Title: Human TIMP-1 antibodies
United States Patent: 7,091,323
Issued: August 15, 2006
Inventors: Pan; Clark
(Castro Valley, CA), Knorr; Andreas M. (Erkrath, DE), Schauer; Michael (Wuppertal,
DE), Hirth-Dietrich; Claudia (Wuppertal, DE), Kraft; Sabine (Planegg, DE),
Krebs; Barbara (Bergisch Gladbach, DE)
Assignee: Bayer Corporation
(Pittsburgh, PA), Bayer Aktiengesellschaft (Leverkusen, DE)
Appl. No.: 10/128,520
Filed: April 24, 2002
Pharm Bus Intell
& Healthcare Studies
Human antibodies that bind to TIMP-1 can
be used as reagents to diagnose and treat disorders in which TIMP-1 is
elevated, such as liver fibrosis, alcoholic liver disease, cardiac
fibrosis, acute coronary syndrome, lupus nephritis, glomerulosclerotic
renal disease, benign prostate hypertrophy, colon cancer, lung cancer, and
idiopathic pulmonary fibrosis.
OF THE INVENTION
The invention provides human antibodies
that bind to TIMP-1. These antibodies are useful for a variety of
therapeutic and diagnostic purposes.
Characteristics of Human TIMP-1 Antibodies
"Antibody" as used herein includes intact immunoglobulin molecules (e.g.,
IgG.sub.1, IgG.sub.2a, IgG.sub.2b, IgG.sub.3, IgM, IgD, IgE, IgA), as well
as fragments thereof, such as Fab, F(ab')2, scFv, and Fv, which are
capable of specific binding to an epitope of a human and/or rat TIMP-1
protein. Antibodies that specifically bind to TIMP-1 provide a detection
signal at least 5-, 10-, or 20-fold higher than a detection signal
provided with other proteins when used in an immunochemical assay.
Preferably, antibodies that specifically bind to human and/or rat TIMP-1
do not detect other proteins in immunochemical assays and can
immunoprecipitate the TIMP-1 from solution.
The K.sub.d of human antibody binding to TIMP-1 can be assayed using any
method known in the art, including technologies such as real-time
Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal.
Chem. 63, 2338 45, 1991, and Szabo et al., Curr. Opin. Struct. Biol. 5,
699 705, 1995). BIA is a technology for studying biospecific interactions
in real time, without labeling any of the interactants (e.g., BIAcore.TM.).
Changes in the optical phenomenon surface plasmon resonance (SPR) can be
used as an indication of real-time reactions between biological molecules.
In a BIAcore.TM. assay, some human antibodies of the invention
specifically bind to human TIMP-1 with a K.sub.d of about 0.1 nM to about
10 .mu.M, about 2 nM to about 1 .mu.M, about 2 nM to about 200 nM, about 2
nM to about 150 nM, about 50 nM to about 100 nM, about 0.2 nM to about 13
nM, about 0.2 nM to about 0.5 nM, about 2 nM to about 13 nM, and about 0.5
nM to about 2 nM. More preferred human antibodies specifically bind to
human TIMP-1 with a K.sub.d selected from the group consisting of about
0.2 nM, about 0.3 nM, about 0.5 M, about 0.6 nM, about 2 nM, about 7 nM,
about 10 nM, about 11 nM, and about 13 nM.
Other human antibodies of the invention specifically bind to rat TIMP-1
with a K.sub.d of about 0.1 nM to about 10 .mu.M, about 2 nM to about 1 .mu.M,
about 2 nM to about 200 nM, about 2 nM to about 150 nM, about 50 nM to
about 100 nM, about 1.3 nM to about 13 nM, about 1.8 nM to about 10 nM,
about 2 nM to about 9 nM, about 1.3 nM to about 9 nM, and about 2 nM to
about 10 nM. Preferred K.sub.d s range from about 0.8 nM, about 1 nM,
about 1.3 nM, about 1.9 nM, about 2 nM, about 3 nM, about 9 nM, about 10
nM, about 13 nM, about 14 nM, and about 15 nM.
