U.S. patent application number 13/538901 was filed with the patent office on 2013-01-03 for anti-c-met antibody formulations.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Barthelemy Demeule, Bruce Kabakoff, Jun Liu, Nicole Piros, Qing Zhu.
Application Number | 20130004484 13/538901 |
Document ID | / |
Family ID | 46513859 |
Filed Date | 2013-01-03 |
United States Patent
Application |
20130004484 |
Kind Code |
A1 |
Demeule; Barthelemy ; et
al. |
January 3, 2013 |
ANTI-C-MET ANTIBODY FORMULATIONS
Abstract
Provided herein are pharmaceutical formulations comprising a
one-armed, anti-c-met antibody and uses of the same.
Inventors: |
Demeule; Barthelemy; (San
Mateo, CA) ; Kabakoff; Bruce; (Pacifica, CA) ;
Liu; Jun; (South San Francisco, CA) ; Piros;
Nicole; (San Francisco, CA) ; Zhu; Qing; (San
Jose, CA) |
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
46513859 |
Appl. No.: |
13/538901 |
Filed: |
June 29, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61503513 |
Jun 30, 2011 |
|
|
|
Current U.S.
Class: |
424/133.1 ;
424/138.1; 435/375 |
Current CPC
Class: |
C07K 16/40 20130101;
C07K 2317/76 20130101; A61K 39/39591 20130101; C07K 16/2863
20130101; C07K 2317/56 20130101; A61P 43/00 20180101; C07K 2317/522
20130101; C07K 2317/52 20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/133.1 ;
424/138.1; 435/375 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C12N 5/09 20100101 C12N005/09; A61P 35/00 20060101
A61P035/00 |
Claims
1. A pharmaceutical formulation comprising: (a) an anti-c-met
antibody, wherein the anti-c-met antibody comprises a HVR-L1
comprising sequence KSSQSLLYTSSQKNYLA (SEQ ID NO:1), a HVR-L2
comprising sequence WASTRES (SEQ ID NO:2), a HVR-L3 comprising
sequence QQYYAYPWT (SEQ ID NO:3), a HVR-H1 comprising sequence
GYTFTSYWLH (SEQ ID NO:4), a HVR-H2 comprising sequence
GMIDPSNSDTRFNPNFKD (SEQ ID NO:5), and a HVR-H3 comprising sequence
ATYRSYVTPLDY (SEQ ID NO:6) and wherein the anti-c-met antibody
comprises a single antigen binding arm and comprises a Fc region,
wherein the Fc region comprises a first and a second Fc
polypeptide, and wherein the first and second Fc polypeptides are
present in a complex; (b) a histidine buffer at pH 5.0-5.4; (c) a
saccharide; and (d) a polysorbate, wherein the polysorbate is
present at greater than 0.02% w/v.
2. The pharmaceutical formulation of claim 1, wherein the
anti-c-met antibody comprises (a) a heavy chain variable domain
comprising the sequence:
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDT
RFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTV SS (SEQ
ID NO:19) and (b) a light chain variable domain comprising the
sequence:
DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTR
ESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR (SEQ ID
NO:20).
3. The pharmaceutical formulation of claim 2, wherein the first and
second Fc polypeptides form a Fc region that increases stability of
said antibody fragment compared to a Fab molecule comprising said
antigen binding arm.
4. The pharmaceutical formulation of any one of claim 1-3, wherein
the anti-c-met antibody comprises (a) a first polypeptide
comprising the amino acid sequence of SEQ ID NO:19, a CH1 sequence,
and a first Fc polypeptide and (b) a second polypeptide comprising
the amino acid sequence of SEQ ID NO:20 and CL1 sequence.
5. The pharmaceutical formulation of claim 4, wherein the
anti-c-met antibody further comprises (c) a third polypeptide
comprising a second Fc polypeptide.
6. The pharmaceutical formulation of any one of claims 1-5, wherein
the first Fc polypeptide comprises the Fc sequence depicted in FIG.
2 (SEQ ID NO: 17) and the second Fc polypeptide comprises the Fc
sequence depicted in FIG. 3 (SEQ ID NO: 18).
7. The pharmaceutical formulation of any one of claims 1-6, wherein
the anti-c-met antibody is onartuzumab.
8. The pharmaceutical formulation of any one of claims 1-7, wherein
the anti-c-met antibody binds the same epitope as onartuzumab.
9. The pharmaceutical formulation of any one of claims 1-8, wherein
the anti-c-met antibody is present at a concentration between about
10 mg/mL and about 100 mg/mL (e.g. about 15 mg/mL and about 75
mg/mL).
10. The pharmaceutical formulation of claim 9, wherein the
anti-c-met antibody is present at a concentration of about 60
mg/mL.
11. The pharmaceutical formulation of any one of claims 1-10,
wherein the saccharide is present at a concentration of about 75 mM
to about 200 mM (e.g., about 100 mM to about 150 mM).
12. The pharmaceutical formulation of claim 11, wherein the
saccharide is present at a concentration of about 120 mM.
13. The pharmaceutical formulation of any one of claims 1-12,
wherein the saccharide is a disaccharide.
14. The pharmaceutical formulation of claim 13, wherein the
disaccharide is trehalose.
15. The pharmaceutical formulation of claim 13, wherein the
disaccharide is sucrose.
16. The pharmaceutical formulation of any one of claims 1-15,
wherein the histidine buffer is at a concentration of about 1 mM to
about 50 mM (e.g. about 1 mM to about 25 mM).
17. The pharmaceutical formulation of claim 16, wherein the
histidine buffer is at a concentration of about 10 mM.
18. The pharmaceutical formulation of any one of claims 1-17,
wherein the histidine buffer is histidine acetate.
19. The pharmaceutical formulation of any one of claims 1-18,
wherein the polysorbate is present at a concentration greater than
0.02% and less than about 0.1%.
20. The pharmaceutical formulation of claim 19, wherein the
polysorbate is present at a concentration of about 0.04%.
21. The pharmaceutical formulation of any one of claims 1-20,
wherein the polysorbate is polysorbate 20.
22. The pharmaceutical formulation of any one of claims 1-21,
wherein the formulation is diluted with a diluent (e.g., 0.9%
NaCl).
23. The pharmaceutical formulation of claim 22, wherein the
anti-c-met antibody is present at a concentration of about 1
mg/mL.
24. A method of inhibiting c-met activated cell proliferation, said
method comprising contacting a cell or tissue with an effective
amount of the pharmaceutical formulation of any one of claims
1-23.
25. A method of modulating a disease associated with dysregulation
of the HGF/c-met signaling axis, said method comprising
administering to a subject an effective amount of the
pharmaceutical formulation of any one of claims 1-23.
26. A method of treating a subject having a proliferative disorder,
said method comprising administering to the subject an effective
amount of the pharmaceutical formulation of any one of claims
1-23.
27. The method of claim 26, wherein the proliferative disorder is
cancer.
28. The method of claim 27, wherein the cancer is lung cancer
(e.g., non-small cell lung cancer (NSCLC)), glioblastoma,
pancreatic cancer, sarcoma, renal cell carcinoma, hepatocellular
carcinoma, gastric cancer, colorectal cancer, and/or breast
cancer.
29. The method of any one of claims 24-28, further comprising a
second therapeutic agent.
30. A method of making a pharmaceutical formulation of any one of
claims 1-23.
31. An article of manufacture comprising a container with the
pharmaceutical formulation of any one of claims 1-23 contained
therein.
32. A method of making the article of manufacture of claim 31.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of priority to U.S. Patent
Application 61/503,513, filed Jun. 30, 2011, the entire contents of
which is incorporated herein by reference in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Feb. 16, 2012, is named P4690R1WO.txt and is 22,553 bytes in
size.
TECHNICAL FIELD
[0003] Provided herein are pharmaceutical formulations comprising
an anti-c-met antibody and uses of the same.
BACKGROUND
[0004] Excipients are added to pharmaceutical formulations to aid
in the stabilization of the active compound. The compatibility of
the excipients with the active compound is crucial for the quality
and stability of the pharmaceutical formulation. While excipients
are important in the stabilization of the active compound,
excipients can cause problems: the excipient may degrade and thus
lose its mechanism of stabilization or it may produce degradants
that interact with the active compound.
[0005] Pharmaceutical formulations in which the active compound is
a polypeptide, e.g. an antibody, can pose special problems as
polypeptides generally are larger and more complex than traditional
organic and inorganic molecules (for example, polypeptides possess
multiple functional groups, in addition to complex
three-dimensional structures). In addition, for a polypeptide to
remain biologically active, the pharmaceutical polypeptide
formulation must preserve intact the conformational integrity of at
least a core sequence of the polypeptide's amino acids, while at
the same time maintaining physical and chemical stability of the
pharmaceutical polypeptide formulation. Excipients are generally
stable in aqueous solution; however, excipients in a pharmaceutical
polypeptide formulation can interact with the polypeptide to
undergo degradation in a formulation and can prevent the
stabilization of the protein or the degradants could interact with
the polypeptide to pose challenges (such as a loss in activity).
Therefore, the evaluation of the interaction of the non-active
components of the pharmaceutical formulation and polypeptide active
agent is crucial for ensuring chemical and physical stability.
[0006] Polysorbates are non-ionic surfactants used to stabilize an
active compound against interface induced aggregation and surface
adsorption. Polysorbates can be effective against various stresses
such as agitation (for example, shaking or stirring),
freeze/thawing, and lyophilization. In pharmaceutical polypeptide
formulations, polysorbates minimize adsorption to surfaces and
reduce the air-liquid interfacial surface tension and thus the rate
of protein denaturation. Loss of polysorbate in the pharmaceutical
formulation can result in instability of the formulation. Further,
polysorbates can be degraded by oxidation and hydrolysis which can
lead to a decrease in the apparent concentration of polysorbate in
the pharmaceutical formulation over long shelf life. Polysorbates
(e.g., polysorbate 20) can be cleaved to produce degradants (e.g.,
free lauric acid and sorbitan polyoxyethylene side chain). These
polysorbate degradants are less surface-active than nondegraded
polysorbate and hence the chemical and physical stability of the
pharmaceutical formulation may be compromised. Further, some
polysorbate degradants are insoluble and may form particles if they
are not solubilized by intact polysorbate, i.e., if the ratio of
degraded polysorbate 20: intact polysorbate 20 is too high.
[0007] The rate and extent of degradation of polysorbate is
influenced by the chemical and physical properties of the active
compound, and the stabilizing ability of polysorbate can vary
between different pharmaceutical formulations comprising different
active compounds. Particularly since polysorbates are included in
protein formulations to stabilize the protein, the decrease in the
concentration of polysorbate and the accumulation of degradant
molecules in a pharmaceutical polypeptide formulation is of
potential concern for protein stability.
[0008] Numerous molecules targeted at the HGF/c-met pathway have
been reported. These molecules include a portion of the
extracellular domain of c-met and anti-c-met antibodies such as
those described in U.S. Pat. No. 5,686,292, Martens, T. et al.,
Clin. Cancer Res. 12 (20 Pt. 1):6144 (2006); U.S. Pat. No.
6,468,529; WO2006/015371; WO2007/063816, and WO2010/045345.
Bivalent forms of anti-c-met antibodies have been shown to promote
dimerization and lead to activation of c-met (agonistic function),
while conversely monovalent antibodies have been shown to inhibit
c-met activity (antagonistic function). For treatment of
pathological conditions requiring an antagonistic function,
bivalency of an anti-c-met antibody could result in an undesirable
agonistic effect, and therefore, the monovalent trait is required
to ensure an antagonistic activity upon binding of the anti-c-met
antibody to the target for treatment of the pathological condition.
Fab fragments and one-armed antibodies are examples of monovalent
antibodies. One-armed antibodies generally have a longer half-life
than Fabs. However, as a one-armed antibody comprises a single
light chain and a single heavy chain (as well as an additional Fc
region), if the one-armed antibody structure is not stabilized, the
polypeptides could potentially form a bivalent antibody with two
heavy chain and two light chains. Aggregation of monovalent
antibodies (formation of multimer and oligomers) and/or failure to
maintain monovalent structure in a pharmaceutical formulation
comprising anti-c-met antibodies could lead to an undesirable
agonistic effect. Minimization of anti-c-met antibody aggregation
in the pharmaceutical formulation is thus particularly important.
Therefore, despite the significant advancement in the molecules
which target the HGF/c-met pathway, stable pharmaceutical
formulations, which minimize aggregation of c-met antibodies, are
still needed.
[0009] All references cited herein, including patent applications
and publications, are incorporated by reference in their
entirety.
DISCLOSURE OF THE INVENTION
[0010] Provided herein are pharmaceutical formulations comprising
an anti-c-met antibody. In some embodiments, the pharmaceutical
formulation is a liquid pharmaceutical formulation. In some
embodiments, the pharmaceutical formulation is a stable
pharmaceutical formulation. In some embodiments, the pharmaceutical
formulation is a stable liquid pharmaceutical formulation. In some
embodiments, the anti-c-met antibody is an antagonist anti-c-met
antibody.
[0011] For example, provided herein are pharmaceutical formulations
comprising: (a) an anti-c-met antibody; (b) a histidine buffer at
pH 5.0-5.4; (c) a saccharide; and (d) a polysorbate, wherein the
polysorbate is present at greater than 0.02% w/v.
[0012] In some embodiments of any of the formulations, the
anti-c-met antibody comprising a HVR-L1 comprising sequence
KSSQSLLYTSSQKNYLA (SEQ ID NO:1), a HVR-L2 comprising sequence
WASTRES (SEQ ID NO:2), a HVR-L3 comprising sequence QQYYAYPWT (SEQ
ID NO:3), a HVR-H1 comprising sequence GYTFTSYWLH (SEQ ID NO:4), a
HVR-H2 comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO:5), and a
HVR-H3 comprising sequence ATYRSYVTPLDY (SEQ ID NO:6). In some
embodiments, the anti-c-met antibody comprises (a) a heavy chain
variable domain comprising the sequence:
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRF
NPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSS (SEQ ID
NO:19) and (b) a light chain variable domain comprising the
sequence:
DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTR
ESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR (SEQ ID
NO:20). In some embodiments, the anti-c-met antibody comprises a
single antigen binding arm and comprises a Fc region, wherein the
Fc region comprises a first and a second Fc polypeptide, and
wherein the first and second Fc polypeptides are present in a
complex. In some embodiments, the first and second Fc polypeptides
form a Fc region that increases stability of said antibody fragment
compared to a Fab molecule comprising said antigen binding arm. In
some embodiments, the anti-c-met antibody comprises (a) a first
polypeptide comprising the amino acid sequence of SEQ ID NO:19, a
CH1 sequence, and a first Fc polypeptide and (b) a second
polypeptide comprising the amino acid sequence of SEQ ID NO:20 and
CL1 sequence. In some embodiments, the anti-c-met antibody further
comprises (c) a third polypeptide comprising a second Fc
polypeptide. In some embodiments, the first Fc polypeptide
comprises the Fc sequence depicted in FIG. 2 (SEQ ID NO: 17) and
the second Fc polypeptide comprises the Fc sequence depicted in
FIG. 3 (SEQ ID NO: 18). In some embodiments, the anti-c-met
antibody is onartuzumab. In some embodiments, the anti-c-met
antibody binds the same epitope as onartuzumab.
[0013] In some embodiments of any of the formulations, the
anti-c-met antibody is present at a concentration between about 10
mg/mL and about 100 mg/mL (e.g. about 15 mg/mL and about 75 mg/mL).
In some embodiments, the anti-c-met antibody is present at a
concentration of about 60 mg/mL.
[0014] In some embodiments of any of the formulations, the
saccharide is present at a concentration of about 75 mM to about
200 mM (e.g., about 100 mM to about 150 mM). In some embodiments,
the saccharide is present at a concentration of about 120 mM. In
some embodiments, the saccharide is a disaccharide. In some
embodiments, the disaccharide is trehalose. In some embodiments,
the disaccharide is sucrose.
[0015] In some embodiments of any of the formulations, the
histidine buffer is at a concentration of about 1 mM to about 50 mM
(e.g. about 1 mM to about 25 mM). In some embodiments, the
histidine buffer is at a concentration of about 10 mM. In some
embodiments, the histidine buffer is histidine acetate.
[0016] In some embodiments of any of the formulations, the
polysorbate is present at a concentration greater than 0.02% and
less than about 0.1%. In some embodiments, the polysorbate is
present at a concentration of about 0.04%. In some embodiments, the
polysorbate is polysorbate 20.
[0017] In some embodiments of any of the formulations, the
formulation is diluted with a diluent (e.g., 0.9% NaCl). In some
embodiments, the anti-c-met antibody is present at a concentration
of about 1 mg/mL.
[0018] Provided herein are methods of inhibiting c-met activated
cell proliferation, said method comprising contacting a cell or
tissue with an effective amount of the pharmaceutical formulation
described herein (e.g., upon dilution).
[0019] Also provided herein are methods of modulating a disease
associated with dysregulation of the HGF/c-met signaling axis, said
method comprising administering to a subject an effective amount of
the pharmaceutical formulation described herein (e.g., upon
dilution).
[0020] Further provided are methods of treating a subject having a
proliferative disorder, said method comprising administering to the
subject an effective amount of the pharmaceutical formulation
described herein (e.g., upon dilution). In some embodiments, the
proliferative disorder is cancer. In some embodiments, the cancer
is lung cancer (non-small cell lung cancer (NSCLC)), glioblastoma,
pancreatic cancer, sarcoma, renal cell carcinoma, hepatocellular
carcinoma, gastric cancer, colorectal cancer, and/or breast cancer.
In some embodiments, the methods further comprise a second
therapeutic agent.
[0021] Provided are methods of making the pharmaceutical
formulation described herein.
[0022] In addition, provided herein are articles of manufacture
comprising a container with a pharmaceutical formulation described
herein (e.g., upon dilution) contained therein. Provided herein are
also methods of making the articles of manufacture comprising a
pharmaceutical formulation described herein (e.g., upon
dilution).
DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 depicts the general structures of short half-life and
long half-life agonists and antagonists of c-Met.
[0024] FIG. 2 depicts amino acid sequences of the framework (FR),
hypervariable region (HVR), first constant domain (CL or CH1) and
Fc region (Fc) of MetMAb (onartuzumab or OA5D5.v2). The Fc sequence
depicted comprises "hole" (cavity) mutations T366S, L368A and
Y407V, as described in WO 2005/063816.
[0025] FIG. 3 depicts sequence of an Fc polypeptide comprising
"knob" (protuberance) mutation T366W, as described in WO
2005/063816. In some embodiments, an Fc polypeptide comprising this
sequence forms a complex with an Fc polypeptide comprising the Fc
sequence of FIG. 1 to generate an Fc region. The sequence disclosed
in FIG. 3 represents residues 6-227 of SEQ ID NO: 18.
[0026] FIG. 4 depicts the rate of aggregate formation as indicated
by the percentage of high molecular weight species (HMWS) over time
(days) at 40.degree. C. for formulations of 20 mg/mL onartuzumab,
10 mM histidine acetate, 120 mM trehalose, and 0.02% polysorbate 20
at pH 5.2, 5.7, and 6.2.
[0027] FIG. 5 depicts the rate of aggregate formation as indicated
by the percentage of high molecular weight species (HMWS) over time
(days) at 25.degree. C. for formulations of 40 mg/mL onartuzumab,
10 mM histidine acetate, 120 mM trehalose, and 0.02% polysorbate 20
at pH 5.1, 5.4, and 5.7.
[0028] FIG. 6 depicts the rate of aggregate formation as indicated
by the percentage of high molecular weight species (HMWS) over time
(days) at 40.degree. C. for formulations of 40 mg/mL onartuzumab,
10 mM histidine acetate, 120 mM trehalose, and 0.02% polysorbate 20
at pH 5.1, 5.4, and 5.7.
[0029] FIG. 7 depicts the chemical stability as measured by
ion-exchange chromatography (IEC) as indicated by the percent main
peak over time (days) at 25.degree. C. and 40.degree. C. for
formulations of 40 mg/mL onartuzumab, 10 mM histidine acetate, 120
mM trehalose, and 0.02% polysorbate 20 at pH 5.1, 5.4, and 5.7.
[0030] FIG. 8 depicts the percentage of intact polysorbate over
time (weeks) at 40.degree. C. for formulations of 60 mg/mL
onartuzumab, 10 mM histidine acetate, pH 5.4, and 120 mM sucrose
with 0.02% polysorbate 20 or 0.04% polysorbate 20.
[0031] FIG. 9 depicts the rate of aggregate formation of
onartuzumab diluted to 1 mg/mL in IV bags containing 0.9% NaCl. The
rate of aggregation is indicated by the percentage of high
molecular weight species (HMWS) over time (hours) of agitation for
diluted formulations of (a) 60 mg/mL onartuzumab, 10 mM histidine
acetate, 120 mM trehalose, and 0.02% polysorbate 20 at pH 5.4 kept
at room temperature as shown by squares and (b) 60 mg/mL
onartuzumab, 10 mM histidine acetate, 120 mM sucrose, and 0.04%
polysorbate 20 at pH 5.4 kept at 30.degree. C. as shown by
circles.
DETAILED DESCRIPTION
[0032] Provided herein are stable pharmaceutical formulations
comprising an anti-c-met antibody. In some embodiments, the
anti-c-met antibody is an antagonist anti-c-met antibody. In some
embodiments, the anti-c-met antibody is a monovalent anti-c-met
antibody. In addition, kits and articles of manufacture comprising
the anti-c-met antibody pharmaceutical formulations and uses of the
anti-c-met antibody pharmaceutical formulations are provided.
I. DEFINITIONS
[0033] The term "pharmaceutical formulation" refers to preparations
which are in such form as to permit the biological activity of the
active compound(s) to be effective, and which contain no additional
components which are toxic to the subjects to which the formulation
is administered. "Pharmaceutically acceptable" excipients
(vehicles, additives) are those which can reasonably be
administered to a subject to provide an effective dose of the
active compound.
[0034] A "stable" formulation is one in which the polypeptide
therein essentially retains its physical stability and/or chemical
stability and/or biological activity upon storage and/or during
administration (e.g., after dilution in an IV bag). Various
analytical techniques for measuring protein stability are available
in the art and are reviewed in Peptide and Protein Drug Delivery,
247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y.,
Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90
(1993), for example. Stability can be measured at a selected
temperature for a selected time period.
[0035] A polypeptide "retains its physical stability" in a
pharmaceutical formulation if the chemical stability at a given
time is such that the protein is considered to still retain its
biological activity and an acceptable safe profile, for example, as
determined by International Conference on Harmonisation of
Technical Requirements for Registration of Pharmaceuticals for
Human Use Guidelines (e.g., commercially acceptable levels of
aggregation, precipitation and/or denaturation, for example, upon
visual examination of color and/or clarity or as measured by UV
light scattering or by size exclusion chromatography).
