U.S. patent application number 13/141608 was filed with the patent office on 2012-05-03 for targeted binding agents directed to a5 1 and uses thereof.
Invention is credited to Avril Alfred, Simon Thomas Barry, Catherine Anne Eberlein, Ian Foltz, Jaspal Singh Kang, Jane Kendrew.
Application Number | 20120107324 13/141608 |
Document ID | / |
Family ID | 22490778 |
Filed Date | 2012-05-03 |
United States Patent
Application |
20120107324 |
Kind Code |
A1 |
Eberlein; Catherine Anne ;
et al. |
May 3, 2012 |
TARGETED BINDING AGENTS DIRECTED TO a5 1 AND USES THEREOF
Abstract
The invention relates to targeted binding agents against
.alpha.5.beta.1 and uses of such agents. More specifically, the
invention relates to fully human monoclonal antibodies directed to
.alpha.5.beta.1. The described targeted binding agents are useful
in the treatment of diseases associated with the activity and/or
overproduction of .alpha.5.beta.1 and as diagnostics.
Inventors: |
Eberlein; Catherine Anne;
(Altrincham Cheshire, GB) ; Foltz; Ian; (Burnaby,
CA) ; Kang; Jaspal Singh; (Surrey, CA) ;
Kendrew; Jane; (Wilmslow Cheshire, GB) ; Alfred;
Avril; (Chiliwack, CA) ; Barry; Simon Thomas;
(Rudyard Staffordshire, GB) |
Family ID: |
22490778 |
Appl. No.: |
13/141608 |
Filed: |
December 21, 2009 |
PCT Filed: |
December 21, 2009 |
PCT NO: |
PCT/EP09/67707 |
371 Date: |
January 19, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60140336 |
Jun 21, 1999 |
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Current U.S.
Class: |
424/155.1 ;
435/320.1; 435/326; 435/69.6; 530/331; 530/350; 530/387.3;
530/388.1; 536/23.53 |
Current CPC
Class: |
B01F 5/0619 20130101;
B01F 5/0613 20130101; B01F 5/0617 20130101; B01F 2005/0639
20130101; B01F 15/00935 20130101; A61P 35/00 20180101; B29B 7/325
20130101 |
Class at
Publication: |
424/155.1 ;
530/331; 530/350; 530/388.1; 530/387.3; 536/23.53; 435/320.1;
435/326; 435/69.6 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 14/435 20060101 C07K014/435; C07K 16/00 20060101
C07K016/00; A61P 35/00 20060101 A61P035/00; C12N 15/62 20060101
C12N015/62; C12N 5/10 20060101 C12N005/10; C12P 21/08 20060101
C12P021/08; C07K 5/08 20060101 C07K005/08; C07H 21/04 20060101
C07H021/04 |
Claims
1. A targeted binding agent that specifically binds to
.alpha.5.beta.1 integrin, wherein said targeted binding agent
contains an RGD tripeptide in any one of the CDRs.
2-3. (canceled)
4. A targeted binding agent of claim 1, wherein said targeted
binding agent binds .alpha.5.beta.1 integrin with a Kd of less than
250 picomolar.
5. A targeted binding agent of claim 1, wherein said targeted
binding agent inhibits binding of fibronectin, fibrin, adhesion
molecule L1-CAM, Tie-2 and/or Flt1 ligands to .alpha.5.beta.1
integrin.
6. A targeted binding agent of claim 1 wherein said targeted
binding agent comprises heavy and light chain variable regions
according to Table 10 and 11.
7. A targeted binding agent claim 1 wherein said targeted binding
agent is MAb 2H12 or 2H12 Variant 1.
8. A targeted binding agent of claim 1, wherein said targeted
binding agent comprises a polypeptide comprising the sequence of
SEQ ID NO.: 12.
9. A targeted binding agent of claim 1, wherein said targeted
binding agent comprises a polypeptide comprising the sequence of
SEQ ID NO.: 10.
10. A targeted binding agent of claim 1, wherein said targeted
binding agent comprises a polypeptide comprising the sequence of
SEQ ID NO.: 20.
11. A targeted binding agent which competes for binding to
.alpha.5.beta.1 integrin with the targeted binding agent of claim
1.
12. A targeted binding agent comprising an amino acid sequence
comprising: a) a CDR3 sequence as shown in Table 10 or 11; b) a
CDR3 sequence as shown in Table 10 and a CDR3 sequence as shown in
Table 11; c) a CDR1, CDR2, and CDR3 sequence as shown in Table 10;
or d) a CDR1, a CDR2 and a CDR3 sequence as shown in Table 11; or
e) a CDR1, a CDR2 and a CDR3 sequence as shown in Table 10 and a
CDR1, a CDR2 and a CDR3 sequence as shown in Table 11; or f) a
CDR1, CDR2 and CDR3 sequence of McAb. 2H12 as shown in Table 10 and
a CDR1, CDR2, CDR3 sequence of McAb. 2H12 as shown in Table 11. g)
a CDR1, CDR2 and CDR3 sequence of McAb. 2H12 Variant 1 as shown in
Table 10 and a CDR1, CDR2, CDR3 sequence of McAb. 2H12 Variant 1 as
shown in Table 11.
13. A targeted binding agent comprising a set of CDRs: HCDR1,
HCDR2, HCDR3, LCDR1, LCDR2, LCDR3, wherein the set of CDRs has 10
or fewer amino acid substitutions from a set of CDRs in which:
HCDR1 is amino acid sequence SEQ ID NO: 13; HCDR2 is amino acid
sequence SEQ ID NO: 14; HCDR3 is amino acid sequence SEQ ID NO: 15;
LCDR1 is amino acid sequence SEQ ID NO: 16; LCDR2 is amino acid
sequence SEQ ID NO: 17; and LCDR3 is amino acid sequence SEQ ID NO:
18.
14. A targeted binding agent according to claim 13, comprising one
or two substitutions in the set of CDRs.
15. A targeted binding agent of claim 12 wherein said targeted
binding agent is a monoclonal antibody.
16. A targeted binding agent of claim 15, wherein said targeted
binding agent is a fragment of a monoclonal antibody selected from
the group consisting of Fab, Fab', F(ab').sub.2, Fv, ScFv, ScFvFc
or dAb.
17. (canceled)
18. A nucleic acid encoding a targeted binding agent according to
claim 12.
19. A vector comprising the nucleic acid molecule of claim 18.
20. A host cell comprising the vector of claim 19.
21. A method of producing an antibody comprising culturing the host
cell of claim 20 and recovering the antibody from the cell
culture.
22. A method of treating a neoplastic disease in a mammal
comprising: selecting an animal in need of treatment for a
neoplastic disease; and administering to said animal a
therapeutically effective dose of a targeted binding agent of claim
12.
23. The method of claim 22, wherein said neoplastic disease is
selected from the group consisting of: melanoma, small cell lung
cancer, non-small cell lung cancer, glioma, hepatocellular
carcinoma, thyroid tumor, gastric cancer, prostate cancer, breast
cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma,
endometrial cancer, kidney cancer, colon cancer, pancreatic cancer,
esophageal carcinoma, head and neck cancers, mesothelioma,
sarcomas, biliary, small bowel adenocarcinoma, pediatric
malignancies, epidermoid carcinoma and gastrointestinal stromal
tumour.
24-28. (canceled)
29. The targeted binding agent of claim 12 in association with a
pharmaceutically acceptable carrier.
30. The targeted binding agent of claim 12 in combination with an
antagonist of vascular endothelial growth factor.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The invention relates to targeted binding agents against the
target antigen .alpha.5.beta.1 integrin (.alpha.5.beta.1) and uses
of such agents. In some embodiments, the invention relates to fully
human monoclonal antibodies directed to .alpha.5.beta.1 and uses of
these antibodies. Aspects of the invention also relate to
hybridomas or other cell lines expressing such targeted binding
agents or antibodies. The described targeted binding agents and
antibodies are useful as diagnostics and for the treatment of
diseases associated with the activity and/or overexpression of
.alpha.5.beta.1.
[0003] 2. Description of the Related Art
[0004] The integrin superfamily includes at least 24 family members
consisting of heterodimers that utilize 18 alpha and 8 beta chains
(Hynes, (2002) Cell 110: 673-87). This family of receptors is
expressed on the cell surface and mediates cell-cell and
cell-extracellular matrix interactions that regulate cell survival,
proliferation, migration, and differentiation as well as tumour
invasion and metastasis (French-Constant and Colognato, (2004)
Trends Cell Biol. 14: 678-86).
[0005] Integrins bind to other cellular receptors, growth factors
and extracellular matrix proteins, with many family members having
overlapping binding specificity for particular proteins. This
redundancy may ensure that important functions continue in the
absence of a particular integrin (Koivisto et al, (2000) Exp. Cell
Res. 255: 10-17). However, temporal and spatial restriction of
expression of individual integrins with similar specificity has
also been reported and may alter the cellular response to ligand
binding (Yokosaki et al, (1996) J. Biol. Chem. 271: 24144-50;
Kemperman et al, (1997) Exp. Cell Res. 234: 156-64; Thomas et al,
(2006) J. Oral Pathol. Med. 35: 1-10).
[0006] The integrin family can be divided into several sub-families
based on ligand specificity of the heterodimers. One subfamily
consists of all of the integrins that recognize and bind the RGD
tripeptide. These receptors include the .alpha.IIb/.beta.3 and all
of the .alpha.V and .alpha.5 heterodimers (Thomas et al, (2006) J.
Oral Pathol. Med. 35: 1-10). The .alpha.5 chain pairs only with the
beta 1 chain, although beta 1 is able to pair with a number of
other alpha chains. The .alpha.5.beta.1 chain heterodimer binds the
extracellular matrix component fibronectin as its primary ligand,
and has been reported to bind fibrin (Suehiro et al (1997) JBC,
272, 5360-5366) the adhesion molecule L1-CAM (Ruppert et al (1995)
JCB, 131, 1881-1891), and to growth factor receptors such as Tie-2
and Flt1 (Cascone et al. (2005) JCB, 170, 993-1004; Orrechia et al
(2003) JCS, 116 3479-3489).
[0007] Expression of .alpha.5 integrin subunit is reported to be
ubiquitous at the mRNA level however the level of expression at the
level of the protein/receptor varies between tissues and cell
types. In addition, it is likely that the integrin is in different
"activation" states within these tissues, with "active"
.alpha.5.beta.1 being associated with active tissue remodeling, or
regulation and pathology in the adult. Of particular interest for
therapeutics is the function of .alpha.5.beta.1 expressed on
angiogenic endothelium, macrophages/monocytes, smooth muscle cells,
fibroblasts and tumour cells. Expression of .alpha.5.beta.1 is
often coincident with its major ligand, fibronectin, which forms
part of the provisional matrix found in many pathological
conditions where vasculature is more permeable, is or where tissue
damage has occurred. Co-expression of the receptor and ligands is
likely to determine the areas where .alpha.5.beta.1 is functionally
active.
[0008] The requirement for .alpha.5.beta.1 in vascular remodeling
is well established (Watt and Hodivala (1994) Current Biology, 4,
270-272). The .alpha.5 Knockout (KO) mice are embryonic lethal due
to a failure to form vasculature (Yang et al (1993) Development,
119, 1093-1105). This alone established a pivotal role for
.alpha.5.beta.1 in vascular remodeling. Consistent with this
observation .alpha.5.beta.1 function plays a pivotal role in
vasculature and embryoid bodies (Francis et al (2002) Arteioscler.
Thromb Vasc Biol, 22, 927-933). Knockout of the primary
.alpha.5.beta.1 ligand, fibronectin, also results in a similar
embryonic lethality as a result of a failure to form vasculature.
This contrasts with KO of other integrin receptors such as
.alpha.v.beta.3, or .alpha.v.beta.5, which do not appear to play
the same pivotal role in the angiogenic process. .alpha.5.beta.1
expression is specifically upregulated on endothelium in response
to various stimuli (Collo and Pepper (1999) JCS, 112, 569-578) and
expression of .alpha.5.beta.1 in endothelial cells plays a role in
promoting expression of genes involved in the regulation of both
inflammation and angiogenesis (Klein et al, (2002) MCB, 22,
5912-5922). Consistent with genetic evidence .alpha.5.beta.1
appears to be a dominant regulatory integrin in the angiogenic
process, when expressed it regulates the activity of other
endothelial cell integrins such as .alpha. .omega..beta.3 (Kim et
al., (2000) JBC 275, 33920-33928), and suppresses apoptosis.