Preferably, antibodies of the invention neutralize an MMP-inhibiting
activity of the TIMP-1. The MMP can be, for example, MMP-1, MMP-2, MMP-3,
MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-19, MMP-20 or
IC.sub.50 for neutralizing MMP-inhibiting activity of TIMP-1 can be
measured by any means known in the art. Preferably, IC.sub.50 is
determined using the high throughput fluorogenic assay described in
Bickett et al., Anal. Biochem. 212, 58 64, 1993. In a typical fluorogenic
assay, the IC.sub.50 of a human antibody for neutralizing human TIMP-1 MMP-inhibiting
activity ranges from about 0.1 nM to about 200 nM, about 1 nM to about 100
nM, about 2 nM to about 50 nM, about 5 nM to about 25 nM, about 10 nM to
about 15 nM, about 0.2 nM to about 11 nM, about 0.2 nM to about 4 nM, and
about 4 nM to about 11 nM. The IC.sub.50 for neutralizing human TIMP-1 MMP-inhibiting
activity of some human antibodies is about 0.2 nM, about 0.3 nM, about 0.4
nM, about 4 nM, about 7 nM, about 9 nM, and about 11 nM.
A typical IC.sub.50 for neutralizing rat TIMP-1 MMP-inhibiting activity
ranges from about 0.1 nM to about 300 nM, about 1 nM to about 100 nM,
about 2 nM to about 50 nM, about 5 nM to about 25 nM, about 10 nM to about
15 nM, about 1.1 nM to about 14 nM, about 1.6 nM to about 11 nM, about 3
nM to about 7 nM, about 1.1 nM to about 7 nM, about 1.1 nM to about 11 nM,
about 3 nM to about 11 nM, and about 3 nM to about 14 nM. The IC.sub.50
for neutralizing rat TIMP-1 MMP-inhibiting activity of some human
antibodies is about 1.1 nM, about 1.6 nM, about 3 nM, about 7 nM, about 11
nM, about 14 nM, about 19 nM, about 20 nM, about 30 nM, and about 100 nM.
Preferred human antibodies of the invention are those for which the
K.sub.d for binding to TIMP-1 and the IC.sub.50 for neutralizing the MMP-inhibiting
activity of the TIMP-1 are approximately equal.
A number of human antibodies having the TIMP-1 binding and MMP-inhibiting
activity neutralizing characteristics described above have been identified
by screening the MorphoSys HuCAL.RTM. Fab 1 library. The CDR cassettes
assembled for the HuCAL.RTM. library were designed to achieve a length
distribution ranging from 5 to 28 amino acid residues, covering the
stretch from position 95 to 102. Knappik et al., J Mol. Biol. 296, 57 86,
2000. Some clones, however, had shorter VHCDR3 regions. In fact, it is a
striking feature of anti-human TIMP-1 human antibodies identified from
this library that they all exhibit the combination VH312 and a relatively
short VHCDR3 region, typically four amino acids.
In some embodiments of the invention, the VHCDR3 region of a human
antibody has an amino acid sequence shown in SEQ ID NOS:1 43. In other
embodiments of the invention, the VLCDR3 region of a human antibody has an
amino acid sequence shown in SEQ ID NOS:44 86. See Tables 2, 3, and 7.
Human antibodies which have TIMP-1 binding and MMP-inhibiting activity
neutralizing characteristics of antibodies such as those described above
and in Tables 2, 3, and 7 also are human antibodies of the invention.
