the
composition. Additional examples of pharmaceutically acceptable
substances are wetting agents or minor amounts of auxiliary
substances such as wetting or emulsifying agents, preservatives or
buffers, which enhance the shelf life or effectiveness of the
antibody.
[0083] The anti-MIF antibody of the invention and the
pharmaceutical compositions comprising them, can be administered in
combination with one or more other therapeutic, diagnostic or
prophylactic agents. Additional therapeutic agents include other
anti-neoplastic, anti-tumor, anti-angiogenic, chemotherapeutic
agents or steroids, depending on the disease to be treated.
[0084] The pharmaceutical compositions of this invention may be in
a variety of forms, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form depends on
the intended mode of administration and therapeutic application.
Typical preferred compositions are in the form of injectable or
infusible solutions, such as compositions similar to those used for
passive immunization of humans. The preferred mode of
administration is parenteral (e.g., intravenous, subcutaneous,
intraperitoneal, intramuscular). In a preferred embodiment, the
antibody is administered by intravenous infusion or injection. In
another preferred embodiment, the antibody is administered by
intramuscular or subcutaneous injection. As will be appreciated by
the skilled artisan, the route and/or mode of administration will
vary depending upon the desired results.
[0085] The anti-MIF antibody may be administered once, but more
preferably is administered multiple times. For example, the
antibody may be administered from three times daily to once every
six months or longer. The administering may be on a schedule such
as three times daily, twice daily, once daily, once every two days,
once every three days, once weekly, once every two weeks, once
every month, once every two months, once every three months and
once every six months.
[0086] The invention also encompass the use of an anti-MIF antibody
or antigen-binding fragment thereof, in the manufacture of a
medicament for the treatment of immunological diseases such as
inflammatory diseases and hyperproliferative disorders.
[0087] The invention further encompass an anti-MIF antibody or
antigen-binding fragment thereof, for use in treating immunological
diseases such as inflammatory diseases and hyperproliferative
disorders.
[0088] The invention also encompass an anti-MIF antibody or
antigen-binding fragment thereof, for use in diagnostic methods. In
one embodiment the anti-MIF antibody or antigen-binding portion
thereof can be used to detect human MIF in a biological sample.
[0089] The anti-MIF antibodies or the antigen-binding portions
thereof can also be used to determine the level of cell surface MIF
in a tissue or in cells derived from the tissue. In some
embodiments, the tissue is diseased tissue. The tissue can then be
used in an immunoassay to determine, e.g., total MIF levels, cell
surface levels of MIF, or localization of MIF.
[0090] The invention further relates to kits comprising an anti-MIF
antibody or an antigen-binding portion of the invention or a
pharmaceutical composition comprising such an antibody or portion.
A kit may include, in addition to the antibody or pharmaceutical
composition, diagnostic or therapeutic agents. A kit also can
include instructions for use in a diagnostic or therapeutic
method.
[0091] The invention further relates to a process for the
identification of anti-MIF antibodies capable of inhibiting human
MIF biological function and inducing a beneficial effect in an
animal model by carrying out the following steps: [0092] a)
selecting an antibody that binds to active MIF and does not bind to
non-active MIF [0093] b) testing said antibody in in-vitro assays,
such as glucocorticoid overriding (GCO) assay, or cell
proliferation assays [0094] c) selecting an antibody, which
inhibits GCO and/or cell proliferation. Results have shown that an
anti-MIF antibody that only binds active MIF and does not bind
non-active MIF and further inhibits GCO and/or cell proliferation
induces a beneficial effect in an animal model (e.g. Example 6)
[0095] The present invention will be further illustrated by
following examples, without any limitation thereto.
EXAMPLES
Example 1
Antibody Selection
[0096] Phage display technology is used to generate human anti-MIF
antibody fragments. Starting from a phage display library,
different screening campaigns are performed, three of them by using
full length MIF (human MIF coated/human MIF in
solution/human-murine MIF alternating). The others by using six MIF
derived peptides alternating with full length MIF. These six
peptides are designed by dividing the MIF protein into six peptides
of approximately 30 amino acids with overlapping stretches of
approximately 15-amino acids. After several selection rounds unique
binders are identified, all unique binders are expressed and
purified as human IgG4 antibodies. These antibodies are tested in
several assays to demonstrate the in-vitro inhibition of MIF. An
epitope mapping to determine the binding region within the MIF
protein is carried. 193 antibodies are tested and categorized
according to their in-vitro activity of inhibiting MIF. In-vitro
assays are described below. Three murine anti-MIF antibodies are
used as control (III.D.9, XIV.14.3 and XIV.15.5).
Example 2
Inhibition of Glucocorticoid Overriding Activity of MIF (GCO)
[0097] This method is based on the inhibition of endogenous MIF,
i.e. MIF that is produced by the cell line used. This method is
applied for antibody screening and for determination of dose
response curves.