Various antagonists of .alpha.5.beta.1 (small molecule, antibody
and peptide inhibitors) have been shown to reduce angiogenesis in
different in vitro and in vivo systems (Kim et al (2000) Am J Path
156, 1345-1362), confirming the pivotal role in regulating vascular
remodeling. Antibodies directed to .alpha.5.beta.1 have been
disclosed in the following International Patent Applications:
WO1999/58139, WO2004/056308, WO2004/089988, WO2005/092073,
WO2007/134876 and WO2008/060645.
[0009] .alpha.5.beta.1 plays a pivotal role in mediating signaling
transduction from the extracellular matrix and also regulating
signaling from growth factor receptors. Engagement of
.alpha.5.beta.1 drives actin polymerization, activation of a
variety of tyrosine kinases, ERK activation, down regulation of
pro-apoptopic drives, and promotes cell cycle progression
(Giancotti and Ruoslahti, (1999) Science, 285, 1028-1032). The
generic role of .alpha.5.beta.1 in signal transduction is
consistent with the receptor regulating function of various cell
types involved in driving disease pathology. In addition to
modulating endothelial cell function, .alpha.5.beta.1 is highly
expressed on white blood cells including monocytes and regulates
the production of angiogenic chemokines from macrophages (White et
al (2001) J. Immunol, 167, 5362-5366). Moreover when engaged to
ligand .alpha.5.beta.1 regulates survival, cell cycle progress and
gene expression in epithelial cells and fibroblasts.
[0010] As a result of the pro-survival signalling and
transcriptional effects mediated by .alpha.5.beta.1, it has also
been implicated in promoting survival and growth of tumour cells.
In particular .alpha.5.beta.1 regulates the growth of astrocytoma
(Maglott et al (2006) Can Res 66, 6002-6007) and breast (Jia et al
(2004) Can Res, 64, 8674-8681; Spangenberg et al (2006) Can Res,
66, 3715-3725) tumour cells.
[0011] Antagonising .alpha.5.beta.1 is likely to modulate many
processes involved in driving pathologies that involve modified or
permeable vasculature, dysfunctional or hyper-proliferative
epithelia, including tumour cells, and diseases of chronic
inflammation driven by leukocytes.
SUMMARY OF THE INVENTION
[0012] The present invention relates to targeted binding agents
that specifically bind to .alpha.5.beta.1 and inhibit the growth of
cells that express .alpha.5.beta.1. Mechanisms by which this can be
achieved can include, and are not limited to, blocking ligand
binding and/or inhibiting cell signaling implicated in tumour cell
growth. The targeted binding agents also inhibit tumour cell
adhesion. The targeted binding agents are useful for reducing
tumour cell growth and angiogenesis.
[0013] In one embodiment of the invention, the targeted binding
agent specifically binds to .alpha.5.beta.1 integrin, wherein said
targeted binding agent contains an RGD tripeptide in any one of the
CDRs. Another embodiment of the invention is a targeted binding
agent that specifically binds to .alpha.5.beta.1 integrin, wherein
said targeted binding agent contains an RGD tripeptide in any one
of the CDR3 domains. Another embodiment of the invention is a
targeted binding agent that specifically binds to .alpha.5.beta.1
integrin, wherein said targeted binding agent contains an RGD
tripeptide in the heavy chain CDR3 domain. Another embodiment of
the invention is a targeted binding agent that specifically binds
to .alpha.5.beta.1 integrin wherein, said targeted binding agent is
a ligand mimetic.
[0014] In one embodiment of the invention, the targeted binding
agent specifically binds to .alpha.5.beta.1 and inhibits binding of
fibronectin, fibrin, adhesion molecule L1-CAM, Tie-2 and/or Flt1
ligands to .alpha.5.beta.1. Another embodiment of the invention is
a targeted binding agent that binds to .alpha.5.beta.1 and inhibits
downstream cell signaling implicated in cell growth.
[0015] In some embodiments, the targeted binding agent binds either
the .alpha.5 chain or the .alpha.5.beta.1 heterodimer and does not
cross-react with the .beta.1 chain alone.
[0016] Another embodiment of the invention is a targeted binding
agent that competes for binding with any of the targeted binding
agents or antibodies described herein.
[0017] In one embodiment, the targeted binding agent binds
.alpha.5.beta.1 with a K.sub.D of less than about 500 picomolar
(pM). In another embodiment, the targeted binding agent binds with
a K.sub.D less than about 400, 300, 200 or 100 pM. In one
embodiment, the targeted binding agent binds with a K.sub.D of less
than about 75, 60, 50, 40, 30, 20, 10 or 5 pM. Affinity and/or
avidity measurements can be measured by FMAT, FACS, and/or
BIACORE.RTM., as described herein.
[0018] In another embodiment, the targeted binding agent binds
.alpha.5.beta.1 with a K.sub.D less than about 400, 300, 200, or
100, 75, 60, 50, 40, 30, 20, 10, or 5 pM as measured in a
monovalent affinity assay. Monovalent affinity may be measured in a
BIACORE.RTM. assay in which soluble receptor is flowed over
immobilized antibody. In comparison with a bivalent affinity assay,
the K.sub.D as reported by a monovalent affinity assay is much less
likely to be affected by experimental artefacts and is thus able to
report a K.sub.D much closer to the true monovalent affinity of the
antibody. In a bivalent affinity assay, the density of immobilized
receptor influences the extent to which single antibody molecules
bind twice and/or rebind immobilized receptor as they are flowed
over. As such, in a bivalent affinity assay, the density of
receptor can directly affect the reported K.sub.D. Thus, a
monovalent affinity assay provides a much more
biologically-relevant measurement of affinity.
[0019] In another embodiment, the targeted binding agent inhibits
receptor-dependent or ligand-induced signaling with an IC50 less
than about 400, 300, 200, or 100, 75, 60, 50, 40, 30, 20, 10, or 5
pM when performed at or close to saturating ligand levels.
[0020] In some embodiments of the invention, the targeted binding
agent inhibits tumour growth and/or metastasis in a mammal. In
other embodiments, the targeted binding agent ameliorates symptoms
associated with inflammatory disorders in a mammal. In one
embodiment, the targeted binding agent ameliorates symptoms
associated with inflammatory disorders selected from rheumatoid
arthritis or psoriasis in a mammal. Symptoms that may be
ameliorated include, but are not limited to, angiogenesis and
synovitis. In still other embodiments, the targeted binding agent
ameliorates symptoms associated with cardiovascular disease in a
mammal. Symptoms that may be ameliorated include, but are not
limited to, inflammation and angiogenesis. In some other
embodiments, the targeted binding agent ameliorates symptoms
associated with sepsis in a mammal Symptoms that may be ameliorated
include, but are not limited to, uncontrolled vascular
permeability, vascular leakage and angiogenesis. In some other
embodiments, the targeted binding agent ameliorates symptoms
associated with ocular disease. In some other embodiments, the
targeted binding agent ameliorates symptoms associated with an
ocular disease, such as ischaemic retinopathy or age-related
macular degeneration. Symptoms that may be ameliorated include, but
are not limited to, uncontrolled vascular permeability and vascular
leakage.
[0021] In one embodiment of the invention, the targeted binding
agent is an antibody. In one embodiment of the invention, the
targeted binding agent is a monoclonal antibody. In one embodiment
of the invention, the targeted binding agent is a fully human
monoclonal antibody. In another embodiment of the invention, the
targeted binding agent is a fully human monoclonal antibody of the
IgG1, IgG2, IgG3 or IgG4 isotype. In another embodiment of the
invention, the targeted binding agent is a fully human monoclonal
antibody of the IgG2 isotype. This isotype has reduced potential to
elicit effector function in comparison with other isotypes, which
may lead to reduced toxicity. In another embodiment of the
invention, the targeted binding agent is a fully human monoclonal
antibody of the IgG1 isotype. The IgG1 isotype has increased
potential to elicit ADCC in comparison with other isotypes, which
may lead to improved efficacy. The IgG1 isotype has improved
stability in comparison with other isotypes, e.g. IgG4, which may
lead to improved bioavailability, or improved ease of manufacture
or a longer half-life. In one embodiment, the fully human
monoclonal antibody of the IgG1 isotype is of the z, za or f
allotype.
[0022] In another embodiment the targeted binding agent or antibody
may comprise a sequence comprising any one, two, three, four, five
or six of the CDR1, CDR2 or CDR3 sequences as shown in Table 10
and/or Table 11. A further embodiment is a targeted binding agent
or an antibody that specifically binds to .alpha.5.beta.1 and
comprises a sequence comprising one of the complementarity
determining regions (CDR) sequences shown in Table 10. Embodiments
of the invention include a targeted binding agent or antibody
comprising a sequence comprising: any one of a CDR1, a CDR2 or a
CDR3 sequence as shown in Table 10. A further embodiment is a
targeted binding agent or an antibody that specifically binds to
.alpha.5.beta.1 and comprises a sequence comprising two of the CDR
sequences shown in Table 10. In another embodiment the targeted
binding agent or antibody comprises a sequence comprising a CDR1, a
CDR2 and a CDR3 sequence as shown in Table 10. In another
embodiment the targeted binding agent or antibody comprises a
sequence comprising one of the CDR sequences shown in Table 11.
Embodiments of the invention include a targeted binding agent or
antibody comprising a sequence comprising: any one of a CDR1, a
CDR2 or a CDR3 sequence as shown in Table 11. In another embodiment
the targeted binding agent or antibody comprises a sequence
comprising two of the CDR sequences shown in Table 11. In another
embodiment the targeted binding agent or antibody comprises a
sequence comprising a CDR1, a CDR2 and a CDR3 sequence as shown in
Table 11. In another embodiment the targeted binding agent or
antibody may comprise a sequence comprising a CDR1, a CDR2 and a
CDR3 sequence as shown in Table 10 and a CDR1, a CDR2 and a CDR3
sequence as shown in Table 11. In some embodiments, the targeted
binding agent is an antibody. In certain embodiments, the targeted
binding agent is a fully human monoclonal antibody. In certain
other embodiments, the targeted binding agent is a binding fragment
of a fully human monoclonal antibody. In certain embodiments the
antibody is a fully human monoclonal antibody. In certain other
embodiments, the targeted binding agent is a binding fragment of a
fully human monoclonal antibody.
[0023] It is noted that those of ordinary skill in the art can
readily accomplish CDR determinations. See for example, Kabat et
al., Sequences of Proteins of Immunological Interest, Fifth
Edition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3.
Kabat provides multiple sequence alignments of immunoglobulin
chains from numerous species antibody isotypes. The aligned
sequences are numbered according to a single numbering system, the
Kabat numbering system. The Kabat sequences have been updated since
the 1991 publication and are available as an electronic sequence
database (latest downloadable version 1997). Any immunoglobulin
sequence can be numbered according to Kabat by performing an
alignment with the Kabat reference sequence. Accordingly, the Kabat
numbering system provides a uniform system for numbering
immunoglobulin chains.
[0024] In one embodiment, the targeted binding agent or antibody
comprises a sequence comprising any one of the heavy chain
sequences shown in Table 10. In another embodiment, the targeted
binding agent or antibody comprises a sequence comprising any one
of the heavy chain sequences of antibodies 7B1.3A1, 1F2.2B7, 2H12,
2H12 Variant 1, 2G2.2B5, 3F12.4A1 or 4G3.3D11. Light-chain
promiscuity is well established in the art, thus, a targeted
binding agent or antibody comprising a sequence comprising any one
of the heavy chain sequences of antibodies 7B1.3A1, 1F2.2B7, 2H12,
2H12 Variant 1, 2G2.2B5, 3F12.4A1 or 4G3.3D11 or another antibody
as disclosed herein, may further comprise any one of the light
chain sequences shown in Table 11 or of antibodies 7B1.3A1,
1F2.2B7, 2H12, 2H12 Variant 1, 2G2.2B5, 3F12.4A1 or 4G3.3D11, or
another antibody as disclosed herein. In some embodiments, the
antibody is a fully human monoclonal antibody.