Obtaining Human Antibodies
Human antibodies with the TIMP-1 binding and MMP-activity neutralizing
characteristics described above can be identified from the MorphoSys
HuCAL.RTM. library as follows. Human or rat TIMP-1, for example, is coated
on a microtiter plate and incubated with the MorphoSys HuCAL.RTM. Fab
phage library (see Example 1, below). Those phage-linked Fabs not binding
to TIMP-1 can be washed away from the plate, leaving only phage which
tightly bind to TIMP-1. The bound phage can be eluted, for example, by a
change in pH or by elution with E. coli and amplified by infection of E.
coli hosts. This panning process can be repeated once or twice to enrich
for a population of antibodies that tightly bind to TIMP-1. The Fabs from
the enriched pool are then expressed, purified, and screened in an ELISA
assay. The identified hits are then screened in the enzymatic assay
described in Bickett et al., 1993, and Bodden et al., 1994. Those Fabs
that lead to the degradation of the peptide are likely the ones which bind
to TIMP-1, thereby blocking its interaction to MMP-1.
The initial panning of the HuCAL.RTM. Fab 1 library also can be performed
with TIMP-1 as the antigen in round one, followed in round 2 by TIMP-1
peptides fused to carrier proteins, such as BSA or transferrin, and in
round 3 by TIMP-1 again. Human TIMP-1 peptides which can be used for
panning include human TIMP-1 residues 2 12 (TCVPPHPQTAF, SEQ ID NO:87;
CTSVPPHPQTAF, SEQ ID NO:88; STCVPPHPQTAF, SEQ ID NO:89; STSVPPHPQTAFC, SEQ
ID NO:90), 28 36 (CEVNQTTLYQ, SEQ ID NO:91), 64 75 (PAMESVCGYFHR, SEQ ID
NO:92), 64 79 (PAMESVCGYFHRSHNR, SEQ ID NO:93; CPAMESVSGYFHRSHNR, SEQ ID
NO:94; PAMESVSGYFHRSHNRC, SEQ ID NO:95), and 145 157 (CLWTDQLLQGSE, SEQ ID
NO:96). These peptide sequences are selected from regions of human TIMP-1
that are predicted to interact with MMPs. See Gomis-Ruth et al., Nature
389, 77 81, 1997. Directing Fabs toward the MMP-interacting region of
human TIMP-1 in round 2 should increase the chance of identifying Fabs
that can block the ability of human TIMP-1 to inhibit human MMP-1
Another method that can be used to improve the likelihood of isolating
neutralizing Fabs is the panning on human TIMP-1 and eluting the binding
Fabs with human MMP-1. This strategy should yield higher affinity
antibodies than would otherwise be obtained.
Details of the screening process are described in the specific examples,
below. Other selection methods for highly active specific antibodies or
antibody fragments can be envisioned by those skilled in the art and used
to identify human TIMP-1 antibodies.
Human antibodies with the characteristics described above also can be
purified from any cell that expresses the antibodies, including host cells
that have been transfected with antibody-encoding expression constructs.
The host cells are cultured under conditions whereby the human antibodies
are expressed. A purified human antibody is separated from other compounds
that normally associate with the antibody in the cell, such as certain
proteins, carbohydrates, or lipids, using methods well known in the art.
Such methods include, but are not limited to, size exclusion
chromatography, ammonium sulfate fractionation, ion exchange
chromatography, affinity chromatography, and preparative gel
electrophoresis. A preparation of purified human antibodies is at least
80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity
of the preparations can be assessed by any means known in the art, such as
SDS-polyacrylamide gel electrophoresis. A preparation of purified human
antibodies of the invention can contain more than one type of human
antibody with the TIMP-1 binding and neutralizing characteristics
Alternatively, human antibodies can be produced using chemical methods to
synthesize its amino acid sequence, such as by direct peptide synthesis
using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149 54,
1963; Roberge et al., Science 269, 202 04, 1995). Protein synthesis can be
performed using manual techniques or by automation. Automated synthesis
can be achieved, for example, using Applied Biosystems 431A Peptide
Synthesizer (Perkin Elmer). Optionally, fragments of human antibodies can
be separately synthesized and combined using chemical methods to produce a
The newly synthesized molecules can be substantially purified by
preparative high performance liquid chromatography (e.g., Creighton,
PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New
York, N.Y., 1983). The composition of a synthetic polypeptide can be
confirmed by amino acid analysis or sequencing (e.g., using Edman
Assessment of Therapeutic Utility of Human Antibodies
To assess the ability of a particular antibody to be therapeutically
useful to treat, liver fibrosis, for example, the antibody can be tested
in vivo in a rat liver fibrosis model. Thus, preferred human antibodies of
the invention are able to block both human and rat TIMP-1 activity. If
desired, human Fab TIMP-1 antibodies can be converted into full
immunoglobulins, for example IgG.sub.1 antibodies, before therapeutic
assessment. This conversion is described in Example 5, below.