GCO-Assay for Antibody Screening:
[0098] A THP1 suspension culture is centrifuged and cells are
resuspended in fresh full medium to a cell density of 10.sup.6
cells per ml. This culture is transferred into wells of a 96-well
microplate (90 .mu.l/well) and anti-MIF antibody is added to give a
final concentration of 75 .mu.g/ml. Each antibody is tested in
triplicate. After o/n incubation at 37.degree. C. dexamethasone is
added to give a concentration of 2 nM and after one hour incubation
at 37.degree. C. LPS is added (3 ng/ml final concentration). After
further six hours incubation at 37.degree. C. the supernatant is
harvested and the IL-6 concentrations are determined in an ELISA
(Cytoset kit, commercially available). The results of the
triplicates are averaged and the percentage of IL-6 secretion is
determined in comparison to the control antibodies. Antibodies that
result in an IL-6 secretion of less than 75% are evaluated as
positive.
Assay for Determination of IC50 Values
[0099] The experimental procedure is carried out as described for
the screening assay with the exception that increasing amounts of
antibody are used (typically from 1-125 nM). The resultant dose
response curve is expressed as % inhibition in comparison to a
negative control antibody. This curve is used for calculation of
the maximum inhibitory effect of the antibody (% Inh max) and the
antibody concentration that shows 50% of the maximum inhibitory
effect (IC50)
[0100] Results are summarized in FIG. 8, column 3 (IC50) and column
4 (maximum inhibition). For comparison, murine antibody XIV.14.3
shows 36% inhibition of GCO only (data not shown).
Example 3
Inhibition of Cell Proliferation
[0101] Serum stimulates secretion of MIF in quiescent NIH/3T3 and
MIF in turn stimulates cell proliferation. Antibodies inhibiting
this endogenous MIF, therefore, decrease the proliferation of
quiescent NIH/3T3 cells. The reduction of proliferation is
determined by the incorporation of .sup.3H-thymidine.
[0102] 1000 NIH/3T3 cells per well are incubated in a 96 well plate
over the weekend at 37.degree. C. in medium containing 10% serum.
Cells are then starved over night at 37.degree. C. by incubation in
medium containing 0.5% serum. The 0.5% medium is removed and
replaced by fresh medium containing 10% serum, 75 .mu.g/ml antibody
and 5 .mu.Ci/ml of 3H-Thymidin. After 16 hours incubation in a
CO.sub.2 incubator at 37.degree. C. cells are washed twice with 150
.mu.l of cold PBS per well. Using a multi-channel pipette 150 .mu.l
of a 5% (w/v) TCA solution per well are added and incubated for 30
minutes at 4.degree. C. Plates are washed with 150 .mu.l PBS. Per
well 75 .mu.l of a 0.5M NaOH solution with 0.5% SDS are added,
mixed and stored at room temperature. Samples are measured in a
.beta.-counter by mixing 5 ml of Ultima Gold (Packard) and 75 .mu.l
sample solution. Each determination is done in triplicate and the
values are compared with the values of the control antibody by a
t-test. Antibodies that significantly reduce proliferation
(P<0.05) are evaluated as positive. Results are summarized in
FIG. 8, column 5.
Example 4
Binding Studies: Epitope Determination of Anti-MIF Antibodies
[0103] Each peptide is diluted in coupling buffer to give a peptide
concentration of typically 5 .mu.g/ml, is added to microplates
(NUNC Immobilizer.TM. Amino Plate F96 Clear) and incubated over
night at 4.degree. C. (100 .mu.l/well). As controls recombinant
full length MIF and PBS are used. The plate is washed 3 times with
200 .mu.l PBST and antibodies (4 .mu.g/ml in PBS) are added (100
.mu.l/well) and incubated for 2 hours at room temperature with
gentle shaking. The plate is washed 3 times with 200 .mu.l PBST and
detection antibody (e.g. Fc specific anti-human IgG/HRP labeled,
Sigma) is added (100 .mu.l/well). After incubation for 1 hour at
room temperature with gentle shaking the plate is washed 3 times
with 200 .mu.l PBST. Each well is incubated with 100 .mu.l TMB
solution (T-0440, Sigma) for 30 minutes in the dark. Staining
reaction is stopped by adding 100 .mu.l of 1.8 M
H.sub.2SO.sub.4-solution per well. Samples are measured at 450
nm.