[0025] In one embodiment, the targeted binding agent or antibody
comprises a sequence comprising any one of the light chain
sequences shown in Table 11. In another embodiment, the targeted
binding agent or antibody comprises a sequence comprising any one
of the light chain sequences of antibodies 7B1.3A1, 1F2.2B7, 2H12,
2H12 Variant 1, 2G2.2B5, 3F12.4A1 or 4G3.3D11. In some embodiments,
the antibody is a fully human monoclonal antibody.
[0026] In one embodiment, the targeting binding agent is monoclonal
antibody 2H12 or 2H12 Variant 1. In certain embodiments, the
targeting binding agent is monoclonal antibody 2H12. In certain
other embodiments, the targeting binding agent is monoclonal
antibody 2H12 Variant 1. In additional embodiments, the targeted
binding agent is derivable from any of the foregoing monoclonal
antibodies.
[0027] In one embodiment a targeted binding agent or an antibody
may comprise a sequence comprising a heavy chain CDR1, CDR2 and
CDR3 selected from any one of the CDRs of antibodies 7B1.3A1,
1F2.2B7, 2H12, 2H12 Variant 1, 2G2.2B5, 3F12.4A1 or 4G3.3D11. In
one embodiment a targeted binding agent or an antibody may comprise
a sequence comprising a light chain CDR1, CDR2 and CDR3 selected
from any one of the CDRs of antibodies 7B1.3A1, 1F2.2B7, 2H12, 2H12
Variant 1, 2G2.2B5, 3F12.4A1 or 4G3.3D11.
[0028] In another embodiment the targeted binding agent or antibody
may comprise a set of CDRS: HCDR1, HCDR2, HCDR3, LCDR1, LCDR2,
LCDR3, wherein the set of CDRS has 10 or fewer amino acid
substitutions from a set of CDRs in which:
[0029] HCDR1 is amino acid sequence SEQ ID NO:13;
[0030] HCDR2 is amino acid sequence SEQ ID NO:14;
[0031] HCDR3 is amino acid sequence SEQ ID NO:15;
[0032] LCDR1 is amino acid sequence SEQ ID NO:16;
[0033] LCDR2 is amino acid sequence SEQ ID NO:17; and
[0034] LCDR3 is amino acid sequence SEQ ID NO:18.
[0035] In another embodiment the targeted binding agent or antibody
may comprise a sequence comprising any one of a CDR1, a CDR2 or a
CDR3 of the fully human monoclonal antibody 2H12 or 2H12 Variant 1,
as shown in Table 10. In another embodiment the targeted binding
agent or antibody may comprise a sequence comprising any one of a
CDR1, a CDR2 or a CDR3 of the fully human monoclonal antibody 2H12
or 2H12 Variant 1 as shown in Table 11. In one embodiment the
targeted binding agent or antibody may comprise a sequence
comprising a CDR1, a CDR2 and a CDR3 of fully human monoclonal
antibody 2H12 or 2H12 Variant 1, as shown in Table 10. In another
embodiment the targeted binding agent or antibody may comprise a
sequence comprising a CDR1, a CDR2 and a CDR3 of fully human
monoclonal antibody 2H12 or 2H12 Variant 1, as shown in Table 11.
In another embodiment the targeted binding agent or antibody may
comprise a sequence comprising a CDR1, a CDR2 and a CDR3 of fully
human monoclonal antibody 2H12 or 2H12 Variant 1, as shown in Table
10, and a CDR1, a CDR2 and a CDR3 sequence of fully human
monoclonal antibody 2H12 or 2H12 Variant 1, as shown in Table 11.
In some embodiments, the antibody is a fully human monoclonal
antibody.
[0036] A further embodiment of the invention is a targeted binding
agent or antibody comprising a sequence comprising the contiguous
sequence spanning the framework regions and CDRs, specifically from
FR1 through FR4 or CDR1 through CDR3, of any one of the sequences
as shown in Table 10 or Table 11. In one embodiment the targeted
binding agent or antibody comprises a sequence comprising the
contiguous sequences spanning the framework regions and CDRs,
specifically from FR1 through FR4 or CDR1 through CDR3, of the
sequence of monoclonal antibody 2H12 or 2H12 Variant 1, as shown in
Table 10 or Table 11. In some embodiments, the antibody is a fully
human monoclonal antibody.
[0037] One embodiment provides a targeted binding agent or
antibody, or binding fragment thereof, wherein the agent or
antibody, or binding fragment thereof, comprises a heavy chain
polypeptide comprising the sequence of SEQ ID NO.:10. In one
embodiment, the agent or antibody, or binding fragment thereof,
further comprises a light chain polypeptide comprising the sequence
of SEQ ID NO.:12. In one embodiment, the targeted binding agent or
antibody, or binding fragment thereof, comprises a heavy chain
polypeptide comprising the sequence of SEQ ID NO: 10 and a light
chain polypeptide comprising the sequence of SEQ ID NO:12. In some
embodiments, the antibody is a fully human monoclonal antibody.
[0038] One embodiment provides a targeted binding agent or
antibody, or binding fragment thereof, wherein the agent or
antibody, or binding fragment thereof, comprises a heavy chain
polypeptide comprising the sequence of SEQ ID NO.:20. In one
embodiment, the agent or antibody, or binding fragment thereof,
further comprises a light chain polypeptide comprising the sequence
of SEQ ID NO.:12. In one embodiment, the targeted binding agent or
antibody, or binding fragment thereof, comprises a heavy chain
polypeptide comprising the sequence of SEQ ID NO: 20 and a light
chain polypeptide comprising the sequence of SEQ ID NO:12. In some
embodiments, the antibody is a fully human monoclonal antibody.
[0039] In one embodiment the targeted binding agent or antibody
comprises as many as twenty, sixteen, ten, nine or fewer, e.g. one,
two, three, four or five, amino acid additions, substitutions,
deletions, and/or insertions within the disclosed CDRs or heavy or
light chain sequences. Such modifications may potentially be made
at any residue within the CDRs. In some embodiments, the antibody
is a fully human monoclonal antibody.
[0040] In one embodiment, the targeted binding agent or antibody
comprises variants or derivatives of the CDRs disclosed herein, the
contiguous sequences spanning the framework regions and CDRs
(specifically from FR1 through FR4 or CDR1 through CDR3), the light
or heavy chain sequences disclosed herein, or the antibodies
disclosed herein. Variants include targeted binding agents or
antibodies comprising sequences which have as many as twenty,
sixteen, ten, nine or fewer, e.g. one, two, three, four, five or
six amino acid additions, substitutions, deletions, and/or
insertions in any of the CDR1, CDR2 or CDR3s as shown in Table 10
or Table 11, the contiguous sequences spanning the framework
regions and CDRs (specifically from FR1 through FR4 or CDR1 through
CDR3) as shown in Table 10 or Table 11, the light or heavy chain
sequences disclosed herein, or with the monoclonal antibodies
disclosed herein. Variants include targeted binding agents or
antibodies comprising sequences which have at least about 60, 70,
80, 85, 90, 95, 98 or about 99% amino acid sequence identity with
any of the CDR1, CDR2 or CDR3s as shown in Table 10 or Table 11,
the contiguous sequences spanning the framework regions and CDRs
(specifically from FR1 through FR4 or CDR1 through CDR3) as shown
in Table 10 or Table 11, the light or heavy chain sequences
disclosed herein, or with the monoclonal antibodies disclosed
herein. The percent identity of two amino acid sequences can be
determined by any method known to one skilled in the art,
including, but not limited to, pairwise protein alignment. In one
embodiment variants comprise changes in the CDR sequences or light
or heavy chain polypeptides disclosed herein that are naturally
occurring or are introduced by in vitro engineering of native
sequences using recombinant DNA techniques or mutagenesis
techniques. Naturally occurring variants include those which are
generated in vivo in the corresponding germline nucleotide
sequences during the generation of an antibody to a foreign
antigen. In one embodiment the derivative may be a heteroantibody,
that is an antibody in which two or more antibodies are linked
together. Derivatives include antibodies which have been chemically
modified. Examples include covalent attachment of one or more
polymers, such as water-soluble polymers, N-linked, or O-linked
carbohydrates, sugars, phosphates, and/or other such molecules. The
derivatives are modified in a manner that is different from the
naturally occurring or starting antibody, either in the type or
location of the molecules attached. Derivatives further include
deletion of one or more chemical groups which are naturally present
on the antibody.
[0041] In one embodiment, the targeted binding agent is a
bispecific antibody. A bispecific antibody is an antibody that has
binding specificity for at least two different epitopes. For
example, bispecific antibodies can be generated that comprise (i)
two antibodies one with a specificity to .alpha.5.beta.1 and
another to a second molecule that are conjugated together, (ii) a
single antibody that has one chain specific to .alpha.5.beta.1 and
a second chain specific to a second molecule or (iii) a single
chain antibody that has specificity to .alpha.5.beta.1 and the
other molecule. Methods for making bispecific antibodies are known
in the art: (See, for example, Millstein et al., Nature,
305:537-539 (1983); Traunecker et al., EMBO J., 10:3655-3659
(1991); Suresh et al., Methods in Enzymology, 121:210 (1986);
Kostelny et al., J. Immunol., 148(5):1547-1553 (1992); Hollinger et
al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993); Gruber et
al., J. Immunol., 152:5368 (1994); U.S. Pat. Nos. 4,474,893;
4,714,681; 4,925,648; 5,573,920; 5,601,81; 95,731,168; 4,676,980;
and 4,676,980, WO 94/04690; WO 91/00360; WO 92/200373; WO 93/17715;
WO 92/08802; and EP 03089.). For example, in connection with (i)
and (ii) see e.g., Fanger et al. Immunol Methods 4:72-81 (1994) and
Wright and Harris, supra. and in connection with (iii) see is e.g.,
Traunecker et al. Int. J. Cancer (Suppl.) 7:51-52 (1992). In each
case, the second specificity can be made to the heavy chain
activation receptors, including, without limitation, CD16 or CD64
(see e.g., Deo et al. Immunol. Today 18:127 (1997)) or CD89 (see
e.g., Valerius et al. Blood 90:4485-4492 (1997)).
[0042] In some embodiments of the invention, the targeted binding
agent or antibody comprises a sequence comprising SEQ ID NO.: 10.
In certain embodiments, SEQ ID NO.: 10 comprises any one of the
combinations of germline and non-germline residues indicated by
each row of Table 9. In some embodiments, SEQ ID NO: 10 comprises
any one, any two, any three, any four, any five, any six or any
seven of the germline residues as indicated in Table 9. In other
embodiments, the targeted binding agent or antibody is derived from
a germline sequence with VH3-33, D6-6 and JH6B domains, wherein one
or more residues has been mutated to yield the corresponding
germline residue at that position.
[0043] In some embodiments of the invention, the targeted binding
agent or antibody comprises a sequence comprising SEQ ID NO.: 12.
In certain embodiments, SEQ ID NO.: 12 comprises any one of the
unique combinations of germline and non-germline residues indicated
by each row of Table 8. In some embodiments, SEQ ID NO: 12
comprises any one, any two, any three or all four of the germline
residues as indicated in Table 8. In other embodiments, the
targeted binding agent or antibody is derived from a germline
sequence with A3 and JK3 domains, wherein one or more residues has
been mutated to yield the corresponding germline residue at that
position.
[0044] In some embodiments of the invention, the targeted binding
agent or antibody comprises a sequence comprising SEQ ID NO.: 10
and SEQ ID NO:12. In certain embodiments, SEQ ID NO.: 10 comprises
any one of the combinations of germline and non-germline residues
indicated by each row of Table 9 and SEQ ID NO:12 comprises any one
of the unique combinations of germline and non-germline residues
indicated by each row of Table 8. In some embodiments, SEQ ID NO:
10 comprises any one, any two, any three, any four, any five, any
six or any seven of the germline residues as indicated in Table 9
and SEQ ID NO:12 comprises any one, any two, any three or all four
of the germline residues as indicated in Table 8.
[0045] A further embodiment of the invention is a targeted binding
agent or antibody which competes or cross-competes for binding to
.alpha.5.beta.1 with the targeted binding agent or antibodies of
the invention. In another embodiment of the invention there is an
antibody which competes or cross-competes for binding to
.alpha.5.beta.1 with the targeted binding agent or antibodies of
the invention. In another embodiment the targeted binding agent or
antibody competes for binding to .alpha.5.beta.1 with any one of
the fully human monoclonal antibodies 2H12 or 2H12 Variant 1.
