U.S. patent application number 13/160444 was filed with the patent office on 2012-03-15 for monoclonal antibodies to hepatocyte growth factor.
This patent application is currently assigned to Galaxy Biotech, LLC. Invention is credited to Kyung Jin Kim, Yi-Chi Su.
Application Number | 20120064066 13/160444 |
Document ID | / |
Family ID | 35320045 |
Filed Date | 2012-03-15 |
United States Patent
Application |
20120064066 |
Kind Code |
A1 |
Kim; Kyung Jin ; et
al. |
March 15, 2012 |
Monoclonal Antibodies to Hepatocyte Growth Factor
Abstract
The present invention is directed toward a neutralizing
monoclonal antibody to hepatocyte growth factor, a pharmaceutical
composition comprising same, and methods of treatment comprising
administering such a pharmaceutical composition to a patient.
Inventors: |
Kim; Kyung Jin; (Cupertino,
CA) ; Su; Yi-Chi; (San Francisco, CA) |
Assignee: |
Galaxy Biotech, LLC
Cupertino
CA
|
Family ID: |
35320045 |
Appl. No.: |
13/160444 |
Filed: |
June 14, 2011 |
Related U.S. Patent Documents
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12342059 |
Dec 22, 2008 |
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13160444 |
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11710009 |
Feb 23, 2007 |
7494650 |
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12342059 |
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10917915 |
Aug 13, 2004 |
7220410 |
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11710009 |
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10825060 |
Apr 15, 2004 |
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10917915 |
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60464061 |
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Current U.S.
Class: |
424/133.1 ;
424/145.1; 435/336; 530/387.3; 530/388.15; 530/388.23 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 43/00 20180101; C07K 2317/73 20130101; A61P 1/16 20180101;
C07K 2317/76 20130101; A61P 35/04 20180101; C07K 2317/92 20130101;
G01N 2333/4753 20130101; A61K 2039/505 20130101; C07K 16/22
20130101 |
Class at
Publication: |
424/133.1 ;
530/388.23; 530/387.3; 530/388.15; 435/336; 424/145.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 5/07 20100101 C12N005/07; A61P 35/04 20060101
A61P035/04; C07K 16/22 20060101 C07K016/22 |
Claims
1. A monoclonal antibody (mAb) that binds and neutralizes human
Hepatocyte Growth Factor (HGF).
2. The mAb of claim 1 which is chimeric.
3. The mAb of claim 1 which is humanized.
4. The mAb of claim 1 which is human.
5. The mAb of claim 1 which inhibits binding of HGF to cMet by at
least 50%.
6. The mAb of claim 1 which inhibits HGF-induced scattering of
Madin-Darby canine kidney cells.
7. The mAb of claim 1 which inhibits HGF-induced angiogenesis.
8. The mAb of claim 1 which neutralizes all biological activities
of HGF.
9. The mAb of claim 1 which inhibits growth of a human tumor
xenograft in a mouse.
10. The mAb of claim 9 which completely inhibits growth of a human
tumor xenograft in a mouse.
11. The mAb of claim 1 which is a Fab or F(ab').sub.2 fragment or
single-chain antibody.
12. The mAb of claim 1 which specifically binds HGF with a binding
affinity of at least 10.sup.8 M.sup.-1.
13. A chimeric or humanized L2G7 mAb.
14. An antibody that competes for binding to human HGF with an
antibody of claim 13.
15. A cell line producing a mAb of claim 1.
16. A pharmaceutical composition comprising a mAb of claim 13.
17. A method of treating cancer in a patient comprising
administering to the patient a pharmaceutical composition
comprising a neutralizing anti-HGF mAb.
18. A method of claim 17 wherein said cancer is glioblastoma.
19. The method of claim 17 wherein said mAb is a chimeric or
humanized L2G7 mAb.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 10/825,060 filed Apr. 15, 2004, which claims
the benefit of the provisional application U.S. Patent Application
No. 60/464,061 filed Apr. 18, 2003, both of which are incorporated
by reference in their entirety for all purposes.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the combination
of monoclonal antibody (mAb) and recombinant DNA technologies for
developing novel biologics, and more particularly, for example, to
the production of monoclonal antibodies that bind to and neutralize
Hepatocyte Growth Factor.
BACKGROUND OF THE INVENTION
[0003] Human Hepatocyte Growth Factor (HGF) is a multifunctional
heterodimeric polypeptide produced by mesenchymal cells. HGF has
been shown to stimulate angiogenesis, morphogenesis and
motogenesis, as well as the growth and scattering of various cell
types (Bussolino et al., J. Cell. Biol. 119: 629, 1992; Zarnegar
and Michalopoulos, J. Cell. Biol. 129:1177, 1995; Matsumoto et al.,
Ciba. Found. Symp. 212:198, 1997; Birchmeier and Gherardi, Trends
Cell. Biol. 8:404, 1998; Xin et al. Am. J. Pathol. 158:1111, 2001).
The pleiotropic activities of HGF are mediated through its
receptor, a transmembrane tyrosine kinase encoded by the
proto-oncogene cMet. In addition to regulating a variety of normal
cellular functions, HGF and its receptor c-Met have been shown to
be involved in the initiation, invasion and metastasis of tumors
(Jeffers et al., J. Mol. Med. 74:505, 1996; Comoglio and Trusolino,
J. Clin. Invest. 109:857, 2002). HGF/cMet are coexpressed, often
over-expressed, on various human solid tumors including tumors
derived from lung, colon, rectum, stomach, kidney, ovary, skin,
multiple myeloma and thyroid tissue (Prat et al., Int. J. Cancer
49:323, 1991; Chan et al., Oncogene 2:593, 1988; Weidner et al.,
Am. J. Respir. Cell. Mol. Biol. 8:229, 1993; Derksen et al., Blood
99:1405, 2002). HGF acts as an autocrine (Rong et al., Proc. Natl.
Acad. Sci. USA 91:4731, 1994; Koochekpour et al., Cancer Res.
57:5391, 1997) and paracrine growth factor (Weidner et al., Am. J.
Respir. Cell. Mol. Biol. 8:229, 1993) and anti-apoptotic regulator
(Gao et al., J. Biol. Chem. 276:47257, 2001) for these tumors.
[0004] HGF is a 102 kDa protein with sequence and structural
similarity to plasminogen and other enzymes of blood coagulation
(Nakamura et al., Nature 342:440, 1989; Weidner et al., Am. J.
Respir. Cell. Mol. Biol. 8:229, 1993, each of which is incorporated
herein by reference) (FIG. 1). Human HGF is synthesized as a 728
amino acid precursor (preproHGF), which undergoes intracellular
cleavage to an inactive, single chain form (proHGF) (Nakamura et
al., Nature 342:440, 1989; Rosen et al., J. Cell. Biol. 127:1783,
1994). Upon extracellular secretion, proHGF is cleaved to yield the
biologically active disulfide-linked heterodimeric molecule
composed of an .alpha.-subunit and .beta.-subunit (Nakamura et al.,
Nature 342:440, 1989; Naldini et al., EMBO J. 11:4825, 1992). The
.alpha.-subunit contains 440 residues (69 kDa with glycosylation),
consisting of the N-terminal hairpin domain and four kringle
domains. The .beta.-subunit contains 234 residues (34 kDa) and has
a serine protease-like domain, which lacks proteolytic activity.
Cleavage of HGF is required for receptor activation, but not for
receptor binding (Hartmann et al., Proc. Natl. Acad. Sci. USA
89:11574, 1992; Lokker et al., J. Biol. Chem. 268:17145, 1992). HGF
contains 4 putative N-glycosylation sites, 1 in the .alpha.-subunit
and 3 in the .beta.-subunit. HGF has 2 unique cell specific binding
sites: a high affinity (Kd=2.times.10.sup.-10 M) binding site for
the cMet receptor and a low affinity (Kd=10.sup.-9 M) binding site
for heparin sulfate proteoglycans (HSPG), which are present on the
cell surface and extracellular matrix (Naldini et al., Oncogene
6:501, 1991; Bardelli et al., J. Biotechnol. 37:109, 1994; Sakata
et al., J. Biol. Chem., 272:9457, 1997). NK2 (a protein
encompassing the N-terminus and first two kringle domains of the
.alpha.-subunit) is sufficient for binding to cMet and activation
of the signal cascade for motility, however the full length protein
is required for the mitogenic response (Weidner et al., Am. J.
Respir. Cell. Mol. Biol. 8:229, 1993). HSPG binds to HGF by
interacting with the N terminus of HGF (Aoyama, et al., Biochem.
36:10286, 1997; Sakata, et al., J. Biol. Chem. 272:9457, 1997).