Example 5
Competition of Human Anti-MIF Antibodies with Murine Anti-MIF
Antibody III.D.9
[0104] Antibody Bax94 is used for competition with mouse anti MIF
antibodies III.D.9. 96 well plates (NUNC Maxisorp) are coated with
recombinant human MIF. The murine anti-MIF antibody II.D.9 and
human anti-MIF antibodies are diluted in TBST/2% BSA and mixed,
whereas the final concentration of III.D9 is kept at 2 .mu.g/ml and
the concentration of human anti-MIF antibodies is increased from 0
.mu.g/ml to typically 32 .mu.g/ml. After washing of the microplate
the antibodies are applied and incubated at room temperature for
typically 2 hours. After washing, the plate is incubated with anti
Mouse IgG (Fc spec.) peroxidase conjugate and incubated for 1 hour
at room temperature. After washing, the plate is incubated with
TMB-solution and the staining reaction is stopped by adding
H.sub.2SO.sub.4-solution. Fitting of the resultant competition
curve enables the calculation of the maximum inhibition of the
III.D.9 binding. The results are summarized in FIG. 8, column
6.
Example 6
Increased Survival of Anti-MIF Antibodies in the Live E. coli
Peritonitis Animal Model
[0105] The experiments are carried out according to Calandra et al.
(Nature Immunology, 2000) using female NMRI mice (25-30 g, 6-10
weeks of age) that are injected intraperitoneally with 6000 CFU of
an E. coli 0111:B4 suspension in 15% mucin and 4% hemoglobin. Two
or three colonies (E. coli 0111:B04) from a nutrient agar plate
culture are inoculated into 10 ml of TSB and incubated overnight at
36.degree. C. with shaking. The culture is diluted in physiological
saline to the required concentration(s)--an overnight the culture
typically reaches 2*10.sup.9 CFU/ml--and mixed with mucin and
hemoglobin (1 volume of diluted inoculum, 2 volumes of 15% mucin, 2
volumes of 4% hemoglobin). As the inoculum mixture tends to
sediment out, it is mixed between injections. A large (e.g. 23
gauge) needle is used for injections to avoid blockage of the
needle by particulates in the injection mixture. Antibody Bax94
(IgG4) and an isotype matching control antibody are given 2 hours
prior to bacterial challenge interperitoneally. The antibody dosage
is typically 800 .mu.g/mouse and 20 mice are used for each group. A
statistically significant effect on survival/time to death could be
shown for the IgG1 and IgG4 isotypes of human anti-MIF antibodies.
FIG. 6 shows the results obtained for antibody Bax94 and antibody
Bax152 (IgG4). Kaplan-Meier statistics is used for evaluation of
the survival curves.
Example 7
Binding Specificity for Active MIF
[0106] The anti-MIF antibodies described in this invention are able
to discriminate between active and non-active MIF, which are
generated by mild oxidation or reduction, respectively.
Discrimination between these conformers is assessed by ELISA or
surface plasmon resonance.
ELISA for Assessing Differential Binding of the Antibodies:
[0107] Transformation of MIF into its active conformation by mild
oxidation. [0108] Recombinant human MIF (0.5 mg/ml in PBS) is
incubated for 3 h at 37.degree. C. with a 3-fold excess (volume) of
a saturated solution of L-Cystine in PBS (.about.0.4-0.5 mM
L-Cystine). The MIF is then dialyzed two times against PBS in a
Slide-A-Lyzer.RTM. Dialysis Cassette with a molecular-weight cutoff
of 7 kDa (Pierce). [0109] Transformation of MIF into its non-active
conformation. [0110] MIF is reduced at a concentration of 0.5 mg/ml
by overnight incubation with 8-16 mM dithiothreitol (final
concentration) at 4.degree. C. [0111] ELISA protocol. [0112] The
anti-MIF antibodies are coated into 96-well microplates (NUNC
Maxisorp.TM.) at a concentration of 5 .mu.g/ml (dilution in coating
buffer). After washing the plate with TBST (Tris-buffered saline
with 0.1% Tween-20 (v/v)) and blocking with TBST/2% BSA (TBST and
2% bovine serum albumin (w/v)), dilution series of either active or
non-active MIF are added and incubated at room temperature for 1-2
h. Bound MIF is detected using a polyclonal rabbit anti-MIF
antibody and a horseradish peroxidase labeled goat-anti-rabbit
antibody (Biorad). TBST/2% BSA is used to dilute MIF, the rabbit
anti-MIF antibody and the peroxidase conjugate to reduce unspecific
binding. FIG. 7 shows the ELISA results obtained with antibody
Bax94.
Assessing Differential Binding of the Antibodies by Biacore.
[0112] [0113] Binding kinetics of active and non-active MIF to
antibody Bax94 are examined by surface plasmon resonance analysis
using a Biacore 3000 System. Therefore, 10000 Response Units of Bax
94 are immobilized on a sensor chip with a CM5 (=carboxymethylated
dextran) matrix and incubated with active or non-active MIF huMIF
in pro-reductive and pro-oxidative Glutathione redox buffers,
ranging from 4.8 mM GSH/0.2 mM GSSG (GSSG=oxidized Glutathione) to
5 mM GSSG in HBS-EP buffer (GE Healthcare). As a control, MIF is
used for binding analysis in a second flow cell containing an
immobilized isotype control antibody. Binding response units of
control antibody and antibody Bax94 are subtracted for
evaluation.