[0036] A polypeptide "retains its chemical stability" in a
pharmaceutical formulation, if the chemical stability at a given
time is such that the protein is considered to still retain its
biological activity. Chemical stability can be assessed by
detecting and quantifying chemically altered forms of the protein.
Chemical alteration may involve size modification (e.g., clipping)
which can be evaluated using size exclusion chromatography,
SDS-PAGE and/or matrix-assisted laser desorption
ionization/time-of-flight mass spectrometry (MALDI/TOF MS), for
example. Other types of chemical alteration include charge
alteration which can be evaluated by ion-exchange chromatography,
for example.
[0037] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0038] A "histidine buffer" is a buffer comprising histidine
ions.
[0039] A "saccharide" herein comprises the general composition
(CH.sub.2O).sub.n and derivatives thereof.
[0040] An "anti-c-met antibody" and "an antibody that binds to
c-met" refer to an antibody that is capable of binding c-met with
sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent in targeting c-met. In some
embodiments, the extent of binding of an anti-c-met antibody to an
unrelated, non-c-met protein is less than about 10% of the binding
of the antibody to c-met as measured, e.g., by a radioimmunoassay
(RIA). In some embodiments, an antibody that binds to c-met has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g., 10.sup.-8 M or less, e.g. from 10.sup.-8 M
to 10.sup.-13 M, e.g., from 10.sup.-9 M to 10.sup.-13 M). In some
embodiments, an anti-c-met antibody binds to an epitope of c-met
that is conserved among c-met from different species.
[0041] The term "antibody" is used in the broadest sense and
specifically covers monoclonal antibodies (including full length
monoclonal antibodies), polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), monovalent antibodies,
multivalent antibodies, and antibody fragments so long as they
exhibit the desired biological activity (e.g., Fab and/or
single-armed antibodies).
[0042] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain
constant domains that correspond to the different classes of
immunoglobulins are called .alpha., .delta., .epsilon., .gamma.,
and .mu., respectively.
[0043] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab').sub.2; diabodies; linear antibodies; single-chain
antibody molecules (e.g., scFv); and multispecific antibodies
formed from antibody fragments.
[0044] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0045] A "blocking" antibody or an "antagonist" antibody is one
which significantly inhibits (either partially or completely) a
biological activity of the antigen it binds.
[0046] An "antibody that binds to the same epitope" as a reference
antibody refers to an antibody that blocks binding of the reference
antibody to its antigen in a competition assay by 50% or more, and
conversely, the reference antibody blocks binding of the antibody
to its antigen in a competition assay by 50% or more. An exemplary
competition assay is provided herein.
[0047] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence changes. In some
embodiments, the number of amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
[0048] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby
Immunology, 6.sup.th ed., W.H. Freeman and Co., page 91 (2007).) A
single VH or VL domain may be sufficient to confer antigen-binding
specificity. Furthermore, antibodies that bind a particular antigen
may be isolated using a VH or VL domain from an antibody that binds
the antigen to screen a library of complementary VL or VH domains,
respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887
(1993); Clarkson et al., Nature 352:624-628 (1991).
[0049] The term "hypervariable region" or "HVR," as used herein,
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops ("hypervariable loops"). Generally, native four-chain
antibodies comprise six HVRs; three in the VH (H1, H2, H3), and
three in the VL (L1, L2, L3). HVRs generally comprise amino acid
residues from the hypervariable loops and/or from the
"complementarity determining regions" (CDRs), the latter being of
highest sequence variability and/or involved in antigen
recognition. Exemplary hypervariable loops occur at amino acid
residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55
(H2), and 96-101 (H3). (Chothia and Lesk, J. Mol. Biol. 196:901-917
(1987).) Exemplary CDRs (CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2,
and CDR-H3) occur at amino acid residues 24-34 of L1, 50-56 of L2,
89-97 of L3, 31-35B of H1, 50-65 of H2, and 95-102 of H3. (Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991).) With the exception of CDR1 in VH, CDRs generally comprise
the amino acid residues that form the hypervariable loops. CDRs
also comprise "specificity determining residues," or "SDRs," which
are residues that contact antigen. SDRs are contained within
regions of the CDRs called abbreviated-CDRs, or a-CDRs. Exemplary
a-CDRs (a-CDR-L1, a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and
a-CDR-H3) occur at amino acid residues 31-34 of L1, 50-55 of L2,
89-96 of L3, 31-35B of H1, 50-58 of H2, and 95-102 of H3. (See
Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).) Unless
otherwise indicated, HVR residues and other residues in the
variable domain (e.g., FR residues) are numbered herein according
to Kabat et al., supra.
[0050] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0051] The phrase "N-terminally truncated heavy chain", as used
herein, refers to a polypeptide comprising parts but not all of a
full length immunoglobulin heavy chain, wherein the missing parts
are those normally located on the N terminal region of the heavy
chain. Missing parts may include, but are not limited to, the
variable domain, CH1, and part or all of a hinge sequence.
Generally, if the wild type hinge sequence is not present, the
remaining constant domain(s) in the N-terminally truncated heavy
chain would comprise a component that is capable of linkage to
another Fc sequence (i.e., the "first" Fc polypeptide as described
herein). For example, said component can be a modified residue or
an added cysteine residue capable of forming a disulfide
linkage.
[0052] The term "Fc region", as used herein, generally refers to a
dimer complex comprising the C-terminal polypeptide sequences of an
immunoglobulin heavy chain, wherein a C-terminal polypeptide
sequence is that which is obtainable by papain digestion of an
intact antibody. The Fc region may comprise native or variant Fc
sequences. Although the boundaries of the Fc sequence of an
immunoglobulin heavy chain may vary, the human IgG heavy chain Fc
sequence is usually defined to stretch from an amino acid residue
at about position Cys226, or from about position Pro230, to the
carboxyl-terminus of the Fc sequence. However, the C-terminal
lysine (Lys447) of the Fc sequence may or may not be present. The
Fc sequence of an immunoglobulin generally comprises two constant
domains, a CH2 domain and a CH3 domain, and optionally comprises a
CH4 domain. By "Fc polypeptide" herein is meant one of the
polypeptides that make up an Fc region. An Fc polypeptide may be
obtained from any suitable immunoglobulin, such as IgG1, IgG2,
IgG3, or IgG4 subtypes, IgA, IgE, IgD or IgM. In some embodiments,
an Fc polypeptide comprises part or all of a wild type hinge
sequence (generally at its N terminus). In some embodiments, an Fc
polypeptide does not comprise a functional or wild type hinge
sequence.
[0053] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. In some embodiments, an FcR is a
native human FcR. In some embodiments, an FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of those
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (see, e.g., Daeron, Annu.
Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example,
in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et
al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab.
Clin. Med. 126:330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR"
herein.
[0054] The term "Fc receptor" or "FcR" also includes the neonatal
receptor, FcRn, which is responsible for the transfer of maternal
IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim
et al., J. Immunol. 24:249 (1994)) and regulation of homeostasis of
immunoglobulins. Methods of measuring binding to FcRn are known
(see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997);
Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton
et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004/92219
(Hinton et al.).
[0055] Binding to human FcRn in vivo and serum half life of human
FcRn high affinity binding polypeptides can be assayed, e.g., in
transgenic mice or transfected human cell lines expressing human
FcRn, or in primates to which the polypeptides with a variant Fc
region are administered. WO 2000/42072 (Presta) describes antibody
variants with improved or diminished binding to FcRs. See also,
e.g., Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).
[0056] The "hinge region," "hinge sequence", and variations
thereof, as used herein, includes the meaning known in the art,
which is illustrated in, for example, Janeway et al., Immuno
Biology: the immune system in health and disease, (Elsevier Science
Ltd., NY) (4th ed., 1999); Bloom et al., Protein Science (1997),
6:407-415; Humphreys et al., J. Immunol. Methods (1997),
209:193-202.
[0057] Unless indicated otherwise, the expression "multivalent
antibody" is used throughout this specification to denote an
antibody comprising three or more antigen binding sites. The
multivalent antibody is preferably engineered to have the three or
more antigen binding sites and is generally not a native sequence
IgM or IgA antibody.
[0058] An "Fv" fragment is an antibody fragment which contains a
complete antigen recognition and binding site. This region consists
of a dimer of one heavy and one light chain variable domain in
tight association, which can be covalent in nature, for example in
scFv. It is in this configuration that the three HVRs of each
variable domain interact to define an antigen binding site on the
surface of the V.sub.H-V.sub.L dimer. Collectively, the six HVRs or
a subset thereof confer antigen binding specificity to the
antibody. However, even a single variable domain (or half of an Fv
comprising only three HVRs specific for an antigen) has the ability
to recognize and bind antigen, although usually at a lower affinity
than the entire binding site.
[0059] The "Fab" fragment contains a variable and constant domain
of the light chain and a variable domain and the first constant
domain (CH1) of the heavy chain. F(ab').sub.2 antibody fragments
comprise a pair of Fab fragments which are generally covalently
linked near their carboxy termini by hinge cysteines between them.
Other chemical couplings of antibody fragments are also known in
the art.
[0060] The phrase "antigen binding arm", as used herein, refers to
a component part of an antibody fragment that has an ability to
specifically bind a target molecule of interest. Generally and
preferably, the antigen binding arm is a complex of immunoglobulin
polypeptide sequences, e.g., HVR and/or variable domain sequences
of an immunoglobulin light and heavy chain.
[0061] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Generally the Fv polypeptide
further comprises a polypeptide linker between the V.sub.H and
V.sub.L domains, which enables the scFv to form the desired
structure for antigen binding. For a review of scFv, see Pluckthun
in The Pharmacology of Monoclonal Antibodies, Vol 113, Rosenburg
and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
[0062] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H and
V.sub.L). By using a linker that is too short to allow pairing
between the two domains on the same chain, the domains are forced
to pair with the complementary domains of another chain and create
two antigen-binding sites. Diabodies are described more fully in,
for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993).
[0063] The expression "linear antibodies" refers to the antibodies
described in Zapata et al., Protein Eng., 8(10):1057-1062 (1995).
Briefly, these antibodies comprise a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which, together with
complementary light chain polypeptides, form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
[0064] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used may be made by a variety of
techniques, including but not limited to the hybridoma method,
recombinant DNA methods, phage-display methods, and methods
utilizing transgenic animals containing all or part of the human
immunoglobulin loci, such methods and other exemplary methods for
making monoclonal antibodies being described herein.
[0065] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0066] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, NIH
Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al., supra. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al., supra.
[0067] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization.
[0068] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0069] A "naked antibody" refers to an antibody that is not
conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or
radiolabel. The naked antibody may be present in a pharmaceutical
formulation.
[0070] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 Daltons, composed of two identical light chains and two
identical heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3). Similarly,
from N- to C-terminus, each light chain has a variable region (VL),
also called a variable light domain or a light chain variable
domain, followed by a constant light (CL) domain. The light chain
of an antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequence of
its constant domain.
[0071] "Affinity" refers to the strength of the sum total of
noncovalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Specific illustrative and
exemplary embodiments for measuring binding affinity are described
in the following.
[0072] An "affinity matured" antibody refers to an antibody with
one or more alterations in one or more HVRs, compared to a parent
antibody which does not possess such alterations, such alterations
resulting in an improvement in the affinity of the antibody for
antigen.
[0073] An antibody having a "biological characteristic" of a
designated antibody is one which possesses one or more of the
biological characteristics of that antibody which distinguish it
from other antibodies that bind to the same antigen.
[0074] A "functional antigen binding site" of an antibody is one
which is capable of binding a target antigen. The antigen binding
affinity of the antigen binding site is not necessarily as strong
as the parent antibody from which the antigen binding site is
derived, but the ability to bind antigen must be measurable using
any one of a variety of methods known for evaluating antibody
binding to an antigen. Moreover, the antigen binding affinity of
each of the antigen binding sites of a multivalent antibody herein
need not be quantitatively the same. For the multimeric antibodies
herein, the number of functional antigen binding sites can be
evaluated using ultracentrifugation analysis as described in
Example 2 of U.S. Patent Application Publication No. 20050186208.
According to this method of analysis, different ratios of target
antigen to multimeric antibody are combined and the average
molecular weight of the complexes is calculated assuming differing
numbers of functional binding sites. These theoretical values are
compared to the actual experimental values obtained in order to
evaluate the number of functional binding sites.
[0075] A "species-dependent antibody" is one which has a stronger
binding affinity for an antigen from a first mammalian species than
it has for a homologue of that antigen from a second mammalian
species. Normally, the species-dependent antibody "binds
specifically" to a human antigen (i.e. has a binding affinity
(K.sub.d) value of no more than about 1.times.10.sup.-7 M,
preferably no more than about 1.times.10.sup.-8 M and most
preferably no more than about 1.times.10.sup.-9 M) but has a
binding affinity for a homologue of the antigen from a second
nonhuman mammalian species which is at least about 50 fold, or at
least about 500 fold, or at least about 1000 fold, weaker than its
binding affinity for the human antigen. The species-dependent
antibody can be any of the various types of antibodies as defined
above. In some embodiments, the species-dependent antibody is a
humanized or human antibody.
[0076] An "isolated" antibody is one which has been separated from
a component of its natural environment. In some embodiments, an
antibody is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE,
isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., ion exchange or reverse phase HPLC). For
review of methods for assessment of antibody purity, see, e.g.,
Flatman et al., J. Chromatogr. B 848:79-87 (2007).
[0077] The term "substantially similar" or "substantially the
same," as used herein, refers to a sufficiently high degree of
similarity between two numeric values (for example, one associated
with an antibody and the other associated with a
reference/comparator antibody), such that one of skill in the art
would consider the difference between the two values to be of
little or no biological and/or statistical significance within the
context of the biological characteristic measured by said values
(e.g., Kd values).
[0078] The phrase "substantially reduced" or "substantially
different," as used herein, refers to a sufficiently high degree of
difference between two numeric values (generally one associated
with a molecule and the other associated with a
reference/comparator molecule) such that one of skill in the art
would consider the difference between the two values to be of
statistical significance within the context of the biological
characteristic measured by said values (e.g., Kd values).
[0079] "Effector functions" refer to those biological activities
attributable to the Fc region of an antibody, which vary with the
antibody isotype. Examples of antibody effector functions include:
C1q binding and complement dependent cytotoxicity (CDC); Fc
receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; down regulation of cell surface receptors
(e.g., B cell receptor); and B cell activation.
[0080] An "immunoconjugate" is an antibody conjugated to one or
more heterologous molecule(s), including but not limited to a
cytotoxic agent.
[0081] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents a cellular function and/or
causes cell death or destruction. Cytotoxic agents include, but are
not limited to, radioactive isotopes (e.g., At211, I131, I125, Y90,
Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of
Lu); chemotherapeutic agents or drugs (e.g., methotrexate,
adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide),
doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or
other intercalating agents); growth inhibitory agents; enzymes and
fragments thereof such as nucleolytic enzymes; antibiotics; toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof; and the various antitumor or anticancer
agents disclosed below.
[0082] A "disorder" is any condition that would benefit from
treatment with a substance/molecule or method described herein.
This includes chronic and acute disorders or diseases including
those pathological conditions which predispose the mammal to the
disorder in question. Non-limiting examples of disorders to be
treated herein include malignant and benign tumors; non-leukemias
and lymphoid malignancies; neuronal, glial, astrocytal,
hypothalamic and other glandular, macrophagal, epithelial, stromal
and blastocoelic disorders; and inflammatory, immunologic and other
angiogenesis-related disorders.
[0083] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one embodiment, the cell
proliferative disorder is cancer.
[0084] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. The terms "cancer",
"cancerous," "cell proliferative disorder," "proliferative
disorder," and "tumor" are not mutually exclusive as referred to
herein.
[0085] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth/proliferation. Examples of cancer
include, but are not limited to, carcinoma, lymphoma (e.g.,
Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and
leukemia. More particular examples of such cancers include squamous
cell cancer, small-cell lung cancer, non-small cell lung cancer,
adenocarcinoma of the lung, squamous carcinoma of the lung, cancer
of the peritoneum, hepatocellular cancer, gastrointestinal cancer,
pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver
cancer, bladder cancer, hepatoma, breast cancer, colon cancer,
colorectal cancer, endometrial or uterine carcinoma, salivary gland
carcinoma, kidney cancer, liver cancer, prostate cancer, vulval
cancer, thyroid cancer, hepatic carcinoma, leukemia and other
lymphoproliferative disorders, and various types of head and neck
cancer. In some embodiments, the cancer is triple-negative (ER-,
PR-, HER2-) cancer. In some embodiments, the cancer is
triple-negative metastatic breast cancer, including any
histologically confirmed triple-negative (ER-, PR-, HER2-)
adenocarcinoma of the breast with locally recurrent or metastatic
disease, e.g., where the locally recurrent disease is not amenable
to resection with curative intent.
[0086] By "metastasis" is meant the spread of cancer from its
primary site to other places in the body. Cancer cells can break
away from a primary tumor, penetrate into lymphatic and blood
vessels, circulate through the bloodstream, and grow in a distant
focus (metastasize) in normal tissues elsewhere in the body.
Metastasis can be local or distant. Metastasis is a sequential
process, contingent on tumor cells breaking off from the primary
tumor, traveling through the bloodstream, and stopping at a distant
site. At the new site, the cells establish a blood supply and can
grow to form a life-threatening mass. Both stimulatory and
inhibitory molecular pathways within the tumor cell regulate this
behavior, and interactions between the tumor cell and host cells in
the distant site are also significant.
[0087] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, antibodies
are used to delay development of a disease or to slow the
progression of a disease.
[0088] An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
[0089] A "therapeutically effective amount" refers to an amount of
a therapeutic agent to treat or prevent a disease or disorder in a
mammal. In the case of cancers, the therapeutically effective
amount of the therapeutic agent may reduce the number of cancer
cells; reduce the primary tumor size; inhibit (i.e., slow to some
extent and preferably stop) cancer cell infiltration into
peripheral organs; inhibit (i.e., slow to some extent and
preferably stop) tumor metastasis; inhibit, to some extent, tumor
growth; and/or relieve to some extent one or more of the symptoms
associated with the disorder. To the extent the drug may prevent
growth and/or kill existing cancer cells, it may be cytostatic
and/or cytotoxic. For cancer therapy, efficacy in vivo can, for
example, be measured by assessing the duration of survival, time to
disease progression (TTP), the response rates (RR), duration of
response, and/or quality of life.
[0090] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g., cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain embodiments, the individual or subject is a
human.
[0091] The term "anti-cancer therapy" refers to a therapy useful in
treating cancer. Examples of anti-cancer therapeutic agents
include, but are limited to, e.g., chemotherapeutic agents, growth
inhibitory agents, cytotoxic agents, agents used in radiation
therapy, anti-angiogenesis agents, apoptotic agents, anti-tubulin
agents, and other agents to treat cancer, anti-CD20 antibodies,
platelet derived growth factor inhibitors (e.g., Gleevec.TM.
(Imatinib Mesylate)), a COX-2 inhibitor (e.g., celecoxib),
interferons, cytokines, antagonists (e.g., neutralizing antibodies)
that bind to one or more of the following targets PDGFR-beta, BlyS,
APRIL, BCMA receptor(s), TRAIL/Apo2, and other bioactive and
organic chemical agents, etc. Combinations thereof are also
included.
[0092] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re188, sm.sup.153, Bi.sup.212, P.sup.32 and
radioactive isotopes of Lu), chemotherapeutic agents e.g.
methotrexate, adriamicin, vinca alkaloids (vincristine,
vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents, enzymes
and fragments thereof such as nucleolytic enzymes, antibiotics, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof, and the various antitumor or anticancer
agents disclosed below. Other cytotoxic agents are described below.
A tumoricidal agent causes destruction of tumor cells.
[0093] A "chemotherapeutic agent" refers to a chemical compound
useful in the treatment of cancer. Examples of chemotherapeutic
agents include alkylating agents such as thiotepa and
cyclosphosphamide (CYTOXAN.RTM.); alkyl sulfonates such as
busulfan, improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylomelamine; acetogenins (especially bullatacin and
bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,
MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic
acid; a camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
chlorophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosoureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gamma1I and calicheamicin omegaI1 (see, e.g., Nicolaou et al.,
Angew. Chem. Intl. Ed. Engl., 33: 183-186 (1994)); CDP323, an oral
alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an
esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antibiotic chromophores), aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin
(including ADRIAMYCIN.RTM., morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin
HCl liposome injection (DOXIL.RTM.), liposomal doxorubicin TLC D-99
(MYOCET.RTM.), pegylated liposomal doxorubicin (CAELYX.RTM.), and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate, gemcitabine (GEMZAR.RTM.), tegafur (UFTORAL.RTM.),
capecitabine (XELODA.RTM.), an epothilone, and 5-fluorouracil
(5-FU); folic acid analogues such as denopterin, methotrexate,
pteropterin, trimetrexate; purine analogs such as fludarabine,
6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such
as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine,
dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens
such as calusterone, dromostanolone propionate, epitiostanol,
mepitiostane, testolactone; anti-adrenals such as
aminoglutethimide, mitotane, trilostane; folic acid replenisher
such as frolinic acid; aceglatone; aldophosphamide glycoside;
aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elformithine; elliptinium acetate; an epothilone; etoglucid;
gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids
such as maytansine and ansamitocins; mitoguazone; mitoxantrone;
mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
losoxantrone; 2-ethylhydrazide; procarbazine; PSK.RTM.
polysaccharide complex (JHS Natural Products, Eugene, Oreg.);
razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; vindesine (ELDISINE.RTM., FILDESIN.RTM.); dacarbazine;
mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;
arabinoside ("Ara-C"); thiotepa; taxoid, e.g., paclitaxel
(TAXOL.RTM.), albumin-engineered nanoparticle formulation of
paclitaxel (ABRAXANE.TM.), and docetaxel (TAXOTERE.RTM.);
chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum
agents such as cisplatin, oxaliplatin (e.g., ELOXATIN.RTM.), and
carboplatin; vincas, which prevent tubulin polymerization from
forming microtubules, including vinblastine (VELBAN.RTM.),
vincristine (ONCOVIN.RTM.), vindesine (ELDISINE.RTM.,
FILDESIN.RTM.), and vinorelbine (NAVELBINE.RTM.); etoposide
(VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid, including bexarotene (TARGRETIN.RTM.);
bisphosphonates such as clodronate (for example, BONEFOS.RTM. or
OSTAC.RTM.), etidronate (DIDROCAL.RTM.), NE-58095, zoledronic
acid/zoledronate (ZOMETA.RTM.), alendronate (FOSAMAX.RTM.),
pamidronate (AREDIA.RTM.), tiludronate (SKELID.RTM.), or
risedronate (ACTONEL.RTM.); troxacitabine (a 1,3-dioxolane
nucleoside cytosine analog); antisense oligonucleotides,
particularly those that inhibit expression of genes in signaling
pathways implicated in aberrant cell proliferation, such as, for
example, PKC-alpha, Raf, H-Ras, and epidermal growth factor
receptor (EGF-R); vaccines such as THERATOPE.RTM. vaccine and gene
therapy vaccines, for example, ALLOVECTIN.RTM. vaccine,
LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine; topoisomerase 1
inhibitor (e.g., LURTOTECAN.RTM.); rmRH (e.g., ABARELIX.RTM.);
BAY439006 (sorafenib; Bayer); SU-11248 (sunitinib, SUTENT.RTM.,
Pfizer); perifosine, COX-2 inhibitor (e.g. celecoxib or
etoricoxib), proteosome inhibitor (e.g., PS341); bortezomib
(VELCADE.RTM.); CCI-779; tipifarnib (R11577); orafenib, ABT510;
Bcl-2 inhibitor such as oblimersen sodium (GENASENSE.RTM.);
pixantrone; EGFR inhibitors (see definition below); tyrosine kinase
inhibitors (see definition below); serine-threonine kinase
inhibitors such as rapamycin (sirolimus, RAPAMUNE.RTM.);
farnesyltransferase inhibitors such as lonafarnib (SCH 6636,
SARASAR.TM.); and pharmaceutically acceptable salts, acids or
derivatives of any of the above; as well as combinations of two or
more of the above such as CHOP, an abbreviation for a combined
therapy of cyclophosphamide, doxorubicin, vincristine, and
prednisolone; and FOLFOX, an abbreviation for a treatment regimen
with oxaliplatin (ELOXATIN.TM.) combined with 5-FU and
leucovorin.