"Competes" indicates that the targeted binding agent or antibody
competes for binding to .alpha.5.beta.1 with any one of the fully
human monoclonal antibodies 2H12 or 2H12 Variant 1, i.e.
competition is unidirectional.
[0046] Embodiments of the invention include a targeted binding
agent or antibody which cross competes with any one of the fully
human monoclonal antibodies 2H12 or 2H12 Variant 1 for binding to
.alpha.5.beta.1. "Cross competes" indicates that the targeted
binding agent or antibody competes for binding to .alpha.5.beta.1
with any one of the fully human monoclonal antibodies 2H12 or 2H12
Variant 1, and vice versa, i.e. competition is bidirectional.
[0047] A further embodiment of the invention is a targeted binding
agent or antibody that binds to the same epitope on .alpha.5.beta.1
as the targeted binding agent or antibodies of the invention.
Embodiments of the invention also include a targeted binding agent
or antibody that binds to the same epitope on .alpha.5.beta.1 as
any one of the fully human monoclonal antibodies 2H12 or 2H12
Variant 1. Other embodiments of the invention include isolated
nucleic acid molecules encoding any of the targeted binding agents
or antibodies described herein, vectors having isolated nucleic
acid molecules encoding the targeted binding agents or antibodies
described herein or a host cell transformed with any of such
nucleic acid molecules. Embodiments of the invention include a
nucleic acid molecule encoding a fully human isolated targeted
binding agent that specifically bind to .alpha.5.beta.1 and inhibit
binding of fibronectin, fibrin, adhesion molecule L1-CAM and growth
factor receptors such as Tie-2 and Flt1 to .alpha.5.beta.1. The
invention also encompasses polynucleotides that hybridize under
stringent or lower stringency hybridization conditions, as defined
herein, to polynucleotides that encode any of the targeted binding
agents or antibodies described herein.
[0048] Embodiments of the invention described herein also provide
cells for producing these antibodies. Examples of cells include
hybridomas, or recombinantly created cells, such as Chinese hamster
ovary (CHO) cells, variants of CHO cells (for example DG44) and NS0
cells that produce antibodies against .alpha.5.beta.1. Additional
information about variants of CHO cells can be found in Andersen
and Reilly (2004) Current Opinion in Biotechnology 15, 456-462
which is incorporated herein in its entirety by reference. The
antibody can be manufactured from a hybridoma that secretes the
antibody, or from a recombinantly engineered cell that has been
transformed or transfected with a gene or genes encoding the
antibody.
[0049] In addition, one embodiment of the invention is a method of
producing an antibody of the invention by culturing host cells
under conditions wherein a nucleic acid molecule is expressed to
produce the antibody followed by recovering the antibody. It should
be realised that embodiments of the invention also include any
nucleic acid molecule which encodes an antibody or fragment of an
antibody of the invention including nucleic acid sequences
optimised for increasing yields of antibodies or fragments thereof
when transfected into host cells for antibody production.
[0050] A further embodiment herein includes a method of producing
antibodies that specifically bind to .alpha.5.beta.1 and inhibit
the biological activity of .alpha.5.beta.1, by immunising a mammal
with cells expressing human .alpha.5.beta.1, isolated cell
membranes containing human .alpha.5.beta.1, purified human
.alpha.5.beta.1, or a fragment thereof, and/or one or more
orthologous sequences or fragments thereof.
[0051] In other embodiments the invention provides compositions,
including a targeted binding agent or antibody of the invention or
binding fragment thereof, and a pharmaceutically acceptable carrier
or diluent.
[0052] Still further embodiments of the invention include methods
of treating an animal suffering from a neoplastic disease by
administering to the animal a therapeutically effective dose of a
targeted binding agent that specifically binds to .alpha.5.beta.1.
In certain embodiments the method further comprises selecting an
animal in need of treatment for a neoplastic disease, and
administering to the animal a therapeutically effective dose of a
targeted binding agent that specifically binds to
.alpha.5.beta.1.
[0053] Still further embodiments of the invention include methods
of treating an animal suffering from a non-neoplastic disease by
administering to the animal a therapeutically effective dose of a
targeted binding agent that specifically binds to .alpha.5.beta.1.
In certain embodiments the method further comprises selecting an
animal in need of treatment for a non-neoplastic disease, and
administering to the animal a therapeutically effective dose of a
targeted binding agent that specifically binds to
.alpha.5.beta.1.
[0054] Still further embodiments of the invention include methods
of treating an animal suffering from a malignant tumour by
administering to the animal a therapeutically effective dose of a
targeted binding agent that specifically binds to .alpha.5.beta.1.
In certain embodiments the method further comprises selecting an
animal in need of treatment for a malignant tumour, and
administering to the animal a therapeutically effective dose of a
targeted binding agent that specifically binds to
.alpha.5.beta.1.
[0055] Still further embodiments of the invention include methods
of treating an animal suffering from an inflammatory disorder by
administering to the animal a therapeutically effective dose of a
targeted binding agent that specifically binds to .alpha.5.beta.1.
In certain embodiments the method further comprises selecting an
animal in need of treatment for an inflammatory disorder, and
administering to the animal a therapeutically effective dose of a
targeted binding agent that specifically binds to
.alpha.5.beta.1.
[0056] Still further embodiments of the invention include methods
of treating an animal suffering from a disease or condition
associated with .alpha.5.beta.1 expression by administering to the
animal a therapeutically effective dose of a targeted binding agent
that specifically binds to .alpha.5.beta.1. In certain embodiments
the method further comprises selecting an animal in need of
treatment for a disease or condition associated with
.alpha.5.beta.1 expression, and administering to the animal a
therapeutically effective dose of a targeted binding agent that
specifically binds to .alpha.5.beta.1.
[0057] A malignant tumour may be selected from the group consisting
of: melanoma, small cell lung cancer, non-small cell lung cancer,
glioma, hepatocellular (liver) carcinoma, thyroid tumour, gastric
(stomach) cancer, prostate cancer, breast cancer, ovarian cancer,
bladder cancer, lung cancer, glioblastoma, endometrial cancer,
kidney cancer, colon cancer, pancreatic cancer, esophageal
carcinoma, head and neck cancers, mesothelioma, sarcomas, biliary
(cholangiocarcinoma), small bowel adenocarcinoma, pediatric
malignancies and epidermoid carcinoma.
[0058] Treatable proliferative, angiogenic, cell adhesion or
invasion-related diseases include neoplastic diseases, such as,
melanoma, small cell lung cancer, non-small cell lung cancer,
glioma, hepatocellular (liver) carcinoma, thyroid tumour, gastric
(stomach) cancer, gallbladder cancer, prostate cancer, breast
cancer, ovarian cancer, bladder cancer, lung cancer, glioblastoma,
endometrial cancer, kidney cancer, colon cancer, pancreatic cancer,
esophageal carcinoma, head and neck cancers, mesothelioma,
sarcomas, biliary (cholangiocarcinoma), small bowel adenocarcinoma,
pediatric malignancies, epidermoid carcinoma and leukaemia,
including chronic myelogenous leukaemia.
[0059] In one embodiment, the neoplastic disease is melanoma, colon
cancer or chronic myelogenous leukaemia.
[0060] Non-neoplastic diseases include inflammatory disorders such
as rheumatoid arthritis or psoriasis, cardiovascular disease such
as atherosclerosis, sepsis, ocular disease such as ischaemic
retinopathy or age-related macular degeneration (AMD).
[0061] Inflammatory disorders include rheumatoid arthritis,
osteoarthritis, asthma, chronic obstructive pulmonary disease
(COPD), allergic rhinitis and psoriasis.
[0062] In one embodiment the present invention is suitable for use
in inhibiting .alpha.5.beta.1, in patients with a tumour which is
dependent alone, or in part, on .alpha.5.beta.1.
[0063] Still further embodiments of the invention include use of a
targeted binding agent or antibody of the invention in the
preparation of a medicament for the treatment of an animal
suffering from a proliferative, angiogenic, cell adhesion or
invasion-related disease. In certain embodiments the use further
comprises selecting an animal in need of treatment for a
proliferative, angiogenic, cell adhesion or invasion-related
disease.
[0064] Still further embodiments of the invention include use of a
targeted binding agent or antibody of the invention in the
preparation of a medicament for the treatment of an animal
suffering from a neoplastic disease. In certain embodiments the use
further comprises selecting an animal in need of treatment for a
neoplastic disease.
[0065] Still further embodiments of the invention include use of a
targeted binding agent or antibody of the invention in the
preparation of a medicament for the treatment of an animal
suffering from a non-neoplastic disease. In certain embodiments the
use further comprises selecting an animal in need of treatment for
a non-neoplastic disease.
[0066] Still further embodiments of the invention include use of a
targeted binding agent or antibody of the invention in the
preparation of a medicament for the treatment of an animal
suffering from a malignant tumour. In certain embodiments the use
further comprises selecting an animal in need of treatment for a
malignant tumour.
[0067] Still further embodiments of the invention include use of a
targeted binding agent or antibody of the invention in the
preparation of a medicament for the treatment of an animal
suffering from an inflammatory disease. In certain embodiments the
use further comprises selecting an animal in need of treatment for
an inflammatory disease.
[0068] Still further embodiments of the invention include use of a
targeted binding agent or antibody of the invention in the
preparation of a medicament for the treatment of an animal
suffering from a disease or condition associated with
.alpha.5.beta.1 expression. In certain embodiments the use further
comprises selecting an animal in need of treatment for a disease or
condition associated with .alpha.5.beta.1 expression.
[0069] Still further embodiments of the invention include a
targeted binding agent or antibody of the invention for the
treatment of an animal suffering from a proliferative, angiogenic,
cell adhesion or invasion-related disease.
[0070] Still further embodiments of the invention include a
targeted binding agent or antibody of the invention for the
treatment of an animal suffering from a neoplastic disease.
[0071] Still further embodiments of the invention include a
targeted binding agent or antibody of the invention for the
treatment of an animal suffering from a non-neoplastic disease.
[0072] Still further embodiments of the invention include a
targeted binding agent or antibody of the invention for the
treatment of an animal suffering from a malignant tumour.
[0073] Still further embodiments of the invention include use of a
targeted binding agent or antibody of the invention for the
treatment of an animal suffering from an inflammatory disease.
[0074] Still further embodiments of the invention include a
targeted binding agent or antibody of the invention for the
treatment of an animal suffering from a disease or condition
associated with .alpha.5.beta.1 expression.
[0075] In one embodiment treatment of a [0076] a proliferative,
angiogenic, cell adhesion or invasion-related disease; [0077] a
neoplastic disease; [0078] a non-neoplastic disease; [0079] a
malignant tumour; [0080] an inflammatory disorder; or [0081] a
disease or condition associated with .alpha.5.beta.1 expression
[0082] comprises managing, ameliorating, preventing, any of the
aforementioned diseases or conditions.
[0083] In one embodiment treatment of a neoplastic disease
comprises inhibition of tumour growth, tumour growth delay,
regression of tumour, shrinkage of tumour, increased time to is
regrowth of tumour on cessation of treatment, increased time to
tumour recurrence, slowing of disease progression.
[0084] In some embodiments of the invention, the animal to be
treated is a human.
[0085] In some embodiments of the invention, the targeted binding
agent is a fully human monoclonal antibody.
[0086] In some embodiments of the invention, the targeted binding
agent is the fully human monoclonal antibody 2H12 or 2H12 Variant
1.
[0087] Embodiments of the invention include a conjugate comprising
the targeted binding agent as described herein, and a therapeutic
agent. In some embodiments of the invention, the therapeutic agent
is a toxin. In other embodiments, the therapeutic agent is a
radioisotope. In still other embodiments, the therapeutic agent is
a pharmaceutical composition.
[0088] In another aspect, a method of selectively killing a
cancerous cell in a patient is provided. The method comprises
administering a fully human antibody conjugate to a patient. The
fully human antibody conjugate comprises an antibody that can bind
to .alpha.5.beta.1 and an agent. The agent is either a toxin, a
radioisotope, or another substance that will kill a cancer cell.
The antibody conjugate thereby selectively kills the cancer
cell.