Example 8
Detection of Active MIF on the Surface of THP-1 Cells
[0114] Cells are incubated with anti-MIF antibody Bax94. Cells are
washed with ice cold PBS and resuspended in cold cell lysis buffer
(Cell Signaling Technology.RTM.). Magnetic Protein G Dynabeads.RTM.
(Invitrogen) are blocked with TBST+5% nonfat dried milk (w/v),
washed and added to the lysed cells. Immunoprecipitation is carried
out at 4.degree. C. overnight. The beads are then washed with cell
lysis buffer and TBST and boiled in SDS PAGE sample buffer (without
reducing agents). Samples are subjected to non-reductive SDS PAGE
for Western Blot analysis.
Example 9
Binding of Anti-MIF Antibodies to Membrane Bound MIF
[0115] THP-1 cells are washed with ice cold PBS and resuspended in
cold cell staining buffer (Biolegend) supplemented with 200
.mu.g/ml mouse IgG. FITC- or TRITC-labeled anti-MIF antibodies are
added to give a final concentration of typically 200-500 nM and
incubation is done at 4.degree. C. Cells are subsequently washed
with ice cold cell staining buffer and resuspended in cell staining
buffer supplemented with the Via-Probe.TM. Cell Viability Solution
(BD Biosciences). Cells are measured in an FACS Canto.TM. II Flow
Cytometry System (BD Biosciences) and the median FITC-/TRITC-shift
of the viable cell populations are compared with the Dye-labeled
isotype control antibody.
Example 10
Affinity Determination of Fab Fragments of Anti-MIF Antibodies by
Biacore
[0116] Typically 40RU Units of human recombinant MIF are
immobilized on a sensor chip with a CM5 (=carboxymethylated
dextran) matrix (Biacore). Fab fragments are injected at a
concentration range of typically 6-100 nM diluted in HBS-EP. After
each cycle the chip is regenerated with 50 mM NaOH+1M NaCl.
Affinities are calculated according to the 1:1 Langmuir model. The
results are summarized in FIG. 8, column 7.
Example 11
Beneficial Effect of Anti-MIF Antibodies in an Animal Model for
Crescentic Glomerulonephritis
[0117] The anti-MIF antibodies are tested in a rat model of
crescentic glomerulonephtitis described by Frederick W. K. Tam et.
al. (Nephrol Dial Transplant, 1999, 1658-1666).
[0118] Nephrotoxic nephritis is induced in male Wistar Kyoto rats
by a single intravenous injection of anti-rat glomerular basement
membrane serum. In the preventive setup of the experiment treatment
with anti-MIF antibodies and an isotype matching control antibody
is started at the time of induction of nephritis (day 0) by
interperitoneal injection of the antibody. Treatment is typically
repeated every second day and animals are culled on day 7 for
histological analyses. Urine is collected prior to the experiment
(baseline) and on prior to the termination of the experiment (day
7). In a therapeutic setup, treatment with anti MIF antibody is
started 4 days after induction of disease and repeated every second
day. Rats are typically culled on day 8. Urine is collected prior
to the experiment (baseline), prior to start of treatment (day 4)
and prior to culling of the animals (day 8). Antibody dosage is
typically 1-20 mg/kg per injection and 6 to 8 rats are used for
each group. Disease severity is determined by measuring
proteinuria, macrophage infiltration into the glomerulus and
histological damage (crescent formation). In a preventive
experiment treatment with anti-MIF antibody Bax69 (10 mg/kg per
dose) for 7 days results in a 47% reduction of proteinuria in
comparison to control antibody treated animals. Treatment of
established disease (therapeutic experiment) results in a dose
dependent reduction of proteinuria by 16% (10 mg/kg Bax69 per dose)
and 34% (20 mg/kg Bax69 per dose) in comparison to control antibody
treated animals.
Example 12
Beneficial Effect of Anti-MIF Antibodies in an Animal Model for
Ulcerative Colitis (Adoptive Transfer of NaiVe T Cells in Rag -/-
Mice)
[0119] C57BL/6 mice were sacrificed and CD45RBhi cells (naive T
cells) are isolated by FACS sorting of the spleen cell population.