[0094] Chemotherapeutic agents as defined herein include
"anti-hormonal agents" or "endocrine therapeutics" which act to
regulate, reduce, block, or inhibit the effects of hormones that
can promote the growth of cancer. They may be hormones themselves,
including, but not limited to: anti-estrogens with mixed
agonist/antagonist profile, including, tamoxifen (NOLVADEX.RTM.),
4-hydroxytamoxifen, toremifene (FARESTON.RTM.), idoxifene,
droloxifene, raloxifene (EVISTA.RTM.), trioxifene, keoxifene, and
selective estrogen receptor modulators (SERMs) such as SERM3; pure
anti-estrogens without agonist properties, such as fulvestrant
(FASLODEX.RTM.), and EM800 (such agents may block estrogen receptor
(ER) dimerization, inhibit DNA binding, increase ER turnover,
and/or suppress ER levels); aromatase inhibitors, including
steroidal aromatase inhibitors such as formestane and exemestane
(AROMASIN.RTM.), and nonsteroidal aromatase inhibitors such as
anastrazole (ARIMIDEX.RTM.), letrozole (FEMARA.RTM.) and
aminoglutethimide, and other aromatase inhibitors include vorozole
(RIVISOR.RTM.), megestrol acetate (MEGASE.RTM.), fadrozole, and
4(5)-imidazoles; lutenizing hormone-releaseing hormone agonists,
including leuprolide (LUPRON.RTM. and ELIGARD.RTM.), goserelin,
buserelin, and tripterelin; sex steroids, including progestines
such as megestrol acetate and medroxyprogesterone acetate,
estrogens such as diethylstilbestrol and premarin, and
androgens/retinoids such as fluoxymesterone, all transretionic acid
and fenretinide; onapristone; anti-progesterones; estrogen receptor
down-regulators (ERDs); anti-androgens such as flutamide,
nilutamide and bicalutamide; and pharmaceutically acceptable salts,
acids or derivatives of any of the above; as well as combinations
of two or more of the above.
[0095] The term "prodrug" as used in this application refers to a
precursor or derivative form of a pharmaceutically active substance
that is less cytotoxic to tumor cells compared to the parent drug
and is capable of being enzymatically activated or converted into
the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer
Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382,
615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A
Chemical Approach to Targeted Drug Delivery," Directed Drug
Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
(1985). The prodrugs include, but are not limited to,
phosphate-containing prodrugs, thiophosphate-containing prodrugs,
sulfate-containing prodrugs, peptide-containing prodrugs, D-amino
acid-modified prodrugs, glycosylated prodrugs,
.beta.-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs which can be converted into the more
active cytotoxic free drug. Examples of cytotoxic drugs that can be
derivatized into a prodrug form for use include, but are not
limited to, those chemotherapeutic agents described above.
[0096] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell (e.g., a
cell whose growth is dependent upon HGF/c-met activation either in
vitro or in vivo). Thus, the growth inhibitory agent may be one
which significantly reduces the percentage of HGF/c-met-dependent
cells in S phase. Examples of growth inhibitory agents include
agents that block cell cycle progression (at a place other than S
phase), such as agents that induce G1 arrest and M-phase arrest.
Classical M-phase blockers include the vincas (vincristine and
vinblastine), taxanes, and topoisomerase II inhibitors such as
doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
Those agents that arrest G1 also spill over into S-phase arrest,
for example, DNA alkylating agents such as tamoxifen, prednisone,
dacarbazine, mechlorethamine, cisplatin, methotrexate,
5-fluorouracil, and ara-C. Further information can be found in The
Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1,
entitled "Cell cycle regulation, oncogenes, and antineoplastic
drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995),
especially p. 13. The taxanes (paclitaxel and docetaxel) are
anticancer drugs both derived from the yew tree. Docetaxel
(TAXOTERE.RTM., Rhone-Poulenc Rorer), derived from the European
yew, is a semisynthetic analogue of paclitaxel (TAXOL.RTM.,
Bristol-Myers Squibb). Paclitaxel and docetaxel promote the
assembly of microtubules from tubulin dimers and stabilize
microtubules by preventing depolymerization, which results in the
inhibition of mitosis in cells.
[0097] By "radiation therapy" is meant the use of directed gamma
rays or beta rays to induce sufficient damage to a cell so as to
limit its ability to function normally or to destroy the cell
altogether. It will be appreciated that there will be many ways
known in the art to determine the dosage and duration of treatment.
Typical treatments are given as a one time administration and
typical dosages range from 10 to 200 units (Grays) per day.
[0098] The term "concurrently" is used herein to refer to
administration of two or more therapeutic agents, where at least
part of the administration overlaps in time. Accordingly,
concurrent administration includes a dosing regimen when the
administration of one or more agent(s) continues after
discontinuing the administration of one or more other agent(s).
[0099] By "reduce or inhibit" is meant the ability to cause an
overall decrease of 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%,
90%, 95%, or greater. Reduce or inhibit can refer to the symptoms
of the disorder being treated, the presence or size of metastases,
or the size of the primary tumor.
[0100] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0101] It is understood that aspect and embodiments of the
invention described herein include "consisting" and/or "consisting
essentially of" aspects and embodiments.
[0102] As used herein, the singular form "a", "an", and "the"
includes plural references unless indicated otherwise.
[0103] As is understood by one skilled in the art, reference to
"about" a value or parameter herein includes (and describes)
embodiments that are directed to that value or parameter per se.
For example, description referring to "about X" includes
description of "X".
II. PHARMACEUTICAL FORMULATIONS
[0104] Provided herein are pharmaceutical formulations comprising
an anti-c-met antibody. In some embodiments, the pharmaceutical
formulation is a liquid pharmaceutical formulation. In some
embodiments, the pharmaceutical formulation is a stable
pharmaceutical formulation. In some embodiments, the pharmaceutical
formulation is a stable liquid pharmaceutical formulation. In some
embodiments, the anti-c-met antibody is an antagonist anti-c-met
antibody. In some embodiments, the anti-c-met antibody is a
monovalent anti-c-met antibody. Minimization in the pharmaceutical
formulation of anti-c-met antibody aggregation is particularly
important. Bivalent forms of anti-c-met antibodies have been shown
to promote dimerization and lead to activation of c-met (agonistic
function), while conversely monovalent antibodies inhibit c-met
activity (antagonistic function). Aggregation of monovalent
antibodies (formation of multimer and oligomers) and/or failure to
maintain monovalent structure in a pharmaceutical formulation
comprising anti-c-met antibodies could lead to an undesirable
agonistic effect.
[0105] In particular, provided herein are pharmaceutical
formulations comprising (a) an anti-c-met antibody and (b) a
polysorbate, wherein the polysorbate concentration is greater than
0.02% w/v. In some embodiments, the pharmaceutical formulation
comprises (a) an anti-c-met antibody; (b) a polysorbate, wherein
the polysorbate concentration is greater than 0.02% w/v; and (c) a
histidine buffer (e.g., a histidine buffer at a pH between 5.0 and
5.4). In some embodiments, the pharmaceutical formulation comprises
(a) an anti-c-met antibody; (b) a histidine buffer at pH 5.0-5.4;
(c) a saccharide; and (d) a polysorbate, wherein the polysorbate
concentration is greater than 0.02% w/v. In some embodiments, the
pharmaceutical formulation is a liquid pharmaceutical formulation.
In some embodiments, the pharmaceutical formulation is a stable
pharmaceutical formulation. In some embodiments, the pharmaceutical
formulation is a stable liquid pharmaceutical formulation.
[0106] Anti-c-met antibodies useful in the pharmaceutical
formulations are described in Section III. In some embodiments, the
anti-c-met antibody is an antagonist anti-c-met antibody. In some
embodiments, the anti-c-met antibody is a monovalent anti-c-met
antibody. For example, in some embodiments, the anti-c-met antibody
comprises a single antigen binding arm. In some embodiments, the
anti-c-met antibody comprises a single antigen binding arm and
comprises a Fc region, wherein the Fc region comprises a first and
a second Fc polypeptide, wherein the first and second Fc
polypeptides are present in a complex. In some embodiments, the
first and second Fc polypeptides form a Fc region that increases
stability of said antibody fragment compared to a Fab molecule
comprising said antigen binding arm. In some embodiments, the
anti-c-met antibody comprises a HVR-L1 comprising the amino acid
sequence of SEQ ID NO:1, a HVR-L2 comprising the amino acid
sequence of SEQ ID NO:2, a HVR-L3 comprising the amino acid
sequence of SEQ ID NO:3, a HVR-H1 comprising the amino acid
sequence of SEQ ID NO:4, a HVR-H2 comprising the amino acid
sequence of SEQ ID NO:5, and a HVR-H3 comprising the amino acid
sequence of SEQ ID NO:6. In some embodiments, the anti-c-met
antibody comprises (a) a heavy chain variable domain comprising the
amino acid sequence of SEQ ID NO:19 and (b) a light chain variable
domain comprising the amino acid sequence of SEQ ID NO:20. In some
embodiments, the anti-c-met antibody comprises (a) a first
polypeptide comprising the amino acid sequence of SEQ ID NO:19, a
CH1 sequence, and a first Fc polypeptide, (b) a second polypeptide
comprising the amino acid sequence of SEQ ID NO:20 and CL1
sequence, and (c) a third polypeptide comprising a second Fc
polypeptide. In some embodiments, the first Fc polypeptide
comprises the Fc sequence depicted in FIG. 2 (SEQ ID NO: 17) and
the second Fc polypeptide comprises the Fc sequence depicted in
FIG. 3 (SEQ ID NO: 18). In some embodiments, the anti-c-met
antibody is onartuzumab.
[0107] In some embodiments of any of the pharmaceutical
formulations described herein, the anti-c-met antibody of the
pharmaceutical formulation is present at a concentration between
about 10 mg/mL and about 100 mg/mL. In some embodiments, the
concentration of the anti-c-met antibody (e.g., onartuzumab) is
between about any of 10 mg/mL to 50 mg/mL, 10 mg/mL to 75 mg/mL, 25
mg/mL to 75 mg/mL, 50 mg/mL to 100 mg/mL, 50 mg/mL to 75 mg/mL,
and/or 75 mg/mL to 100 mg/mL. In some embodiments, the
concentration of the anti-c-met antibody (e.g., onartuzumab) is
greater than about any of 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL,
60 mg/mL, 70 mg/mL, or 100 mg/mL. For example, in some embodiments,
the pharmaceutical formulation comprises (a) an anti-c-met antibody
(e.g., onartuzumab), wherein the anti-c-met antibody is present at
a concentration between about 50 mg/mL and about 75 mg/mL; (b) a
histidine buffer at pH 5.0-5.4; (c) a saccharide; and (d) a
polysorbate, wherein the polysorbate concentration is greater than
0.02% w/v. In some embodiments, the concentration of the anti-c-met
antibody (e.g., onartuzumab) is less than about any of 150 mg/mL,
125 mg/mL, 100 mg/mL, or 75 mg/mL. In some embodiments, the
concentration of the anti-c-met antibody (e.g., onartuzumab) is
about any of 20 mg/mL, 30 mg/mL, 40 mg/mL, 50 mg/mL, 60 mg/mL, 70
mg/mL, or 80 mg/mL. In some embodiments, the concentration of the
anti-c-met antibody (e.g., onartuzumab) is about 60 mg/mL.
[0108] The pharmaceutical formulation preferably comprises a
polysorbate. The polysorbate is generally included in an amount
which reduces aggregate formation (such as that which occurs upon
shaking or shipping). Examples of polysorbate include, but are not
limited to, polysorbate 20 (polyoxyethylene (20) sorbitan
monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan
monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan
monostearate), and/or polysorbate 80 (polyoxyethylene (20) sorbitan
monooleate). In some embodiments, the polysorbate is polysorbate 20
(polyoxyethylene (20) sorbitan monolaurate). In some embodiments of
any of the pharmaceutical formulations described herein, the
polysorbate concentration is sufficient to minimize aggregation
and/or maintain stability upon long term storage and/or during
administration (e.g., after dilution in an IV bag). In some
embodiments, the polysorbate concentration is greater than 0.02%
w/v, greater than or equal to about 0.03% w/v, or greater than or
equal to about 0.04% w/v. In some embodiments, the polysorbate
concentration is greater than 0.02% w/v and less than about 0.1%
w/v. In some embodiments, the polysorbate concentration is greater
than 0.03% w/v and less than about 0.1% w/v. In some embodiments,
the polysorbate concentration is about any of 0.03% w/v, 0.04% w/v,
or 0.05% w/v. In some embodiments, the polysorbate is present at a
concentration of about 0.04% w/v. For example, in some embodiments,
the pharmaceutical formulation comprises (a) an anti-c-met antibody
(e.g., onartuzumab); (b) a histidine buffer at pH 5.0-5.4; (c)
saccharide; and (d) polysorbate 20, wherein the polysorbate 20
concentration is about 0.04% w/v.
[0109] The pharmaceutical formulation preferably comprises a
saccharide. Saccharides include monosaccharides, disaccharides,
trisaccharides, polysaccharides, sugar alcohols, reducing sugars,
nonreducing sugars, etc. Further examples of saccharides include,
but are not limited to, glucose, sucrose, trehalose, lactose,
fructose, maltose, dextran, glycerin, dextran, erythritol,
glycerol, arabitol, sylitol, sorbitol, mannitol, mellibiose,
melezitose, raffinose, mannotriose, stachyose, maltose, lactulose,
maltulose, glucitol, maltitol, lactitol, iso-maltulose, etc. In
some embodiments, the saccharide is a disaccharide. In some
embodiments, the saccharide is a nonreducing disaccharide. In some
embodiments, the saccharide is trehalose. In some embodiments, the
saccharide is sucrose. For example, in some embodiments, the
pharmaceutical formulation comprises (a) an anti-c-met antibody
(e.g., onartuzumab); (b) a histidine buffer at pH 5.0-5.4; (c)
sucrose; and (d) a polysorbate, wherein the polysorbate
concentration is greater than 0.02% w/v.
[0110] The saccharide is generally included in an amount which
reduces aggregate formation. In some embodiments of any of the
pharmaceutical formulations described herein, the saccharide is
present at a concentration of between about any of 50 mM to 250 mM,
75 mM to 200 mM, 75 mM to 150 mM, 100 mM to 150 mM, or 110 mM to
130 mM. In some embodiments, the saccharide is present at a
concentration greater than about any of 50 mM, 75 mM, 100 mM, 110
mM, or 115 mM. In some embodiments, the saccharide is present at a
concentration of about any of 100 mM, 110 mM, 120 mM, 130 mM, or
140 mM. In some embodiments, the saccharide is present at a
concentration of about 120 mM. For example, in some embodiments,
the pharmaceutical formulation comprises (a) an anti-c-met antibody
(e.g., onartuzumab); (b) a histidine buffer at pH 5.0-5.4; (c)
sucrose, wherein the sucrose is present at a concentration of about
120 mM; and (d) a polysorbate, wherein the polysorbate
concentration is greater than 0.02% w/v.
[0111] The pharmaceutical formulation preferably comprises a
histidine buffer. Examples of histidine buffers include, but are
not limited to, histidine chloride, histidine succinate, histidine
acetate, histidine phosphate, histidine sulfate. In some
embodiments, the histidine buffer is histidine acetate. In some
embodiments of any of the pharmaceutical formulations described
herein, the histidine buffer concentration is between about any of
1 mM to 50 mM, 1 mM to 35 mM, 1 mM to 25 mM, 1 mM to 20 mM, 7.5 mM
to 12.5 mM, or 5 mM to 15 mM. In some embodiments, the histidine
buffer concentration is greater than or equal to about any of 5 mM,
7.5 mM, or 10 mM. In some embodiments, the histidine buffer
concentration is about any of 5 mM, 7.5 mM, 10 mM, 12.5 mM, or 15
mM. In some embodiments, the histidine buffer concentration is
about 10 mM. In some embodiments of any of the pharmaceutical
formulations described herein, the histidine buffer is at a pH of
between pH 5.0 and 5.4, for example, about any of pH 5.0, pH 5.1,
pH 5.2, pH 5.3, or pH 5.4. In some embodiments, the pH is between
pH 5.1 and 5.4. For example, in some embodiments, the
pharmaceutical formulation comprises (a) an anti-c-met antibody
(e.g., onartuzumab); (b) a histidine acetate buffer at pH 5.4,
wherein the histidine acetate buffer is at a concentration of about
10 mM; (c) saccharide; and (d) a polysorbate, wherein the
polysorbate concentration is greater than 0.02% w/v.
[0112] In some embodiments, the pharmaceutical formulation
comprises (a) an anti-c-met antibody (e.g., onartuzumab), wherein
the anti-c-met antibody is present at a concentration between about
50 mg/mL and about 75 mg/mL; (b) a histidine acetate buffer at pH
5.0-5.4, wherein the histidine acetate buffer is at a concentration
between about 1 mM and about 20 mM; (c) sucrose, wherein the
sucrose is at a concentration between about 100 mM to about 150 mM;
and (d) polysorbate 20, wherein the polysorbate 20 concentration is
greater than 0.02% w/v. In some embodiments, the pharmaceutical
formulation comprises (a) an anti-c-met antibody (e.g.,
onartuzumab), wherein the anti-c-met antibody is present at a
concentration of about 60 mg/mL; (b) a histidine acetate buffer at
pH 5.4, wherein the histidine acetate buffer is at a concentration
of about 10 mM; (c) sucrose, wherein the sucrose is at a
concentration of about 120 mM; and (d) polysorbate 20, wherein the
polysorbate 20 concentration is about 0.04% w/v.
[0113] The pharmaceutical formulation herein may also contain more
than one active compound as necessary for the particular indication
being treated, preferably those with complementary activities that
do not adversely affect each other. Such molecules are suitably
present in combination in amounts that are effective for the
purpose intended.
[0114] Further, provided herein are vials and methods of filing a
vial comprising a pharmaceutical formulation described herein. In
some embodiments, the pharmaceutical formulation is provided inside
a vial with a stopper pierceable by a syringe, preferably in
aqueous form. The vial is desirably stored at about 2-8.degree. C.
as well as up to 30.degree. C. for 24 hours until it is
administered to a subject in need thereof. The vial may for example
be a 15 cc vial (for example for a 600 mg dose) or 20 cc vial (for
example for a 900 mg dose).
[0115] The pharmaceutical formulation for administration is
preferably a liquid formulation (not lyophilized) and has not been
subjected to prior lyophilization. While the pharmaceutical
formulation may be lyophilized, preferably it is not. In some
embodiments of any of the pharmaceutical formulations, the
pharmaceutical formulation, the pharmaceutical formulation is a
lyophilized pharmaceutical formulation. In some embodiments, the
pharmaceutical formulation is a liquid formulation. In some
embodiments, the pharmaceutical formulation does not contain a
tonicifying amount of a salt such as sodium chloride. In some
embodiments of any of the pharmaceutical formulations, the
pharmaceutical formulation is diluted.
[0116] In some embodiments of any of the pharmaceutical
formulations, the pharmaceutical formulation comprising the
anti-c-met antibody is stable. In some embodiments, the
pharmaceutical formulation comprising the anti-c-met antibody is
physically stable. In some embodiments, the pharmaceutical
formulation comprising the anti-c-met antibody is chemically
stable. In some embodiments, the pharmaceutical formulation
comprising the anti-c-met antibody is physically stable and
chemically stable. In some embodiments, the pharmaceutical
formulation comprises an antagonistic anti-c-met antibody and
agonistic activity of the pharmaceutical formulation is
substantially undetectable. Methods of detecting agonistic and/or
agonistic activity are known in the art, for example, U.S. Pat. No.
6,207,152, which is incorporated by reference in its entirety. In
some embodiments, the pharmaceutical formulation is substantially
nonimmunogenic.
[0117] In some embodiments of any of the pharmaceutical
formulations, the pharmaceutical formulation does not significantly
result in increased aggregate formation after storage at about
40.degree. C. for about two weeks or about four weeks, at about
25.degree. C. for about one month or about three months; at about
5.degree. C. for about six months, about one year, or about two
years, and/or about -20.degree. C. for about three months, about
six months, or about a year. In some embodiments, the
pharmaceutical formulation has reduced or lower levels of aggregate
formation after storage at about 40.degree. C. for about two weeks
or about four weeks, at about 25.degree. C. for about one month or
about three months; at about 5.degree. C. for about six months,
about one year, or about two years, and/or about -20.degree. C. for
about three months, about six months, or about a year (e.g.,
compared to a similar formulation at pH 5.7).
[0118] High Molecular Weight Species (HMWS) are generally larger
than the reference molecule. For example, onartuzumab is about 100
kDa (99,161 Daltons), therefore, a HMWS is greater than about 100
kDa. The size of a bivalent antibody is approximately 150 kDa and a
dimer of onartuzumab is about 200 kDa. In some embodiments of any
of the pharmaceutical formulations, the pharmaceutical formulation
comprises less than about any of 1.5%, 1.25%, 1%, 0.75%, 0.5%,
0.25%, 0.20% or 0.15% HMWS (e.g., upon storage). In some
embodiments of any of the pharmaceutical formulations, the
pharmaceutical formulation does not significantly increase the
percentage of HMWS after storage at about 40.degree. C. for about
two weeks or about four weeks, at about 25.degree. C. for about one
month or about three months; at about 5.degree. C. for about six
months, about one year, or about two years, and/or about
-20.degree. C. for about three months, about six months, or about a
year. In some embodiments, the pharmaceutical formulation has
reduced or lower levels of HMWS after storage at about 40.degree.