Postulated roles for the HSPG-HGF interaction include the
enhancement of HGF bioavailability, biological activity and
oligomerization (Bardelli, et al., J. Biotechnol. 37:109,1994;
Zioncheck et al., J. Biol. Chem. 270:16871, 1995).
[0005] cMet is a member of the class IV protein tyrosine kinase
receptor family. The full length cMet gene was cloned and
identified as the cMet proto-oncogene (Cooper et al., Nature
311:29, 1984; Park et al., Proc. Natl. Acad. Sci. USA 84:6379,
1987). The cMet receptor is initially synthesized as a single
chain, partially glycosylated precursor, p170.sup.(MET) (FIG. 1)
(Park et al., Proc. Natl. Acad. Sci. USA 84:6379, 1987; Giordano et
al., Nature 339:155, 1989; Giordano et al., Oncogene 4:1383, 1989;
Bardelli et al., J. Biotechnol. 37:109, 1994). Upon further
glycosylation, the protein is proteolytically cleaved into a
heterodimeric 190 kDa mature protein (1385 amino acids), consisting
of the 50 kDa .alpha.-subunit (residues 1-307) and the 145 kDa
.beta.-subunit. The cytoplasmic tyrosine kinase domain of the
.beta.-subunit is involved in signal transduction.
[0006] Several different approaches have been investigated to
obtain an antagonistic molecule of the HGF/cMet interaction:
truncated HGF proteins such as NK1 (N terminal domain plus kringle
domain 1; Lokker et al., J. Biol. Chem. 268:17145, 1993), NK2 (N
terminal domain plus kringle domains 1 and 2; Chan et al., Science
254:1382, 1991) and NK4 (N-terminal domain plus four kringle
domains; Kuba et al., Cancer Res. 60:6737, 2000), anti-cMet mAbs
(Dodge, Master's Thesis, San Francisco State University, 1998) and
anti-HGF mAbs (Cao et al., Proc. Natl. Acad. Sci. USA 98:7443,
2001, which is incorporated herein by reference).
[0007] NK1 and NK2 can compete effectively with the binding of HGF
to its receptor, but have been shown to have partial agonistic
activities in vitro (Cioce et al., J. Biol. Chem. 271:13110, 1996;
Schwall et al., J. Cell Biol. 133:709, 1996), rather than purely
antagonist activities as desired. More recently, Kuba et al.,
Cancer Res. 60:6737, 2000, demonstrated that NK4 could partially
inhibit the primary growth (FIG. 2) and metastasis of murine lung
tumor LLC in a nude mouse model by continuous infusion of NK4. The
fact that NK4 had to administered continuously to obtain a partial
growth inhibition of primary tumors indicates a potentially short
half-life of the NK4 molecule and/or lack of potency. Compared to
NK4, the approach of using antibodies will benefit from their
favorable pharmacokinetics and the possibility of obtaining
antibodies with much higher potency.
[0008] As another approach, Dodge (Master's Thesis, San Francisco
State University, 1998) generated antagonistic anti-cMet monoclonal
antibodies (mAbs). One mAb, 5D5, exhibited strong antagonistic
activity in ELISA, but induced a proliferative response of
cMet-expressing BAF-3 cells, presumably due to dimerization of the
membrane receptors. Prat et al., J. Cell Sci. 111:237, 1998, also
reported such agonistic activities of anti-cMet mAbs. Zaccolo et
al., Eur. J. Immunol 27:618, 1997, used phage display methods do
develop human Fab fragments against mouse and human hepatocyte
growth factor. These Fab fragments had no effect on the activity of
HGF when used alone. When one of the anti-human HGF Fab fragments
was combined with an antibody that bound to the Fab fragment
itself, it actually enhanced the activity of HGF in a biological
assay.
[0009] Cao et al., Proc. Natl. Acad. Sci. USA 98:7443, 2001,
demonstrated that the administration of a cocktail of three
anti-HGF mAbs, which were selected based upon their ability to
inhibit the scattering activity of HGF in vitro, were able to
inhibit the growth of human tumors in the xenograft nude mouse
model (FIG. 3). They postulated that three mAbs recognizing three
different binding sites on HGF were required to inhibit the
bioactivities of HGF in vivo: two mAbs inhibited the binding of HGF
to cMet and one mAb inhibited the binding of HGF to heparin.
However, it is impractical for commercial and regulatory reasons to
develop a drug combining three novel mAbs, e.g., because some
clinical activity of each antibody would need to be demonstrated
independently.
[0010] Thus, there is a need for a single monoclonal antibody that
blocks biological activity of HGF in vitro and in vivo. The present
invention fulfills this and other needs.
BRIEF SUMMARY OF THE INVENTION
[0011] In one embodiment, the invention provides a neutralizing mAb
to human Hepatocyte Growth Factor (HGF). The mAb inhibits at least
one, and preferably several or all biological activities of HGF
including binding to its receptor cMet, inducing scattering of
cells such as Madin-Darby canine kidney cells, inducing
proliferation of 4MBr-5 monkey epithelial cells and/or hepatocytes
and/or HUVEC, and inducing angiogenesis. The Anti-HGF mAb can
inhibit such an activity when used as a single agent. A preferred
anti-HGF mAb inhibits, most preferably completely inhibits, growth
of a human tumor xenograft in a mouse. Preferably, the mAb of the
invention is chimeric, humanized, human-like or human. Exemplary
antibodies are L2G7 and its chimeric and humanized forms. Cell
lines producing such antibodies are also provided. In another
embodiment, a pharmaceutical composition comprising a neutralizing
anti-HGF antibody, e.g., chimeric or humanized L2G7, is provided.
In a third embodiment, the pharmaceutical composition is
administered to a patient to treat cancer or other disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1. Schematic models of HGF and cMet.
[0013] FIG. 2. Graph showing that NK4 partially inhibits the
primary growth of murine lung tumor LLC in nude mice (from Kuba et
al., Cancer Res. 60:6737, 2000). NK4 was infused continuously for
14 days from 4.sup.th day after tumor implantation s.c. in nude
mice.
[0014] FIG. 3. Graph showing that a cocktail of three anti-HGF mAbs
is required to inhibit the growth of human brain tumor U-118 cells
in nude mice (from Cao et al., Proc. Natl. Acad. Sci. USA 98:7443,
2001). U-118 tumor cells were injected s.c. into nude mice. From
day 1 anti-HGF mAbs A-1, -5, and -7, or mAbs 7-2 and -3 were
administered at 200 .mu.g/injection, twice/wk for 10 wks.
[0015] FIG. 4. Determination of relative binding epitopes of mAbs
L1H4, L2C7, L2G7 using competitive binding ELISA. Plates were
coated with recombinant HGF (rHGF), blocked with skim milk and
incubated with suboptimal concentration of biotinylated mAbs in the
presence of 100.times. excess amounts of unlabeled mAbs.
Biotinylated mAb bound was detected by the addition of
HRP-Strepavidin.
[0016] FIG. 5. Binding of anti-HGF mAbs to rHGF as determined in a
direct HGF binding ELISA. Plate was coated with the HI-F11
supernatant containing rHGF, blocked by 2% skim milk and incubated
with mAbs, followed by the addition of HRP-G.alpha.MIgG (as
described under Examples).
[0017] FIG. 6. Abilities of anti-HGF mAbs to capture rHGF-Flag in
solution. Anti-HGF mAbs were captured on a goat anti-mouse IgG
coated ELISA plate. Plates were then blocked with 2% skim milk and
incubated with rHGF-Flag, followed by HRP-M2 anti-Flag mAb (as
described under Examples).
[0018] FIG. 7. Inhibition of rHGF-Flag binding to cMet-Fc by
anti-HGF mAbs in a capture ELISA. cMet-Fc captured on goat
anti-human IgG coated plate is incubated with HGF-Flag preincubated
with/without mAbs. The bound rHGF-Flag was detected by the addition
of HRP-M2 anti-Flag mAb (as described under Examples).
[0019] FIG. 8. Neutralization of HGF induced MDCK scattering by
anti-HGF mAb L2G7. (A) Control without any treatment. (B) rHGF+IgG.
(C) rHGF+mAb L2G7. MDCK cells were incubated with a 1:20 dilution
of H1-F11 culture supernatant (.about.3 .mu.g/ml of HGF) in the
presence of 10 .mu.g/ml of mAbs. Photos were taken at 100.times.
magnification.
[0020] FIG. 9. Inhibition of HGF-induced proliferation of My 1 LU
cells by L2G7 mAb. The fold molar excess of mAb over HGF is shown
on the horizontal axis, and the cpm.times.10.sup.-2 incorporated is
shown on the vertical axis. Data points were obtained in
triplicate.