CD45RBhi cells (5.times.10.sup.5) are injected i.p. in Rag-/-
C57BL/6 mice (7-9 weeks old), which develop of Ulcerative colitis
after approx. 2 weeks. (de Jong et al., Nature immunology., 2001,
1061-1066). Anti-MIF antibodies and the isotype control antibody
are injected intraperitoneally twice a week (1 mg/mouse/dose). In a
preventive setup treatment is started at the time of injection of
T-cells. In a therapeutic setup, treatment is started 4 weeks after
induction of the disease. Mice are monitored weekly for weight and
disease development. Typically eight weeks after the transfer of
CD4CD45RBhi cells into Rag-/- C57BL/6 recipients the disease
activity index (DAI) is calculated and colon sections are collected
for histology index (HI) score. Diseases activity index (DAI) and
histology index (HI) are determined at the end of the animal model
(DAI is based on four parameters: hunching and wasting (scored 0 or
1), colon thickening (0-3) and stool consistency (0-3)). In a
therapeutic experiment anti-MIF antibodies Bax69 and BaxA10 are
used for treatment of established disease and the mean DAI is
significantly reduced by approx. 60% (Bax69) and approx 40%
(BaxA10) in comparison to isotype control treated mice.
Furthermore, the mean HI score is reduced by approximately 33%
after treatment with Bax69.
Example 13
Beneficial Effect of Anti-MIF Antibodies in an Animal Model for
Ulcerative Colitis (Agonistic Anti-CD40 Model)
[0120] This model is based on the activation of macrophages and
dendritic cells by an agonistic anti-CD40 antibody, which induces
intestinal pathology that resembles IBD in Rag 1-/- mice.
[0121] Age/sex matched Rag-1-/- mice (4-5 wks) are purchased form
Jackson Laboratories and kept for two weeks prior to the experiment
in the animal facility. The agonist-CD40 monoclonal antibody
(FGK45, IgG2a) or Isotype control Rat IgG2a are dissolved in PBS at
1 mg/ml. Five groups (10 mice each group) are injected i.p with 200
.mu.g of agonist anti-CD40 monoclonal antibody and out of that four
groups are treated with anti-MIF antibodies on day 0 and day 1
(2.times.1 mg/mouse). The sixth group (10 mice) is injected only
with isotype control (Rat IgG2a, healthy control). Mice are weighed
for the next 7 days. On Day 7, disease activity index (DAI) was
calculated and colon sections collected for histology index (HI)
score. The DAI score is based on: hunching (0-1); wasting (0-1),
stool consistency (0-3) and colon thickening (0-3). Histology score
was based on thickness (0-3), crypt elongation, inflammation (0-3)
and abscess (0-1). Treatment with anti-MIF antibodies Bax94, BaxA10
and Bax69 significantly reduces the DAI score (BaxA10: .about.48%
reduction; Bax94.about.62% reduction; Bax69.about.73% reduction)
compared to isotype control treated mice. Furthermore, the mean HI
scores is also reduced by the these antibodies.
Example 14
Inhibition of Tumor Growth in Mf1 Nude Mice by Anti MIF
Antibodies
[0122] Human prostate adenocarcinoma cells (PC-3) are harvested
from exponentially growing cultures and mixed with growth
factor-depleted matrigel. 2*10.sup.6 cells in 0.25 ml matrigel are
inoculated subcutaneously into the right flank of Mf1 nude mice.
Treatment with anti-MIF antibody Bax94 and the isotype control C3
is started one day after inoculation (0.6 mg antibody/mouse/day)
and is repeated every second day. Measurement of the sizes of the
tumors is typically started two weeks after cell injection and done
every second day. The volumes are calculated using the formula
V=0.5*a*b.sup.2 (where "a" is the longest diameter and "b" is the
shortest diameter). Tumor growth of mice treated with Bax94 is
significantly reduced and the mean volume of the tumors analyzed 28
days after tumor induction is 4.3 fold higher within the isotype
control treated group in comparison to the Bax94 treated group.
[0123] In a therapeutic setup of the experiment antibody treatment
was started one week after tumor engraftment. 50 mg/kg per dose of
the isotype control antibody C3 and the anti-MIF antibody Bax69 are
injected intraperitoneally every second day. After 22 days of
treatment the median of the tumor volume was determined to be 2.7
fold higher within the C3 treated group in comparison to the of
Bax69 treated group.
Example 15
Pro-Apoptotic Effects of Anti-MIF Antibodies
[0124] Pro-apoptotic effects of anti-MIF antibody Bax94 are shown
in a cell based caspase-3 assay using the human prostate cancer
cell line PC-3. PC-3 cells are seeded on 10 cm culture dishes
(.about.10.sup.6 cells/dish) in the presence of 10% FCS. Fresh
medium containing 100 nM antibody Bax94 or 100 nM control antibody
C3 is added after 24 h. After another incubation period of 48 h
cellular lysates are prepared and caspase-3 activity is measured by
adding a fluorescent labeled caspase substrate. (FIG. 9).
Example 16
Inhibition of Tumor Cell Invasion
[0125] Anti MIF antibodies Bax94 and Bax69 are tested in
Transwell.TM. invasion assays, using the human prostate cancer cell
line PC-3.