C. for about two weeks or about four weeks, at about 25.degree. C.
for about one month or about three months; at about 5.degree. C.
for about six months, about one year, or about two years, and/or
about -20.degree. C. for about three months, about six months, or
about a year (e.g., compared to a similar formulation at pH 5.7).
In some embodiments of any of the pharmaceutical formulations, the
pharmaceutical formulation does not significantly increase the
percentage of HMWS after storage at about 40.degree. C. for about
any of 15 days, 30 days, 45 days, or 60 days or at about 25.degree.
C. for about 30 days or about 60 days. In some embodiments, the
pharmaceutical formulation has reduced or lower levels of HMWS
after storage at about 40.degree. C. for about any of 15 days, 30
days, 45 days, or 60 days or at about 25.degree. C. for about 30
days or about 60 days (e.g., compared to a similar formulation at
pH 5.7).
[0119] Low Molecular Weight Species (LMWS) are generally smaller
than the reference molecule. For example, onartuzumab is about 100
kDa (99,161 Daltons), therefore, a LMWS is less than about 100 kDa.
In some embodiments of any of the pharmaceutical formulations, the
pharmaceutical formulation does not significantly increase
degradation and/or percentage of LMWS after storage at about
40.degree. C. for about two weeks or about four weeks, at about
25.degree. C. for about one month or about three months; at about
5.degree. C. for about six months, about one year, or about two
years, and/or about -20.degree. C. for about three months, about
six months, or about a year (e.g., compared to a similar
formulation at pH 5.7). In some embodiments of any of the
pharmaceutical formulations, the pharmaceutical formulation does
not significantly increase degradation and/or percentage of LMWS
after storage at about 40.degree. C. for about any of 15 days, 30
days, 45 days, or 60 days, at about 25.degree. C. for a for about
30 days or about 60 days (e.g., compared to a similar formulation
at pH 5.7).
[0120] In some embodiments of any of the pharmaceutical
formulations, the percentage of intact polysorbate in the
pharmaceutical formulation is greater than about any of 75%, 80%,
85%, or 90% after storage at about 40.degree. C. for about two
weeks or about four weeks, at about 25.degree. C. for about one
month or about three months; at about 5.degree. C. for about six
months, about one year, or about two years, and/or about
-20.degree. C. for about three months, about six months, or about a
year. In some embodiments of, the percentage of intact polysorbate
in the pharmaceutical formulation is greater than about any of 75%,
80%, 85%, or 90% after storage at about 40.degree. C. for about any
of one, two, three, four, five, six, seven, or eight weeks.
[0121] In some embodiments of any of the pharmaceutical
formulations, the percentage of degraded polysorbate in the
pharmaceutical formulation is less than about any of 25%, 20%, 15%,
or 10% after storage at about 40.degree. C. for about two weeks or
about four weeks, at about 25.degree. C. for about one month or
about three months; at about 5.degree. C. for about six months,
about one year, or about two years, and/or about -20.degree. C. for
about three months, about six months, or about a year. In some
embodiments, the percentage of degraded polysorbate in the
pharmaceutical formulation is less than about any of 25%, 20%, 15%,
or 10% after storage at about 40.degree. C. for one, two, three,
four, five, six, seven, or eight weeks.
[0122] In some embodiments, the ratio of degraded polysorbate to
intact polysorbate in the pharmaceutical formulation is less than
about any of 0.25, 0.20, 0.15 or 0.10 after storage at about
40.degree. C. for about two weeks or about four weeks, at about
25.degree. C. for about one month or about three months; at about
5.degree. C. for about six months, about one year, or about two
years, and/or about -20.degree. C. for about three months, about
six months, or about a year. In some embodiments, the ratio of
degraded polysorbate to intact polysorbate in the pharmaceutical
formulation is less than about any of 0.25, 0.20, 0.15 or 0.10
after storage at about 40.degree. C. for about any of one, two,
three, four, five, six, seven, or eight weeks. In some embodiments,
the pharmaceutical formulation comprising the anti-c-met antibody
is more stable than a similar formulation at pH 5.7 and/or with a
polysorbate concentration of 0.02% or less.
[0123] Moreover, the pharmaceutical formulation is desirably one
which has been demonstrated to be stable upon storage and/or during
administration (e.g., after dilution in an IV bag). Various
stability assays are available to the skilled practitioner for
confirming the stability of the formulation. For example, the
formulation may be one which is found to be stable upon storage: at
about 25.degree. C. for at least about one month or at least about
three months, about 5.degree. C. for at least about six months or
at least about one year; and/or about -20.degree. C. for at least
about six months or at least about one year. Furthermore, the
pharmaceutical formulation is preferably stable following freezing
(to, e.g., -70.degree. C.) and thawing of the pharmaceutical
formulation. Freezing of the aqueous pharmaceutical formulation,
without simultaneous drying that occurs during freeze-drying, is
specifically contemplated herein, facilitating longer term storage
thereof, for instance in a stainless steel tank. Freezing of the
pharmaceutical formulation is specifically contemplated herein.
Hence, the pharmaceutical formulation can be tested for stability
upon freezing and thawing. In another embodiment, the formulation
is provided inside a stainless steel tank. The formulation in the
stainless steel tank is optionally frozen and not freeze-dried.
[0124] The pharmaceutical formulation to be used for in vivo
administration should be sterile. This can be achieved according to
the procedures known to the skilled person for generating sterile
pharmaceutical formulations suitable for administration to human
subjects, including filtration through sterile filtration
membranes, prior to, or following, preparation of the
formulation.
[0125] In some embodiments of any of the pharmaceutical
formulations, the pharmaceutical formulation comprising the
anti-c-met antibody is stable upon dilution with a diluent (e.g.,
saline). For example, provided herein are IV bags comprising a
diluted pharmaceutical formulation described herein. In some
embodiments, the diluent is saline (e.g., 0.9% sodium chloride). In
some embodiments, the concentration of the anti-c-met antibody is
diluted to about any of 0.5 mg/mL, 1 mg/mL, 1.5 mg/mL, 2 mg/mL, 3
mg/mL, 4 mg/mL or 5 mg/mL. In some embodiments, the concentration
of the anti-c-met antibody is diluted to between about any of 0.5-5
mg/mL, 0.5-2.5 mg/mL, or 0.5-1.5 mg/mL. Therefore, for example,
provided herein are pharmaceutical formulations comprising a) an
anti-c-met antibody, wherein the antibody concentration is about 1
mg/mL, and (b) a polysorbate, wherein the polysorbate concentration
is greater than 0.00033% w/v. In some embodiments, the
pharmaceutical formulation comprises (a) an anti-c-met antibody,
wherein the antibody concentration is about 1 mg/mL; (b) a
polysorbate, wherein the polysorbate concentration is greater than
0.00033% w/v; (c) a histidine buffer (e.g., a histidine buffer at a
pH between 5.0 and 5.4). In some embodiments of any of the
pharmaceutical formulations, the pharmaceutical formulation is
stable (e.g., physically stable) in an IV bag and/or IV
administration set.
[0126] In some embodiments of any of the pharmaceutical
formulations, the pharmaceutical formulation (for example, after
dilution) is stable upon agitation for any of about thirty minutes,
one hour, 1.5 hours, or two hours at about any of 75, 100, 125, or
150 rpm. In some embodiments of any of the pharmaceutical
formulations, the pharmaceutical formulation (for example, after
dilution) comprises less than about any of 1.5%, 1.25%, 1%, 0.75%,
0.5%, 0.25%, 0.20% or 0.15% HMWS (for example, upon agitation).
[0127] Provided herein are also methods of making a pharmaceutical
formulation comprising preparing the formulation as described
herein. In some embodiments, the methods further comprise
evaluating physical stability, chemical stability, or biological
activity of the anti-c-met antibody in the formulation.
[0128] Stability can be tested by evaluating physical stability,
chemical stability, and/or biological activity of the antibody in
the formulation around the time of formulation as well as following
storage, during administration, and/or upon agitation at the noted
temperatures. Physical and/or stability can be evaluated
qualitatively and/or quantitatively in a variety of different ways,
including evaluation of aggregate formation (for example using size
exclusion chromatography, by measuring turbidity, and/or by visual
inspection); by assessing charge heterogeneity using cation
exchange chromatography or capillary zone electrophoresis;
amino-terminal or carboxy-terminal sequence analysis; mass
spectrometric analysis; SDS-PAGE analysis to compare reduced and
intact antibody; peptide map (for example tryptic or LYS-C)
analysis; evaluating biological activity or antigen binding
function of the antibody; etc. Instability may result in
aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g.,
Met oxidation), isomerization (e.g., Asp isomerization),
clipping/hydrolysis/fragmentation (e.g., hinge region
fragmentation), succinimide formation, unpaired cysteine(s),
N-terminal extension, C-terminal processing, glycosylation
differences, etc. Biological activity or antigen binding function
can be evaluated using various techniques available to the skilled
practitioner.
[0129] One or more other pharmaceutically acceptable carriers,
excipients or stabilizers such as those described in Remington's
Pharmaceutical Sciences 18th edition, Gennaro, A. Ed. (1990) may be
included in the formulation provided that they do not adversely
affect the desired characteristics of the formulation. Acceptable
carriers, excipients or stabilizers are nontoxic to recipients at
the dosages and concentrations employed and include; additional
buffering agents; co-solvents; antioxidants including ascorbic acid
and methionine; chelating agents such as EDTA; metal complexes
(e.g., Zn-protein complexes); biodegradable polymers such as
polyesters; preservatives; and/or salt-forming counterions such as
sodium.
III. ANTI-C-MET ANTIBODIES
[0130] Provided herein are anti-c-met antibodies for use in the
pharmaceutical formulations described herein. Useful anti-c-met
antibodies include antibodies that bind with sufficient affinity
and specificity to c-met and can reduce or inhibit one or more
c-met activities. Anti-c-met antibodies in the pharmaceutical
formulations can be used to modulate one or more aspects of
HGF/c-met-associated effects, including but not limited to c-met
activation, downstream molecular signaling (e.g., mitogen activated
protein kinase (MAPK) phosphorylation), cell proliferation, cell
migration, cell survival, cell morphogenesis and angiogenesis.
These effects can be modulated by any biologically relevant
mechanism, including disruption of ligand (e.g., HGF) binding to
c-met, c-met phosphorylation and/or c-met multimerization. In some
embodiments, the anti-c-met antibody is an antagonist anti-c-met
antibody. In some embodiments, the anti-c-met antibody interferes
with diseases or conditions wherein c-met/HGF activity is
involved.
[0131] In some embodiments of any of the anti-c-met antibody
formulations described herein, the anti-c-met antibody is an
anti-c-met antibody fragment. In some embodiments, the anti-c-met
antibody is an antagonist anti-c-met antibody. In some embodiments,
the anti-c-met antibody is monovalent. In some embodiments, the
anti-c-met antibody fragment may comprise a single antigen binding
arm and an Fc region. Anti-c-met antibody fragments are described
herein and are known in the art, in the one-armed format.
Accordingly, in some embodiments, the anti-c-met antibody fragment
is a one-armed antibody (i.e., the heavy chain variable domain and
the light chain variable domain form a single antigen binding arm)
comprising an Fc region, wherein the Fc region comprises a first
and a second Fc polypeptide, wherein the first and second Fc
polypeptides are present in a complex. In some embodiments, the
first and second Fc polypeptides form a Fc region that increases
stability of the anti-c-met antibody compared to a Fab molecule
comprising said antigen binding arm. In some embodiments, the
anti-c-met antibody comprises (a) a first polypeptide comprising
the amino acid sequence of SEQ ID NO:19, a CH1 sequence, and a
first Fc polypeptide and (b) a second polypeptide comprising the
amino acid sequence of SEQ ID NO:20 and CL1 sequence. In some
embodiments, the anti-c-met antibody further comprises (c) a third
polypeptide comprising a second Fc polypeptide.
[0132] In some embodiments, the anti-c-met antibody fragment of the
pharmaceutical formulation described herein comprises an antigen
binding site of the bivalent antibody and thus retains the ability
to bind antigen. In some embodiments, the anti-c-met antibody
fragment comprises the Fc region and retains at least one of the
biological functions normally associated with the Fc region when
present in an bivalent antibody, such as FcRn binding, antibody
half life modulation, ADCC function and complement binding. In some
embodiments, the anti-c-met antibody fragment does not have ADCC
function and/or complement binding activity. In some embodiments,
the anti-c-met antibody fragment is a monovalent antibody that has
an in vivo half life substantially similar to a bivalent antibody.
For example, such an antibody fragment may comprise on antigen
binding arm linked to an Fc sequence capable of conferring in vivo
stability to the fragment. In some embodiments, an Fc polypeptide
comprises part or all of a wild type hinge sequence (generally at
its N terminus). In some embodiments, an Fc polypeptide does not
comprise a functional or wild type hinge sequence. In some
embodiments, the anti-c-met antibody fragment is a one-armed
antibody as described in WO2005/063816. In some embodiments, the
one-armed antibody comprises Fc mutations constituting "knobs" and
"holes" as described in WO2005/063816; Ridgeway, J et al, Prot Eng
(1996) 9:617-21; and Zhu Z et al. Prot Sci (1997) 6:781-8. In some
embodiments, the Fc region comprises at least one protuberance
(knob) and at least one cavity (hole), wherein presence of the
protuberance and cavity enhances formation of a complex between an
Fc polypeptide comprising the protuberance and an Fc polypeptide
comprising the cavity, for example as described in WO 2005/063816.
In some embodiments, the Fc region of the anti-c-met antibodies
comprises a first and a second Fc polypeptide, wherein the first
and second polypeptide each comprises one or more mutations with
respect to wild type human Fc. In some embodiments, a cavity
mutation is T366S, L368A and/or Y407V. In some embodiments, a
protuberance mutation is T366W. In some embodiments, the first
polypeptide comprises the Fc sequence depicted in FIG. 2 and the
second polypeptide comprises the Fc sequence depicted in FIG. 3. In
some embodiments, the anti-c-met antibody may comprise at least one
characteristic that promotes heterodimerization, while minimizing
homodimerization, of the Fc sequences within the antibody
fragment.
[0133] In some embodiments of any of the anti-c-met antibodies
described herein, the anti-c-met antibody is an antagonist
anti-c-met antibody. In some embodiments, blocking anti-c-met
antibodies or antagonist anti-c-met antibodies completely inhibit
the biological activity of the antigen. For treatment of
pathological conditions requiring an antagonistic function and
where bivalency of an anti-c-met antibody results in an undesirable
agonistic effect upon binding to a target antigen (even though it
is an antagonistic anti-c-met antibody as a Fab fragment), the
monovalent trait of a one-armed antibody (i.e., an antibody
comprising a single antigen binding arm) results in and/or ensures
an antagonistic function upon binding of the anti-c-met antibody to
a target molecule. Furthermore, the one-armed antibody comprising a
Fc region is characterized by superior pharmacokinetic attributes
(such as an enhanced half life and/or reduced clearance rate in
vivo) compared to Fab forms having similar/substantially identical
antigen binding characteristics, thus overcoming a major drawback
in the use of conventional monovalent Fab antibodies.
[0134] Anti-c-met antibodies (which may be provided as one-armed
antibodies) useful in the pharmaceutical formulation described
herein include those known in the art (see, e.g., Martens, T. et
al., Clin. Cancer Res. 12 (20 Pt. 1):6144 (2006); U.S. Pat. No.
6,468,529; WO2006/015371; WO2007/063816, and WO2010/045345, which
are incorporated by reference in their entirety). In some
embodiments, the anti-c-met antibody for use in the pharmaceutical
formulations described herein comprises one or more of the HVR
sequences of the monoclonal antibody produced by the hybridoma cell
line deposited under American Type Culture Collection (ATCC)
Accession Number ATCC HB-11894 (hybridoma 1A3.3.13) or HB-11895
(hybridoma 5D5.11.6). In some embodiments, the anti-c-met antibody
is a one-armed antibody comprising one or more of the HVRs of the
light chain variable domain and/or one or more of the HVRs of the
heavy chain variable domain of ATCC Accession Number ATCC HB-11894
(hybridoma 1A3.3.13) or HB-11895 (hybridoma 5D5.11.6) and an Fc
polypeptide.
[0135] In some embodiments of the anti-c-met antibody
pharmaceutical formulation, the anti-c-met antibody comprises a
light chain variable domain comprising one or more of HVR1-LC,
HVR2-LC and HVR3-LC sequence depicted in FIG. 2 (SEQ ID NOs:1-3).
In some embodiments, the anti-c-met antibody comprises a heavy
chain variable domain comprising one or more of HVR1-HC, HVR2-HC
and HVR3-HC sequence depicted in FIG. 2 (SEQ ID NOs:4-6). In some
embodiments, the anti-c-met antibody comprises a light chain
variable domain comprising one or more of HVR1-LC, HVR2-LC and
HVR3-LC sequence depicted in FIG. 2 (SEQ ID NOs:1-3) and one or
more of HVR1-HC, HVR2-HC and HVR3-HC sequence depicted in FIG. 2
(SEQ ID NOs:4-6). In some embodiments, the heavy chain variable
domain comprises one or more of HVR1-HC, HVR2-HC and HVR3-HC
sequence depicted in FIG. 2 (SEQ ID NOs:4-6) and one or more of
FR1-HC, FR2-HC, FR3-HC and FR4-HC sequence depicted in FIG. 2 (SEQ
ID NOs:11-14). In some embodiments, the light chain variable domain
comprises one or more of HVR1-LC, HVR2-LC and HVR3-LC sequence
depicted in FIG. 2 (SEQ ID NOs:1-3) and one or more of FR1-LC,
FR2-LC, FR3-LC and FR4-LC sequence depicted in FIG. 2 (SEQ ID
NOs:7-10). In some embodiments, the anti-c-met antibody is a
one-armed antibody comprising one or more of the HVRs of the light
chain variable domain (SEQ ID NOs:1-3) and/or one or more of the
HVRs of the heavy chain variable domain (SEQ ID NOs:4-6) and an Fc
polypeptide.
[0136] In some embodiments of the anti-c-met antibody
pharmaceutical formulation, the anti-c-met antibody comprises: (a)
at least one, two, three, four, or five HVR sequences selected from
the group consisting of: (i) HVR-L1 comprising sequence A1-A17,
wherein A1-A17 is KSSQSLLYTSSQKNYLA (SEQ ID NO:23) (ii) HVR-L2
comprising sequence B1-B7, wherein B1-B7 is WASTRES (SEQ ID NO:24);
(iii) HVR-L3 comprising sequence C1-C9, wherein C1-C9 is QQYYAYPWT
(SEQ ID NO:25); (iv) HVR-H1 comprising sequence D1-D10, wherein
D1-D10 is GYTFTSYWLH (SEQ ID NO:26); (v) HVR-H2 comprising sequence
E1-E18, wherein E1-E18 is GMIDPSNSDTRFNPNFKD (SEQ ID NO:27); and
(vi) HVR-H3 comprising sequence F1-F11, wherein F1-F11 is
XYGSYVSPLDY (SEQ ID NO:28) and X is not R; and (b) at least one
variant HVR, wherein the variant HVR sequence comprises
modification of at least one residue of the sequence depicted in
SEQ ID NOs:23, 24, 25, 26, 27, or 28. In some embodiments, HVR-L1
of the anti-c-met antibody comprises the sequence of SEQ ID NO:23.
In some embodiments, HVR-L2 comprises the sequence of SEQ ID NO:24.
In some embodiments, HVR-L3 comprises the sequence of SEQ ID NO:25.
In some embodiments, HVR-H1 comprises the sequence of SEQ ID NO:26.
In some embodiments, HVR-H2 comprises the sequence of SEQ ID NO:27.
In some embodiments, HVR-H3 the sequence of SEQ ID NO:28. In some
embodiments, HVR-H3 comprises TYGSYVSPLDY (SEQ ID NO: 29). In some
embodiments, HVR-H3 comprises SYGSYVSPLDY (SEQ ID NO:30). In some
embodiments, the anti-c-met antibody comprising these sequences (in
combination as described herein) is humanized or human. In some
embodiments, the anti-c-met antibody is a one-armed antibody
comprising one or more of the HVRs of the light chain variable
domain (SEQ ID NOs:23-25) and/or one or more of the HVRs of the
heavy chain variable domain (SEQ ID NOs:26-30) and an Fc
polypeptide.
[0137] Provided herein are also anti-c-met antibodies for use in
the pharmaceutical formulation comprising one, two, three, four,
five or six HVRs, wherein each HVR comprises, consists or consists
essentially of a sequence selected from the group consisting of SEQ
ID NOs:23, 24, 25, 26, 27, 28, and 29, and wherein SEQ ID NO:23
corresponds to an HVR-L1, SEQ ID NO:24 corresponds to an HVR-L2,
SEQ ID NO:25 corresponds to an HVR-L3, SEQ ID NO:26 corresponds to
an HVR-H1, SEQ ID NO:27 corresponds to an HVR-H2, and SEQ ID
NOs:26, 27, or 28 corresponds to an HVR-H3. In some embodiments,
the anti-c-met antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1,
HVR-H2, and HVR-H3, wherein each, in order, comprises SEQ ID
NOs:23, 24, 25, 26, 27 and 29. In some embodiments, the anti-c-met
antibody comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and
HVR-H3, wherein each, in order, comprises SEQ ID NOs:23, 24, 25,
26, 27 and 30.
[0138] Variant HVRs can have modifications of one or more residues
within the HVR. In some embodiments, a HVR-L2 variant comprises 1-5
(1, 2, 3, 4 or 5) substitutions in any combination of the following
positions: B1 (M or L), B2 (P, T, G or S), B3 (N, G, R or T), B4
(I, N or F), B5 (P, I, L or G), B6 (A, D, T or V) and B7 (R, I, M
or G). In some embodiments, a HVR-H1 variant comprises 1-5 (1, 2,
3, 4 or 5) substitutions in any combination of the following
positions: D3 (N, P, L, S, A, I), D5 (I, S or Y), D6 (G, D, T, K,
R), D7 (F, H, R, S, T or V) and D9 (M or V). In some embodiments, a
HVR-H2 variant comprises 1-4 (1, 2, 3 or 4) substitutions in any
combination of the following positions: E7 (Y), E9 (I), E 10 (I),
E14 (T or Q), E15 (D, K, S, T or V), E16 (L), E17 (E, H, N or D)
and E18 (Y, E or H). In some embodiments, a HVR-H3 variant
comprises 1-5 (1, 2, 3, 4 or 5) substitutions in any combination of
the following positions: F1 (T, S), F3 (R, S, H, T, A, K), F4 (G),
F6 (R, F, M, T, E, K, A, L, W), F7 (L, I, T, R, K, V), F8 (S, A),
F10 (Y, N) and F11 (Q, S, H, F). Letter(s) in parenthesis following
each position indicates an illustrative substitution (i.e.,
replacement) amino acid; as would be evident to one skilled in the
art, suitability of other amino acids as substitution amino acids
in the context described herein can be routinely assessed using
techniques known in the art and/or described herein. In some
embodiments, a HVR-L1 comprises the sequence of SEQ ID NO:23. In
some embodiments, F1 in a variant HVR-H3 is T. In some embodiments,
F1 in a variant HVR-H3 is S. In some embodiments, F3 in a variant
HVR-H3 is R. In some embodiments, F3 in a variant HVR-H3 is S. In
some embodiments, F7 in a variant HVR-H3 is T. In some embodiments,
the anti-c-met antibody comprises a variant HVR-H3 wherein F1 is T
or S, F3 is R or S, and F7 is T.