To identify antibodies that cross-react with human and rat TIMP-1, an
ELISA can be carried out using rat TIMP-1. Functional cross-reactivity can
be confirmed in an enzymatic assay, as described in Bickett et al., Anal.
Biochem. 212, 58 64, 1993. The assay uses human or rat TIMP-1, human MMP-1
or rat MMP-13 (the rat counterpart of human MMP-1), and a synthetic
fluorogenic peptide substrate. Enzyme activity of uncomplexed MMP-1 (or
MMP-13) is assessed by observing an increase in a fluorescence signal.
Antibodies that block human and/or rat TIMP-1 activity can be screened in
an ELISA assay that detects the decrease of TIMP-1/MMP-1 complex formation
in cultures of HepG2 cells. Antibodies that meet this criteria can then be
tested in a rat liver fibrosis model to assess therapeutic efficacy and
correlate this efficacy with the ability of the antibodies to block TIMP-1
inhibition of MMP-1 in vitro.
Antibodies that demonstrate therapeutic efficacy in the rat liver fibrosis
model can then be tested for binding to and blockade of TIMP-2,-3, and -4
in an in vitro enzymatic assay. Blocking the minimum number of TIMPs
necessary for efficacy in liver fibrosis or other TIMP-associated
pathology is preferable to minimize potential side effects.
Polynucleotides Encoding Human TIMP-1 Antibodies
The invention also provides polynucleotides encoding human TIMP-1
antibodies. These polynucleotides can be used, for example, to produce
quantities of the antibodies for therapeutic or diagnostic use.
Polynucleotides that can be used to encode the VHCDR3 regions shown in SEQ
ID NOS: 1 43 are shown in SEQ ID NOS:226 268, respectively.
Polynucleotides that can be used to encode the VLCDR3 region shown in SEQ
ID NOS:44 86 are shown in SEQ ID NOS: 183 225, respectively.
Polynucleotides that encode heavy chains (SEQ ID NOS:140 182) and light
chains (SEQ ID NOS:97 139) of human antibodies of the invention that have
been isolated from the MorphoSys HuCAL.RTM. library are shown in SEQ ID
NOS:269 311 and SEQ ID NOS:312 354, respectively.
Polynucleotides of the invention present in a host cell can be isolated
free of other cellular components such as membrane components, proteins,
and lipids. Polynucleotides can be made by a cell and isolated using
standard nucleic acid purification techniques, or synthesized using an
amplification technique, such as the polymerase chain reaction (PCR), or
by using an automatic synthesizer. Methods for isolating polynucleotides
are routine and are known in the art. Any such technique for obtaining a
polynucleotide can be used to obtain isolated polynucleotides encoding
antibodies of the invention. For example, restriction enzymes and probes
can be used to isolate polynucleotides which encode the antibodies.
Isolated polynucleotides are in preparations that are free or at least 70,
80, or 90% free of other molecules.
Human antibody-encoding DNA molecules of the invention can be made with
standard molecular biology techniques, using mRNA as a template.
Thereafter, DNA molecules can be replicated using molecular biology
techniques known in the art and disclosed in manuals such as Sambrook et
al. (1989). An amplification technique, such as PCR, can be used to obtain
additional copies of the polynucleotides.