[0126] 5*10.sup.4 PC-3 cells are seeded per well in 24
well-Transwell.TM. dishes (8 .mu.m pore size), which are coated
with polyD-lysine on the bottom face of the polycarbonate membrane
and with growth-factor depleted matrigel on the Transwell.TM.
insert surface. Cells are allowed to attach for 4 h in the presence
of 10% FCS. Thereafter, the medium was changed to serum-free medium
and cells are starved overnight (i.e. for 16 h). Subsequently,
compounds (10 nM MIF, 500 nM antibodies) are added to the lower
chamber. Cells are allowed to migrate through the porous membrane
for 24 h. After this incubation period, attached migrated cells of
the lower face of the membrane are stained with Giemsa solution.
The number of cells adhering to the lower face of the membrane is
counted in independent visual fields at 400-fold magnification
(FIG. 10).
Sequence CWU 1
1
241107PRTArtificial Sequenceanti macrophage migration inhibitory
factor (MIF) monoclonal antibody Bax8 light chain variable region
(V-L) 1Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val
Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile
Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr
Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Trp 85 90 95 Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 105 2107PRTArtificial
Sequenceanti macrophage migration inhibitory factor (MIF)
monoclonal antibody Bax69 light chain variable region (V-L) 2Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Arg Ile Met Thr Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Phe Val Ala Ser His Ser Gln Ser Gly Val Pro Ser
Arg Phe Arg Gly 50 55 60 Ser Gly Ser Glu Thr Asp Phe Thr Leu Thr
Ile Ser Gly Leu Gln Pro65 70 75 80 Glu Asp Ser Ala Thr Tyr Tyr Cys
Gln Gln Ser Phe Trp Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105 3107PRTArtificial Sequenceanti
macrophage migration inhibitory factor (MIF) monoclonal antibody
Bax74 light chain variable region (V-L) 3Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Pro Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Thr Tyr 20 25 30 Leu Ser
Trp Tyr Gln His Lys Pro Gly Asn Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Ala Thr Ser Arg Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Gly Gly Ser Gly Thr Arg Phe Thr Leu Ala Ile Ser Ser Leu Gln
Pro65 70 75 80 Asp Asp Phe Ala Thr Tyr Phe Cys Gln Gln Thr Tyr Ser
Thr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys
100 105 4107PRTArtificial Sequenceanti macrophage migration
inhibitory factor (MIF) monoclonal antibody Bax94 light chain
variable region (V-L) 4Asp Ile Gln Met Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Gly Val Ser Ser Ser 20 25 30 Ser Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Thr Ser
Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Ala
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Gln65 70 75 80 Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Arg Ser Leu 85 90
95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105
5107PRTArtificial Sequenceanti macrophage migration inhibitory
factor (MIF) monoclonal antibody Bax152 light chain variable region
(V-L) 5Asp Ile Gln Met Thr Gln Ser Pro Val Thr Leu Ser Leu Ser Pro
Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
Arg Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Thr Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Asn Arg Ala Thr
Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Arg Leu Glu65 70 75 80 Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Tyr Gly Asn Ser Leu 85 90 95 Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys 100 105 6107PRTArtificial
Sequenceanti macrophage migration inhibitory factor (MIF)
monoclonal antibody BaxA10 light chain variable region (V-L) 6Asp
Ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Val Ser Ser Ser
20 25 30 Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg
Leu Leu 35 40 45 Ile Tyr Gly Thr Ser Ser Arg Ala Thr Gly Ile Pro
Asp Arg Phe Ser 50 55 60 Gly Ser Ala Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Arg Leu Gln65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Tyr Gly Arg Ser Leu 85 90 95 Thr Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys 100 105 7126PRTArtificial Sequenceanti
macrophage migration inhibitory factor (MIF) monoclonal antibody
Bax8 heavy chain variable region (V-H) 7Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Thr Met Asp
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Tyr Ile Ser Pro Ser Gly Gly Asn Thr Ser Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Ser Arg Gln Tyr Val Leu Arg Tyr Phe Asp