[0089] In one aspect, a conjugated fully human antibody that
specifically binds to .alpha.5.beta.1 is provided. Attached to the
antibody is an agent, and the binding of the antibody to a cell
results in the delivery of the agent to the cell. In one
embodiment, the above conjugated fully human antibody binds to an
extracellular domain of .alpha.5.beta.1. In another embodiment, the
antibody and conjugated toxin are internalised by a cell that
expresses .alpha.5.beta.1. In another embodiment, the agent is a
cytotoxic agent. In another embodiment, the agent is, for example
saporin, or auristatin, pseudomonas exotoxin, gelonin, ricin,
calicheamicin or maytansine-based immunoconjugates, and the like.
In still another embodiment, the agent is a radioisotope.
[0090] The targeted binding agent or antibody of the invention can
be administered alone, or can be administered in combination with
additional antibodies or chemotherapeutic drugs or radiation
therapy. For example, a monoclonal, oligoclonal or polyclonal
mixture of .alpha.5.beta.1 antibodies that block cell adhesion,
invasion, angiogenesis or proliferation can be administered in
combination with a drug shown to inhibit tumour cell
proliferation.
[0091] Another embodiment of the invention includes a method of
diagnosing diseases or conditions in which an antibody as disclosed
herein is utilised to detect the level of .alpha.5.beta.1 in a
patient or patient sample. In one embodiment, the patient sample is
blood or blood serum or urine. In further embodiments, methods for
the identification of risk factors, diagnosis of disease, and
staging of disease is presented which involves the identification
of the expression and/or overexpression of .alpha.5.beta.1 using
anti-.alpha.5.beta.1 antibodies. In some embodiments, the methods
comprise administering to a patient a fully human antibody
conjugate that selectively binds to .alpha.5.beta.1 on a cell. The
antibody conjugate comprises an antibody that specifically binds to
.alpha.5.beta.1 and a label. The methods further comprise observing
the presence of the label in the patient. A relatively high amount
of the label on specific cell types will indicate a relatively high
risk of the disease and a relatively low amount of the label will
indicate a relatively low risk of the disease. In one embodiment,
the label is a green fluorescent protein.
[0092] The invention further provides methods for assaying the
level of .alpha.5.beta.1 in a patient sample, comprising contacting
an antibody as disclosed herein with a biological sample from a
patient, and detecting the level of binding between said antibody
and .alpha.5.beta.1 in said sample. In more specific embodiments,
the biological sample is blood, plasma or serum.
[0093] Another embodiment of the invention includes a method for
diagnosing a condition associated with the expression of
.alpha.5.beta.1 in a cell by contacting the serum or a cell with an
antibody as disclosed herein, and thereafter detecting the presence
of .alpha.5.beta.1. In one embodiment the condition can be a
proliferative, angiogenic, cell adhesion or invasion-related
disease including, but not limited to, a neoplastic disease.
[0094] In another embodiment, the invention includes an assay kit
for detecting .alpha.5.beta.1 in mammalian tissues, cells, or body
fluids to screen for .alpha.5.beta.1-related diseases. The kit
includes an antibody as disclosed herein and a means for indicating
the reaction of the antibody with .alpha.5.beta.1, if present. In
one embodiment the antibody is a monoclonal antibody. In one
embodiment, the antibody that binds .alpha.5.beta.1 is labelled. In
another embodiment the antibody is an unlabelled primary antibody
and the kit further includes a means for detecting the primary
antibody. In one embodiment, the means for detecting includes a
labelled second antibody that is an anti-immunoglobulin. The
antibody may be labelled with a marker selected from the group
consisting of a fluorochrome, an enzyme, a radionuclide and a
radiopaque material.
[0095] In some embodiments, the targeted binding agents or
antibodies as disclosed herein can be modified to enhance their
capability of fixing complement and participating in
complement-dependent cytotoxicity (CDC). In other embodiments, the
targeted binding agents or antibodies can be modified to enhance
their capability of activating effector cells and participating in
antibody-dependent cytotoxicity (ADCC). In yet other embodiments,
the targeted binding agents or antibodies as disclosed herein can
be modified both to enhance their capability of activating effector
cells and participating in antibody-dependent cytotoxicity (ADCC)
and to enhance their capability of fixing complement and
participating in complement-dependent cytotoxicity (CDC).
[0096] In some embodiments, the targeted binding agents or
antibodies as disclosed herein can be modified to reduce their
capability of fixing complement and participating in
complement-dependent cytotoxicity (CDC). In other embodiments, the
targeted binding agents or antibodies can be modified to reduce
their capability of activating effector cells and participating in
antibody-dependent cytotoxicity (ADCC). In yet other embodiments,
the targeted binding agents or antibodies as disclosed herein can
be modified both to reduce their capability of activating effector
cells and participating in antibody-dependent cytotoxicity (ADCC)
and to reduce their capability of fixing complement and
participating in complement-dependent cytotoxicity (CDC).
[0097] In certain embodiments, the half-life of a targeted binding
agent or antibody as disclosed herein and of compositions of the
invention is at least about 4 to 7 days. In certain embodiments,
the mean half-life of a targeted binding agent or antibody as
disclosed herein and of compositions of the invention is at least
about 2 to 5 days, 3 to 6 days, 4 to 7 days, 5 to 8 days, 6 to 9
days, 7 to 10 days, 8 to 11 days, 8 to 12, 9 to 13, 10 to 14, 11 to
15, 12 to 16, 13 to 17, 14 to 18, 15 to 19, or 16 to 20 days. In
other embodiments, the mean half-life of a targeted binding agent
or antibody as disclosed herein and of compositions of the
invention is at least about 17 to 21 days, 18 to 22 days, 19 to 23
days, 20 to 24 days, 21 to 25, days, 22 to 26 days, 23 to 27 days,
24 to 28 days, 25 to 29 days, or 26 to 30 days. In still further
embodiments the half-life of a targeted binding agent or antibody
as disclosed herein and of compositions of the invention can be up
to about 50 days. In certain embodiments, the half-lives of
antibodies and of compositions of the invention can be prolonged by
methods known in the art. Such prolongation can in turn reduce the
amount and/or frequency of dosing of the antibody compositions.
Antibodies with improved in vivo half-lives and methods for
preparing them are disclosed in U.S. Pat. No. 6,277,375; and
International Publication Nos. WO 98/23289 and WO 97/3461.
[0098] In another embodiment, the invention provides an article of
manufacture including a container. The container includes a
composition containing a targeted binding agent or antibody as
disclosed herein, and a package insert or label indicating that the
composition can be used to treat cell adhesion, invasion,
angiogenesis, and/or proliferation-related diseases, including, but
not limited to, diseases characterised by the expression or
overexpression of .alpha.5.beta.1.
[0099] In other embodiments, the invention provides a kit
comprising a composition containing a targeted binding agent or
antibody as disclosed herein, and instructions to administer the
composition to a subject in need of treatment.
[0100] The present invention provides formulation of proteins
comprising a variant Fc region. That is, a non-naturally occurring
Fc region, for example an Fc region comprising one or more non
naturally occurring amino acid residues, i.e. an amino acid other
than the amino acid normally found at a particular position. Also
encompassed by the variant Fc regions of present invention are Fc
regions which comprise amino acid deletions, additions and/or
modifications.
[0101] The serum half-life of proteins comprising Fc regions may be
increased by increasing the binding affinity of the Fc region for
FcRn. In one embodiment, the Fc variant protein has enhanced serum
half life relative to comparable molecule.
[0102] In another embodiment, the present invention provides an Fc
variant, wherein the Fc region comprises at least one non naturally
occurring amino acid at one or more positions selected from the
group consisting of 239, 330 and 332, as numbered by the EU index
as set forth in Kabat. In a specific embodiment, the present
invention provides an Fc variant, wherein the Fc region comprises
at least one non naturally occurring amino acid selected from the
group consisting of 239D, 330L and 332E, as numbered by the EU
index as set forth in Kabat. Optionally, the Fc region may further
comprise additional non-naturally occurring amino acid at one or
more positions selected from the group consisting of 252, 254, and
256, as numbered by the EU index as set forth in Kabat. In a
specific embodiment, the present invention provides an Fc variant,
wherein the Fc region comprises at least one non naturally
occurring amino acid selected from the group consisting of 239D,
330L and 332E, as numbered by the EU index as set forth in Kabat
and at least one non naturally occurring amino acid at one or more
positions selected from the group consisting of 252Y, 254T and
256E, as numbered by the EU index as set forth in Kabat.
[0103] In another embodiment, the present invention provides an Fc
variant, wherein the Fc region comprises at least one non naturally
occurring amino acid at one or more positions selected from the
group consisting of 234, 235 and 331, as numbered by the EU index
as set forth in Kabat. In a specific embodiment, the present
invention provides an Fc variant, wherein the Fc region comprises
at least one non naturally occurring amino acid selected from the
group consisting of 234F, 235F, 235Y, and 331S, as numbered by the
EU index as set forth in Kabat. In a further specific embodiment,
an Fc variant of the invention comprises the 234F, 235F, and 331S
non naturally occurring amino acid residues, as numbered by the EU
index as set forth in Kabat. In another specific embodiment, an Fc
variant of the invention comprises the 234F, 235Y, and 331S non
naturally occurring amino acid residues, as numbered by the EU
index as set forth in Kabat. Optionally, the Fc region may further
comprise additional non naturally occurring amino acid at one or
more positions selected from the group consisting of 252, 254, and
256, as numbered by the EU index as set forth in Kabat. In a
specific embodiment, the present invention provides an Fc variant,
wherein the Fc region comprises at least one non naturally
occurring amino acid selected from the group consisting of 234F,
235F, 235Y, and 331S, as numbered by the EU index as set forth in
Kabat; and at least one non naturally occurring amino acid at one
or more positions are selected from the group consisting of 252Y,
254T and 256E, as numbered by the EU index as set forth in
Kabat.
[0104] In another embodiment, the present invention provides an Fc
variant protein formulation, wherein the Fc region comprises at
least a non naturally occurring amino acid at one or more positions
selected from the group consisting of 239, 330 and 332, as numbered
by the EU index as set forth in Kabat. In a specific embodiment,
the present invention provides an Fc variant protein formulation,
wherein the Fc region comprises at least one non naturally
occurring amino acid selected from the group consisting of 239D,
330L and 332E, as numbered by the EU index as set forth in Kabat.
Optionally, the Fc region may further comprise additional non
naturally occurring amino acid at one or more positions selected
from the group consisting of 252, 254, and 256, as numbered by the
EU index as set forth in Kabat. In a specific embodiment, the
present invention provides an Fc variant protein formulation,
wherein the Fc region comprises at least one non naturally
occurring amino acid selected from the group consisting of 239D,
330L and 332E, as numbered by the EU index as set forth in Kabat
and at least one non naturally occurring amino acid at one or more
positions are selected from the group consisting of 252Y, 254T and
256E, as numbered by the EU index as set forth in Kabat.
[0105] In another embodiment, the present invention provides an Fc
variant protein formulation, wherein the Fc region comprises at
least one non naturally occurring amino acid at one or more
positions selected from the group consisting of 234, 235 and 331,
as numbered by the EU index as set forth in Kabat. In a specific
embodiment, the present invention provides an Fc variant protein
formulation, wherein the Fc region comprises at least one non
naturally occurring amino acid selected from the group consisting
of 234F, 235F, 235Y, and 331S, as numbered by the EU index as set
forth in Kabat. Optionally, the Fc region may further comprise
additional non naturally occurring amino acid at one or more
positions selected from the group consisting of 252, 254, and 256,
as numbered by the EU index as set forth in Kabat. In a specific
embodiment, the present invention provides an Fc variant protein
formulation, wherein the Fc region comprises at least one non
naturally occurring amino acid selected from the group consisting
of 234F, 235F, 235Y, and 331S, as numbered by the EU index as set
forth in Kabat; and at least one non naturally occurring amino acid
at one or more positions are selected from the group consisting of
252Y, 254T and 256E, as numbered by the EU index as set forth in
Kabat.
[0106] Methods for generating non naturally occurring Fc regions
are known in the art. For example, amino acid substitutions and/or
deletions can be generated by mutagenesis methods, including, but
not limited to, site-directed mutagenesis (Kunkel, Proc. Natl.