[0139] In some embodiments, the anti-c-met antibody of the
pharmaceutical formulation comprises a variant HVR-H3 wherein F1 is
T, F3 is R and F7 is T. In some embodiments, the anti-c-met
antibody comprises a variant HVR-H3 wherein F1 is S. In some
embodiments, the anti-c-met antibody comprises a variant HVR-H3
wherein F1 is T, and F3 is R. In some embodiments, the anti-c-met
antibody comprises a variant HVR-H3 wherein F1 is S, F3 is R and F7
is T. In some embodiments, the anti-c-met antibody comprises a
variant HVR-H3 wherein F1 is T, F3 is S, F7 is T, and F8 is S. In
some embodiments, the anti-c-met antibody comprises a variant
HVR-H3 wherein F1 is T, F3 is S, F7 is T, and F8 is A. In some
embodiments, said variant HVR-H3 antibody further comprises HVR-L1,
HVR-L2, HVR-L3, HVR-H1 and HVR-H2 wherein each comprises, in order,
the sequence depicted in SEQ ID NOs:1, 2, 3, 4 and 5. In some
embodiments, these antibodies further comprise a human subgroup III
heavy chain framework consensus sequence. In some embodiments of
these antibodies, the framework consensus sequence comprises
substitution at position 71, 73 and/or 78. In some embodiments of
these antibodies, position 71 is A, 73 is T and/or 78 is A. In some
embodiments of these antibodies, these antibodies further comprise
a human .kappa.I light chain framework consensus sequence.
[0140] In some embodiments, the anti-c-met antibody of the
pharmaceutical formulation comprises a variant HVR-L2 wherein B6 is
V. In some embodiments, said variant HVR-L2 anti-c-met antibody
further comprises HVR-L1, HVR-L3, HVR-H1, HVR-H2 and HVR-H3,
wherein each comprises, in order, the sequence depicted in SEQ ID
NOs:23, 25, 26, 27 and 28. In some embodiments, said variant HVR-L2
anti-c-met antibody further comprises HVR-L1, HVR-L3, HVR-H1,
HVR-H2 and HVR-H3, wherein each comprises, in order, the sequence
depicted in SEQ ID NOs:23, 25, 26, 27 and 29. In some embodiments,
said variant HVR-L2 anti-c-met antibody further comprises HVR-L1,
HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein each comprises, in
order, the sequence depicted in SEQ ID NOs:23, 25, 26, 27 and 30.
In some embodiments, these anti-c-met antibodies further comprise a
human subgroup III heavy chain framework consensus sequence. In
some embodiments of these anti-c-met antibodies, the framework
consensus sequence comprises substitution at position 71, 73 and/or
78. In some embodiments of these anti-c-met antibodies, position 71
is A, 73 is T and/or 78 is A. In some embodiments of these
anti-c-met antibodies, these antibodies further comprise a human
.kappa.I light chain framework consensus sequence.
[0141] In some embodiments, the anti-c-met antibody of the
pharmaceutical formulation comprises a variant HVR-H2 wherein E14
is T, E15 is K and E17 is E. In some embodiments, the anti-c-met
antibody comprises a variant HVR-H2 wherein E17 is E. In some
embodiments, said variant HVR-H3 anti-c-met antibody further
comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, and HVR-H3 wherein each
comprises, in order, the sequence depicted in SEQ ID NOs:23, 24,
25, 26, and 28. In some embodiments, said variant HVR-H2 anti-c-met
antibody further comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1, and
HVR-H3, wherein each comprises, in order, the sequence depicted in
SEQ ID NOs:23, 24, 25, 26, and 29. In some embodiments, said
variant HVR-H2 anti-c-met antibody further comprises HVR-L1,
HVR-L2, HVR-L3, HVR-H1, and HVR-H3, wherein each comprises, in
order, the sequence depicted in SEQ ID NOs:23, 24, 25, 26 and 30.
In some embodiments, these anti-c-met antibodies further comprise a
human subgroup III heavy chain framework consensus sequence. In
some embodiments of these anti-c-met antibodies, the framework
consensus sequence comprises substitution at position 71, 73 and/or
78. In some embodiments of these anti-c-met antibodies, position 71
is A, 73 is T and/or 78 is A. In some embodiments of these
antibodies, these anti-c-met antibodies further comprise a human
.kappa.I light chain framework consensus sequence.
[0142] In some embodiments, the anti-c-met antibody of the
pharmaceutical formulation comprises (a) a heavy chain variable
domain comprising the sequence:
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTR
FNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVS S (SEQ
ID NO:19) and/or (b) a light chain variable domain comprising the
sequence:
DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTR
ESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR (SEQ ID
NO:20). In some embodiments, the anti-c-met antibody is a one-armed
antibody comprising (a) the light chain variable domain (SEQ ID
NO:20) and/or (b) the heavy chain variable domain (SEQ ID NO:19)
and (c) a Fc polypeptide.
[0143] In some embodiments, the anti-c-met antibody of the
pharmaceutical formulation comprises (a) HVR-H1, HVR-H2, and HVR-H3
of a heavy chain variable domain comprising the sequence:
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTR
FNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVS S (SEQ
ID NO:19) and/or (b) HVR-L1, HVR-L2, and HVR-L3 of a light chain
variable domain comprising the sequence:
DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTR
ESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR (SEQ ID
NO:20). In some embodiments, the anti-c-met antibody is a one-armed
antibody comprising (a) the light chain variable domain (SEQ ID
NO:20) and/or (b) the heavy chain variable domain (SEQ ID NO:19)
and (c) a Fc polypeptide.
[0144] In some embodiments, the anti-c-met antibody of the
pharmaceutical formulation is an anti-c-met antibody fragment,
wherein the antibody fragment comprises (a) a first polypeptide
comprising a heavy chain variable domain comprising SEQ ID NO:19,
CH1 sequence (e.g., SEQ ID NO:16), and a first Fc polypeptide; and
(b) a second polypeptide comprising a light chain variable domain
comprising SEQ ID NO:20, and CL1 sequence (e.g., SEQ ID NO:15).
[0145] In some embodiments, the anti-c-met antibody of the
pharmaceutical formulation is an anti-c-met antibody fragment,
wherein the antibody fragment comprises (a) a first polypeptide
comprising a heavy chain variable domain comprising SEQ ID NO:19,
CH1 sequence (e.g., SEQ ID NO:16), and a first Fc polypeptide; (b)
a second polypeptide comprising a light chain variable domain
comprising SEQ ID NO:20, and CL1 sequence (e.g., SEQ ID NO:15); and
(c) a third polypeptide comprising a second Fc polypeptide, wherein
the heavy chain variable domain and the light chain variable domain
are present as a complex and form a single antigen binding arm and
wherein the first and second Fc polypeptides are present in a
complex. In some embodiments, the first and second Fc polypeptides
form a Fc region that increases stability of said antibody fragment
compared to a Fab molecule comprising said antigen binding arm. In
some embodiments, the Fc region is that of a human IgG (e.g., IgG1,
2, 3 or 4). In some embodiments, the first Fc polypeptide comprises
the Fc sequence depicted in FIG. 2 (SEQ ID NO:17) and the second Fc
polypeptide comprises the Fc sequence depicted in FIG. 3 (SEQ ID
NO:18). In some embodiments, the first Fc polypeptide comprises the
Fc sequence depicted in FIG. 3 (SEQ ID NO:18) and the second Fc
polypeptide comprises the Fc sequence depicted in FIG. 2 (SEQ ID
NO:17).
[0146] In some embodiments, the anti-c-met antibody is an
anti-c-met antibody or antibody fragment thereof, wherein the
antibody comprises (a) a first polypeptide comprising a heavy chain
variable domain comprising SEQ ID NO:19, CH1 sequence, and a first
Fc polypeptide; (b) a second polypeptide comprising a light chain
variable domain comprising SEQ ID NO:20, and CL1 sequence; and (c)
a third polypeptide comprising a second Fc polypeptide, wherein the
heavy chain variable domain and the light chain variable domain are
present as a complex and form a single antigen binding arm, wherein
the first and second Fc polypeptides are present in a complex and
form a Fc region that increases stability of said antibody fragment
compared to a Fab molecule comprising said antigen binding arm.
[0147] In some embodiments, the anti-c-met antibody comprises (a) a
first polypeptide comprising a heavy chain variable domain, said
polypeptide comprising the sequence: EVQLVESGGGLVQPGGSLRLSCAASGYTFT
SYWLHWVRQAPGKGLEWVGMIDPSNS
FNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMT
KNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:21); (b) a second
polypeptide comprising a light chain variable domain, the
polypeptide comprising the sequence
DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRE
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:22); and a third
polypeptide comprising a Fc sequence, the polypeptide comprising
the sequence
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPSREEMTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:18),
wherein the heavy chain variable domain and the light chain
variable domain are present as a complex and form a single antigen
binding arm and wherein the first and second Fc polypeptides are
present in a complex. In some embodiments, the first and second Fc
polypeptides form a Fc region that increases stability of said
antibody fragment compared to a Fab molecule comprising said
antigen binding arm.
[0148] In some embodiments, polynucleotides encoding any of the
anti-c-met antibodies described herein are expressed such that the
anti-c-met antibody is produced. In some embodiments,
polynucleotides encoding any of the anti-c-met antibody are
expressed in vitro or in vivo (for example, in CHO cells or E. coli
cells).
[0149] In some embodiments, the anti-c-met antibody for use in the
pharmaceutical formulation described herein is onartuzumab
(interchangeably termed MetMAb), a one-armed antibody comprising a
Fc region. A sequence of MetMAb is shown in FIGS. 2 and 3. MetMAb
(also termed OA5D5v2 and onartuzumab) is also described in, e.g.,
WO2006/015371; WO2010/04345; and Jin et al, Cancer Res (2008)
68:4360. Biosimilar version of MetMAb are also contemplated and
encompassed herein for use in the formulation.
[0150] In some embodiments, the anti-c-met antibody of the
pharmaceutical formulation specifically binds at least a portion of
c-met Sema domain or variant thereof. In some embodiments, the
anti-c-met antibody is an antagonist. In some embodiments, the
anti-c-met antagonist antibody specifically binds at least one of
the sequences selected from the group consisting of LDAQT (SEQ ID
NO:31) (e.g., residues 269-273 of c-met), LTEKRKKRS (SEQ ID NO:32)
(e.g., residues 300-308 of c-met), KPDSAEPM (SEQ ID NO: 33) (e.g.,
residues 350-357 of c-met) and NVRCLQHF (SEQ ID NO:34) (e.g.,
residues 381-388 of c-met). In some embodiments, the anti-c-met
antagonist antibody specifically binds a conformational epitope
formed by part or all of at least one of the sequences selected
from the group consisting of LDAQT (SEQ ID NO:31) (e.g., residues
269-273 of c-met), LTEKRKKRS (SEQ ID NO:32) (e.g., residues 300-308
of c-met), KPDSAEPM (SEQ ID NO: 33) (e.g., residues 350-357 of
c-met) and NVRCLQHF (SEQ ID NO:34) (e.g., residues 381-388 of
c-met). In some embodiments, an antagonist antibody specifically
binds an amino acid sequence having at least 50%, 60%, 70%, 80%,
90%, 95%, 98% sequence identity or similarity with the sequence
LDAQT (SEQ ID NO:31), LTEKRKKRS (SEQ ID NO:32), KPDSAEPM (SEQ ID
NO:33) and/or NVRCLQHF (SEQ ID NO:34). In some embodiments, the
anti-c-met antibody is an antagonist anti-c-met antibody. In some
embodiments, the anti-c-met antibody is a one-armed antibody. In
order to screen for antibodies which bind to an epitope on an
antigen bound by an antibody of interest, a routine cross-blocking
assay such as that described in Antibodies, A Laboratory Manual,
Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can
be performed.
[0151] In some embodiments of any of the anti-c-met antibodies
described herein, the anti-c-met antibody may interfere with
HGF/c-met activation, including but not limited to interfering with
HGF binding to the extracellular portion of c-met and receptor
multimerization. In some embodiments, the anti-c-met antibody are
useful in treating or diagnosing pathological conditions associated
with abnormal or unwanted signaling of the HGF/c-met pathway. In
some embodiments, the anti-c-met antibody may modulate the
HGF/c-met pathway, including modulation of c-met ligand binding,
c-met dimerization, activation, and other biological/physiological
activities associated with HGF/c-met signaling. In some
embodiments, the anti-c-met antibody may disrupt HGF/c-met
signaling pathway. In some embodiments of any of the anti-c-met
antibodies described herein, binding of the anti-c-met antibody to
c-met inhibits c-met activation by HGF. In some embodiments of any
of the anti-c-met antibodies described herein, binding of the
anti-c-met antibody to c-met in a cell inhibits proliferation,
survival, scattering, morphogenesis and/or motility of the
cell.
[0152] In some instances, it may be advantageous to have an
anti-c-met antibody that does not interfere with binding of a
ligand (such as HGF) to c-met. Accordingly, in some embodiments,
the anti-c-met antibody does not bind an HGF binding site on c-met.
In some embodiment, the anti-c-met antibody does not substantially
inhibit HGF binding to c-met. In some embodiments, the anti-c-met
antibody does not substantially compete with HGF for binding to
c-met. In one example, the anti-c-met antibody can be used in
conjunction with one or more other antagonists, wherein the
antagonists are targeted at different processes and/or functions
within the HGF/c-met axis. Thus, in some embodiments, the
anti-c-met antibody binds to an epitope on c-met distinct from an
epitope bound by another c-met antagonist (such as the Fab fragment
of the monoclonal antibody produced by the hybridoma cell line
deposited under American Type Culture Collection Accession Number
ATCC HB-11894 (hybridoma 1A3.3.13)). In another embodiment, the
anti-c-met antibody is distinct from (i.e., it is not) a Fab
fragment of the monoclonal antibody produced by the hybridoma cell
line deposited under American Type Culture Collection Accession
Number ATCC HB-11894 (hybridoma 1A3.3.13).
[0153] In some embodiments, the anti-c-met antibody binds to c-met
of a first animal species, and does not specifically bind to c-met
of a second animal species. In some embodiments, the first animal
species is human and/or primate (e.g., cynomolgus monkey), and the
second animal species is murine (e.g., mouse) and/or canine. In
some embodiments, the first animal species is human. In some
embodiments, the first animal species is primate, for example
cynomolgus monkey. In some embodiments, the second animal species
is murine, for example mouse. In some embodiments, the second
animal species is canine.
[0154] In some embodiments, the anti-c-met antibody elicits little
to no immunogenic response in said subject. In some embodiments,
the anti-c-met antibody elicits an immunogenic response at or less
than a clinically-acceptable level.
[0155] In some embodiments of any of the anti-c-met antibodies
described herein, an altered antibody that possesses some but not
all effector functions. In some embodiments, the anti-c-met
antibody does not possess complement depletion and/or ADCC
activity. In some embodiments, the Fc activities of the produced
immunoglobulin are measured to ensure that only the desired
properties are maintained (e.g., half-life but not complement
depletion and/or ADCC activity). In vitro and/or in vivo
cytotoxicity assays can be conducted to confirm the
reduction/depletion of CDC and/or ADCC activities. For example, Fc
receptor (FcR) binding assays can be conducted to ensure that the
antibody lacks Fc.gamma.R binding (hence likely lacking ADCC
activity), but retains FcRn binding ability. The primary cells for
mediating ADCC, NK cells, express Fc.gamma.RIII only, whereas
monocytes express Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR
expression on hematopoietic cells is summarized in Table 3 on page
464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). An
example of an in vitro assay to assess ADCC activity of a molecule
of interest is described in U.S. Pat. No. 5,500,362 or 5,821,337.
Useful effector cells for such assays include peripheral blood
mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in a animal model such as
that disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998). C1q
binding assays may also be carried out to confirm that the antibody
is unable to bind C1q and hence lacks CDC activity. To assess
complement activation, a CDC assay, e.g. as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed. FcRn binding and in vivo clearance/half life
determinations can also be performed using methods known in the
art. In some embodiments, the anti-c-met antibody is glycosylated.
In some embodiments, the anti-c-met antibody is substantially
aglycosylated.
[0156] The anti-c-met antibodies of the formulations described
herein can be characterized for their physical/chemical properties
and biological functions by various assays known in the art. The
purified anti-c-met antibodies can be further characterized by a
series of assays including, but not limited to, N-terminal
sequencing, amino acid analysis, non-denaturing size exclusion high
pressure liquid chromatography (HPLC), mass spectrometry, ion
exchange chromatography and papain digestion.
[0157] In some embodiments of any of the anti-c-met antibodies
described herein, the anti-c-met antibody may be purified (1) to
greater than 95% by weight of antibody as determined by the Lowry
method, and most preferably more than 99% by weight, (2) to a
degree sufficient to obtain at least 15 residues of N-terminal or
internal amino acid sequence by use of a spinning cup sequenator,
or (3) to homogeneity by SDS-PAGE under reducing or nonreducing
conditions using Coomassie blue, or silver stain.
[0158] Further, in some embodiments, the anti-c-met antibody
according to any of the above embodiments may incorporate any of
the features, singly or in combination, as described in Sections
1-8 below:
[0159] 1. Antibody Affinity
[0160] In some embodiments, the anti-c-met antibody provided herein
has a dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100
nM, .ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM,
or .ltoreq.0.001 nM (e.g. 10.sup.-8M or less, e.g. from 10.sup.-8M
to 10.sup.-13 M, e.g., from 10.sup.-9M to 10.sup.-13 M).
[0161] Binding affinity of a ligand to its receptor can be
determined using any of a variety of assays, and expressed in terms
of a variety of quantitative values. Antigen binding assays are
known in the art and can be used herein include without limitation
any direct or competitive binding assays using techniques such as
western blots, radioimmunoassays, enzyme-linked immunoabsorbent
assay (ELISA), "sandwich" immunoassays, surface plasmon resonance
based assay (such as the BIAcore assay as described in PCT
Application Publication No. WO2005/012359), immunoprecipitation
assays, fluorescent immunoassays, and protein A immunoassays.
[0162] Accordingly, in some embodiments, the binding affinity is
expressed as Kd values and reflects intrinsic binding affinity
(e.g., with minimized avidity effects). The anti-c-met antibody
selected will normally have a sufficiently strong binding affinity
for c-met, for example, the antibody may bind human c-met with a Kd
value of between 100 nM.sup.-1 pM.
[0163] 2. Antibody Fragments
[0164] In some embodiments, the anti-c-met antibody of the
pharmaceutical formulation described herein is an antibody
fragment. Antibody fragments include, but are not limited to, Fab,
Fab', Fab'-SH, F(ab').sub.2, Fv, one-armed antibodies, and scFv
fragments, and other fragments described below. For a review of
certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134
(2003). For a review of scFv fragments, see, e.g., Pluckthun, in
The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and
Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see
also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab').sub.2 fragments comprising salvage
receptor binding epitope residues and having increased in vivo
half-life, see U.S. Pat. No. 5,869,046. Other monovalent antibody
forms are described in, e.g., WO2007048037, WO2008145137,
WO2008145138, and WO2007059782. One-armed antibodies are described,
e.g., in WO2005/063816. Diabodies are antibody fragments with two
antigen-binding sites that may be bivalent or bispecific. See, for
example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med.
9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA
90: 6444-6448 (1993). Triabodies and tetrabodies are also described
in Hudson et al., Nat. Med. 9:129-134 (2003).
[0165] Single-domain antibodies are antibody fragments comprising
all or a portion of the heavy chain variable domain or all or a
portion of the light chain variable domain of an antibody. In some
embodiments, a single-domain antibody is a human single-domain
antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No.
6,248,516 B1).
[0166] Antibody fragments can be made by various techniques,
including but not limited to proteolytic digestion of an intact
antibody as well as production by recombinant host cells (e.g. E.
coli or phage), as described herein.
[0167] 3. Chimeric and Humanized Antibodies
[0168] In some embodiments, the anti-c-met antibody of the
pharmaceutical formulation described herein is a chimeric antibody.
Certain chimeric antibodies are described, e.g., in U.S. Pat. No.
4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,
81:6851-6855 (1984)). In one example, a chimeric antibody comprises
a non-human variable region (e.g., a variable region derived from a
mouse, rat, hamster, rabbit, or non-human primate, such as a
monkey) and a human constant region. In a further example, a
chimeric antibody is a "class switched" antibody in which the class
or subclass has been changed from that of the parent antibody.
Chimeric antibodies include antigen-binding fragments thereof.
[0169] In some embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the CDR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0170] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro and Fransson, Front. Biosci.
13:1619-1633 (2008), and are further described, e.g., in Riechmann
et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad.
Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods
36:25-34 (2005) (describing SDR (a-HVR) grafting); Padlan, Mol.
Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua
et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and
Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J.
Cancer, 83:252-260 (2000) (describing the "guided selection"
approach to FR shuffling).
[0171] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296
(1993)); framework regions derived from the consensus sequence of
human antibodies of a particular subgroup of light or heavy chain
variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci.
USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623
(1993)); human mature (somatically mutated) framework regions or
human germline framework regions (see, e.g., Almagro and Fransson,
Front. Biosci. 13:1619-1633 (2008)); and framework regions derived
from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem.
272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.
271:22611-22618 (1996)).
[0172] 4. Human Antibodies
[0173] In some embodiments, the anti-c-met antibody of the
pharmaceutical formulation described herein is a human antibody.
Human antibodies can be produced using various techniques known in
the art. Human antibodies are described generally in van Dijk and
van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg,
Curr. Opin. Immunol. 20:450-459 (2008).
[0174] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos.
6,075,181 and 6,150,584 describing XENOMOUSE.TM. technology; U.S.
Pat. No. 5,770,429 describing HuMAB.RTM. technology; U.S. Pat. No.
7,041,870 describing K-M MOUSE.RTM. technology, and U.S. Patent
Application Publication No. US 2007/0061900, describing
VELocIMousE.RTM. technology). Human variable regions from intact
antibodies generated by such animals may be further modified, e.g.,
by combining with a different human constant region.