[0021] FIG. 10. Inhibition of HGF-induced proliferation of HUVEC by
L2G7 mAb and control mouse antibody (mIgG). Data points were
obtained in triplicate.
[0022] FIG. 11. Effect on HGF-induced proliferation of HCT 116
colon tumor cells by L2G7 and L1H4 antibodies. Data point were
obtained in triplicate.
[0023] FIG. 12. Effect of treatment with L2G7 mAb or PBS (control)
on growth of U-118 tumors in groups of NIH III Beige/Nude mice
(n=6). Arrow indicates when injections began. (A) Tumor size vs day
from tumor implantation. (B) Tumor mass at end of experiment.
DETAILED DESCRIPTION OF THE INVENTION
[0024] The invention provides neutralizing anti-HGF monoclonal
antibodies, pharmaceutical compositions comprising them, and
methods of using them for the treatment of disease.
1. Antibodies
[0025] Antibodies are very large, complex molecules (molecular
weight of .about.150,000 or about 1320 amino acids) with intricate
internal structure. A natural antibody molecule contains two
identical pairs of polypeptide chains, each pair having one light
chain and one heavy chain. Each light chain and heavy chain in turn
consists of two regions: a variable ("V") region involved in
binding the target antigen, and a constant ("C") region that
interacts with other components of the immune system. The light and
heavy chain variable regions fold up together in 3-dimensional
space to form a variable region that binds the antigen (for
example, a receptor on the surface of a cell). Within each light or
heavy chain variable region, there are three short segments
(averaging 10 amino acids in length) called the complementarity
determining regions ("CDRs"). The six CDRs in an antibody variable
domain (three from the light chain and three from the heavy chain)
fold up together in 3-D space to form the actual antibody binding
site which locks onto the target antigen. The position and length
of the CDRs have been precisely defined. Kabat, E. et al.,
Sequences of Proteins of Immunological Interest, U.S. Department of
Health and Human Services, 1983, 1987. The part of a variable
region not contained in the CDRs is called the framework, which
forms the environment for the CDRs.
[0026] A monoclonal antibody (mAB) is a single molecular species of
antibody and therefore does not encompass polyclonal antibodies
produced by injecting an animal (such as a rodent, rabbit or goat)
with an antigen, and extracting serum from the animal. A humanized
antibody is a genetically engineered (monoclonal) antibody in which
the CDRs from a mouse antibody ("donor antibody", which can also be
rat, hamster or other similar species) are grafted onto a human
antibody ("acceptor antibody"). Humanized antibodies can also be
made with less than the complete CDRs from a mouse antibody (e.g.,
Pascalis et al., J. Immunol. 169:3076, 2002). Thus, a humanized
antibody is an antibody having CDRs from a donor antibody and
variable region framework and constant regions from a human
antibody. Thus, typically a humanized antibody comprises (i) a
light chain comprising three CDRs from a mouse antibody, e.g.,
L2G7, a variable region framework from a human antibody, and a
human constant region, and (ii) a heavy chain comprising three CDRs
from a mouse antibody, e.g., L2G7, a variable region framework from
a human antibody and a human constant region. In addition, in order
to retain high binding affinity, at least one of two additional
structural elements can be employed. See, U.S. Pat. Nos. 5,530,101
and 5,585,089, each of which is incorporated herein by reference,
which provide detailed instructions for construction of humanized
antibodies.
[0027] In the first structural element, the framework of the heavy
chain variable region of the humanized antibody is chosen to have
maximal sequence identity (between 65% and 95%) with the framework
of the heavy chain variable region of the donor antibody, by
suitably selecting the acceptor antibody from among the many known
human antibodies. Sequence identity is determined when antibody
sequences being compared are aligned according to the Kabat
numbering convention. In the second structural element, in
constructing the humanized antibody, selected amino acids in the
framework of the human acceptor antibody (outside the CDRs) are
replaced with corresponding amino acids from the donor antibody, in
accordance with specified rules. Specifically, the amino acids to
be replaced in the framework are chosen on the basis of their
ability to interact with the CDRs. For example, the replaced amino
acids can be adjacent to a CDR in the donor antibody sequence or
within 4-6 angstroms of a CDR in the humanized antibody as measured
in 3-dimensional space.
[0028] A chimeric antibody is an antibody in which the variable
region of a mouse (or other rodent) antibody is combined with the
constant region of a human antibody; their construction by means of
genetic engineering is well-known. Such antibodies retain the
binding specificity of the mouse antibody, while being about
two-thirds human. The proportion of nonhuman sequence present in
mouse, chimeric and humanized antibodies suggests that the
immunogenicity of chimeric antibodies is intermediate between mouse
and humanized antibodies. Other types of genetically engineered
antibodies that may have reduced immunogenicity relative to mouse
antibodies include human antibodies made using phage display
methods (Dower et al., WO91/17271; McCafferty et al., WO92/001047;
Winter, WO92/20791; and Winter, FEBS Lett. 23:92, 1998, each of
which is incorporated herein by reference) or using transgenic
animals (Lonberg et al., WO93/12227; Kucherlapati WO91/10741, each
of which is incorporated herein by reference).
[0029] As used herein, the term "human-like" antibody refers to a
Mab in which a substantial portion of the amino acid sequence of
one or both chains (e.g., about 50% or more) originates from human
immunoglobulin genes. Hence, human-like antibodies encompass but
are not limited to chimeric, humanized and human antibodies. As
used herein, a "reduced-immunogenicity" antibody is one expected to
have significantly less immunogenicity than a mouse antibody when
administered to human patients. Such antibodies encompass chimeric,
humanized and human antibodies as well as antibodies made by
replacing specific amino acids in mouse antibodies that may
contibute to B- or T-cell epitopes, for example exposed residues
(Padlan, Mol. Immunol. 28:489, 1991). As used herein, a
"genetically engineered" antibody is one for which the genes have
been constructed or put in an unnatural environment (e.g., human
genes in a mouse or on a bacteriophage) with the help of
recombinant DNA techniques, and would therefore, e.g., not
encompass a mouse mAb made with conventional hybridoma
technology.
[0030] The epitope of a mAb is the region of its antigen to which
the mAb binds. Two antibodies bind to the same or overlapping
epitope if each competitively inhibits (blocks) binding of the
other to the antigen. That is, a 1.times., 5.times., 10.times.,
20.times. or 100.times. excess of one antibody inhibits binding of
the other by at least 50% but preferably 75%, 90% or even 99% as
measured in a competitive binding assay compared to a control
lacking the competing antibody (see, e.g., Junghans et al., Cancer
Res. 50:1495, 1990, which is incorporated herein by reference).
Alternatively, two antibodies have the same epitope if essentially
all amino acid mutations in the antigen that reduce or eliminate
binding of one antibody reduce or eliminate binding of the other.
Two antibodies have overlapping epitopes if some amino acid
mutations that reduce or eliminate binding of one antibody reduce
or eliminate binding of the other.
2. Neutralizing Anti-HGF Antibodies
[0031] A monoclonal antibody (mAb) that binds HGF (i.e., an
anti-HGF mAb) is said to neutralize HGF, or be neutralizing, if the
binding partially or completely inhibits one or more biological
activities of HGF (i.e., when the mAb is used as a single agent).
Among the biological properties of HGF that a neutralizing antibody
may inhibit are the ability of HGF to bind to its cMet receptor, to
cause the scattering of certain cell lines such as Madin-Darby
canine kidney (MDCK) cells; to stimulate proliferation of (i.e., be
mitogenic for) certain cells including hepatocytes, 4MBr-5 monkey
epithelial cells, and various human tumor cells; or to stimulate
angiogenesis, for example as measured by stimulation of human
vascular endothelial cell (HUVEC) proliferation or tube formation
or by induction of blood vessels when applied to the chick embryo
chorioallantoic membrane (CAM). Antibodies of the invention
preferably bind to human HGF, i.e., to the protein encoded by the
GenBank sequence with Accession number D90334 (incorporated by
reference).