Acad. Sci. USA 82:488-492 (1985)), PCR mutagenesis (Higuchi, in
"PCR Protocols: A Guide to Methods and Applications", Academic
Press, San Diego, pp. 177-183 (1990)), and cassette mutagenesis
(Wells et al., Gene 34:315-323 (1985)). Preferably, site-directed
mutagenesis is performed by the overlap-extension PCR method
(Higuchi, in "PCR Technology: Principles and Applications for DNA
Amplification", Stockton Press, New York, pp. 61-70 (1989)). The
technique of overlap-extension PCR (Higuchi, ibid.) can also be
used to introduce any desired mutation(s) into a target sequence
(the starting DNA). For example, the first round of PCR in the
overlap-extension method involves amplifying the target sequence
with an outside primer (primer 1) and an internal mutagenesis
primer (primer 3), and separately with a second outside primer
(primer 4) and an internal primer (primer 2), yielding two PCR
segments (segments A and B). The internal mutagenesis primer
(primer 3) is designed to contain mismatches to the target sequence
specifying the desired mutation(s). In the second round of PCR, the
products of the first round of PCR (segments A and B) are amplified
by PCR using the two outside primers (primers 1 and 4). The
resulting full-length PCR segment (segment C) is digested with
restriction enzymes and the resulting restriction fragment is
cloned into an appropriate vector. As the first step of
mutagenesis, the starting DNA (e.g., encoding an Fc fusion protein,
an antibody or simply an Fc region), is operably cloned into a
mutagenesis vector. The primers are designed to reflect the desired
amino acid substitution. Other methods useful for the generation of
variant Fc regions are known in the art (see, e.g., U.S. Pat. Nos.
5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046;
6,121,022; 5,624,821; 5,648,260; 6,528,624; 6,194,551; 6,737,056;
6,821,505; 6,277,375; U.S. Patent Publication Nos. 2004/0002587 and
PCT Publications WO 94/29351; WO 99/58572; WO 00/42072; WO
02/060919; WO 04/029207; WO 04/099249; WO 04/063351).
[0107] In some embodiments of the invention, the glycosylation
patterns of the antibodies provided herein are modified to enhance
ADCC and CDC effector function. See Shields R L et al., (2002) JBC.
277:26733; Shinkawa T et al., (2003) JBC. 278:3466 and Okazaki A et
al., (2004) J. Mol. Biol., 336: 1239. In some embodiments, an Fc
variant protein comprises one or more engineered glycoforms, i.e.,
a carbohydrate composition that is covalently attached to the
molecule comprising an Fc region. Engineered glycoforms may be
useful for a variety of purposes, including but not limited to
enhancing or reducing effector function. Engineered glycoforms may
be generated by any method known to one skilled in the art, for
example by using engineered or variant expression strains, by
co-expression with one or more enzymes, for example DI
N-acetylglucosaminyltransferase III (GnTI11), by expressing a
molecule comprising an Fc region in various organisms or cell lines
from various organisms, or by modifying carbohydrate(s) after the
molecule comprising Fc region has been expressed. Methods for
generating engineered glycoforms are known in the art, and include
but are not limited to those described in Umana et al, 1999, Nat.
Biotechnol 17:176-180; Davies et al., 20017 Biotechnol Bioeng
74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740;
Shinkawa et al., 2003, J Biol Chem 278:3466-3473) U.S. Pat. No.
6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; PCT
WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1; PCT WO
02/30954A1; Potillegent.TM. technology (Biowa, Inc. Princeton,
N.J.); GlycoMAb.TM. glycosylation engineering technology (GLYCART
biotechnology AG, Zurich, Switzerland). See, e.g., WO is 00061739;
EA01229125; US 20030115614; Okazaki et al., 2004, JMB, 336:
1239-49.
[0108] It is also known in the art that the glycosylation of the Fc
region can be modified to increase or decrease effector function
(see for examples, Umana et al, 1999, Nat. Biotechnol 17:176-180;
Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al,
2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol
Chem 278:3466-3473) U.S. Pat. No. 6,602,684; U.S. Ser. No.
10/277,370; U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO
01/292246A1; PCT WO 02/311140A1; PCT WO 02/30954A1; Potillegent.TM.
technology (Biowa, Inc. Princeton, N.J.); GlycoMAb.TM.
glycosylation engineering technology (GLYCART biotechnology AG,
Zurich, Switzerland). Accordingly, in one embodiment the Fc regions
of the antibodies of the invention comprise altered glycosylation
of amino acid residues. In another embodiment, the altered
glycosylation of the amino acid residues results in lowered
effector function. In another embodiment, the altered glycosylation
of the amino acid residues results in increased effector function.
In a specific embodiment, the Fc region has reduced fucosylation.
In another embodiment, the Fc region is afucosylated (see for
examples, U.S. Patent Application Publication No.
2005/0226867).
BRIEF DESCRIPTION OF THE DRAWINGS
[0109] FIG. 1 is a bar chart showing the effect of inhibitory
.alpha.5.beta.1 antibodies on the attachment of endothelial cells
to fibronectin, in the presence of 10 ug/ml L230. Antibody
treatments are indicated on the X-axis, and compared to a no
treatment control. IgG1 control, 2H12 were used at 10 .mu.g/ml and
25 .mu.g/ml as indicated. The mean cell adhesion as measured by
cell count is indicated on the Y-axis, together with the standard
deviation of the mean (error bars).
[0110] FIG. 2 is a bar chart showing the effect of inhibitory
.alpha.5.beta.1 antibodies on endothelial cell tube formation in an
endothelial tube formation co-culture assay. Antibodies are
indicated on the X-axis and concentrations from left to right in
each group of bars are 5 .mu.g/mL, 1 .mu.g/mL, 0.2 .mu.g/mL and
0.04 .mu.g/mL. The degree of tube formation in terms of length (mm)
and bifurcations is shown on the Y-axis. The values represented are
the mean+/-the standard deviation. Vessel length (mm) is
represented in black bars and bifurcations in grey bars.
[0111] FIG. 3 is a graph showing the that 2H12 IgG4, 2H12 IgG2 WT
and 2H12 IgG2 Variant 1 inhibit binding of K562 cells to
fibronectin with similar potencies. 2H12 IgG2 wild type (circle,
solid line), 2H12 IgG2 Variant 1 (triangle, dashed line) and 2H12
IgG4 WT (inverse triangle, dash dot line) were incubated with K562
cells at a range of concentrations represented on the X-axis in
mg/ml. Total cell binding is represented on the Y axis (optical
density (OD)).
[0112] FIG. 4 is a bar chart showing the effect of inhibitory
.alpha.5.beta.1 antibodies on angiogenesis in vivo. Treatments are
represented on the X-axis: Control vehicle twice weekly; 2H12 20
mg/kg twice weekly (checked bars); 2H12 10 mg/kg twice weekly
(diagonal striped bars); The Y axis shows mean vessel density+/-the
standard error.
[0113] FIG. 5 is a graph showing the binding profile of
biotinylated .alpha.5.beta.1 binding antibodies 3C5 (IgG2) and 2H12
Variant 1 (IgG2), versus IgG2 control, to monocytes in the whole
human blood. 2H12 Variant (small squares, solid line) show much
lower binding to monocytes than 2H12 (large square solid line).
IgG2 control antibody is represented by diamonds and dot/dash line.
Antibody concentrations are represented on X axis in .mu.g/ml whole
blood. Total binding of the antibody is represented Y axis in mean
fluorescence units.
[0114] FIG. 6 provides a graph showing the growth of U87MG tumour
xenografts over time. Control tumour treated with PBS vehicle
control is shown as filled squares. Tumour treated with IgG1TM
control antibody is shown as filled diamonds. Tumours treated with
2H12 IgG1TM are show as filled triangles. Time (days) is indicated
on the x axis and size (cm3) is shown in the y axis.
[0115] FIG. 7 provides a bar graph showing the binding of K562
cells to fibonectin in the presence of either 2H12 in the IgG1TM or
IgG2 formats. Antibody concentration is represented on the x-axis
in .mu.g/ml. Total adhesion measured by absorbance (OD units) is
represented on the y-axis.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0116] Embodiments of the invention relate to targeted binding
agents that bind to .alpha.5.beta.1. In some embodiments, the
targeted binding agents bind to .alpha.5.beta.1 and inhibit the
binding of fibronectin, fibrin, the adhesion molecule L1-CAM and
growth factor receptors such as Tie-2 and Flt1 to .alpha.5.beta.1.
In one embodiment, the targeted binding agents are monoclonal
antibodies, or binding fragments thereof. Such monoclonal
antibodies may be referred to as anti-.alpha.5.beta.1 antibodies
herein.
[0117] Other embodiments of the invention include fully human
anti-.alpha.5.beta.1 antibodies, and antibody preparations that are
therapeutically useful. In one embodiment, preparations of the
anti-.alpha.5.beta.1 antibody of the invention have desirable
therapeutic properties, including strong binding affinity for
.alpha.5.beta.1 and the ability to inhibit .alpha.5.beta.1-induced
cell activity in vitro and in vivo.
[0118] In addition, embodiments of the invention include methods of
using these antibodies for treating diseases. Anti-.alpha.5.beta.1
antibodies of the invention are useful for preventing
.alpha.5.beta.1-mediated tumourigenesis and tumour invasion of
healthy tissue. In addition .alpha.5.beta.1 antibodies can be
useful for treating diseases associated with angiogenesis such as
ocular disease such as AMD, inflammatory disorders such as
rheumatoid arthritis, and cardiovascular disease and sepsis as well
as neoplastic diseases. Diseases that are treatable through this
inhibition mechanism include, but are not limited to a neoplastic
disease. Any disease that is characterized by any type of malignant
tumour, including metastatic cancers, lymphatic tumours, and blood
cancers, can also be treated by this inhibition mechanism.
Exemplary cancers in humans include a bladder tumour, breast
tumour, prostate tumour, basal cell carcinoma, biliary tract
cancer, bladder cancer, bone cancer, brain and CNS cancer (e.g.,
glioma tumour), cervical cancer, choriocarcinoma, colon and rectum
cancer, connective tissue cancer, cancer of the digestive system;
endometrial cancer, esophageal cancer; eye cancer; cancer of the
head and neck; gastric cancer; intra-epithelial neoplasm; kidney
cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g.
small cell and non-small cell); lymphoma including Hodgkin's and
Non-Hodgkin's lymphoma; melanoma; myeloma, neuroblastoma, oral
cavity cancer (e.g., lip, tongue, mouth, and pharynx); ovarian
cancer; pancreatic cancer, retinoblastoma; rhabdomyosarcoma; rectal
cancer, renal cancer, cancer of the respiratory system; sarcoma,
skin cancer; stomach cancer, testicular cancer, thyroid cancer;
uterine cancer, cancer of the urinary system, as well as other
carcinomas and sarcomas. Malignant disorders commonly diagnosed in
dogs, cats, and other pets include, but are not limited to,
lymphosarcoma, osteosarcoma, mammary tumours, mastocytoma, brain
tumour, melanoma, adenosquamous carcinoma, carcinoid lung tumour,
bronchial gland tumour, bronchiolar adenocarcinoma, fibroma,
myxochondroma, pulmonary sarcoma, neurosarcoma, osteoma, papilloma,
retinoblastoma, Ewing's sarcoma, Wilm's tumour, Burkitt's lymphoma,
microglioma, neuroblastoma, osteoclastoma, oral neoplasia,
fibrosarcoma, osteosarcoma and rhabdomyosarcoma, genital squamous
cell carcinoma, transmissible venereal tumour, testicular tumour,
seminoma, Sertoli cell tumour, hemangiopericytoma, histiocytoma,
chloroma (e.g., granulocytic sarcoma), corneal papilloma, corneal
squamous cell carcinoma, hemangiosarcoma, pleural mesothelioma,
basal cell tumour, thymoma, stomach tumour, adrenal gland
carcinoma, oral papillomatosis, hemangioendothelioma and
cystadenoma, follicular lymphoma, intestinal lymphosarcoma,
fibrosarcoma and pulmonary squamous cell carcinoma. In rodents,
such as a ferret, exemplary cancers include insulinoma, lymphoma,
sarcoma, neuroma, pancreatic islet cell tumour, gastric MALT
lymphoma and gastric adenocarcinoma. Neoplasias affecting
agricultural livestock include leukemia, hemangiopericytoma and
bovine ocular neoplasia (in cattle); preputial fibrosarcoma,
ulcerative squamous cell carcinoma, preputial carcinoma, connective
tissue neoplasia and mastocytoma (in horses); hepatocellular
carcinoma (in swine); lymphoma and pulmonary adenomatosis (in
sheep); pulmonary sarcoma, lymphoma, Rous sarcoma,
reticulo-endotheliosis, fibrosarcoma, nephroblastoma, B-cell
lymphoma and lymphoid leukosis (in avian species); retinoblastoma,
hepatic neoplasia, lymphosarcoma (lymphoblastic lymphoma),
plasmacytoid leukemia and swimbladder sarcoma (in fish), caseous
lumphadenitis (CLA): chronic, infectious, contagious disease of
sheep and goats caused by the bacterium Corynebacterium
pseudotuberculosis, and contagious lung tumour of sheep caused by
jaagsiekte.