Trp Ser Ala Asp Ala 100 105 110 Phe Asp Ile Trp Gly Gln Gly Thr Met
Val Thr Val Ser Ser 115 120 125 8118PRTArtificial Sequenceanti
macrophage migration inhibitory factor (MIF) monoclonal antibody
Bax69 heavy chain variable region (V-H) 8Glu Val Gln Leu Leu Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Ser Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ser Ser Ile Gly Ser Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Gly Ser Gln Trp Leu Tyr Gly Met Asp Val
Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
9121PRTArtificial Sequenceanti macrophage migration inhibitory
factor (MIF) monoclonal antibody Bax74 heavy chain variable region
(V-H) 9Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Lys Tyr 20 25 30 Tyr Met Ile Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Gly Pro Ser Gly Gly Phe
Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly
Thr Pro Asp Tyr Gly Gly Asn Ser Leu Asp His Trp Gly 100 105 110 Gln
Gly Thr Leu Val Thr Val Ser Ser 115 120 10127PRTArtificial
Sequenceanti macrophage migration inhibitory factor (MIF)
monoclonal antibody Bax94 heavy chain variable region (V-H) 10Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr
20 25 30 Ala Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Ser Gly Ile Val Pro Ser Gly Gly Phe Thr Lys Tyr
Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Asn Val Ile
Ala Val Ala Gly Thr Gly Tyr Tyr Tyr Tyr 100 105 110 Gly Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125
11127PRTArtificial Sequenceanti macrophage migration inhibitory
factor (MIF) monoclonal antibody Bax152 heavy chain variable region
(V-H) 11Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ile Tyr 20 25 30 Ala Met Asp Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Val Pro Ser Gly Gly Phe
Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val
Asn Val Ile Ala Val Ala Gly Thr Gly Tyr Tyr Tyr Tyr 100 105 110 Gly
Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125
12127PRTArtificial Sequenceanti macrophage migration inhibitory
factor (MIF) monoclonal antibody BaxA10 heavy chain variable region
(V-H) 12Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Trp Tyr 20 25 30 Ala Met Asp Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Tyr Pro Ser Gly Gly Arg
Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80 Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val
Asn Val Ile Ala Val Ala Gly Thr Gly Tyr Tyr Tyr Tyr 100 105 110 Gly
Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125
13321DNAArtificial Sequenceanti macrophage migration inhibitory
factor (MIF) monoclonal antibody Bax8 light chain variable region
(V-L) 13gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
cagagtcacc 60atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca
gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccagtt
tgcaaagtgg ggtcccatca 180aggttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta
ctgtcaacag agttacagta ccccttggac gttcggccaa 300gggaccaagg
tggaaatcaa a 32114321DNAArtificial Sequenceanti macrophage
migration inhibitory factor (MIF) monoclonal antibody Bax69 light
chain variable region (V-L) 14gacatccaga tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc ggtcaagtca gagaattatg
acttatttaa attggtatca gcaaaaaccg 120gggaaagccc ctaaactcct
gatctttgtt gcatcccatt cacaaagtgg ggtcccatcc 180aggttcagag
gcagtgggtc tgagacagat ttcactctca ccatcagcgg tctgcaacct
240gaagattctg caacttacta ctgtcaacaa agtttttgga cccccctcac
tttcggcgga 300gggaccaagg tggagatcaa a 32115321DNAArtificial
Sequenceanti macrophage migration inhibitory factor (MIF)
monoclonal antibody Bax74 light chain variable region (V-L)
15gacatccaga tgacccagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc
60ctctcctgca gggccagtca gggtgttagc agcagctcct tagcctggta ccagcagaaa
120cctggccagg ctcccaggct cctcatctat ggtacatcca gcagggccac
tggcatccca 180gacaggttca gtggcagtgc gtctgggaca gacttcactc
tcaccatcag cagactgcag 240cctgaagatt ttgcagtgta ttactgtcag
cagtatggta ggtcactcac tttcggcgga 300gggaccaagg tggagatcaa a
32116321DNAArtificial Sequenceanti macrophage migration inhibitory
factor (MIF) monoclonal antibody Bax94 light chain variable region
(V-L) 16gacatccaga tgacccagtc tccaggcacc ctgtctttgt ctccagggga
aagagccacc 60ctctcctgca gggccagtca gggtgttagc agcagctcct tagcctggta
ccagcagaaa 120cctggccagg ctcccaggct cctcatctat ggtacatcca
gcagggccac tggcatccca 180gacaggttca gtggcagtgc gtctgggaca
gacttcactc tcaccatcag cagactgcag 240cctgaagatt ttgcagtgta