[0032] A neutralizing mAb of the invention at a concentration of,
e.g., 0.01, 0.1, 0.5, 1, 2, 5, 10, 20 or 50 .mu.g/ml will inhibit a
biological function of HGF (e.g., stimulation of proliferation or
scattering) by about at least 50% but preferably 75%, more
preferably by 90% or 95% or even 99%, and most preferably
approximately 100% (essentially completely) as assayed by methods
described under Examples or known in the art. Inhibition is
considered complete if the level of activity is within the margin
of error for a negative control lacking HGF. Typically, the extent
of inhibition is measured when the amount of HGF used is just
sufficient to fully stimulate the biological activity, or is 0.05,
0.1, 0.5, 1, 3 or 10 .mu.g/ml. Preferably, at least 50%, 75%, 90%,
or 95% or essentially complete inhibition will be achieved when the
molar ratio of antibody to HGF is 0.5.times., 1.times., 2.times.,
3.times., 5.times. or 10.times.. Preferably, the mAb will be
neutralizing, i.e., inhibit the biological activity, when used as a
single agent, but possible 2 mAbs will be needed together to give
inhibition. Most preferably, the mAb will neutralize not just one
but several of the biological activities listed above; for purposes
herein, an anti-HGF mAb that used as a single agent neutralizes all
the biological activities of HGF will be called "fully
neutralizing", and such mAbs are most preferable. MAbs of the
invention will preferably be specific for HGF, that is they will
not bind, or only bind to a much lesser extent (e.g., Ka at least
ten-fold less), proteins that are related to HGF such as fibroblast
growth factor (FGF) and vascular endothelial growth factor (VEGF).
Preferred antibodies lack agonistic activity toward HGF. That is,
the antibodies block interaction of HGH with cMet without
stimulating cells bearing HGF directly. MAbs of the invention
typically have a binding affinity (K.sub.a) for HGF of at least
10.sup.7 M.sup.-1 but preferably 10.sup.8 M.sup.-1 or higher, and
most preferably 10.sup.9 M.sup.-1 or higher or even 10.sup.10
M.sup.-1 or higher.
[0033] MAbs of the invention include anti-HGF antibodies in their
natural tetrameric form (2 light chains and 2 heavy chains) and may
be of any of the known isotypes IgG, IgA, IgM, IgD and IgE and
their subtypes, i.e., human IgG1, IgG2, IgG3, IgG4 and mouse IgG1,
IgG2a, IgG2b, and IgG3. The mAbs of the invention are also meant to
include fragments of antibodies such as Fv, Fab and F(ab').sub.2;
bifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J.
Immunol. 17:105, 1987), single-chain antibodies (Huston et al.,
Proc. Natl. Acad. Sci. USA 85:5879, 1988; Bird et al., Science
242:423, 1988); and antibodies with altered constant regions (e.g.,
U.S. Pat. No. 5,624,821). The mAbs may be of animal (e.g., mouse,
rat, hamster or chicken) origin, or they may be genetically
engineered. Rodent mAbs are made by standard methods well-known in
the art, comprising multiple immunization with HGF in appropriate
adjuvant i.p., i.v., or into the footpad, followed by extraction of
spleen or lymph node cells and fusion with a suitable immortalized
cell line, and then selection for hybridomas that produce antibody
binding to HGF, e.g., see under Examples. Chimeric and humanized
mAbs, made by art-known methods mentioned supra, are preferred
embodiments of the invention. Human antibodies made, e.g., by phage
display or transgenic mice methods are also preferred (see e.g.,
Dower et al., McCafferty et al., Winter, Lonberg et al.,
Kucherlapati, supra). More generally, human-like, reduced
immunogenicity and genetically engineered antibodies as defined
herein are all preferred.
[0034] The neutralizing anti-HGF mAbs L1H4, L2C7 and L2G7 mAbs
described infra are examples of the invention, with L2G7 a
preferred example. Neutralizing mAbs with the same or overlapping
epitope as any of these mAbs, e.g., as L2G7, provide other
examples. A chimeric or humanized form of L2G7 or with LGF is an
especially preferred embodiment. A mAb (including chimeric,
humanized and human antibodies) that competes with L2G7 for binding
to HGF and neutralizes HGF in at least one, and preferably all, in
vitro or in vivo assays described herein is also preferred. MAbs
that are 90%, 95%, 99% or 100% identical (determined by aligning
antibody sequences according to the Kabat convention) to L2G7 in
amino acid sequence, at least in the CDRs are included in the
invention. Preferably such antibodies differ from L2G7 by a small
number of functionally inconsequential amino acid substitutions
(e.g., conservative substitutions), deletions, or insertions.
Preferably such antibodies retain the functional properties of
L2G7, i.e., such antibodies neutralize HGF in at least one, and
preferably all, in vitro or in vivo assays described herein. For
purposes of classifying amino acids substitutions as conservative
or nonconservative, amino acids may be grouped as follows: Group I
(hydrophobic sidechains): norleucine, met, ala, val, leu, ile;
Group II (neutral hydrophilic side chains): cys, ser, thr; Group
III (acidic side chains): asp, glu; Group IV (basic side chains):
asn, gln, his, lys, arg; Group V (residues influencing chain
orientation): gly, pro; and Group VI (aromatic side chains): trp,
tyr, phe. Conservative substitutions involve substitutions between
amino acids in the same class. Non-conservative substitutions
constitute exchanging a member of one of these classes for a member
of another.
[0035] Native mAbs of the invention may be produced from their
hybridomas. Genetically engineered mAbs, e.g., chimeric or
humanized mAbs, may be expressed by a variety of art-known methods.
For example, genes encoding their light and heavy chain V regions
may be synthesized from overlapping oligonucleotides and inserted
together with available C regions into expression vectors (e.g.,
commercially available from Invitrogen) that provide the necessary
regulatory regions, e.g., promoters, enhancers, poly A sites, etc.
Use of the CMV promoter-enhancer is preferred. The expression
vectors may then be transfected using various well-known methods
such as lipofection or electroporation into a variety of mammalian
cell lines such as CHO or non-producing myelomas including Sp2/0
and NS0, and cells expressing the antibodies selected by
appropriate antibiotic selection. See, e.g., U.S. Pat. No.
5,530,101. Larger amounts of antibody may be produced by growing
the cells in commercially available bioreactors.
[0036] Once expressed, the mAbs or other antibodies of the
invention may be purified according to standard procedures of the
art such as microfiltration, ultrafiltration, protein A or G
affinity chromatography, size exclusion chromatography, anion
exchange chromatography, cation exchange chromatography and/or
other forms of affinity chromatography based on organic dyes or the
like. Substantially pure antibodies of at least about 90 or 95%
homogeneity are preferred, and 98% or 99% or more homogeneity most
preferred, for pharmaceutical uses.
3. Therapeutic Methods
[0037] In a preferred embodiment, the present invention provides a
pharmaceutical formulation comprising the antibodies described
herein. That is, the antibodies can be used in the manufacture of a
medicament for treatment of disease. Pharmaceutical formulations
(i.e., medicaments) of the antibodies contain the mAb in a
physiologically acceptable carrier, optionally with excipients or
stabilizers, in the form of lyophilized or aqueous solutions.
Acceptable carriers, excipients or stabilizers are nontoxic to
recipients at the dosages and concentrations employed, and include
buffers such as phosphate, citrate, or acetate at a pH typically of
5.0 to 8.0, most often 6.0 to 7.0; salts such as sodium chloride,
potassium chloride, etc. to make isotonic; antioxidants,
preservatives, low molecular weight polypeptides, proteins,
hydrophilic polymers such as polysorbate 80, amino acids,
carbohydrates, chelating agents, sugars, and other standard
ingredients known to those skilled in the art (Remington's
Pharmaceutical Science 16.sup.th edition, Osol, A. Ed. 1980). The
mAb is typically present at a concentration of 1-100 mg/ml, e.g.,
10 mg/ml.
[0038] Antibodies of the invention are typically substantially pure
from undesired contaminant. This means that the antibody is
typically at least about 50% w/w (weight/weight) pure, as well as
being substantially free from interfering proteins and
contaminants. Preferably the antibodies are at least 90, 95% or 99%
w/w pure. Pharmaceutical compositions for parenteral administration
are usually sterile, substantially isotonic and prepared in
accordance with Good Manufacturing Practices of the FDA or similar
body.
[0039] In another preferred embodiment, the invention provides a
method of treating a patient with a disease using an anti-HGF mAb
in a pharmaceutical formulation. The mAb prepared in a
pharmaceutical formulation can be administered to a patient by any
suitable route, especially parentally by intravenous infusion or
bolus injection, intramuscularly or subcutaneously. Intravenous
infusion can be given over as little as 15 minutes, but more often
for 30 minutes, or over 1, 2 or even 3 hours. The mAb can also be
injected directly into the site of disease (e.g., a tumor), or
encapsulated into carrying agents such as liposomes. The dose given
will be sufficient to alleviate the condition being treated
("therapeutically effective dose") and is likely to be 0.1 to 5
mg/kg body weight, for example 1, 2, 3 or 4 mg/kg, but may be as
high as 10 mg/kg or even 15 or 20 mg/kg. A fixed unit dose may also
be given, for example, 50, 100, 200, 500 or 1000 mg, or the dose
may be based on the patient's surface area, e.g., 100 mg/m.sup.2.