[0175] Human antibodies can also be made by hybridoma-based
methods. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described.
(See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.
Immunol., 147: 86 (1991).) Human antibodies generated via human
B-cell hybridoma technology are also described in Li et al., Proc.
Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods
include those described, for example, in U.S. Pat. No. 7,189,826
(describing production of monoclonal human IgM antibodies from
hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268
(2006) (describing human-human hybridomas). Human hybridoma
technology (Trioma technology) is also described in Vollmers and
Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and
Vollmers and Brandlein, Methods and Findings in Experimental and
Clinical Pharmacology, 27(3):185-91 (2005).
[0176] Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain. Techniques for selecting human
antibodies from antibody libraries are described below.
[0177] 5. Library-Derived Antibodies
[0178] The anti-c-met antibody of the pharmaceutical formulation
described herein may be isolated by screening combinatorial
libraries for antibodies with the desired activity or activities.
For example, a variety of methods are known in the art for
generating phage display libraries and screening such libraries for
antibodies possessing the desired binding characteristics. Such
methods are reviewed, e.g., in Hoogenboom et al. in Methods in
Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press,
Totowa, N.J., 2001) and further described, e.g., in the McCafferty
et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628
(1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and
Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed.,
Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.
338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093
(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472
(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132
(2004).
[0179] In some phage display methods, repertoires of V.sub.H and
V.sub.L genes are separately cloned by polymerase chain reaction
(PCR) and recombined randomly in phage libraries, which can then be
screened for antigen-binding phage as described in Winter et al.,
Ann. Rev. Immunol., 12: 433-455 (1994). Phage typically display
antibody fragments, either as single-chain Fv (scFv) fragments or
as Fab fragments. Libraries from immunized sources provide
high-affinity antibodies to the immunogen without the requirement
of constructing hybridomas. Alternatively, the naive repertoire can
be cloned (e.g., from human) to provide a single source of
antibodies to a wide range of non-self and also self antigens
without any immunization as described by Griffiths et al., EMBO J,
12: 725-734 (1993). Finally, naive libraries can also be made
synthetically by cloning unrearranged V-gene segments from stem
cells, and using PCR primers containing random sequence to encode
the highly variable CDR3 regions and to accomplish rearrangement in
vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227:
381-388 (1992). Patent publications describing human antibody phage
libraries include, for example: U.S. Pat. No. 5,750,373, and US
Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,
2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and
2009/0002360.
[0180] Antibodies or antibody fragments isolated from human
antibody libraries are considered human antibodies or human
antibody fragments herein.
[0181] 6. Multispecific Antibodies
[0182] In some embodiments, the anti-c-met antibody of the
pharmaceutical formulation described herein is a multispecific
antibody, e.g. a bispecific antibody. Multispecific antibodies are
monoclonal antibodies that have binding specificities for at least
two different sites. In some embodiments, one of the binding
specificities is for an antigen and the other is for any other
antigen. In some embodiments, bispecific antibodies may bind to two
different epitopes of an antigen. Bispecific antibodies may also be
used to localize cytotoxic agents to cells which express an
antigen. Bispecific antibodies can be prepared as full length
antibodies or antibody fragments.
[0183] Techniques for making multispecific antibodies include, but
are not limited to, recombinant co-expression of two immunoglobulin
heavy chain-light chain pairs having different specificities (see
Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and
Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole"
engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific
antibodies may also be made by engineering electrostatic steering
effects for making antibody Fc-heterodimeric molecules (WO
2009/089004A1); cross-linking two or more antibodies or fragments
(see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science,
229: 81 (1985)); using leucine zippers to produce bi-specific
antibodies (see, e.g., Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992)); using "diabody" technology for making
bispecific antibody fragments (see, e.g., Hollinger et al., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain
Fv (scFv) dimers (see, e.g. Gruber et al., J. Immunol., 152:5368
(1994)); and preparing trispecific antibodies as described, e.g.,
in Tutt et al. J. Immunol. 147: 60 (1991).
[0184] Engineered antibodies with three or more functional antigen
binding sites, including "Octopus antibodies," are also included
herein (see, e.g. US 2006/0025576A1).
[0185] The antibody or fragment herein also includes a "Dual Acting
FAb" or "DAF" comprising an antigen binding site that binds to
c-met as well as another, different antigen (see, US 2008/0069820,
for example).
[0186] 7. Antibody Variants
[0187] In some embodiments, amino acid sequence variants of the
anti-c-met antibody for use in the pharmaceutical formulation
described herein are contemplated. For example, it may be desirable
to improve the binding affinity and/or other biological properties
of the antibody. Amino acid sequence variants of an antibody may be
prepared by introducing appropriate modifications into the
nucleotide sequence encoding the antibody, or by peptide synthesis.
Such modifications include, for example, deletions from, and/or
insertions into and/or substitutions of residues within the amino
acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
[0188] a. Substitution, Insertion, and Deletion Variants
[0189] In some embodiments, anti-c-met antibody variants having one
or more amino acid substitutions for use in the pharmaceutical
formulation described herein are provided. Sites of interest for
substitutional mutagenesis include the HVRs and FRs. Conservative
substitutions are shown in Table 1 under the heading of
"conservative substitutions." More substantial changes are provided
in Table 1 under the heading of "exemplary substitutions," and as
further described below in reference to amino acid side chain
classes. Amino acid substitutions may be introduced into an
antibody of interest and the products screened for a desired
activity, e.g., retained/improved antigen binding, decreased
immunogenicity, or improved ADCC or CDC.
TABLE-US-00001 TABLE 1 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met;
Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
[0190] Amino acids may be grouped according to common side-chain
properties:
[0191] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0192] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0193] (3) acidic: Asp, Glu;
[0194] (4) basic: H is, Lys, Arg;
[0195] (5) residues that influence chain orientation: Gly, Pro;
[0196] (6) aromatic: Trp, Tyr, Phe.
[0197] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0198] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further study will have modifications (e.g.,
improvements) in certain biological properties (e.g., increased
affinity, reduced immunogenicity) relative to the parent antibody
and/or will have substantially retained certain biological
properties of the parent antibody. An exemplary substitutional
variant is an affinity matured antibody, which may be conveniently
generated, e.g., using phage display-based affinity maturation
techniques such as those described herein. Briefly, one or more HVR
residues are mutated and the variant antibodies displayed on phage
and screened for a particular biological activity (e.g. binding
affinity).
[0199] Alterations (e.g., substitutions) may be made in HVRs, e.g.,
to improve antibody affinity. Such alterations may be made in HVR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or SDRs
(a-CDRs), with the resulting variant V.sub.H or V.sub.L being
tested for binding affinity. Affinity maturation by constructing
and reselecting from secondary libraries has been described, e.g.,
in Hoogenboom et al. in Methods in Molecular Biology 178:1-37
(O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) In some
embodiments of affinity maturation, diversity is introduced into
the variable genes chosen for maturation by any of a variety of
methods (e.g., error-prone PCR, chain shuffling, or
oligonucleotide-directed mutagenesis). A secondary library is then
created. The library is then screened to identify any antibody
variants with the desired affinity. Another method to introduce
diversity involves HVR-directed approaches, in which several HVR
residues (e.g., 4-6 residues at a time) are randomized. HVR
residues involved in antigen binding may be specifically
identified, e.g., using alanine scanning mutagenesis or modeling.
CDR-H3 and CDR-L3 in particular are often targeted.
[0200] In some embodiments, substitutions, insertions, or deletions
may occur within one or more HVRs so long as such alterations do
not substantially reduce the ability of the antibody to bind
antigen. For example, conservative alterations (e.g., conservative
substitutions as provided herein) that do not substantially reduce
binding affinity may be made in HVRs. Such alterations may be
outside of HVR "hotspots" or SDRs. In some embodiments of the
variant V.sub.H and V.sub.L sequences provided above, each HVR
either is unaltered, or contains no more than one, two or three
amino acid substitutions.
[0201] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham and Wells (1989)
Science, 244:1081-1085. In this method, a residue or group of
target residues (e.g., charged residues such as arg, asp, his, lys,
and glu) are identified and replaced by a neutral or negatively
charged amino acid (e.g., alanine or polyalanine) to determine
whether the interaction of the antibody with antigen is affected.
Further substitutions may be introduced at the amino acid locations
demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an
antigen-antibody complex to identify contact points between the
antibody and antigen. Such contact residues and neighboring
residues may be targeted or eliminated as candidates for
substitution. Variants may be screened to determine whether they
contain the desired properties.
[0202] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0203] b. Glycosylation Variants
[0204] In some embodiments, the anti-c-met antibody of the
pharmaceutical formulation described herein is altered to increase
or decrease the extent to which the antibody is glycosylated.
Addition or deletion of glycosylation sites to an antibody may be
conveniently accomplished by altering the amino acid sequence such
that one or more glycosylation sites is created or removed.
[0205] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.
TIBTECH 15:26-32 (1997). The oligosaccharide may include various
carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc),
galactose, and sialic acid, as well as a fucose attached to a
GlcNAc in the "stem" of the biantennary oligosaccharide structure.
In some embodiments, modifications of the oligosaccharide in an
antibody may be made in order to create antibody variants with
certain improved properties.
[0206] In some embodiments, antibody variants are provided having a
carbohydrate structure that lacks fucose attached (directly or
indirectly) to an Fc region. For example, the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%
or from 20% to 40%. The amount of fucose is determined by
calculating the average amount of fucose within the sugar chain at
Asn297, relative to the sum of all glycostructures attached to Asn
297 (e.g. complex, hybrid and high mannose structures) as measured
by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for
example. Asn297 refers to the asparagine residue located at about
position 297 in the Fc region (Eu numbering of Fc region residues);
however, Asn297 may also be located about .+-.3 amino acids
upstream or downstream of position 297, i.e., between positions 294
and 300, due to minor sequence variations in antibodies. Such
fucosylation variants may have improved ADCC function. See, e.g.,
US Patent Publication Nos. US 2003/0157108 (Presta, L.); US
2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications
related to "defucosylated" or "fucose-deficient" antibody variants
include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US
2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US
2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO
2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742;
WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004);
Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of
cell lines capable of producing defucosylated antibodies include
Lec13 CHO cells deficient in protein fucosylation (Ripka et al.
Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US
2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,
especially at Example 11), and knockout cell lines, such as
alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,
e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda,
Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and
WO2003/085107).
[0207] Antibodies variants are further provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or
improved ADCC function. Examples of such antibody variants are
described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.
No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
Antibody variants with at least one galactose residue in the
oligosaccharide attached to the Fc region are also provided. Such
antibody variants may have improved CDC function. Such antibody
variants are described, e.g., in WO 1997/30087 (Patel et al.); WO
1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
[0208] c. Fc Region Variants
[0209] In some embodiments, one or more amino acid modifications
may be introduced into the Fc region of the anti-c-met antibody of
the pharmaceutical formulation described herein, thereby generating
an Fc region variant. The Fc region variant may comprise a human Fc
region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region)
comprising an amino acid modification (e.g. a substitution) at one
or more amino acid positions.
[0210] In some embodiments, contemplated are antibody variants that
possesses some but not all effector functions, which make it a
desirable candidate for applications in which the half life of the
antibody in vivo is important yet certain effector functions (such
as complement and ADCC) are unnecessary or deleterious. In vitro
and/or in vivo cytotoxicity assays can be conducted to confirm the
reduction/depletion of CDC and/or ADCC activities. For example, Fc
receptor (FcR) binding assays can be conducted to ensure that the
antibody lacks Fc.gamma.R binding (hence likely lacking ADCC
activity), but retains FcRn binding ability. The primary cells for
mediating ADCC, NK cells, express Fc.gamma.RIII only, whereas
monocytes express Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR
expression on hematopoietic cells is summarized in Table 3 on page
464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).
Non-limiting examples of in vitro assays to assess ADCC activity of
a molecule of interest is described in U.S. Pat. Nos. 5,500,362
(see, e.g. Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA
83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad.
Sci. USA 82:1499-1502 (1985); 5,821,337 (see Bruggemann, M. et al.,
J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive
assays methods may be employed (see, for example, ACTI.TM.
non-radioactive cytotoxicity assay for flow cytometry
(CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96.RTM.
non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Useful
effector cells for such assays include peripheral blood mononuclear
cells (PBMC) and Natural Killer (NK) cells. Alternatively, or
additionally, ADCC activity of the molecule of interest may be
assessed in vivo, e.g., in a animal model such as that disclosed in
Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q
binding assays may also be carried out to confirm that the antibody
is unable to bind C1q and hence lacks CDC activity. See, e.g., C1q
and C3c binding ELISA in WO 2006/029879 and WO 2005/100402. To
assess complement activation, a CDC assay may be performed (see,
for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163
(1996); Cragg, M. S. et al., Blood 101:1045-1052 (2003); and Cragg,
M. S. and M. J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding
and in vivo clearance/half life determinations can also be
performed using methods known in the art (see, e.g., Petkova, S. B.
et al., Int'l. Immunol. 18(12):1759-1769 (2006)).
[0211] Antibodies with reduced effector function include those with
substitution of one or more of Fc region residues 238, 265, 269,
270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants
include Fc mutants with substitutions at two or more of amino acid
positions 265, 269, 270, 297 and 327, including the so-called
"DANA" Fc mutant with substitution of residues 265 and 297 to
alanine (U.S. Pat. No. 7,332,581).
[0212] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604
(2001).)
[0213] In some embodiments, an antibody variant comprises an Fc
region with one or more amino acid substitutions which improve
ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the
Fc region (EU numbering of residues).
[0214] In some embodiments, alterations are made in the Fc region
that result in altered (i.e., either improved or diminished) C1q
binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as
described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et
al. J. Immunol. 164: 4178-4184 (2000).
[0215] Antibodies with increased half lives and improved binding to
the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.
117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are
described in US2005/0014934A1 (Hinton et al.). Those antibodies
comprise an Fc region with one or more substitutions therein which
improve binding of the Fc region to FcRn. Such Fc variants include
those with substitutions at one or more of Fc region residues: 238,
256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360,
362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc
region residue 434 (U.S. Pat. No. 7,371,826).
[0216] See also Duncan & Winter, Nature 322:738-40 (1988); U.S.
Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351
concerning other examples of Fc region variants.
[0217] d. Cysteine Engineered Antibody Variants
[0218] In some embodiments, it may be desirable to create cysteine
engineered antibodies, e.g., "thioMAbs," in which one or more
residues of the anti-c-met antibody of the pharmaceutical
formulation described herein are substituted with cysteine
residues. In particular embodiments, the substituted residues occur
at accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as drug moieties or linker-drug
moieties, to create an immunoconjugate, as described further
herein. In some embodiments, any one or more of the following
residues may be substituted with cysteine: V205 (Kabat numbering)
of the light chain; A118 (EU numbering) of the heavy chain; and
5400 (EU numbering) of the heavy chain Fc region. Cysteine
engineered antibodies may be generated as described, e.g., in U.S.
Pat. No. 7,521,541.
[0219] e. Antibody Derivatives
[0220] In some embodiments, the anti-c-met antibody of the
pharmaceutical formulation described herein may be further modified
to contain additional nonproteinaceous moieties that are known in
the art and readily available. The moieties suitable for
derivatization of the antibody include but are not limited to water
soluble polymers. Non-limiting examples of water soluble polymers
include, but are not limited to, polyethylene glycol (PEG),
copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer is attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0221] In another embodiment, conjugates of the anti-c-met antibody
and nonproteinaceous moiety that may be selectively heated by
exposure to radiation are provided. In some embodiments, the
nonproteinaceous moiety is a carbon nanotube (Kam et al., Proc.
Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation may be
of any wavelength, and includes, but is not limited to, wavelengths
that do not harm ordinary cells, but which heat the
nonproteinaceous moiety to a temperature at which cells proximal to
the antibody-nonproteinaceous moiety are killed.
[0222] 8. Immunoconjugates
[0223] Immunoconjugates comprising the anti-c-met antibody
conjugated to one or more cytotoxic agents, such as
chemotherapeutic agents or drugs, growth inhibitory agents, toxins
(e.g., protein toxins, enzymatically active toxins of bacterial,
fungal, plant, or animal origin, or fragments thereof), or
radioactive isotopes are contemplated for use in the pharmaceutical
formulation described herein.
[0224] In some embodiments, an immunoconjugate is an antibody-drug
conjugate (ADC) in which an antibody is conjugated to one or more
drugs, including but not limited to a maytansinoid (see U.S. Pat.
Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an
auristatin such as monomethylauristatin drug moieties DE and DF
(MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and
7,498,298); a dolastatin; a calicheamicin or derivative thereof
(see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285,
5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al.,
Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res.
58:2925-2928 (1998)); an anthracycline such as daunomycin or
doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523
(2006); Jeffrey et al., Bioorganic & Med. Chem. Letters
16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005);
Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000);
Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532
(2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S.
Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as
docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a
trichothecene; and CC1065.
[0225] In some embodiments, an immunoconjugate comprises the
anti-c-met antibody as described herein conjugated to an
enzymatically active toxin or fragment thereof, including but not
limited to diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin, and the
tricothecenes.
[0226] In some embodiments, an immunoconjugate comprises the
anti-c-met antibody as described herein conjugated to a radioactive
atom to form a radioconjugate. A variety of radioactive isotopes
are available for the production of radioconjugates. Examples
include At.sup.211, I.sup.131, I.sup.125, Y.sup.90, Re.sup.186,
Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32, Pb.sup.212 and
radioactive isotopes of Lu. When the radioconjugate is used for
detection, it may comprise a radioactive atom for scintigraphic
studies, for example Tc99m or I123, or a spin label for nuclear
magnetic resonance (NMR) imaging (also known as magnetic resonance
imaging, MRI), such as iodine-123 again, iodine-131, indium-111,
fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,
manganese or iron.
[0227] Conjugates of the anti-c-met antibody and cytotoxic agent
may be made using a variety of bifunctional protein coupling agents
such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of a cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No.
5,208,020) may be used.
[0228] The immunuoconjugates or ADCs herein expressly contemplate,
but are not limited to such conjugates prepared with cross-linker
reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS,
LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,
sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and
sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which
are commercially available (e.g., from Pierce Biotechnology, Inc.,
Rockford, Ill., U.S.A).
IV. RECOMBINANT METHODS AND COMPOSITIONS
[0229] The anti-c-met antibody for use in any of the pharmaceutical
formulations described herein may be produced recombinant methods
and compositions, e.g., as described in U.S. Pat. No. 4,816,567. In
one embodiment, isolated nucleic acid encoding an antibody is
provided. Such nucleic acid may encode an amino acid sequence
comprising the VL and/or an amino acid sequence comprising the VH
of the antibody (e.g., the light and/or heavy chains of the
antibody). In a further embodiment, one or more vectors (e.g.,
expression vectors) comprising such nucleic acid are provided. In a
further embodiment, a host cell comprising such nucleic acid is
provided. In one such embodiment, a host cell comprises (e.g., has
been transformed with): (1) a vector comprising a nucleic acid that
encodes an amino acid sequence comprising the VL of the antibody
and an amino acid sequence comprising the VH of the antibody, or
(2) a first vector comprising a nucleic acid that encodes an amino
acid sequence comprising the VL of the antibody and a second vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VH of the antibody. Production of a one-armed
antibody is described, e.g., in WO2005/063816.
[0230] In one embodiment, the host cell is eukaryotic, e.g. a
Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,
Sp20 cell). In one embodiment, a method of making an antibody is
provided, wherein the method comprises culturing a host cell
comprising a nucleic acid encoding the antibody, as provided above,
under conditions suitable for expression of the antibody, and
optionally recovering the antibody from the host cell (or host cell
culture medium).
[0231] For recombinant production of an antibody, nucleic acid
encoding an antibody, e.g., as described above, is isolated and
inserted into one or more vectors for further cloning and/or
expression in a host cell. Such nucleic acid may be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody).
[0232] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells
described herein. For example, antibodies may be produced in
bacteria, in particular when glycosylation and Fc effector function
are not needed. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523, WO/05/063816. (See also Charlton, Methods
in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press,
Totowa, N.J., 2003), pp. 245-254, describing expression of antibody
fragments in E. coli.) After expression, the antibody may be
isolated from the bacterial cell paste in a soluble fraction and
can be further purified.
[0233] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized," resulting in
the production of an antibody with a partially or fully human
glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414
(2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
[0234] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells.
[0235] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,
and 6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants).
[0236] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or 293 cells as described, e.g., in Graham et al.,
J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse
sertoli cells (TM4 cells as described, e.g., in Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African
green monkey kidney cells (VERO-76); human cervical carcinoma cells
(HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL
3A); human lung cells (W138); human liver cells (Hep G2); mouse
mammary tumor (MMT 060562); TR1 cells, as described, e.g., in
Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5
cells; and FS4 cells. Other useful mammalian host cell lines
include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells
(Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and
myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of
certain mammalian host cell lines suitable for antibody production,
see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248
(B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268
(2003).
V. USES AND METHODS OF TREATMENT
[0237] The pharmaceutical formulations provided herein comprising
an anti-c-met antibody are useful for modulating disease states
associated with dysregulation of the HGF/c-met signaling axis. The
HGF/c-met signaling pathway is involved in multiple biological and
physiological functions, including, e.g., cell proliferation and
angiogenesis.
[0238] Provided herein are methods of inhibiting c-met activated
cell proliferation, said method comprising contacting a cell or
tissue with a pharmaceutical formulation described herein (e.g.,
upon dilution (e.g., a diluted pharmaceutical formulation described
herein)) comprising an effective amount of an anti-c-met antibody,
whereby cell proliferation associated with c-met activation is
inhibited. In some embodiments, the cell proliferative disorder is
associated with increased expression or activity of c-met or
hepatocyte growth, or both. In some embodiments, the cancer is
c-met positive (expresses high levels of c-met, for example, by
immunohistochemistry). In some embodiments, the cell proliferation
is cancer. In some embodiments, the cancer is non-small cell lung
cancer (NSCLC), glioblastoma, pancreatic cancer, sarcoma, renal
cell carcinoma, hepatocellular carcinoma, gastric cancer,
colorectal cancer, or breast cancer. In some embodiments, the
cancer is stage IIIb and/or stage IV. In some embodiments, the
cancer is locally advanced or metastatic cancer. In some
embodiments, the therapy is second line or third line therapy
(e.g., second line or third line NSCLC therapy). In some
embodiments, the cancer is EGFR mutant. In some embodiments, the
cancer is EGFR wild-type. In some embodiments, the cancer is c-met
positive (expresses high levels of c-met, for example, by
immunohistochemistry (IHC)). In some embodiments, the
pharmaceutical formulation comprises (a) an anti-c-met antibody
(e.g., onartuzumab), wherein the anti-c-met antibody is present at
a concentration between about 50 mg/mL and about 75 mg/mL; (b) a
histidine acetate buffer at pH 5.0-5.4, wherein the histidine
acetate buffer is at a concentration between about 1 mM and about
20 mM; (c) sucrose, wherein the sucrose is at a concentration
between about 100 mM to about 150 mM; and (d) a polysorbate,
wherein the polysorbate concentration is greater than 0.02%
w/v.