[0119] Other embodiments of the invention include diagnostic assays
for specifically determining the quantity of .alpha.5.beta.1 in a
biological sample. The assay kit can include a targeted binding
agent or antibody as disclosed herein along with the necessary
labels for detecting such antibodies. These diagnostic assays are
useful to screen for cell adhesion, invasion, angiogenesis or
proliferation-related diseases including, but not limited to,
neoplastic diseases.
[0120] Another aspect of the invention is an antagonist of the
biological activity of .alpha.5.beta.1 wherein the antagonist binds
to .alpha.5.beta.1. In one embodiment, the antagonist is a targeted
binding agent, such as an antibody. In one embodiment the
antagonist is able to antagonize the biological activity of
.alpha.5.beta.1 in vitro and in vivo. The antagonist may be
selected from an antibody described herein, for example, antibody
2H12 or 2H12 Variant 1.
[0121] In one embodiment the antagonist of the biological activity
of .alpha.5.beta.1 binds to .alpha.5.beta.1 thereby inhibiting cell
adhesion and/or invasion and/or angiogenesis and/or proliferation.
The mechanism of action of this inhibition may include binding of
the antagonist to .alpha.5.beta.1 and inhibiting the binding of a
native .alpha.5.beta.1-specific ligand, such as, for example
fibronectin, to .alpha.5.beta.1. Without wishing to be bound by any
particular theoretical considerations, mechanisms by which
antagonism of the biological activity of .alpha.5.beta.1 can be
achieved include, but are not limited to, inhibition of binding of
fibronectin to .alpha.5.beta.1, and/or inhibition of
.alpha.5.beta.1-fibronectin mediated signaling activity.
[0122] One embodiment is a hybridoma that produces the targeted
binding agent as described hereinabove. In one embodiment is a
hybridoma that produces the light chain and/or the heavy chain of
the antibodies as described hereinabove. In one embodiment the
hybridoma produces the light chain and/or the heavy chain of a
fully human monoclonal antibody. In another embodiment the
hybridoma produces the light chain and/or the heavy chain of fully
human monoclonal antibody 2H12 or 2H12 Variant 1. Alternatively the
hybridoma may produce an antibody which binds to the same epitope
or epitopes as fully human monoclonal antibody 2H12 or 2H12 Variant
1.
[0123] Another embodiment is a nucleic acid molecule encoding the
targeted binding agent as described hereinabove. In one embodiment
is a nucleic acid molecule encoding the light chain or the heavy
chain of an antibody as described hereinabove. In one embodiment
the nucleic acid molecule encodes the light chain or the heavy
chain of a fully human monoclonal antibody. Still another
embodiment is a nucleic acid molecule encoding the light chain or
the heavy chain of a fully human monoclonal antibody 2H12 or 2H12
Variant 1.
[0124] Another embodiment of the invention is a vector comprising a
nucleic acid molecule or molecules as described hereinabove,
wherein the vector encodes a targeted binding agent as defined
hereinabove. In one embodiment of the invention is a vector
comprising a nucleic acid molecule or molecules as described
hereinabove, wherein the vector encodes a light chain and/or a
heavy chain of an antibody as defined hereinabove.
[0125] Yet another embodiment of the invention is a host cell
comprising a vector as described hereinabove. Alternatively the
host cell may comprise more than one vector.
[0126] In addition, one embodiment of the invention is a method of
producing a targeted binding agent of the invention by culturing
host cells under conditions wherein a nucleic acid molecule is
expressed to produce the targeted binding agent, followed by
recovery of the targeted binding agent. In one embodiment of the
invention is a method of producing an antibody of the invention by
culturing host cells under conditions wherein a nucleic acid
molecule is expressed to produce the antibody, followed by recovery
of the antibody.
[0127] In one embodiment the invention includes a method of making
an targeted binding agent by transfecting at least one host cell
with at least one nucleic acid molecule encoding the targeted
binding agent as described hereinabove, expressing the nucleic acid
molecule in the host cell and isolating the targeted binding agent.
In one embodiment the invention includes a method of making an
antibody by transfecting at least one host cell with at least one
nucleic acid molecule encoding the antibody as described
hereinabove, expressing the nucleic acid molecule in the host cell
and isolating the antibody.
[0128] According to another aspect, the invention includes a method
of antagonising the biological activity of .alpha.5.beta.1 by
administering an antagonist as described herein. The method may
include selecting an animal in need of treatment for
disease-related cell adhesion and/or invasion and/or angiogenesis
and/or proliferation, and administering to the animal a
therapeutically effective dose of an antagonist of the biological
activity of .alpha.5.beta.1.
[0129] Another aspect of the invention includes a method of
antagonising the biological activity of .alpha.5.beta.1 by
administering a targeted binding agent as described hereinabove.
The method may include selecting an animal in need of treatment for
disease-related cell adhesion and/or invasion and/or angiogenesis
and/or proliferation, and administering to the animal a
therapeutically effective dose of a targeted binding agent which
antagonises the biological activity of .alpha.5.beta.1.
[0130] Another aspect of the invention includes a method of
antagonising the biological activity of .alpha.5.beta.1 by
administering an antibody as described hereinabove. The method may
include selecting an animal in need of treatment for
disease-related cell adhesion and/or invasion and/or angiogenesis
and/or proliferation, and administering to the animal a
therapeutically effective dose of an antibody which antagonises the
biological activity of .alpha.5.beta.1.
[0131] According to another aspect there is provided a method of
treating disease-related cell adhesion and/or invasion and/or
angiogenesis and/or proliferation in an animal by administering a
therapeutically effective amount of an antagonist of the biological
activity of .alpha.5.beta.1. The method may include selecting an
animal in need of treatment for disease-related cell adhesion
and/or invasion and/or angiogenesis and/or proliferation, and
administering to the animal a therapeutically effective dose of an
antagonist of the biological activity of .alpha.5.beta.1.
[0132] According to another aspect there is provided a method of
treating disease-related cell adhesion and/or invasion and/or
angiogenesis and/or proliferation in an animal by administering a
therapeutically effective amount of a targeted binding agent which
antagonizes the biological is activity of .alpha.5.beta.1. The
method may include selecting an animal in need of treatment for
disease-related cell adhesion and/or invasion and/or angiogenesis
and/or proliferation, and administering to the animal a
therapeutically effective dose of a targeted binding agent which
antagonises the biological activity of .alpha.5.beta.1. The
targeted binding agent can be administered alone, or can be
administered in combination with additional antibodies or
chemotherapeutic drugs or radiation therapy.
[0133] According to another aspect there is provided a method of
treating disease-related cell adhesion and/or invasion and/or
angiogenesis and/or proliferation in an animal by administering a
therapeutically effective amount of an antibody which antagonizes
the biological activity of .alpha.5.beta.1. The method may include
selecting an animal in need of treatment for disease-related cell
adhesion and/or invasion and/or angiogenesis and/or proliferation,
and administering to the animal a therapeutically effective dose of
an antibody which antagonises the biological activity of
.alpha.5.beta.1. The antibody can be administered alone, or can be
administered in combination with additional antibodies or
chemotherapeutic drugs or radiation therapy.
[0134] According to another aspect there is provided a method of
treating cancer in an animal by administering a therapeutically
effective amount of an antagonist of the biological activity of
.alpha.5.beta.1. The method may include selecting an animal in need
of treatment for cancer, and administering to the animal a
therapeutically effective dose of an antagonist which antagonises
the biological activity of .alpha.5.beta.1. The antagonist can be
administered alone, or can be administered in combination with
additional antibodies or chemotherapeutic drugs or radiation
therapy.
[0135] According to another aspect there is provided a method of
treating cancer in an animal by administering a therapeutically
effective amount of a targeted binding agent which antagonizes the
biological activity of .alpha.5.beta.1. The method may include
selecting an animal in need of treatment for cancer, and
administering to the animal a therapeutically effective dose of a
targeted binding agent which antagonises the biological activity of
.alpha.5.beta.1. The targeted binding agent can be administered
alone, or can be administered in combination with additional
antibodies or chemotherapeutic drugs or radiation therapy.
[0136] According to another aspect there is provided a method of
treating cancer in an animal by administering a therapeutically
effective amount of an antibody which antagonizes the biological
activity of .alpha.5.beta.1. The method may include selecting an
animal in need of treatment for cancer, and administering to the
animal a therapeutically effective dose of an antibody which
antagonises the biological activity of .alpha.5.beta.1. The
antibody can be administered alone, or can be administered in
combination with additional antibodies or chemotherapeutic drugs or
radiation therapy.
[0137] According to another aspect there is provided a method of
reducing or inhibiting tumour cell proliferation, adhesion,
invasion and/or angiogenesis, in an animal by administering a
therapeutically effective amount of an antibody which antagonizes
the biological activity of .alpha.5.beta.1. The method may include
selecting an animal in need of a reduction or inhibition of
proliferation, cell adhesion, invasion and/or angiogenesis, and
administering to the animal a therapeutically effective dose of an
antibody which antagonises the biological activity of
.alpha.5.beta.1. The antibody can be administered alone, or can be
administered in combination with additional antibodies or
chemotherapeutic drugs or radiation therapy.
[0138] According to another aspect there is provided a method of
reducing tumour growth and/or metastasis, in an animal by
administering a therapeutically effective amount of an antibody
which antagonizes the biological activity of .alpha.5.beta.1. The
method may include selecting an animal in need of a reduction of
tumour growth and/or metastasis, and administering to the animal a
therapeutically effective dose of an antibody which antagonises the
biological activity of .alpha.5.beta.1. The antibody can be
administered alone, or can be administered in combination with
additional antibodies or chemotherapeutic drugs or radiation
therapy.
[0139] According to another aspect of the invention there is
provided the use of an antagonist of the biological activity of
.alpha.5.beta.1 for the manufacture of a medicament for the
treatment of disease-related cell adhesion and/or invasion and/or
angiogenesis and/or proliferation. In one embodiment the antagonist
of the biological activity of .alpha.5.beta.1 is a targeted binding
agent of the invention. In one embodiment the antagonist of the
biological activity of .alpha.5.beta.1 is an antibody of the
invention.
[0140] According to another aspect of the invention there is
provided an antagonist of the biological activity of
.alpha.5.beta.1 for use as a medicament for the treatment of
disease-related cell adhesion and/or invasion and/or angiogenesis
and/or proliferation. In one embodiment the antagonist of the
biological activity of .alpha.5.beta.1 is a targeted binding agent
of the invention. In one embodiment the antagonist of the
biological activity of .alpha.5.beta.1 is an antibody of the
invention.
[0141] According to another aspect of the invention there is
provided the use of a targeted is binding agent or an antibody
which antagonizes the biological activity of .alpha.5.beta.1 for
the manufacture of a medicament for the treatment of
disease-related cell adhesion and/or invasion and/or angiogenesis
and/or proliferation.
[0142] According to another aspect of the invention there is
provided a targeted binding agent or an antibody which antagonizes
the biological activity of .alpha.5.beta.1 for use as a medicament
for the treatment of disease-related cell adhesion and/or invasion
and/or angiogenesis and/or proliferation.
[0143] According to another aspect of the invention there is
provided the use of a targeted binding agent or an antibody which
antagonizes the biological activity of .alpha.5.beta.1 for the
manufacture of a medicament for the treatment of disease-related
cell adhesion and/or invasion and/or angiogenesis and/or
proliferation.
[0144] According to another aspect of the invention there is
provided an antibody which antagonizes the biological activity of
.alpha.5.beta.1 for use as a medicament for the treatment of
disease-related cell adhesion and/or invasion and/or angiogenesis
and/or proliferation.