Usually between 1 and 8 doses, (e.g., 1, 2, 3, 4, 5, 6, 7 or 8) are
administered to treat cancer, but 10, 20 or more doses may be
given. The mAb can be administered daily, biweekly, weekly, every
other week, monthly or at some other interval, depending, e.g. on
the half-life of the mAb, for 1 week, 2 weeks, 4 weeks, 8 weeks,
3-6 months or longer. Repeated courses of treatment are also
possible, as is chronic administration. A regime of a dosage and
intervals of administration that alleviates or at least partially
arrests the symptoms of the disease (biochemical, histologic and/or
clinical), including its complications and intermediate
pathological phenotypes in development of the disease is referred
to as a therapeutically effective regime.
[0040] The pharmaceutical compositions of the invention can also be
used in prophylaxis of a patient at risk of cancer. Such patients
include those having genetic susceptibility to cancer, patients who
have undergone exposure to carcinogenic agents, such as radiation
or toxins, and patients who have undergone previous treatment for
cancer and are at risk of recurrence. A prophylactic dosage is an
amount sufficient to eliminate or reduce the risk, lessen the
severity, or delay the outset of the disease, including
biochemical, histologic and/or clinical symptoms of the disease,
its complications and intermediate pathological phenotypes
presenting during development of the disease. Administration of a
pharmaceutical composition in an amount and at intervals effective
to effect one or more of these objects is referred to as a
prophylactically effective regime.
[0041] Diseases especially susceptible to therapy with the anti-HGF
mAbs of this invention include solid tumors known or suspected to
require angiogenesis or to be associated with elevated levels of
HGF, for example ovarian cancer, breast cancer, lung cancer (small
cell or non-small cell), colon cancer, prostate cancer, pancreatic
cancer, renal cancer, gastric cancer, liver cancer, head-and-neck
tumors, melanoma, sarcomas, and brain tumors (e.g., glioblastomas),
of children or adults. Treatment can also be administered to
patients having leukemias or lymphomas. In a preferred embodiment,
the anti-HGF mAb can be administered together with (i.e., before,
during or after) other anti-cancer therapy. For example, the
anti-HGF mAb may be administered together with any one or more of
the chemotherapeutic drugs known to those of skill in the art of
oncology, for example Taxol (paclitaxel) or its derivatives,
platinum compounds such as carboplatin or cisplatin, anthrocyclines
such as doxorubicin, alkylating agents such as cyclophosphamide,
anti-metabolites such as 5-fluorouracil, or etoposide. The anti-HGF
mAb can be administered in combination with two, three or more of
these agents in a standard chemotherapeutic regimen, for example
taxol and carboplatin, e.g. for breast and ovarian cancer. Other
agents with which the anti-HGF mAb can be administered include
biologics such as monoclonal antibodies, including Herceptin.TM.
against the HER2 antigen, Avastin.TM. against VEGF, or antibodies
to the EGF receptor, as well as small molecule anti-angiogenic or
EGF receptor antagonist drugs. In addition, the anti-HGF mAb can be
used together with radiation therapy or surgery.
[0042] Treatment (e.g., standard chemotherapy) including the
anti-HGF mAb antibody may increase the median progression-free
survival or overall survival time of patients with these tumors
(e.g., ovarian, breast, lung, pancreas, brain and colon, especially
when relapsed or refractory) by at least 30% or 40% but preferably
50%, 60% to 70% or even 100% or longer, compared to the same
treatment (e.g., chemotherapy) but without anti-HGF mAb. In
addition or alternatively, treatment (e.g., standard chemotherapy)
including the anti-HGF mAb may increase the complete response rate,
partial response rate, or objective response rate
(complete+partial) of patients with these tumors (e.g., ovarian,
breast, lung, pancreas, brain and colon, especially when relapsed
or refractory) by at least 30% or 40% but preferably 50%, 60% to
70% or even 100% compared to the same treatment (e.g.,
chemotherapy) but without the anti-HGF mAb. Optionally, treatment,
can inhibit tumor invasion, or metastasis.
[0043] Typically, in a clinical trial (e.g., a phase II, phase
II/III or phase III trial), the aforementioned increases in median
progression-free survival and/or response rate of the patients
treated with chemotherapy plus the anti-HGF mAb, relative to the
control group of patients receiving chemotherapy alone (or plus
placebo), will be statistically significant, for example at the p
=0.05 or 0.01 or even 0.001 level. It will also be understood by
one of skill that the complete and partial response rates are
determined by objective criteria commonly used in clinical trials
for cancer, e.g., as listed or accepted by the National Cancer
Institute and/or Food and Drug Administration.
4. Other Methods
[0044] The anti-HGF mAbs of the invention also find use in
diagnostic, prognostic and laboratory methods. They may be used to
measure the level of HGF in a tumor or in the circulation of a
patient with a tumor, and therefore to follow and guide treatment
of the tumor. For example, a tumor associated with high levels of
HGF would be especially susceptible to treatment with an anti-HGF
mAb. In particular embodiments, the mAbs can be used in an ELISA or
radioimmunoassay to measure the level of HGF, e.g., in a tumor
biopsy specimen or in serum or in media supernatant of
HGF-secreting cells in cell culture. The use of two anti-HGF mAbs
binding to different epitopes (i.e., not competing for binding)
will be especially useful in developing a sensitive "sandwich"
ELISA to detect HGF. For various assays, the mAb may be labeled
with fluorescent molecules, spin-labeled molecules, enzymes or
radioisotypes, and may be provided in the form of kit with all the
necessary reagents to perform the assay for HGF. In other uses, the
anti-HGF mAbs will be used to purify HGF, e.g., by affinity
chromatography.
EXAMPLES
1. Generation of Anti-HGF mAbs
[0045] To generate mAbs which bind to and block the activities of
human HGF, recombinant human HGF (rHGF) was first produced in a
mammalian expression system. cDNAs encoding the recombinant human
HGF (rHGF) or rHGF-Flag peptide (8 amino acid residues of Flag
attached to the c-terminus of HGF) were constructed in a
pIND-inducible expression vector (No et al., Proc. Natl. Acad. Sci.
USA. 93:3346, 1996). These cDNAs were then transfected into EcR-293
human kidney fibroblast cells (Invitrogen) using Fugene
transfection reagent (Roche). Stable cell lines, H1-F11 and 24.1,
secreting HGF and HGF-Flag respectively, were selected in the
presence of 600 .mu.g/ml of G418 and 400 .mu.g/ml of Zeocin
(Invitrogen). H1-F11 and 24.1 were induced to secrete HGF and
HGF-Flag by treatment with 4 .mu.M of Ponasterone A (Invitrogen)
for 4-5 days in serum free DMEM containing glutamine and
antibiotics. After aggregates were removed by centrifugation at
15,000 rpm for 30 min at 4.degree. C., HGF secreted into the
culture supernatant was concentrated approximately 100-fold using a
membrane ultrafiltration cartridge with an MW 50,000 cut-off filter
[amicon Centriprep YM-50 filter followed by microcon YM-50 filter
(Millipore)]. Such concentrated H1F11 culture supernatant contains
.about.100 .mu.g/ml of HGF and .about.120 .mu.g/ml of bovine serum
albumin.
[0046] Balb/c mice were immunized in each hind foot pad >10
times at one week intervals, with 1-2 .mu.g of purified rHGF (Pepro
Tech) or 1-2 .mu.g of rHGF plus 1-2 .mu.g of BSA (concentrated
H1-F11 culture supernatant) resuspended in MPL-TDM (Ribi
Immunochem. Research). Three days after the final boost, popliteal
lymph node cells were fused with murine myeloma cells, P3X63AgU.1
(ATCC CRL1597), using 35% polyethylene glycol. Hybridomas were
selected in HAT medium as described (Chuntharapai and Kim, J.
Immunol. 163:766, 1997, which is incorporated herein by reference).
Ten days after the fusion, hybridoma culture supernatants were
screened in a direct HGF binding ELISA as well as in an HGF-Flag
capture ELISA. The latter assay was used to further confirm the
specificity of anti-HGF mAbs selected using the direct HGF binding
ELISA and to select mAbs that can bind to HGF in solution phase.