[0239] Provided herein are methods of treating a pathological
condition associated with dysregulation of c-met activation in a
subject, said method comprising administering to the subject a
pharmaceutical formulation described herein (e.g., upon dilution
(e.g., a diluted pharmaceutical formulation described herein))
comprising an effective amount of the c-met antibody, whereby said
condition is treated. In some embodiments, the pathological
condition is cancer. In some embodiments, the cancer is non-small
cell lung cancer (NSCLC), glioblastoma, pancreatic cancer, sarcoma,
renal cell carcinoma, hepatocellular carcinoma, gastric cancer,
colorectal cancer, or breast cancer. In some embodiments, the
cancer is stage IIIb and/or stage IV cancer. In some embodiments,
the cancer is locally advanced or metastatic cancer. In some
embodiments, the therapy is second line or third line therapy
(e.g., second line or third line NSCLC therapy). Dysregulation of
c-met activation (and thus signaling) can result from a number of
cellular changes, including, for example, overexpression of HGF
(c-met's cognate ligand) and/or c-met itself. In some embodiments,
the cancer is EGFR mutant. In some embodiments, the cancer is EGFR
wild-type. In some embodiments, the cancer is c-met positive
(expresses high levels of c-met, for example, by IHC). In some
embodiments, the pharmaceutical formulation comprises (a) an
anti-c-met antibody (e.g., onartuzumab), wherein the anti-c-met
antibody is present at a concentration between about 50 mg/mL and
about 75 mg/mL; (b) a histidine acetate buffer at pH 5.0-5.4,
wherein the histidine acetate buffer is at a concentration between
about 1 mM and about 20 mM; (c) sucrose, wherein the sucrose is at
a concentration between about 100 mM to about 150 mM; and (d)
polysorbate 20, wherein the polysorbate 20 concentration is greater
than 0.02% w/v.
[0240] Also provided herein are methods of inhibiting the growth of
a cell that expresses c-met or hepatocyte growth factor, or both,
said method comprising contacting said cell with a pharmaceutical
formulation described herein (e.g., upon dilution (e.g., a diluted
pharmaceutical formulation described herein)) comprising an
anti-c-met antibody thereby causing an inhibition of growth of said
cell. In some embodiments, the growth of said cell is at least in
part dependent upon a growth potentiating effect of c-met or
hepatocyte growth factor, or both. In some embodiments, the cell is
contacted by HGF expressed by a different cell (e.g., through a
paracrine effect). In some embodiments, the pharmaceutical
formulation comprises (a) an anti-c-met antibody (e.g.,
onartuzumab), wherein the anti-c-met antibody is present at a
concentration between about 50 mg/mL and about 75 mg/mL; (b) a
histidine acetate buffer at pH 5.0-5.4, wherein the histidine
acetate buffer is at a concentration between about 1 mM and about
20 mM; (c) sucrose, wherein the sucrose is at a concentration
between about 100 mM to about 150 mM; and (d) polysorbate 20,
wherein the polysorbate 20 concentration is greater than 0.02%
w/v.
[0241] Provided herein are also methods for treating or preventing
cancer comprising administering a pharmaceutical formulation
comprising (a) an anti-c-met antibody (e.g., onartuzumab), wherein
the anti-c-met antibody is present at a concentration between about
50 mg/mL and about 75 mg/mL; (b) a histidine acetate buffer at pH
5.0-5.4, wherein the histidine acetate buffer is at a concentration
between about 1 mM and about 20 mM; (c) sucrose, wherein the
sucrose is at a concentration between about 100 mM to about 150 mM;
and (d) polysorbate 20, wherein the polysorbate 20 concentration is
greater than 0.02% w/v (e.g., upon dilution to about any of 0.75,
1, or 1.25 mg/mL (e.g., a diluted pharmaceutical formulation
described herein)). In some embodiments, the pharmaceutical
formulation comprises (a) an anti-c-met antibody (e.g.,
onartuzumab), wherein the anti-c-met antibody is present at a
concentration of about 60 mg/mL; (b) a histidine acetate buffer at
pH 5.4, wherein the histidine acetate buffer is at a concentration
of about 10 mM; (c) sucrose, wherein the sucrose is at a
concentration of about 120 mM; and (d) polysorbate 20, wherein the
polysorbate 20 concentration is about 0.04% w/v. In some
embodiments, the cancer is non-small cell lung cancer (NSCLC),
glioblastoma, pancreatic cancer, sarcoma, renal cell carcinoma,
hepatocellular carcinoma, gastric cancer, colorectal cancer, or
breast cancer. In some embodiments, the cancer is stage IIIb and/or
stage IV cancer. In some embodiments, the cancer is locally
advanced or metastatic cancer. In some embodiments, the therapy is
second line or third line therapy (e.g., second line or third line
NSCLC therapy). In some embodiments, the cancer is EGFR mutant. In
some embodiments, the cancer is EGFR wild-type. In some
embodiments, the cancer is c-met positive (expresses high levels of
c-met, for example, by IHC). In some embodiments, the dose of
anti-c-met antibody is about 15 mg/kg. In some embodiments, the
dose of anti-c-met antibody is about 15 mg/kg administered day one
of a 21 day cycle. In some embodiments, the dose of anti-c-met
antibody is about 10 mg/kg. In some embodiments, the dose of
anti-c-met antibody is about 10 mg/kg administered on day 1 and 15
of a 28 day cycle.
[0242] Methods described herein can be used to affect any suitable
pathological state, for example, cells and/or tissues associated
with dysregulation of the HGF/c-met signaling pathway. In some
embodiments of any of the methods described herein, a cell that is
targeted in a method described herein is a cancer cell. For
example, a cancer cell can be one selected from the group
consisting of a breast cancer cell, a colorectal cancer cell, a
lung cancer cell, a papillary carcinoma cell (e.g., of the thyroid
gland), a colon cancer cell, a pancreatic cancer cell, an ovarian
cancer cell, a cervical cancer cell, a central nervous system
cancer cell, an osteogenic sarcoma cell, a renal carcinoma cell, a
hepatocellular carcinoma cell, a bladder cancer cell, a gastric
carcinoma cell, a head and neck squamous carcinoma cell, a melanoma
cell and a leukemia cell. In some embodiments, a cell that is
targeted in a method described herein is a hyperproliferative
and/or hyperplastic cell. In some embodiments, a cell that is
targeted in a method described herein is a dysplastic cell. In yet
another embodiment, a cell that is targeted in a method described
herein is a metastatic cell.
[0243] In some embodiments of any of the methods described herein,
the method further comprises additional treatment steps. For
example, in some embodiments, the method further comprises a step
wherein a targeted cell and/or tissue (e.g., a cancer cell) is
exposed to radiation treatment or a second therapeutic agent (e.g.,
chemotherapeutic agent). For example, methods are provided for
treating or preventing cancer comprising administering (i) a
pharmaceutical formulation comprising (a) an anti-c-met antibody
(e.g., onartuzumab), wherein the anti-c-met antibody is present at
a concentration between about 50 mg/mL and about 75 mg/mL; (b) a
histidine acetate buffer at pH 5.0-5.4, wherein the histidine
acetate buffer is at a concentration between about 1 mM and about
20 mM; (c) sucrose, wherein the sucrose is at a concentration
between about 100 mM to about 150 mM; and (d) polysorbate 20,
wherein the polysorbate 20 concentration is greater than 0.02% w/v
(e.g., upon dilution to about any of 0.75, 1, or 1.25 mg/mL (e.g.,
a diluted pharmaceutical formulation described herein)) and (ii) a
second therapeutic agent. In some embodiments, the second
therapeutic agent is an EGFR inhibitor (e.g., erlotinib), VEGF
inhibitor (e.g., bevacizumab), taxane (e.g., paclitaxel).
[0244] In some embodiments of any of the methods described herein,
the method further comprises administering an effective amount of a
second therapeutic agent. In some embodiments, the dose of
anti-c-met antibody is about 15 mg/kg. In some embodiments, the
dose of anti-c-met antibody is about 10 mg/kg.
[0245] In some embodiments, the second therapeutic agent is an EGFR
inhibitor. In some embodiments, the EGFR inhibitor is erlotinib
(N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine).
In some embodiments, the dose of anti-c-met antibody is about 15
mg/kg administered day one of a 21 day cycle. For example, provided
are methods of treating cancer (e.g., NSCLC) comprising
administering (i) a pharmaceutical formulation comprising (a) an
anti-c-met antibody (e.g., onartuzumab), wherein the anti-c-met
antibody is present at a concentration between about 50 mg/mL and
about 75 mg/mL; (b) a histidine acetate buffer at pH 5.0-5.4,
wherein the histidine acetate buffer is at a concentration between
about 1 mM and about 20 mM; (c) sucrose, wherein the sucrose is at
a concentration between about 100 mM to about 150 mM; and (d)
polysorbate 20, wherein the polysorbate 20 concentration is greater
than 0.02% w/v (e.g., upon dilution to about any of 0.75, 1, or
1.25 mg/mL (e.g., a diluted pharmaceutical formulation described
herein)), wherein the anti-c-met antibody is administered at a dose
of 15 mg/kg every three weeks; and (ii) erlotinib
(N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine),
wherein erlotinib is administered at a dose of 150 mg, each day of
a three week cycle.
[0246] In some embodiments, the second therapeutic agent is a
taxane (e.g., paclitaxel). In some embodiments, the cancer is
breast cancer. In some embodiments, the breast cancer is an
ER-negative, PR-negative, and HER2-negative (ER-, PR-, and HER2-;
or triple-negative) metastatic breast cancer. In some embodiments,
the dose of anti-c-met antibody is about 10 mg/kg. on day 1 and day
15 of a 28-day cycle. For example, provided are methods for
treating cancer (e.g., breast cancer) comprising administering (i)
a pharmaceutical formulation comprising (a) an anti-c-met antibody
(e.g., onartuzumab), wherein the anti-c-met antibody is present at
a concentration between about 50 mg/mL and about 75 mg/mL; (b) a
histidine acetate buffer at pH 5.0-5.4, wherein the histidine
acetate buffer is at a concentration between about 1 mM and about
20 mM; (c) sucrose, wherein the sucrose is at a concentration
between about 100 mM to about 150 mM; and (d) polysorbate 20,
wherein the polysorbate 20 concentration is greater than 0.02% w/v
(e.g., upon dilution to about any of 0.75, 1, or 1.25 mg/mL (e.g.,
a diluted pharmaceutical formulation described herein)), wherein
the anti-c-met antibody is administered at a dose of 10 mg/kg on
day 1 and day 15 of a 28-day cycle; and (ii) paclitaxel, wherein
paclitaxel is administered at a dose of 90 mg/m.sup.2 by IV
infusion on day 1, day 8, and day 15 of the 28-day cycle. In some
embodiments, the method increases survival of the patient,
decreases the patient's risk of cancer recurrence and/or to
increases the patient's likelihood of survival. In some
embodiments, the method further comprises administration of an
anti-VEGF antibody (e.g., bevacizumab). For example, provided are
methods for treating cancer (e.g., breast cancer) comprising
administering (i) a pharmaceutical formulation comprising (a) an
anti-c-met antibody (e.g., onartuzumab), wherein the anti-c-met
antibody is present at a concentration between about 50 mg/mL and
about 75 mg/mL; (b) a histidine acetate buffer at pH 5.0-5.4,
wherein the histidine acetate buffer is at a concentration between
about 1 mM and about 20 mM; (c) sucrose, wherein the sucrose is at
a concentration between about 100 mM to about 150 mM; and (d)
polysorbate 20, wherein the polysorbate 20 concentration is greater
than 0.02% w/v (e.g., upon dilution to about any of 0.75, 1, or
1.25 mg/mL (e.g., a diluted pharmaceutical formulation described
herein)), wherein the anti-c-met antibody is administered at a dose
of 10 mg/kg on day 1 and day 15 of a 28-day cycle; (ii) an
anti-VEGF antibody (e.g., bevacizumab), wherein the anti-VEGF
antibody is administered at a dose of 10 mg/kg on Day 1 and Day 15
of the 28-day cycle; and (iii) paclitaxel, wherein paclitaxel is
administered at a dose of 90 mg/m.sup.2 by IV infusion on Day 1,
Day 8, and Day 15 of the 28-day cycle.
[0247] Pharmaceutical formulation comprising the anti-c-met
antibody can be used either alone or in combination with other
agents in a therapy. For instance, pharmaceutical formulation
comprising the anti-c-met antibody may be co-administered with a
second therapeutic agent (e.g., another antibody, chemotherapeutic
agent(s) (including cocktails of chemotherapeutic agents), other
cytotoxic agent(s), anti-angiogenic agent(s), cytokines, and/or
growth inhibitory agent(s)). In some embodiments, the second
therapeutic agent is administered concurrently or sequentially. The
second therapeutic agent can be administered separately from the
pharmaceutical formulation comprising the anti-c-met antibody, but
as a part of the same treatment regimen. Where the anti-c-met
antibody of the pharmaceutical formulation inhibits tumor growth,
it may be particularly desirable to combine it with one or more
other therapeutic agent(s) which also inhibits tumor growth. For
instance, pharmaceutical formulation comprising the anti-c-met
antibody may be combined with an EGFR inhibitor, an anti-VEGF
antibody and/or anti-ErbB antibodies in a treatment scheme, e.g. in
treating any of the diseases described herein, including colorectal
cancer, metastatic breast cancer and kidney cancer.
[0248] Such combined therapies noted above encompass combined
administration (where two or more agents are included in the same
or separate formulations), simultaneously, and separate
administration, in which case, administration of the pharmaceutical
formulation can occur prior to, and/or following, administration of
the additional therapeutic agent and/or adjuvant.
[0249] Accordingly, in some embodiments of any of the methods
described herein, the method comprises targeting a cell wherein
c-met or hepatocyte growth factor, or both, is more abundantly
expressed by said cell (e.g., a cancer cell) as compared to a
normal cell of the same tissue origin. A c-met-expressing cell can
be regulated by HGF from a variety of sources, i.e. in an autocrine
or paracrine manner. C-met activation and/or signaling can also
occur independent of ligand. Hence, in some embodiments of any of
the methods, c-met activation in a targeted cell occurs independent
of ligand.
[0250] The pharmaceutical formulation comprising the anti-c-met
antibody can be administered to a human subject for therapeutic
purposes. Moreover, pharmaceutical formulation comprising the
anti-c-met antibody can be administered to a non-human mammal
expressing an antigen with which the immunoglobulin cross-reacts
(e.g., a primate, pig or mouse) for veterinary purposes or as an
animal model of human disease.
[0251] The pharmaceutical formulation comprising the anti-c-met
antibody can be used to treat, inhibit, delay progression of,
prevent/delay recurrence of, ameliorate, or prevent diseases,
disorders or conditions associated with abnormal expression and/or
activity of one or more antigen molecules, including but not
limited to malignant and benign tumors; non-leukemias and lymphoid
malignancies; neuronal, glial, astrocytal, hypothalamic and other
glandular, macrophagal, epithelial, stromal and blastocoelic
disorders; and inflammatory, angiogenic and immunologic
disorders.
[0252] In some embodiments of any of the methods, a pharmaceutical
formulation comprising an immunoconjugate comprising the anti-c-met
antibody conjugated with a cytotoxic agent is administered to the
patient. In some embodiments, the immunoconjugate and/or antigen to
which it is bound is/are internalized by the cell, resulting in
increased therapeutic efficacy of the immunoconjugate in killing
the target cell to which it binds. In some embodiments, the
cytotoxic agent targets or interferes with nucleic acid in the
target cell.
[0253] The pharmaceutical formulation comprising the anti-c-met
antibody (and any additional therapeutic agent) can be administered
by any suitable means, including parenteral, intrapulmonary, and
intranasal, and, if desired for local treatment, intralesional
administration. Parenteral infusions include intramuscular,
intravenous, intraarterial, intraperitoneal, or subcutaneous
administration. In some embodiments, the pharmaceutical formulation
is administered intravenously. Dosing can be by any suitable route,
e.g. by injections, such as intravenous or subcutaneous injections,
depending in part on whether the administration is brief or
chronic. Various dosing schedules including, but not limited to,
single or multiple administrations over various time-points, bolus
administration, and pulse infusion are contemplated herein.
[0254] Pharmaceutical formulation comprising the anti-c-met
antibody are dosed and administered in a fashion consistent with
good medical practice. Factors for consideration in this context
include the particular disorder being treated, the particular
mammal being treated, the clinical condition of the individual
patient, the cause of the disorder, the site of delivery of the
agent, the method of administration, the scheduling of
administration, and other factors known to medical practitioners.
The pharmaceutical formulation comprising the anti-c-met antibody
need not be, but is optionally formulated with one or more agents
currently used to prevent or treat the disorder in question. The
effective amount of such other agents depends on the amount of
antibodies present in the formulation, the type of disorder or
treatment, and other factors discussed above. These are generally
used in the same dosages and with administration routes as used
hereinbefore or about from 1 to 99% of the heretofore employed
dosages described herein, or any dosage and by any route that is
empirically/clinically determined to be appropriate.
[0255] For the prevention or treatment of disease, the appropriate
dosage of the anti-c-met antibody in the pharmaceutical formulation
(when used alone or in combination with one or more additional
therapeutic agents) will depend on the type of disease to be
treated, the type of antibody, the severity and course of the
disease, whether the anti-c-met antibody in the pharmaceutical
formulation is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the anti-c-met antibody, and the discretion of the attending
physician. The pharmaceutical formulation comprising the anti-c-met
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 10 mg/kg, about 15 mg/kg or greater (e.g., 15-20
mg/kg) dosage of the anti-c-met antibody is administered to the
patient, whether, for example, by one or more separate
administrations, or by continuous infusion. In some embodiments,
the dose of anti-c-met antibody is about 15 mg/kg. In some
embodiments, the dose of anti-c-met antibody is about 15 mg/kg
administered day one of a 21 day cycle. In some embodiments, the
dose of anti-c-met antibody is about 10 mg/kg. In some embodiments,
the dose of anti-c-met antibody is about 10 mg/kg administered on
day 1 and 15 of a 28 day cycle.
[0256] Doses may be administered intermittently, e.g. about any of
every week, every two weeks, every three weeks, or every four
weeks.
[0257] For repeated administrations over several days or longer,
depending on the condition, the treatment would generally be
sustained until a desired suppression of disease symptoms occurs.
However, other dosage regimens may be useful. The progress of this
therapy is easily monitored by conventional techniques and
assays.
VI. ARTICLES OF MANUFACTURE
[0258] Article of manufacture comprising the pharmaceutical
formulation comprising an anti-c-met antibody described herein for
the treatment, prevention and/or diagnosis of the disorders
described above is provided. The article of manufacture comprises a
container and a label or package insert on or associated with the
container. Suitable containers include, for example, bottles,
vials, syringes, IV solution bags etc. The containers may be formed
from a variety of materials such as glass or plastic. The container
holds the pharmaceutical formulation comprising the anti-c-met
antibody which is by itself or when combined with another
composition effective for treating, preventing and/or diagnosing
the condition and may have a sterile access port (for example the
container may be an intravenous solution bag or a vial having a
stopper pierceable by a hypodermic injection needle). For example,
provided herein are articles of manufacture and kits comprising a
container with a pharmaceutical formulation comprising (a) an
anti-c-met antibody (e.g., onartuzumab), wherein the anti-c-met
antibody is present at a concentration between about 50 mg/mL and
about 75 mg/mL; (b) a histidine acetate buffer at pH 5.0-5.4,
wherein the histidine acetate buffer is at a concentration between
about 1 mM and about 20 mM; (c) sucrose, wherein the sucrose is at
a concentration between about 100 mM to about 150 mM; and (d)
polysorbate 20, wherein the polysorbate 20 concentration is greater
than 0.02% w/v contained therein. In some embodiments, the
pharmaceutical formulation comprises (a) an anti-c-met antibody
(e.g., onartuzumab), wherein the anti-c-met antibody is present at
a concentration of about 60 mg/mL; (b) a histidine acetate buffer
at pH 5.4, wherein the histidine acetate buffer is at a
concentration of about 10 mM; (c) sucrose, wherein the sucrose is
at a concentration of about 120 mM; and (d) a polysorbate, wherein
the polysorbate concentration is about 0.04% w/v. The label or
package insert indicates that the composition is used for treating
the condition of choice, such as cancer. In some embodiments, the
cancer is non-small cell lung cancer (NSCLC), glioblastoma,
pancreatic cancer, sarcoma, renal cell carcinoma, gastric cancer,
colorectal cancer, or breast cancer. In some embodiments, the
cancer is stage IIIb and/or stage IV cancer. In some embodiments,
the cancer is locally advanced or metastatic cancer. In some
embodiments, the therapy is second line or third line therapy
(e.g., second line or third line NSCLC therapy). In some
embodiments, the cancer is EGFR mutant. In some embodiments, the
cancer is EGFR wild-type. In some embodiments, the cancer is c-met
positive (expresses high levels of c-met, for example, by
immunohistochemistry). In some embodiments, the dose of anti-c-met
antibody is about 15 mg/kg. In some embodiments, the dose of
anti-c-met antibody is about 15 mg/kg administered day one of a 21
day cycle. In some embodiments, the dose of anti-c-met antibody is
about 10 mg/kg. In some embodiments, the dose of anti-c-met
antibody is about 10 mg/kg administered on day 1 and 15 of a 28 day
cycle.
[0259] The article of manufacture in this embodiment may further
comprise a package insert indicating that the first and second
antibody compositions can be used to treat a particular condition,
e.g. cancer. In some embodiments, the cancer is non-small cell lung
cancer (NSCLC), glioblastoma, pancreatic cancer, sarcoma, renal
cell carcinoma, gastric cancer, colorectal cancer, or breast
cancer. In some embodiments, the cancer is stage IIIb and/or stage
IV. In some embodiments, the cancer is locally advanced or
metastatic cancer. In some embodiments, the therapy is second line
or third line therapy (e.g., second line or third line NSCLC
therapy). In some embodiments, the cancer is EGFR mutant. In some
embodiments, the cancer is EGFR wild-type. In some embodiments, the
cancer is c-met positive (expresses high levels of c-met, for
example, by immunohistochemistry). In some embodiments, the dose of
anti-c-met antibody is about 15 mg/kg. In some embodiments, the
dose of anti-c-met antibody is about 15 mg/kg administered day one
of a 21 day cycle.