Alternatively, synthetic chemistry techniques can be used to synthesize
polynucleotides encoding antibodies of the invention. The degeneracy of
the genetic code allows alternate nucleotide sequences to be synthesized
that will encode an antibody having, for example, one of the VHCDR3,
VLCDR3, light chain, or heavy chain amino acid sequences shown in SEQ ID
NOS:1 43, 44 86, 97 139, or 140 182, respectively.
Expression of Polynucleotides
To express a polynucleotide encoding a human antibody of the invention,
the polynucleotide can be inserted into an expression vector that contains
the necessary elements for the transcription and translation of the
inserted coding sequence. Methods that are well known to those skilled in
the art can be used to construct expression vectors containing sequences
encoding human antibodies and appropriate transcriptional and
translational control elements. These methods include in vitro recombinant
DNA techniques, synthetic techniques, and in vivo genetic recombination.
Such techniques are described, for example, in Sambrook et al. (1989) and
in Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley &
Sons, New York, N.Y., 1995. See also Examples 1 3, below.
A variety of expression vector/host systems can be utilized to contain and
express sequences encoding a human antibody of the invention. These
include, but are not limited to, microorganisms, such as bacteria
transformed with recombinant bacteriophage, plasmid, or cosmid DNA
expression vectors; yeast transformed with yeast expression vectors,
insect cell systems infected with virus expression vectors (e.g.,
baculovirus), plant cell systems transformed with virus expression vectors
(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with
bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell
The control elements or regulatory sequences are those non-translated
regions of the vector--enhancers, promoters, 5' and 3' untranslated
regions--which interact with host cellular proteins to carry out
transcription and translation. Such elements can vary in their strength
and specificity. Depending on the vector system and host utilized, any
number of suitable transcription and translation elements, including
constitutive and inducible promoters, can be used. For example, when
cloning in bacterial systems, inducible promoters such as the hybrid lacZ
promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or
pSPORT1 plasmid (Life Technologies) and the like can be used. The
baculovirus polyhedrin promoter can be used in insect cells. Promoters or
enhancers derived from the genomes of plant cells (e.g., heat shock,
RUBISCO, and storage protein genes) or from plant viruses (e.g., viral
promoters or leader sequences) can be cloned into the vector. In mammalian
cell systems, promoters from mammalian genes or from mammalian viruses are
preferable. If it is necessary to generate a cell line that contains
multiple copies of a nucleotide sequence encoding a human antibody,
vectors based on SV40 or EBV can be used with an appropriate selectable
Large scale production of human TIMP-1 antibodies can be carried out using
methods such as those described in Wurm et al., Ann. N.Y. Acad. Sci. 782,
70 78, 1996, and Kim et al., Biotechnol. Bioengineer. 58, 73 84, 1998.
Any of the human TIMP-1 antibodies described above can be provided in a
pharmaceutical composition comprising a pharmaceutically acceptable
carrier. The pharmaceutically acceptable carrier preferably is non-pyrogenic.
The compositions can be administered alone or in combination with at least
one other agent, such as stabilizing compound, which can be administered
in any sterile, biocompatible pharmaceutical carrier, including, but not
limited to, saline, buffered saline, dextrose, and water. A variety of
aqueous carriers may be employed, e.g., 0.4% saline, 0.3% glycine, and the
like. These solutions are sterile and generally free of particulate
matter. These solutions may be sterilized by conventional, well known
sterilization techniques (e.g., filtration). The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and buffering
agents, etc. The concentration of the antibody of the invention in such
pharmaceutical formulation can vary widely, i.e., from less than about
0.5%, usually at or at least about 1% to as much as 15 or 20% by weight
and will be selected primarily based on fluid volumes, viscosities, etc.,
according to the particular mode of administration selected. See U.S. Pat.