Blocking activities of selected mAbs were then determined in the
HGF-Flag/cMet-Fc binding ELISA and in the MDCK scatter assay as
described (Jeffers et al., Proc. Natl. Acad. Sci. USA 95:14417,
1998). Selected hybridomas were cloned twice using limiting
dilution techniques. The isotype of mAbs were determined using an
isotyping kit (Zymed). Ascites of selected mAbs were raised and
purified using ImmunoPure (A/G) IgG Purification Kit (Pierce). Also
biotinylated mAbs were prepared using EZ-sulfo-NHS-LC-Biotin
according to the instructions provided by Pierce. Each of the
assays referred to here is described in more detail below. For the
direct HGF binding ELISA, microtiter plates (Maxisorb; Nunc) are
coated with 50 .mu.l/well of H1-F11 culture supernatants containing
rHGF, diluted in PBS at a 1:2 ratio of HGF/PBS, overnight at
4.degree. C. After washing the plate, the nonspecific binding sites
are blocked with PBS containing 2% skim milk for 1 hr at room
temperature (RT). After washing the plate, 50 .mu.l/well of
purified mAbs or hybridoma culture supernatants are added to each
well for 1 hr. After washing, plates are then incubated with 50
.mu.l/well of 1 .mu.g/ml of HRP-goat anti-mouse IgG
(HRP-G.alpha.MIgG, Cappel) for 1 hr. The bound HRP-G.alpha.MIgG is
detected by the addition of the tetramethylbenzidine substrate
(Sigma). The reaction is stopped by the addition of 1N
H.sub.2SO.sub.4 and the plates are then read at 450 nm using an
ELISA plate reader. Washes are carried out 3 times in wash buffer
(PBS containing 0.05% Tween 20).
[0047] For the HGF-Flag capture ELISA, microtiter plates are coated
with 50 .mu.l/well of 2 .mu.g/ml of goat antibodies specific to the
Fc portion of mouse IgG (G.alpha.MIgG-Fc) in PBS overnight at
4.degree. C. and blocked with 2% skim milk for 1 hr at RT. After
washing, the plates are incubated with 50 .mu.l/well of purified
mAbs or hybridoma culture supernatants for 1 hr. After washing,
plates are then incubated with 50 .mu.l/well of 24.1 cell culture
supernatant containing rHGF-Flag. After washing, plates are then
incubated with 50 82 l/well of HRP-M2 anti-Flag mAb (Invitrogen) in
the presence of 15 .mu.g/ml of murine IgG. The bound HRP-anti-Flag
M2 is detected by the addition of the substrate as described above.
Washes are carried out 3 times in wash buffer.
[0048] At least three mAbs, designated L1H4, L2C7 and L2G7,
obtained from hybridomas generated by immunizing the Balb/c mice
with rHGF in concentrated H1-F11 culture supernatant as described
above, showed binding in both the direct rHGF binding ELISA and the
HGF-Flag capture ELISA and were selected for further study. These
hybridomas were then cloned twice, ascites were raised in mice by
standard methods, and mAbs were purified using a protein G/A
column. Their isotypes were determined using an isotyping kit
(Zymed Lab). The L2G7 hybridoma has been deposited on Apr. 29, 2003
with the American Type Culture Collection, P.O. Box 1549 Manassas,
Va. 20108, as ATCC Number PTA-5162 under the Budapest Treaty. These
deposit will be maintained at an authorized depository and replaced
in the event of mutation, nonviability or destruction for a period
of at least five years after the most recent request for release of
a sample was received by the depository, for a period of at least
thirty years after the date of the deposit, or during the
enforceable life of the related patent, whichever period is
longest. All restrictions on the availability to the public of
these cell lines will be irrevocably removed upon the issuance of a
patent from the application.
[0049] Once a single, archtypal anti-human-HGF mAb, for example
L2G7, has been isolated that has the desired properties described
herein of neutralizing HGF in vitro and/or inhibiting (e.g.,
completely) tumor growth in vivo, it is straightforward to generate
other mAbs with similar properties, by using art-known methods. For
example, mice may be immunized with HGF as described above,
hybridomas produced, and the resulting mAbs screened for the
ability to compete with the archtypal mAb for binding to HGF.
Alternatively, the method of Jespers et al., Biotechnology 12:899,
1994, which is incorporated herein by reference, may be used to
guide the selection of mAbs having the same epitope and therefore
similar properties to the archtypal mAb, e.g., L2G7. Using phage
display, first the heavy chain of the archtypal antibody is paired
with a repertoire of (preferably human) light chains to select an
HGF-binding mAb, and then the new light chain is paired with a
repertoire of (preferably human) heavy chains to select a
(preferably human) HGF-binding mAb having the same epitope as the
archtypal mAb.
2. Characterization of Anti-HGF mAbs In Vitro
[0050] The binding epitopes of the antibodies were partially
characterized by a competitive binding ELISA in which a 100.times.
excess of unlabeled mAb was used to compete with the binding of the
same or another biotinylated mAb in the HGF binding ELISA. FIG. 4
shows that the binding of the anti-HGF mAbs, L1H4 and L2G7, was
inhibited only by themselves, suggesting that they recognize unique
epitopes. The binding of L2C7 was inhibited by L2G7 but not by
L1H4. This suggests that the L2C7 epitope overlaps with that of
L2G7 but not of L1H4. However, L2C7 was not able to inhibit the
binding of L2G7, suggesting that the L2C7 and L2G7 epitopes overlap
but are distinct, and/or the affinity of L2C7 is much lower than
that of L2G7. The epitopes of L1H4, L2C7 and L2G7 are respectively
designated A, B and C.
[0051] The relative binding abilities of the three anti-HGF mAbs
were measured using purified antibodies in the direct HGF binding
ELISA, in which rHGF is first bound to the plate. In this assay,
L2C7 and L2G7 bound better than L1H4 (FIG. 5). The ability of the
mAbs to bind rHGF-Flag in solution was also determined, using the
HGF-Flag capture ELISA. All three mAbs were able to capture
rHGF-Flag in solution phase but mAb L2G7 was more effective than
the others (FIG. 6). These results suggest that mAb L2G7 has the
highest binding affinity to HGF among the three mAbs.
[0052] One of the biological activities of HGF is the ability to
bind to its receptor cMet, so the ability of the anti-HGF mAbs to
inhibit binding of HGF to cMET was assayed. For this assay, cMet-Fc
was first produced by transfecting human fibroblast 293 cells with
cDNA encoding residues 1-929 ECD of cMet linked with the Fc portion
of human IgG1 (residues 216 to 446) as described by Mark et al., J.
Biol. Chem. 267:26166, 1992 in the pDisplay expression vector
(Invitrogen). Microtiter plates are coated with 50 .mu.l/well of 2
.mu.g/ml of goat antibodies specific to the Fc portion of human IgG
(G.alpha.HIgG-Fc) in PBS overnight at 4.degree. C. and blocked with
2% BSA for 1 hr at RT. After washing the plates, 50 .mu.l of
culture supernatant of 293 transfected with cMet-Fc cDNA is added
to each well for 1 hr at RT. After washing the plates, 50
.mu.l/well of 24.1 cell culture supernatant containing rHGF-Flag,
preincubated with various concentrations of mAbs, is added to each
well for 1 hr. After washing, plates are then incubated with 50
.mu.l/well of HRP-M2 anti-Flag mAb (Invitrogen). The bound
HRP-anti-Flag M2 is detected by the addition of the substrate as
described above. Washes are carried out 3 times in wash buffer.
[0053] In this HGF-Flag/cMet-Fc binding inhibition assay, all three
mAbs demonstrated some degrees of inhibition while an Ig control
antibody did not (FIG. 7). MAb L2G7 at .gtoreq.1 .mu.g/ml and mAb
L1H4 at 50 .mu.g/ml completely abolished the binding of rHGF-Flag
to cMet-Fc; mAb L2C7 even at 50 .mu.g/ml gave only 85% inhibition.
Hence, mAb L2G7 was much more potent in inhibiting the interaction
of rHGF-Flag with cMet-Fc (and therefore presumably HGF with its
receptor cMet) than the other antibodies, consistent with its
putatively greater affinity for HGF.
[0054] Since the receptor protein used in cMet-Fc/HGF-Flag binding
ELISA is a soluble receptor protein, its conformation may be
different from that of the natural membrane bound receptor.
Furthermore, HGF binds to HSPG in addition to cMet and it is known
that the HSPG-HGF interaction enhances various HGF activities.
Thus, mAbs blocking the interaction of HGF with soluble cMet may
not necessarily have the capacity to neutralize HGF bioactivities
on the cells. Thus, it is important to further confirm the blocking
activities of mAbs in selected biological systems. HGF is known to
be a potent scattering factor. Thus, the neutralizing activity of
the anti-HGF mAbs was also determined using the Madin-Darby canine
kidney (MDCK cells obtained from ATCC) scatter assay as described
(Jeffers et al., Proc. Natl. Acad. Sci. USA 95:14417, 1998). MDCK
cells grown in DMEM supplemented with 5% FCS are plated at 10.sup.3
cells/100 .mu.l/well in the presence of predetermined
concentrations of rHGF with or without mAbs in DMEM with 5% FCS.