ttactgtcag cagtatggta ggtcactcac tttcggcgga 300gggaccaagg
tggagatcaa a 32117321DNAArtificial Sequenceanti macrophage
migration inhibitory factor (MIF) monoclonal antibody Bax152 light
chain variable region (V-L) 17gacatccaga tgacccagtc tccagtcacc
ctgtctttgt ctccagggga aagagccacc 60ctctcttgca gggccagtca gagtgttcgg
agtagttact tagcctggta ccagcagaaa 120cccggccaga ctcccaggct
cctcatctat ggtgcctcca acagggccac tggcatccca 180gacaggttca
gtggcagtgg gtctgggaca gacttcactc tcaccatcag cagactggag
240cctgaagatt ttgcagtcta ttactgtcag cagtatggta actcactcac
tttcggcgga 300gggaccaagg tggagatcaa a 32118321DNAArtificial
Sequenceanti macrophage migration inhibitory factor (MIF)
monoclonal antibody BaxA10 light chain variable region (V-L)
18gacatccaga tgacccagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc
60ctctcctgca gggccagtca gggtgttagc agcagctcct tagcctggta ccagcagaaa
120cctggccagg ctcccaggct cctcatctat ggtacatcca gcagggccac
tggcatccca 180gacaggttca gtggcagtgc gtctgggaca gacttcactc
tcaccatcag cagactgcag 240cctgaagatt ttgcagtgta ttactgtcag
cagtatggta ggtcactcac tttcggcgga 300gggaccaagg tggagatcaa a
32119378DNAArtificial Sequenceanti macrophage migration inhibitory
factor (MIF) monoclonal antibody Bax8 heavy chain variable region
(V-H) 19gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc
tttacgtctt 60tcttgcgctg cttccggatt cactttctct atttacacta tggattgggt
tcgccaagct 120cctggtaaag gtttggagtg ggtttcttat atctctcctt
ctggtggcaa tacttcttat 180gctgactccg ttaaaggtcg cttcactatc
tctagagaca actctaagaa tactctctac 240ttgcagatga acagcttaag
ggctgaggac acggccgtgt attactgtgc gagtagacaa 300tacgtattac
gatattttga ctggtcggca gatgcttttg atatctgggg ccaagggaca
360atggtcaccg tctcaagc 37820354DNAArtificial Sequenceanti
macrophage migration inhibitory factor (MIF) monoclonal antibody
Bax69 heavy chain variable region (V-H) 20gaagttcaat tgttagagtc
tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt
cactttctct atttactcta tgaattgggt tcgccaagct 120cctggtaaag
gtttggagtg ggtttcttct atcggttctt ctggtggcac tacttattat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa
tactctctac 240ttgcagatga acagcttaag ggctgaggac acggccgtgt
attactgtgc gggctcacag 300tggctgtacg gtatggacgt ctggggccaa
gggaccacgg tcaccgtctc aagc 35421363DNAArtificial Sequenceanti
macrophage migration inhibitory factor (MIF) monoclonal antibody
Bax74 heavy chain variable region (V-H) 21gaagttcaat tgttagagtc
tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt
cactttctct aagtactata tgatttgggt tcgccaagct 120cctggtaaag
gtttggagtg ggtttcttgg atcggtcctt ctggtggctt tactttttat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa
tactctctac 240ttgcagatga acagcttaag ggctgaggac acggccgtgt
attactgtgc gagagggacg 300cccgactacg
gtggtaactc ccttgaccac tggggccagg gcaccctggt caccgtctca 360agc
36322381DNAArtificial Sequenceanti macrophage migration inhibitory
factor (MIF) monoclonal antibody Bax94 heavy chain variable region
(V-H) 22gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc
tttacgtctt 60tcttgcgctg cttccggatt cactttctct atttacgcta tggattgggt
tcgccaagct 120cctggtaaag gtttggagtg ggtttctggt atcgttcctt
ctggtggctt tactaagtat 180gctgactccg ttaaaggtcg cttcactatc
tctagagaca actctaagaa tactctctac 240ttgcagatga acagcttaag
ggctgaggac acggccgtgt attactgtgc gagagtgaac 300gttatagcag
tggctggtac tggatactac tactacggta tggacgtctg gggccaaggg
360accacggtca ccgtctcaag c 38123381DNAArtificial Sequenceanti
macrophage migration inhibitory factor (MIF) monoclonal antibody
Bax152 heavy chain variable region (V-H) 23gaagttcaat tgttagagtc
tggtggcggt cttgttcagc ctggtggttc tttacgtctt 60tcttgcgctg cttccggatt
cactttctct atttacgcta tggattgggt tcgccaagct 120cctggtaaag
gtttggagtg ggtttctggt atcgttcctt ctggtggctt tactaagtat
180gctgactccg ttaaaggtcg cttcactatc tctagagaca actctaagaa
tactctctac 240ttgcagatga acagcttaag ggctgaggac acggccgtgt
attactgtgc gagagtgaac 300gttatagcag tggctggtac tggatactac
tactacggta tggacgtctg gggccaaggg 360accacggtca ccgtctcaag c
38124381DNAArtificial Sequenceanti macrophage migration inhibitory
factor (MIF) monoclonal antibody BaxA10 heavy chain variable region
(V-H) 24gaagttcaat tgttagagtc tggtggcggt cttgttcagc ctggtggttc
tttacgtctt 60tcttgcgctg cttccggatt cactttctct tggtacgcta tggattgggt
tcgccaagct 120cctggtaaag gtttggagtg ggtttctggt atctatcctt
ctggtggccg tactaagtat 180gctgactccg ttaaaggtcg cttcactatc
tctagagaca actctaagaa tactctctac 240ttgcagatga acagcttaag
ggctgaggac acggccgtgt attactgtgc gagagtgaac 300gttatagcag
tggctggtac tggatactac tactacggta tggacgtctg gggccaaggg
360accacggtca ccgtctcaag c 381
* * * * *