No. 5,851,525. If desired, more than one type of human antibody, for
example with different K.sub.d for TIMP-1 binding or with different
IC.sub.50s for MMP-inhibiting activity neutralization, can be included in
a pharmaceutical composition.
The compositions can be administered to a patient alone, or in combination
with other agents, drugs or hormones. In addition to the active
ingredients, these pharmaceutical compositions can contain suitable
pharmaceutically-acceptable carriers comprising excipients and auxiliaries
that facilitate processing of the active compounds into preparations which
can be used pharmaceutically. Pharmaceutical compositions of the invention
can be administered by any number of routes including, but not limited to,
oral, intravenous, intramuscular, intra-arterial, intramedullary,
intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal,
intranasal, parenteral, topical, sublingual, or rectal means.
After pharmaceutical compositions have been prepared, they can be placed
in an appropriate container and labeled for treatment of an indicated
condition. Such labeling would include amount, frequency, and method of
Methods of Decreasing MMP-Inhibiting Activity of Human TIMP-1
The invention provides methods of decreasing an MMP-inhibiting activity of
human or rat TIMP-1. Such methods can be used therapeutically, as
described below, or in a research setting. Thus, the methods can be
carried out in a cell-free system, in a cell culture system, or in vivo.
In vivo methods of decreasing MMP-inhibiting activity of human or rat
TIMP-1 are described below.
Human TIMP-1 is contacted with a human antibody that binds to the human
TIMP-1, thereby decreasing the MMP-inhibiting activity of the human TIMP-1
relative to human TIMP-1 activity in the absence of the antibody. The
antibody can be added directly to the cell-free system, cell culture
system, or to an animal subject or patient, or can be provided by means of
an expression vector encoding the antibody.
The invention also provides diagnostic methods, with which human or rat
TIMP-1 can be detected in a test preparation, including without limitation
a sample of serum, lung, liver, heart, kidney, colon, a cell culture
system, or a cell-free system (e.g., a tissue homogenate). Such diagnostic
methods can be used, for example, to diagnose disorders in which TIMP-1 is
elevated. Such disorders include, but are not limited to, liver fibrosis,
alcoholic liver disease, cardiac fibrosis, acute cardiac syndrome, lupus
nephritis, glomerulosclerotic renal disease, benign prostate hypertrophy,
lung cancer, colon cancer, and idiopathic pulmonary fibrosis. When used
for diagnosis, detection of an amount of the antibody-TIMP-1 complex in a
test sample from a patient which is greater than an amount of the complex
in a normal sample identifies the patient as likely to have the disorder.
The test preparation is contacted with a human antibody of the invention,
and the test preparation is then assayed for the presence of an
antibody-TIMP-1 complex. If desired, the human antibody can comprise a
detectable label, such as a fluorescent, radioisotopic, chemiluminescent,
or enzymatic label, such as horseradish peroxidase, alkaline phosphatase,
Optionally, the antibody can be bound to a solid support, which can
accommodate automation of the assay. Suitable solid supports include, but
are not limited to, glass or plastic slides, tissue culture plates,
microtiter wells, tubes, silicon chips, or particles such as beads
(including, but not limited to, latex, polystyrene, or glass beads). Any
method known in the art can be used to attach the antibody to the solid
support, including use of covalent and non-covalent linkages, passive
absorption, or pairs of binding moieties attached to the antibody and the
solid support. Binding of TIMP-1 and the antibody can be accomplished in
any vessel suitable for containing the reactants. Examples of such vessels
include microtiter plates, test tubes, and microcentrifuge tubes.
The invention also provides methods of ameliorating symptoms of a disorder
in which TIMP-1 is elevated. These disorders include, without limitation,
liver fibrosis alcoholic liver disease, cardiac fibrosis, acute coronary
syndrome, lupus nephritis, glomerulosclerotic renal disease, idiopathic
pulmonary fibrosis, benign prostate hypertrophy, lung cancer, colon
cancer, and scarring. See, e.g., Inokubo et al., Am. Heart J. 141, 211 17,
2001; Ylisirnio et al., Anticancer Res. 20, 1311 16, 2000; Holten-Andersen
et al., Clin. Cancer Res. 6, 4292 99, 2000; Holten-Andersen et al., Br. J.