After 2 days incubation at 37.degree. C. in 5% CO.sub.2, cells are
then washed in PBS, fixed in 2% formaldehyde for 10 min at RT.
After washing in PBS cells are stained with 0.5% crystal violet in
50% ethanol (v/v) for 10 min at RT. Scattering activity is
determined by microscopic examination.
[0055] Culture supernatant of the H1-F11 clone secreting HGF,
described above, was used as the source of HGF in the scatter
assay. As little as 1:80 dilution of H1-F11 culture supernatant
induced the scattering and growth of MDCK cells. However, the
scattering assays were carried out using a 1:20 dilution of H1-F11
culture supernatant (.about.3 .mu.g/ml). MAb L2G7 even at a 1:5
molar ratio of HGF/mAb inhibited the HGF induced scattering of MDCK
by itself (FIG. 8), conclusively demonstrating that mAb L2G7 is
indeed a neutralizing mAb. mAb L1H4 at .gtoreq.20 .mu.g/ml could
also neutralize scattering of MDCK induced by HGF, while mAb L2C7
even at 20 .mu.g/ml gave only a partial neutralizing activity (data
not shown).
[0056] The various characteristics of the three anti-HGF antibodies
determined in the assays above are summarized in the Table 1.
TABLE-US-00001 TABLE 1 Characterization of mAbs to HGF Binding
Block Block mAb Isotype Epitope HGF/cMet-Fc binding MDCK scattering
L1H4 G1, .kappa. A Weak Block + L2C7 G2b, .kappa. B Partial Block
+/- L2G7 G2a, .kappa. C Strong Block +++
[0057] HGF is a member of the heparin binding growth factor family
including fibroblast growth factor (FGF) and vascular endothelial
growth factor (VEGF). Also, HGF has .about.40% overall sequence
similarity with plasminogen (Nakamura et al., Nature. 342:440,
1989) and shares a similar domain structure with macrophage
stimulating protein (MSF, Wang et al., Scand. J. Immunol. 56:545,
2002). Thus, the binding specificity of the anti-HGF antibodies
must be determined. The binding of anti-HGF mAbs to these HGF
related proteins (available from R&D systems) is assayed using
a direct binding ELISA similar to the one for HGF described above.
MAb L2G7, mAb L2C7 and mAb L1H4 will not significantly bind to
these proteins, demonstrating their specificity for HGF.
3. Ability of Anti-HGF MAbs to Inhibit Tumor-Promoting Biological
Activities of HGF
[0058] HGF has a number of biological activities that make it
likely that it plays a role in the growth and invasiveness of
certain human tumors. One such activity of HGF is as a powerful
mitogen for hepatocytes and other epithelial cells (Rubin et al.,
Proc. Natl. Acad. Sci. USA. 88:415, 1991). Thus, to further prove
the neutralizing activity of the anti-HGF mAbs, the effects of the
mAbs on the HGF-induced proliferation of 4MBr-5 monkey epithelial
cells (ATCC) or rat hepatocytes are determined. Hepatocytes are
isolated according to a method described by Garrison and Haynes, J.
Biol. Chem. 269:4264, 1985. Cells are resuspended at
5.times.10.sup.4 cells/ml in DMEM containing 5% FCS and stimulated
with a predetermined concentration of HGF with various
concentration of mAbs. After 21/2 days incubation at 37.degree. C.
in 5% CO.sub.2, the level of cell proliferation is determined by
the addition of .sup.3H-thymidine for 4 hrs. Cells are harvested
using an automated cell harvester and the level of
.sup.3H-thymidine incorporated is determined on a scintillation
counter. At sufficient concentrations, mAb L2G7 may largely or
completely inhibit HGF-induced proliferation of the cells, and mAbs
L2C7 and L1H4 may at least partially inhibit proliferation. These
antibodies may also inhibit the HGF-induced proliferation of other
epithelial cell lines.
[0059] For example, the inhibitory activity of L2G7 on the
HGF-induced proliferation of mink lung Mv 1 Lu cells was determined
(Borset et al., J. Immunol. Methods 189:59, 1996). Cells grown in
DMEM containing 10% FCS are harvested by treatment with
EDTA/trysin. After washing, the cells are resuspended at
5.times.10.sup.4 cells/ml in serum free DMEM with a predetermined
concentration (50 ng/ml) of HGF+/-various concentrations of mAb.
After 1 day incubation at 37.degree. C. in 5% CO.sub.2, the level
of cell proliferation is determined by the addition of 1 .mu.Ci of
.sup.3H-thymidine for an additional 24 hr. Cells are harvested onto
glass-fiber filters using an automated cell harvester and the level
of .sup.3H-thymidine incorporated is determined on a scintillation
counter. FIG. 9 shows that the addition of 100-fold higher molar
concentration of L2G7 mAb completely inhibited the proliferative
response of Mv 1 Lu cells. Indeed, L2G7 even at a 3-fold molar
ratio of mAb to HGF showed complete inhibition, while control IgG
showed no inhibition even at 100-fold molar excess.
[0060] HGF is also reported to be a potent angiogenesis factor
(Bussolino et al., J. Cell Biol. 119:629, 1992; Cherrington et al.,
Adv. Cancer Res. 79:1, 2000), and angiogenesis, the formation of
new blood vessels, is believed to be essential to the growth of
tumors. Therefore, the ability of the anti-HGF mAbs to inhibit the
angiogenic properties of HGF is shown in three assays: (i)
proliferation of human vascular endothelial cells (HUVEC), (ii)
tube formation of HUVEC, and (iii) development of new blood vessels
on the chick embryo chorioallantoic membrane (CAM). Since HGF has
been shown to synergize with VEGF in angiogenesis (Xin et al., Am.
J. Pathol. 158:1111, 2001), these assays may be performed both in
the presence and absence of VEGF.
[0061] The HUVEC proliferation assay is performed as described with
a modification (Conn et al., Proc. Natl. Acad. Sci. USA 87:1323,
1990). HUVEC cells obtained from Clonetics are grown in Endothelial
Growth Medium (EBM-2) containing 10% FCS plus endothelial cell
growth supplements provided by Clonetics. Preferably cells from
passages 4 to 7 are used in this study. The cells are resuspended
to be 10.sup.5 cells/ml in medium-199 containing antibiotics, 10 mM
HEPES and 10% FCS (assay medium). HUVEC cells (50 .mu.l/well) are
added to microtiter wells containing a suitable concentration of
HGF with various concentrations of anti-HGF mAbs for 1 hr at
37.degree. C. After cells are incubated for 72 hr at 37.degree. C.
in 5% CO.sub.2, the level of cell proliferation is determined by
incorporation of .sup.3H-thymidine for 4 hrs. At sufficient
concentrations, mAb L2G7 will largely or completely inhibit
HGF-induced proliferation of the HUVEC, and mAbs L2C7 and L1H4 may
at least partially inhibit proliferation.
[0062] Alternatively, the level of cell proliferation may be
determined by the well-known colorimetric MTT assay. The HUVECs
(10.sup.4 cells/100 .mu.l/well) are grown in serum free medium for
24 hr, and then incubated with 100 .mu.l of of 50 ng/ml of HGF
(predetermined to be a suboptimal amount) with various
concentrations of mAb L2G7 for 72 hr. MTT solution (5 mg/ml) is
added to each well (20 .mu.l/200 .mu.l medium) for 4 hr. Then 100
.mu.l medium/well is removed and mixed with 100 .mu.l/well of
acidified isopropyl alcohol (0.04N HCl in isopropyl). The plates
are read on an ELISA reader at 560 nm. The % maximum response is
calculated as follows: [OD of HGF+mAB treated cells-OD of untreated
cells]/[OD of HGF treated cells-OD of untreated cells].times.100.
FIG. 10 shows that even a 2-fold molar excess of L2G7 mAB largely
blocks the proliferation of HUVEC in response to HGF.
[0063] The endothelial tube assay is carried out essentially as
described (Matsumura, et al., J. Immunol. 158: 3408, 2001; Xin et
al., Am. J. Pathol. 158:1111, 2001). HUVEC (Clonetics) from passage
4-7 are grown in Clonetics EGM medium supplemented with 10% FBS and
endothelial cell growth supplements. Plates are coated with
Matrigel (BD Biosciences) according to the manufacture's
instructions at 37.degree. C. for 30 min, and the cells are seeded
as 3.times.10.sup.6 cells/ml in 1.times. basal medium with HGF and
various concentrations of anti-HGF mAbs. Tube formation is
evaluated under microscope at low-power (10.times.) magnification.