[0260] Alternatively, or additionally, in some embodiments of any
of the articles of manufacture, the article of manufacture may
further comprise a second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0261] Moreover, the article of manufacture may comprise (a) a
first container with a pharmaceutical formulation described herein
comprising an anti-c-met antibody contained therein; and (b) a
second container with a composition contained therein, wherein the
composition comprises a further cytotoxic agent. For example, the
article of manufacture may comprise (i) a first container with a
pharmaceutical formulation comprising (a) an anti-c-met antibody
(e.g., onartuzumab), wherein the anti-c-met antibody is present at
a concentration between about 50 mg/mL and about 75 mg/mL; (b) a
histidine acetate buffer at pH 5.0-5.4, wherein the histidine
acetate buffer is at a concentration between about 1 mM and about
20 mM; (c) sucrose, wherein the sucrose is at a concentration
between about 100 mM to about 150 mM; and (d) polysorbate 20,
wherein the polysorbate 20 concentration is greater than 0.02% w/v;
and (ii) a second container with a composition contained therein,
wherein the composition comprises a second therapeutic agent.
[0262] In some embodiments, the second therapeutic agent is an EGFR
inhibitor. In some embodiments, the EGFR inhibitor is erlotinib
(N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)-4-quinazolinamine).
In some embodiments, the article of manufacture comprises
instructions for administration of about 15 mg/kg administered day
one of a 21 day cycle of anti-c-met antibody formulation and 150
mg, each day of a three week cycle of erlotinib. In some
embodiments, the article of manufacture comprises instructions for
the treatment of cancer (e.g., NSCLC).
[0263] In some embodiments, the second therapeutic agent is a
taxane (e.g., paclitaxel). In some embodiments, the article of
manufacture comprises instructions for administration of about 10
mg/kg. on day 1 and day 15 of a 28-day cycle of the anti-c-met
antibody formulation and 90 mg/m.sup.2 by IV infusion on day 1, day
8, and day 15 of the 28-day cycle of paclitaxel. In some
embodiments, the article of manufacture comprises a third container
with a composition contained therein, wherein the composition
comprises a third therapeutic agent, wherein the third therapeutic
agent is an anti-VEGF antibody (e.g., bevacizumab). In some
embodiments, the article of manufacture comprises instructions for
administration of about 10 mg/kg. on day 1 and day 15 of a 28-day
cycle of the anti-c-met antibody formulation, 90 mg/m.sup.2 by IV
infusion on day 1, day 8, and day 15 of the 28-day cycle of
paclitaxel, and 10 mg/kg on Day 1 and Day 15 of the 28-day cycle of
the anti-VEGF antibody (e.g., bevacizumab). In some embodiments,
the article of manufacture comprises instructions for the treatment
of cancer. In some embodiments, the cancer is breast cancer (e.g.,
ER-negative, PR-negative, and HER2-negative (ER-, PR-, and HER2-;
or triple-negative) metastatic breast cancer). In some embodiments,
the method increases survival of the patient, decreases the
patient's risk of cancer recurrence and/or to increases the
patient's likelihood of survival.
[0264] It is understood that any of the above articles of
manufacture may include an immunoconjugate of the anti-c-met
antibody disclosed herein in place or in addition to the anti-c-met
antibody.
[0265] Further provided herein are methods of making any of the
articles of manufacture described herein.
Exemplary Embodiments
[0266] 1. A pharmaceutical formulation comprising:
[0267] (a) an anti-c-met antibody;
[0268] (b) a histidine buffer at pH 5.0-5.4;
[0269] (c) a saccharide; and
[0270] (d) a polysorbate, wherein the polysorbate is present at
greater than 0.02% w/v.
2. The pharmaceutical formulation of embodiment 1, wherein the
anti-c-met antibody comprises a HVR-L1 comprising sequence
KSSQSLLYTSSQKNYLA (SEQ ID NO:1), a HVR-L2 comprising sequence
WASTRES (SEQ ID NO:2), a HVR-L3 comprising sequence QQYYAYPWT (SEQ
ID NO:3), a HVR-H1 comprising sequence GYTFTSYWLH (SEQ ID NO:4), a
HVR-H2 comprising sequence GMIDPSNSDTRFNPNFKD (SEQ ID NO:5), and a
HVR-H3 comprising sequence ATYRSYVTPLDY (SEQ ID NO:6). 3. The
pharmaceutical formulation of any one of embodiments 1-2, wherein
the anti-c-met antibody comprises (a) a heavy chain variable domain
comprising the sequence:
EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDT
RFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTV SS (SEQ
ID NO:19) and (b) a light chain variable domain comprising the
sequence:
DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTR
ESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKR (SEQ ID
NO:20). 4. The pharmaceutical formulation of any one of embodiments
1-3, wherein the anti-c-met antibody comprises a single antigen
binding arm and comprises a Fc region, wherein the Fc region
comprises a first and a second Fc polypeptide, and wherein the
first and second Fc polypeptides are present in a complex. 5. The
pharmaceutical formulation of embodiment 4, wherein the first and
second Fc polypeptides form a Fc region that increases stability of
said antibody fragment compared to a Fab molecule comprising said
antigen binding arm. 6. The pharmaceutical formulation of any one
of embodiment 1-5, wherein the anti-c-met antibody comprises (a) a
first polypeptide comprising the amino acid sequence of SEQ ID
NO:19, a CH1 sequence, and a first Fc polypeptide and (b) a second
polypeptide comprising the amino acid sequence of SEQ ID NO:20 and
CL1 sequence. 7. The pharmaceutical formulation of embodiment 6,
wherein the anti-c-met antibody further comprises (c) a third
polypeptide comprising a second Fc polypeptide. 8. The
pharmaceutical formulation of any one of embodiments 1-7, wherein
the first Fc polypeptide comprises the Fc sequence depicted in FIG.
2 (SEQ ID NO: 17) and the second Fc polypeptide comprises the Fc
sequence depicted in FIG. 3 (SEQ ID NO: 18). 9. The pharmaceutical
formulation of any one of embodiments 1-8, wherein the anti-c-met
antibody is onartuzumab. 10. The pharmaceutical formulation of any
one of embodiments 1-8, wherein the anti-c-met antibody binds the
same epitope as onartuzumab. 11. The pharmaceutical formulation of
any one of embodiments 1-10, wherein the anti-c-met antibody is
present at a concentration between about 10 mg/mL and about 100
mg/mL (e.g. about 15 mg/mL and about 75 mg/mL). 12. The
pharmaceutical formulation of embodiment 11, wherein the anti-c-met
antibody is present at a concentration of about 60 mg/mL. 13. The
pharmaceutical formulation of any one of embodiments 1-12, wherein
the saccharide is present at a concentration of about 75 mM to
about 200 mM (e.g., about 100 mM to about 150 mM). 14. The
pharmaceutical formulation of embodiment 13, wherein the saccharide
is present at a concentration of about 120 mM. 15. The
pharmaceutical formulation of any one of embodiments 1-14, wherein
the saccharide is a disaccharide. 16. The pharmaceutical
formulation of embodiment 15, wherein the disaccharide is
trehalose. 17. The pharmaceutical formulation of embodiment 15,
wherein the disaccharide is sucrose. 18. The pharmaceutical
formulation of any one of embodiments 1-17, wherein the histidine
buffer is at a concentration of about 1 mM to about 50 mM (e.g.
about 1 mM to about 25 mM). 19. The pharmaceutical formulation of
embodiment 18, wherein the histidine buffer is at a concentration
of about 10 mM. 20. The pharmaceutical formulation of any one of
embodiments 1-19, wherein the histidine buffer is histidine
acetate. 21. The pharmaceutical formulation of any one of
embodiments 1-20, wherein the polysorbate is present at a
concentration greater than 0.02% and less than about 0.1%. 22. The
pharmaceutical formulation of embodiment 21, wherein the
polysorbate is present at a concentration of about 0.04%. 23. The
pharmaceutical formulation of any one of embodiments 1-22, wherein
the polysorbate is polysorbate 20. 24. The pharmaceutical
formulation of any one of embodiments 1-23, wherein the formulation
is diluted with a diluent (e.g., 0.9% NaCl). 25. The pharmaceutical
formulation of embodiment 24, wherein the anti-c-met antibody is
present at a concentration of about 1 mg/mL. 26. A method of
inhibiting c-met activated cell proliferation, said method
comprising contacting a cell or tissue with an effective amount of
the pharmaceutical formulation of any one of embodiments 1-25. 27.
A method of modulating a disease associated with dysregulation of
the HGF/c-met signaling axis, said method comprising administering
to a subject an effective amount of the pharmaceutical formulation
of any one of embodiments 1-25. 28. A method of treating a subject
having a proliferative disorder, said method comprising
administering to the subject an effective amount of the
pharmaceutical formulation of any one of embodiments 1-25. 29. The
method of embodiment 28, wherein the proliferative disorder is
cancer. 30. The method of embodiment 29, wherein the cancer is lung
cancer (e.g., non-small cell lung cancer (NSCLC)), glioblastoma,
pancreatic cancer, sarcoma, renal cell carcinoma, hepatocellular
carcinoma, gastric cancer, colorectal cancer, and/or breast cancer.
31. The method of any one of embodiments 26-30, further comprising
a second therapeutic agent. 32. A method of making a pharmaceutical
formulation of any one of embodiments 1-25. 33. An article of
manufacture comprising a container with the pharmaceutical
formulation of any one of embodiments 1-25 contained therein. 34. A
method of making the article of manufacture of embodiment 33.
[0271] The following are examples of the pharmaceutical
formulations. It is understood that various other embodiments may
be practiced, given the general description provided above.
EXAMPLES
Example 1
Effect of pH and Buffer on Aggregation and Chemical Stability in
Liquid Onartuzumab Formulations
[0272] Initial anti-c-met antibody, onartuzumab, formulations
showed increased low molecule weight species (LMWS) detected by
size exclusion chromatography (SEC) when stored at 5.degree. C.
over time. Because of the increase in LMWS, a liquid formulation
was not initially pursued, and a lyophilized formulation was
developed with 20 mg/mL of onartuzumab, 10 mM histidine succinate,
4% trehalose dihydrate, 0.02% polysorbate 20, pH 5.7.
[0273] In an effort to develop and determine feasibility of a
liquid formulation and increase antibody concentration, pH and
excipient screens were performed.
[0274] The viscosity and stability of onartuzumab at various
concentrations (20-100 mg/mL) was evaluated in different buffers:
a) 10 mM histidine-acetate, 0.02% polysorbate 20, and 120 mM
trehalose, b) 10 mM histidine-succinate, 0.02% polysorbate 20, and
120 mM trehalose, and c) 200 mM arginine-succinate and 0.02%
polysorbate 20. The formulation including arginine succinate had
faster aggregate formation at accelerated temperatures (data not
shown). The viscosity was acceptable for all formulations
evaluated.
[0275] The effect of pH on onartuzumab aggregation and chemical
stability was evaluated with the following liquid formulations: a)
20 mg/mL of onartuzumab in 10 mM histidine acetate, 120 mM
trehalose, 0.02% polysorbate 20, pH 5.2, b) 20 mg/mL of onartuzumab
in 10 mM histidine acetate, 120 mM trehalose, 0.02% polysorbate 20,
pH 5.7, and c) 20 mg/mL of onartuzumab in 10 mM histidine acetate,
120 mM trehalose, 0.02% polysorbate 20, pH 6.2. Samples were kept
at 25.degree. C. or 40.degree. C. over time, and aggregate
formulation and chemical stability was evaluated approximately
every fifteen days. Aggregate formation was measured using size
exclusion chromatography which characterizes changes in size
heterogeneity using a TSK G3000 SWXL size-exclusion column.
[0276] As shown in FIG. 4, the higher pH of 6.2 resulted in
increased aggregate formation as indicated by % of high molecule
weight species (HMWS) at 40.degree. C. compared to either pH 5.2 or
5.7. The formulation with a pH 5.2 displayed the lowest aggregate
formation as indicated by % HMWS.
[0277] The effect of pH on onartuzumab aggregation and chemical
stability was further evaluated using a higher antibody
concentration with the following liquid formulations: a) 40 mg/mL
of onartuzumab in 10 mM histidine acetate, 120 mM trehalose, 0.02%
polysorbate 20, pH 5.1, b) 40 mg/mL of onartuzumab in 10 mM
histidine acetate, 120 mM trehalose, 0.02% polysorbate 20, pH 5.4,
and c) 40 mg/mL of onartuzumab in 10 mM histidine acetate, 120 mM
trehalose, 0.02% polysorbate 20, pH 5.7. Samples were kept at
25.degree. C. or 40.degree. C. over time, and aggregate formulation
and chemical stability was evaluated approximately every four
weeks. Aggregate formation was measured as detailed above. As shown
in FIGS. 5 and 6, lower pH (e.g., pH 5.4 and 5.1) resulted in
decreased aggregate formation as indicated by % HMWS at accelerated
temperatures (25.degree. C. and 40.degree. C.) compared to pH
5.7.
[0278] Chemical stability was measured using cation exchange
chromatography which characterizes changes in charge heterogeneity
using a Dionex WCX-10 ion-exchange column (IEC) and elution with a
salt gradient.
[0279] As shown in FIG. 7, while the lower pH resulted in decreased
aggregate formation, there was similar chemical stability as
measured by IEC between pH 5.1 and 5.7 at accelerated temperatures
at 25.degree. C. and 40.degree. C. Based on these findings, a
formulation including histidine buffer (e.g., histidine acetate)
and pH 5.4 was utilized for further experimentation.
Example 2
Effect of Polysorbate Concentration on Rate and Extent of
Polysorbate Degradation
[0280] Polysorbate is utilized in polypeptide formulations to
minimize adsorption to surfaces and reduce the air-liquid
interfacial surface tension and thus the rate of protein
denaturation. Loss of polysorbate in a pharmaceutical formulation
can result in instability of the formulation. Further, polysorbates
can be degraded by oxidation and hydrolysis which can lead to a
decrease in the apparent concentration of polysorbate in the
pharmaceutical formulation over long shelf life. These polysorbate
degradants are less surface-active than nondegraded polysorbate and
hence the chemical and physical stability of the pharmaceutical
formulation may be compromised. Further, some polysorbate
degradants are insoluble and may form particles if they are not
solubilized by intact polysorbate, i.e., if the ratio of degraded
polysorbate 20: intact polysorbate 20 is too high.
[0281] The rate and extent of degradation of polysorbate was
evaluated in an onartuzumab formulation including 60 mg/mL of
onartuzumab in 10 mM histidine acetate, 120 mM sucrose, 0.02%
polysorbate 20, pH 5.4 at 40.degree. C. over time. The
concentration of polysorbate 20 was assessed with an assay based on
the retention of polysorbate by a mixed-mode ion-exchange column
and subsequent elution with a step gradient. Detection was
performed using an evaporative light scattering detector (ELSD).
The rate of degradation and the ratio of degraded polysorbate
20:intact polysorbate 20 was higher than expected in the
onartuzumab formulation at 40.degree. C. over time. The higher than
expected rate of degradation and the ratio of degraded polysorbate
20:intact polysorbate 20 could result in instability of the
onartuzumab formulation and particulate formation after extended
storage.
[0282] The percentage of polysorbate 20 was increased in the
onartuzumab formulation and the rate and extent of degradation of
polysorbate was evaluated in onartuzumab formulations: a) 60 mg/mL
of onartuzumab in 10 mM histidine acetate, 120 mM sucrose, 0.02%
polysorbate 20, pH 5.4 and b) 60 mg/mL onartuzumab in 10 mM
histidine acetate, 120 mM sucrose, 0.04% polysorbate 20, pH 5.4 at
40.degree. C. over time as described above. Surprisingly, as shown
in FIG. 8, the increase in polysorbate 20 concentration not only
increased the overall levels of polysorbate 20 available to
stabilize the onartuzumab formulation, but further decreased the
ratio of degraded polysorbate 20:intact polysorbate 20 at
40.degree. C. over time, thereby further increasing the stability
of the onartuzumab formulation after extended storage.
Example 3
Effect of Polysorbate Concentration on % High Molecular Weight
Species Upon Dilution and Agitation
[0283] Prior to administration, the onartuzumab formulation is
diluted with a diluent (e.g., saline solution) for intravenous
infusion. Upon dilution, the polysorbate 20 concentration is
significantly reduced, and the stability of the onartuzumab in the
formulation could be compromised, for example, by polypeptide
aggregation as evidenced by % HMWS, when the diluted onartuzumab
formulation (e.g., IV bag and/or IV administration set) is agitated
(e.g., during transportation). As previously discussed, aggregation
of the monovalent onartuzumab (formation of multimer and oligomers)
and/or failure to maintain monovalent structure could lead to an
undesirable agonistic effect.
[0284] To evaluate the stability of the polypeptide and extent of
aggregation upon dilution, onartuzumab formulations, a) 60 mg/mL of
onartuzumab in 10 mM histidine acetate, 120 mM trehalose, 0.02%
polysorbate 20, pH 5.4 and b) 60 mg/mL onartuzumab in 10 mM
histidine acetate, 120 mM sucrose, 0.04% polysorbate 20, pH 5.4,
were diluted to 1 mg/mL in IV bags (PVC) with 0.9% NaCl. The bags
were agitated (orbital shaker, 100 rpm) at room temperature for
formulation (a) and 30.degree. C. for formulation (b).
[0285] As shown in FIG. 9, the % HMWS upon agitation was
significantly reduced in formulation (b) comprising 0.04%
polysorbate 20 compared to formulation (a) comprising 0.02%
polysorbate.
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[0351] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, the descriptions and examples should not be
construed as limiting the scope of the invention. The disclosures
of all patent and scientific literature cited herein are expressly
incorporated in their entirety by reference.
Sequence CWU 1
1
34117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Lys Ser Ser Gln Ser Leu Leu Tyr Thr Ser Ser Gln
Lys Asn Tyr Leu1 5 10 15Ala27PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 2Trp Ala Ser Thr Arg Glu Ser1
539PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Gln Gln Tyr Tyr Ala Tyr Pro Trp Thr1
5410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Gly Tyr Thr Phe Thr Ser Tyr Trp Leu His1 5
10518PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 5Gly Met Ile Asp Pro Ser Asn Ser Asp Thr Arg Phe
Asn Pro Asn Phe1 5 10 15Lys Asp612PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 6Ala Thr Tyr Arg Ser Tyr
Val Thr Pro Leu Asp Tyr1 5 10723PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 7Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys 20815PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 8Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile Tyr1 5 10 15932PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 9Gly Val Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr1 5 10 15Leu Thr Ile Ser Ser
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys 20 25
301011PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg1 5
101125PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser 20
251213PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val1 5 101330PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 13Arg Phe Thr Ile Ser Ala Asp Thr
Ser Lys Asn Thr Ala Tyr Leu Gln1 5 10 15Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 20 25 301411PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 14Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser1 5 1015106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
15Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln1
5 10 15Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr 20 25 30Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser 35 40 45Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr 50 55 60Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala
Asp Tyr Glu Lys65 70 75 80His Lys Val Tyr Ala Cys Glu Val Thr His
Gln Gly Leu Ser Ser Pro 85 90 95Val Thr Lys Ser Phe Asn Arg Gly Glu
Cys 100 10516108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 16Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr 100
10517222PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 17Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe1 5 10 15Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro 20 25 30Glu Val Thr Cys Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val 35 40 45Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr 50 55 60Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val65 70 75 80Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 85 90 95Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 100 105 110Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 115 120
125Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val
130 135 140Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly145 150 155 160Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp 165 170 175Gly Ser Phe Phe Leu Val Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp 180 185 190Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His 195 200 205Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly Lys 210 215 22018227PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
18Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1
5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140Leu Trp Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155
160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly Lys22519119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
19Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1
5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30Trp Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45Gly Met Ile Asp Pro Ser Asn Ser Asp Thr Arg Phe Asn
Pro Asn Phe 50 55 60Lys Asp Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys
Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Tyr Arg Ser Tyr Val Thr Pro
Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu Val Thr Val Ser Ser
11520114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 20Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ser Ser
Gln Ser Leu Leu Tyr Thr 20 25 30Ser Ser Gln Lys Asn Tyr Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys 35 40 45Ala Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Ala Tyr
Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110Lys
Arg21449PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 21Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Leu His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Gly Met Ile Asp Pro Ser Asn Ser
Asp Thr Arg Phe Asn Pro Asn Phe 50 55 60Lys Asp Arg Phe Thr Ile Ser
Ala Asp Thr Ser Lys Asn Thr Ala Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Tyr Arg
Ser Tyr Val Thr Pro Leu Asp Tyr Trp Gly Gln Gly 100 105 110Thr Leu
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120
125Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
130 135 140Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp145 150 155 160Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu 165 170 175Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser 180 185 190Ser Ser Leu Gly Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro225 230 235
240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp 260 265 270Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu305 310 315 320Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu
Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Ser 355 360
365Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu385 390 395 400Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys
Leu Thr Val Asp Lys 405 410 415Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440
445Lys22220PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 22Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ser Ser
Gln Ser Leu Leu Tyr Thr 20 25 30Ser Ser Gln Lys Asn Tyr Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys 35 40 45Ala Pro Lys Leu Leu Ile Tyr Trp
Ala Ser Thr Arg Glu Ser Gly Val 50 55 60Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr65 70 75 80Ile Ser Ser Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95Tyr Tyr Ala Tyr
Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110Lys Arg
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120
125Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn
130 135 140Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn
Ala Leu145 150 155 160Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu
Gln Asp Ser Lys Asp 165 170 175Ser Thr Tyr Ser Leu Ser Ser Thr Leu
Thr Leu Ser Lys Ala Asp Tyr 180 185 190Glu Lys His Lys Val Tyr Ala
Cys Glu Val Thr His Gln Gly Leu Ser 195 200 205Ser Pro Val Thr Lys
Ser Phe Asn Arg Gly Glu Cys 210 215 2202317PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 23Lys
Ser Ser Gln Ser Leu Leu Tyr Thr Ser Ser Gln Lys Asn Tyr Leu1 5 10
15Ala247PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 24Trp Ala Ser Thr Arg Glu Ser1 5259PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 25Gln
Gln Tyr Tyr Ala Tyr Pro Trp Thr1 52610PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 26Gly
Tyr Thr Phe Thr Ser Tyr Trp Leu His1 5 102718PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 27Gly
Met Ile Asp Pro Ser Asn Ser Asp Thr Arg Phe Asn Pro Asn Phe1 5 10
15Lys Asp2811PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 28Xaa Tyr Gly Ser Tyr Val Ser Pro Leu
Asp Tyr1 5 102911PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 29Thr Tyr Gly Ser Tyr Val Ser Pro Leu
Asp Tyr1 5 103011PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 30Ser Tyr Gly Ser Tyr Val Ser Pro Leu
Asp Tyr1 5 10315PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 31Leu Asp Ala Gln Thr1 5329PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 32Leu
Thr Glu Lys Arg Lys Lys Arg Ser1 5338PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 33Lys
Pro Asp Ser Ala Glu Pro Met1 5348PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 34Asn Val Arg Cys Leu Gln
His Phe1 5
* * * * *