Cancer 80, 495 503, 1999; Peterson et al., Cardiovascular Res. 46, 307 15,
2000; Arthur et al., Alcoholism: Clinical and Experimental Res. 23, 840
43, 1999; Iredale et al., Hepatol. 24, 176 84, 1996.
Human antibodies of the invention are particularly useful for treating
liver fibrosis. All chronic liver diseases cause the development of
fibrosis in the liver. Fibrosis is a programmed uniform wound healing
response. Toxic damage or injury caused by foreign proteins cause the
deposition of extracellular matrix such as collagen, fibronectin, and
laminin. Liver fibrosis and cirrhosis can be caused by chronic
degenerative diseases of the liver such as viral hepatitis, alcohol
hepatitis, autoimmune hepatitis, primary biliary cirrhosis, cystic
fibrosis, hemochromatosis, Wilson's disease, and non-alcoholic steato-hepatitis,
as well as chemical damage.
Altered degradation and synthesis of extracellular matrix (particularly
collagens) play central roles in pathogenesis of liver fibrosis. In the
early phases, hepatic stellate cells (HSC) are initially activated and
release matrix metalloproteases with the ability to degrade the normal
liver matrix. When HSC are fully activated, there is a net down-regulation
of matrix degradation mediated by increased synthesis and extracellular
release of tissue inhibitors of metalloprotease (TIMP)-1 and -2. The
dynamic regulation of activity of metalloproteases during liver fibrosis
makes them and their inhibitors targets for therapeutic intervention.
Human antibodies of the invention are also particularly useful for
treating lung fibrosis. Lung airway fibrosis is a hallmark of airway
remodeling in patients with chronic asthma, so human antibodies of the
invention are also particularly useful for chronic asthma. Airway
remodeling is a well-recognized feature in patients with chronic asthma.
TIMP-1 but not TIMP-2 levels were significantly higher in untreated
asthmatic subjects than in glucocorticoid-treated subjects or controls
(p<0.0001), and were far greater than those of MMP-1, MMP-2, MMP-3, and
MMP-9 combined (Mautino et al., Am J Respir Crit Care Med 1999 160:324
330). TIMP-1 mRNA and protein expression are selectively and markedly
increased in a murine model of bleomycin-induced pulmonary fibrosis (Am.
J. Respir. Cell Mol. Biol. 24:599 607, 2001). This specific elevation of
TIMP-1 without increase in MMPs in asthma patients suggests that
inhibition of TIMP-1 by an antibody can restore normal collagen
degradation in the lung.
Human antibodies of the invention are also particularly useful for
treating cancer. TIMP-1 protein has been found to be elevated in plasma of
colon (Holten-Andersen et al., Br J Cancer 1999, 80:495 503) and prostate
(Jung et al., Int J Cancer, 1997, 74:220 223) cancer patients, and high
TIMP-1 plasma level correlates with poor clinical outcome of colon cancer
(Holten-Andersen et al., Clin Cancer Res 2000 6:4292 4299). TIMP-1 induces
dose-dependent proliferation of breast tumorigenic clonal cell line and
tyrosine phosphorylation (Luparello et al, Breast Cancer Res Treat, 1999,
54:235 244). Therefore, the use of antibody against TIMP-1 may block its
ability to induce cancer.
Human TIMP-1 antibodies can be used to prevent or diminish scar formation,
such as scar formation after surgery (particularly ophthalmic surgery) or
injury (such as a burn, scrape, crush, cut or tear injury).