At sufficient concentrations, mAb L2G7 will largely or completely
inhibit HGF-induced endothelial tube formation, and mAbs L2C7 and
L1H4 may at least partially inhibit it.
[0064] The chick embryo chorioallantoic membrane (CAM) assay is
performed essentially as described (Kim et al, Nature 362:841,
1992). Three-day old chicken embryos are removed from their shells
and grown in petri dishes in 5% CO.sub.2 at 37.degree. C. Seven
days later, dried methylcellulose discs containing HGF with various
concentrations of anti-HGF mAbs are layered onto the CAM. The
methylcellulose discs are prepared by mixing 5 .mu.l of 1.5%
methylcellulose in PBS with 5 .mu.l of HGF preincubated with mAbs.
Three days later the development of blood vessels around
methylcellulose discs are examined. At sufficient concentrations,
mAb L2G7 will largely or completely inhibit such blood vessel
formation, and mAbs L2C7 and L1H4 may at least partially inhibit
it.
[0065] HGF is also reported to promote tumor growth (Comoglio and
Trusolino, J. Clin. Invest. 109:857, 2002). The ability of the
anti-HGF antibodies to inhibit this activity is shown in two steps.
First, a number of tumor cell lines are examined for their ability
to secrete HGF and proliferate in response to HGF since HGF may be
an autocrine growth factor for some of these cells. These cell
lines include a panel of human tumor cell lines known to express
HGF and cMet (Koochekpour et al., Cancer Res. 57:5391, 1997; Wang
et al., J. Cell Biol. 153:1023, 2001). Specific cell lines to be
tested include U-118 glioma, HCT116 colon carcinoma, A549 lung
carcinoma and A431 epidermoid carcinoma cells, all available from
the ATCC. Once such tumor cell lines are identified, the effect of
anti-HGF mAbs on the proliferative response to HGF of these cells
is determined, using methods similar to those described above. At
sufficient concentrations, mAb L2G7 will largely or completely
inhibit HGF-induced proliferation of many or all of these cell
lines, and mAbs L2C7 and L1H4 may at least partially inhibit
proliferation.
[0066] For example, human HCT116 tumor cells are seeded into
96-well microtiter plates at 5.times.10.sup.3 cells/well in 200
.mu.l of DMEM plus 5% FCS. After 24 hr incubation at 37.degree. C.
in 5% CO.sub.2, cells are washed with PBS and incubated in serum
free DMEM for 48 hr. Cells are then incubated with 100 ng/ml of
HGF+/-20 .mu.g/ml of mAbs in DMEM for another 20 hr. As controls,
cells grown in DMEM alone or DMEM plus 10% FCS are included. At the
end of the incubation, levels of cell proliferation are determined
by incorporation of .sup.3H-thymidine for 4 hr. The result of such
an experiment was carried out in triplicates is shown in FIG. 11.
HGF induced a moderate proliferation of the HCT116 cells, which was
completely abolished by addition of L2G7 antibody (but not by the
less potent L1H4 antibody).
[0067] In all the assays described above, each anti-HGF antibody
will neutralize or inhibit activity when used alone without other
antagonists of HGF, i.e., as a single agent, but additive or
synergistic effects may be achieved by administering the antibody
in conjunction with other anti-HGF antibodies or other active
agents.
4. Ability of Anti-HGF mAbs to Inhibit Tumor Growth In Vivo
[0068] The ability of the anti-HGF antibodies to inhibit human
tumor growth is demonstrated in xenograft models in immunodeficient
mice or other rodents such as rat. Illustrative but not limiting
examples of immunodeficient strains of mice that can be used are
nude mice such as CD-1 nude, Nu/Nu, Balb/c nude, NIH-III
(NIH-bg-nu-xid BR); scid mice such as Fox Chase SCID (C.B-17 SCID),
Fox Chase outbred SCID and SCID Beige; mice deficient in RAG
enzyme; as well as nude rats. Experiments are carried out as
described previously (Kim et al., Nature 362:841, 1992, which is
incorporated herein by reference). Human tumor cells grown in
complete DMEM medium are harvested in HBSS. Female immunodeficient,
e.g., athymic nude mice (4-6 wks old) are injected s.c. with
typically 5.times.10.sup.6 cells in 0.2 ml of HBSS in the dorsal
areas. When the tumor size reaches 50-100 mm.sup.3, the mice are
grouped randomly and appropriate amounts of the anti-HGF and
control mAbs (typically between 0.1 and 1.0 mg, e.g. 0.5 mg) are
administered i.p. once, twice or three times per week in a volume
of, e.g., 0.1 ml, for e.g., 1, 2, 3, or 4 weeks or the duration of
the experiment. Tumor sizes are determined typically twice a week
by measuring in two dimensions [length (a) and width (b)]. Tumor
volume is calculated according to V=ab.sup.2/2 and expressed as
mean tumor volume.+-.SEM. The number of mice in each treatment
group is at least 3, but more often between 5 and 10, e.g., 7.
Statistical analysis may be performed using, e.g., Student's t
test. In a variation of this experiment, administration of the
antibody begins simultaneously or shortly after injection of the
tumor cells. The effect of the antibody may also be measured by
prolongation of the survival of the mice, or increase in percent of
the mice surviving.
[0069] Various tumor cell lines known to secrete or respond to HGF
are used in separate experiments, for example U118 human
glioblastoma cells, and/or HCT116 human colon tumor cells.
Preferred antibodies of the invention, such as human-like and
reduced-immunogenicity antibodies and the L2G7 antibody and its
chimeric and humanized forms and antibodies with the same epitope
as L2G7, when used as a single agent, will inhibit growth of tumors
by at least 25%, but possibly 40% or 50%, and as much as 75% or 90%
or greater, or even completely inhibit tumor growth after some
period of time or cause tumor regression or disappearance. This
inhibition will take place for at least tumor cell line such as
U118 in at least one mouse strain such as NIH III Beige/Nude, but
preferably will occur for 2, 3, several, many, or even essentially
all HGF-expressing tumor cell lines of a particular (e.g., glioma)
or any type, when tested in one or more immunodeficient mouse
strains that do not generate a neutralizing antibody response
against the injected antibody. Treatment with some preferred
antibodies in one or more of the xenograft models leads to the
indefinite survival of 50%, 75%, 90% or even essentially all mice,
who would otherwise die or need to be sacrificed because of growth
of their tumor.
[0070] For example, such an experiment was performed with U-118
glioblastoma cells, grown in DMEM medium with FCS and harvested in
HBSS. Female NIH III Beige/Nude mice (4-6 wks old) are injected
s.c. with 10.sup.6 cells in 0.2 ml of HBSS in the dorsal areas.
When the tumor size reaches .about.50 mm.sup.3, the mice are
grouped randomly into 2 groups of 6 mice each, and 200 .mu.g of the
L2G7 mAb (treatment group) or of PBS (control group) are given i.p.
twice a week in a volume of 0.1 ml. Tumor sizes are determined
twice a week as described above. At the end of the experiment, the
tumors are excised and weighed. FIG. 12 shows that treatment with
L2G7 completely inhibited tumor growth.
[0071] Similar tumor inhibition experiments are performed with the
anti-HGF antibody administered in combination one or more
chemotherapeutic agents such as 5-FU (5-fluorouracil) or CPT-11
(Camptosar) to which the tumor type is expected to be responsive,
as described by Ashkenize et al., J. Clin. Invest. 104:155, 1999.
The combination of the antibody and chemotherapeutic drug may
produce a greater inhibition of \tumor growth than either agent
alone. The effect may be additive or synergistic, and strongly
inhibit growth, e.g. by 80% or 90% or more, or even cause tumor
regression or disappearance. The anti-HGF antibody may also be
administered in combination with an antibody against another growth
or angiogenic factor, for example anti-VEGF, and additive or
synergistic growth inhibition and/or tumor regression or
disappearance is expected.
[0072] Although the invention has been described with reference to
the presently preferred embodiments, it should be understood that
various modifications can be made without departing from the
invention. Unless otherwise apparent from the context any step,
element, embodiment, feature or aspect of the invention can be used
with any other.
[0073] All publications, patents and patent applications cited are
herein incorporated by reference in their entirety for all purposes
to the same extent as if each individual publication, patent and
patent application was specifically and individually indicated to
be incorporated by reference in its entirety for all purposes.
* * * * *