In one embodiment of the invention, a therapeutically effective dose of a
human antibody of the invention is administered to a patient having a
disorder in which TIMP-1 is elevated, such as those disorders described
above. Symptoms of the disorder, including deposition of extracellular
matrix, as well as loss of tissue or organ function, are thereby
Determination of a Therapeutically Effective Dose
The determination of a therapeutically effective dose is well within the
capability of those skilled in the art. A therapeutically effective dose
refers to that amount of human antibody that reduces MMP-inhibiting
activity of the TIMP-1 relative to the activity which occurs in the
absence of the therapeutically effective dose.
The therapeutically effective dose can be estimated initially either in
cell culture assays or in animal models, usually rats, mice, rabbits,
dogs, or pigs. The animal model also can be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes for
administration in humans. A rat liver fibrosis model is described in
Therapeutic efficacy and toxicity, e.g., ED.sub.50 (the dose
therapeutically effective in 50% of the population) and LD.sub.50 (the
dose lethal to 50% of the population) of a human antibody, can be
determined by standard pharmaceutical procedures in cell cultures or
experimental animals. The dose ratio of toxic to therapeutic effects is
the therapeutic index, and it can be expressed as the ratio,
Pharmaceutical compositions that exhibit large therapeutic indices are
preferred. The data obtained from cell culture assays and animal studies
is used in formulating a range of dosage for human use. The dosage
contained in such compositions is preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no toxicity. The
dosage varies within this range depending upon the dosage form employed,
sensitivity of the patient, and the route of administration.
The exact dosage will be determined by the practitioner, in light of
factors related to the patient who requires treatment. Dosage and
administration are adjusted to provide sufficient levels of the human
antibody or to maintain the desired effect. Factors that can be taken into
account include the severity of the disease state, general health of the
subject, age, weight, and gender of the subject, diet, time and frequency
of administration, drug combination(s), reaction sensitivities, and
tolerance/response to therapy. Long-acting pharmaceutical compositions can
be administered every 3 to 4 days, every week, or once every two weeks
depending on the half-life and clearance rate of the particular
Polynucleotides encoding human antibodies of the invention can be
constructed and introduced into a cell either ex vivo or in vivo using
well-established techniques including, but not limited to,
transferrin-polycation-mediated DNA transfer, transfection with naked or
encapsulated nucleic acids, liposome-mediated cellular fusion,
intracellular transportation of DNA-coated latex beads, protoplast fusion,
viral infection, electroporation, "gene gun," and DEAE- or calcium
Effective in vivo dosages of an antibody are in the range of about 5 mg to
about 50 mg/kg, about 50 mg to about 5 mg/kg, about 100 mg to about 500
mg/kg of patient body weight, and about 200 to about 250 mg/kg of patient
body weight. For administration of polynucleotides encoding the
antibodies, effective in vivo dosages are in the range of about 100 ng to
about 200 ng, 500 ng to about 50 mg, about 1 mg to about 2 mg, about 5 mg
to about 500 mg, and about 20 mg to about 100 mg of DNA.
The mode of administration of human antibody-containing pharmaceutical
compositions of the invention can be any suitable route which delivers the
antibody to the host. Pharmaceutical compositions of the invention are
particularly useful for parenteral administration, i.e., subcutaneous,
intramuscular, intravenous, or intranasal administration.
Claim 1 of 12 Claims
1. A purified human antibody,
wherein the antibody: binds to a tissue inhibitor of metalloprotease-1 (TIMP-I);
neutralizes a matrix metalloprotease (MMP)-inhibiting activity of the
TIMP-1; and comprises: a VHCDR1 region comprising an amino acid sequence as
set forth in SEQ ID NO:355; a VHCDR2 region comprising an amino acid
sequence as set forth in SEQ ID NO:359; a VHCDR3 region comprising an amino
acid sequence as set forth in SEQ ID NO:360; a VLCDR1 region comprising an
amino acid sequence as set forth in SEQ ID NO:363; a VLCDR2 region
comprising an amino acid sequence as set forth in SEQ ID NO:364; and a
VLCDR3 region comprising an amino acid sequence as set forth in SEQ ID
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