[0145] According to another aspect of the invention there is
provided the use of an antagonist of the biological activity of
.alpha.5.beta.1 for the manufacture of a medicament for the
treatment of cancer in a mammal. In one embodiment the antagonist
of the biological activity of .alpha.5.beta.1 is a targeted binding
agent of the invention. In one embodiment the antagonist of the
biological activity of .alpha.5.beta.1 is an antibody of the
invention.
[0146] According to another aspect of the invention there is
provided an antagonist of the biological activity of
.alpha.5.beta.1 for use as a medicament for the treatment of cancer
in a mammal. In one embodiment the antagonist of the biological
activity of .alpha.5.beta.1 is a targeted binding agent of the
invention. In one embodiment the antagonist of the biological
activity of .alpha.5.beta.1 is an antibody of the invention.
[0147] According to another aspect of the invention there is
provided the use of a targeted binding agent which antagonizes the
biological activity of .alpha.5.beta.1 for the manufacture of a
medicament for the treatment of cancer in a mammal.
[0148] According to another aspect of the invention there is
provided a targeted binding agent which antagonizes the biological
activity of .alpha.5.beta.1 for use as a medicament for the
treatment of cancer in a mammal.
[0149] According to another aspect of the invention there is
provided the use of an antibody which antagonizes the biological
activity of .alpha.5.beta.1 for the manufacture of a medicament for
the treatment of cancer in a mammal.
[0150] According to another aspect of the invention there is
provided an antibody which antagonizes the biological activity of
.alpha.5.beta.1 for use as a medicament for the treatment of cancer
in a mammal.
[0151] According to another aspect there is provided the use of a
targeted binding agent or an antibody which antagonizes the
biological activity of .alpha.5.beta.1 for the manufacture of a
medicament for the reduction or inhibition proliferation, cell
adhesion, invasion and/or angiogenesis in an animal.
[0152] According to another aspect there is provided a targeted
binding agent or an antibody which antagonizes the biological
activity of .alpha.5.beta.1 for use as a medicament for the
reduction or inhibition proliferation, cell adhesion, invasion
and/or angiogenesis in an animal.
[0153] According to another aspect there is provided the use of a
targeted binding agent or an antibody which antagonizes the
biological activity of .alpha.5.beta.1 for the manufacture of a
medicament for reducing tumour growth and/or metastasis, in an
animal.
[0154] According to another aspect there is provided a targeted
binding agent or an antibody which antagonizes the biological
activity of .alpha.5.beta.1 for use as a medicament for reducing
tumour growth and/or metastasis, in an animal.
[0155] In one embodiment the present invention is particularly
suitable for use in antagonizing .alpha.5.beta.1, in patients with
a tumour which is dependent alone, or in part on .alpha.5.beta.1.
According to another aspect of the invention there is provided a
pharmaceutical composition comprising an antagonist of the
biological activity of .alpha.5.beta.1, and a pharmaceutically
acceptable carrier. In one embodiment the antagonist comprises an
antibody. According to another aspect of the invention there is
provided a pharmaceutical composition comprising an antagonist of
the biological activity of .alpha.5.beta.1, and a pharmaceutically
acceptable carrier. In one embodiment the antagonist comprises an
antibody.
[0156] In some embodiments, following administration of the
antibody that specifically binds to .alpha.5.beta.1, a clearing
agent is administered, to remove excess circulating antibody from
the blood.
[0157] Anti-.alpha.5.beta.1 antibodies are useful in the detection
of .alpha.5.beta.1 in patient samples and accordingly are useful as
diagnostics for disease states as described herein. In addition,
based on their ability to significantly inhibit
.alpha.5.beta.1-mediated signaling activity (as demonstrated in the
Examples below), anti-.alpha.5.beta.1 antibodies have therapeutic
effects in treating symptoms and conditions resulting from
.alpha.5.beta.1 expression. In specific embodiments, the antibodies
and methods herein relate to the treatment of symptoms resulting
from .alpha.5.beta.1 induced cell adhesion, invasion, angiogenesis,
proliferation and/or intracellular signaling. Further embodiments
involve using the antibodies and methods described herein to treat
cell adhesion, invasion, angiogenesis and/or proliferation-related
diseases including neoplastic diseases, such as, melanoma, small
cell lung cancer, non-small cell lung cancer, glioma,
hepatocellular (liver) carcinoma, thyroid tumour, gastric (stomach)
cancer, prostate cancer, breast cancer, ovarian cancer, bladder
cancer, lung cancer, glioblastoma, endometrial cancer, kidney
cancer, colon cancer, and pancreatic cancer. The antibodies may
also be useful in treating cell adhesion and/or invasion in
arthritis, atherosclerosis and diseases involving angiogenesis.
[0158] Another embodiment of the invention includes an assay kit
for detecting .alpha.5.beta.1 in mammalian tissues, cells, or body
fluids to screen for cell adhesion-, invasion-, angiogenesis- or
proliferation related diseases. The kit includes a targeted binding
agent that binds to .alpha.5.beta.1 and a means for indicating the
reaction of the targeted binding agent with .alpha.5.beta.1, if
present. In one embodiment, the targeted binding agent that binds
.alpha.5.beta.1 is labeled. In another embodiment the targeted
binding agent is an unlabeled and the kit further includes a means
for detecting the targeted binding agent. Preferably the targeted
binding agent is labeled with a marker selected from the group
consisting of a fluorochrome, an enzyme, a radionuclide and a
radio-opaque material.
[0159] Another embodiment of the invention includes an assay kit
for detecting .alpha.5.beta.1 in mammalian tissues, cells, or body
fluids to screen for cell adhesion-, invasion-, angiogenesis or
proliferation-related diseases. The kit includes an antibody that
binds to .alpha.5.beta.1 and a means for indicating the reaction of
the antibody with .alpha.5.beta.1, if present. The antibody may be
a monoclonal antibody. In one embodiment, the antibody that binds
.alpha.5.beta.1 is labeled. In another embodiment the antibody is
an unlabeled primary antibody and the kit further includes a means
for detecting the primary antibody. In one embodiment, the means
includes a labeled second antibody that is an anti-immunoglobulin.
Preferably the antibody is labeled with a marker selected from the
group consisting of a fluorochrome, an enzyme, a radionuclide and a
radio-opaque material.
[0160] Further embodiments, features, and the like regarding the
antibodies as disclosed herein are provided in additional detail
below.
Sequence Listing
[0161] Embodiments of the invention include the specific antibodies
listed below in Table 1. This table reports the identification
number of each anti-.alpha.5.beta.1 antibody, along with the SEQ ID
number of the variable domain of the corresponding heavy chain and
light chain genes and polypeptides, respectively. Each antibody has
been given an identification number.
TABLE-US-00001 TABLE 1 MAb ID SEQ ID No.: Sequence NO: 7B1.3A1
Nucleotide sequence encoding the variable region of the heavy chain
1 Amino acid sequence encoding the variable region of the heavy
chain 2 Nucleotide sequence encoding the variable region of the
light chain 3 Amino acid sequence encoding the variable region of
the light chain 4 1F2.2B7 Nucleotide sequence encoding the variable
region of the heavy chain 5 Amino acid sequence encoding the
variable region of the heavy chain 6 Nucleotide sequence encoding
the variable region of the light chain 7 Amino acid sequence
encoding the variable region of the light chain 8 2H12 Nucleotide
sequence encoding the variable region of the heavy chain 9 Amino
acid sequence encoding the variable region of the heavy chain 10
Nucleotide sequence encoding the variable region of the light chain
11 Amino acid sequence encoding the variable region of the light
chain 12 Amino acid sequence encoding the CDR1 region of the heavy
chain 13 Amino acid sequence encoding the CDR2 region of the heavy
chain 14 Amino acid sequence encoding the CDR3 region of the heavy
chain 15 Amino acid sequence encoding the CDR1 region of the light
chain 16 Amino acid sequence encoding the CDR2 region of the light
chain 17 Amino acid sequence encoding the CDR3 region of the light
chain 18 2H12 Nucleotide sequence encoding the variable region of
the heavy chain 19 Variant 1 Amino acid sequence encoding the
variable region of the heavy chain 20 Nucleotide sequence encoding
the variable region of the light chain 21 Amino acid sequence
encoding the variable region of the light chain 22 2G2.2B5
Nucleotide sequence encoding the variable region of the heavy chain
23 Amino acid sequence encoding the variable region of the heavy
chain 24 Nucleotide sequence encoding the variable region of the
light chain 25 Amino acid sequence encoding the variable region of
the light chain 26 3F12.4A1 Nucleotide sequence encoding the
variable region of the heavy chain 27 Amino acid sequence encoding
the variable region of the heavy chain 28 Nucleotide sequence
encoding the variable region of the light chain 29 Amino acid
sequence encoding the variable region of the light chain 30
4G3.3D11 Nucleotide sequence encoding the variable region of the
heavy chain 31 Amino acid sequence encoding the variable region of
the heavy chain 32 Nucleotide sequence encoding the variable region
of the light chain 33 Amino acid sequence encoding the variable
region of the light chain 34 Germline sequence VH3-33, D6-6, JH6B
35 Germline sequence A3, JK3 36
DEFINITIONS
[0162] Unless otherwise defined, scientific and technical terms
used herein shall have the meanings that are commonly understood by
those of ordinary skill in the art. Further, unless otherwise
required by context, singular terms shall include pluralities and
plural terms shall include the singular. Generally, nomenclatures
utilized in connection with, and techniques of, cell and tissue
culture, molecular biology, and protein and oligo- or
polynucleotide chemistry and hybridization described herein are
those well known and commonly used in the art.
[0163] Standard techniques are used for recombinant DNA,
oligonucleotide synthesis, and tissue culture and transformation
(e.g., electroporation, lipofection). Enzymatic reactions and
purification techniques are performed according to manufacturer's
specifications or as commonly accomplished in the art or as
described herein. The foregoing techniques and procedures are
generally performed according to conventional methods well known in
the art and as described in various general and more specific
references that are cited and discussed throughout the present
specification. See e.g., Sambrook et al. Molecular Cloning: A
Laboratory Manual (3rd ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (2001)), which is incorporated herein by
reference. The nomenclatures utilized in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well known and commonly used
in the art. Standard techniques are used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
[0164] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0165] An antagonist or inhibitor may be a polypeptide, nucleic
acid, carbohydrate, lipid, small molecular weight compound, an
oligonucleotide, an oligopeptide, RNA interference (RNAi),
antisense, a recombinant protein, an antibody, or fragments thereof
or conjugates or fusion proteins thereof. For a review of RNAi see
Milhavet O, Gary D S, Mattson M P. (Pharmacol Rev. 2003 December;
55(4):629-48. Review) and antisense (see Opalinska J B, Gewirtz A
M. (Sci STKE. 2003 Oct. 28; 2003 (206):pe47.)
[0166] Disease-related cell adhesion and/or invasion and/or
angiogenesis and/or proliferation may be any abnormal, undesirable
or pathological cell adhesion and/or invasion and/or angiogenesis
and/or proliferation, for example tumour-related cell adhesion
and/or invasion and/or angiogenesis and/or proliferation. Cell
adhesion- and/or invasion and/or angiogenesis- and/or
proliferation-related diseases include, but are not limited to,
non-solid tumours such as leukemia, multiple myeloma or lymphoma,
and also solid tumours such as melanoma, small cell lung cancer,
non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, glioblastoma, carcinoma of the thyroid, bile duct, bone,
gastric, brain/CNS, head and neck, hepatic system, stomach,
prostate, breast, renal, testicle, ovary, skin, cervix, lung,
muscle, neuron, esophageal, bladder, lung, uterus, vulva,
endometrium, kidney, colorectum, pancreas, pleural/peritoneal
membranes, salivary gland, and epidermous.
[0167] A compound refers to any small molecular weight compound
with a molecular weight of less than about 2000 Daltons.
[0168] The term ".alpha.5.beta.1" refers to the molecule that is
.alpha.5.beta.1 protein.
[0169] The term "allotype" is used with respect to antigenic
determinants specified by allelic forms of antibody genes.
Allotypes represent slight differences in the amino acid sequences
of heavy or light chains of different individuals and are sequence
differences between alleles of a subclass whereby an antisera
recognize only the allelic differences. The most important types
are Gm (heavy chain) and Km (light chain). Gm polymorphism is
determined by IGHG1, IGHG2 and IGHG3 genes which have alleles
encoding allotypic antigenic determinants referred to as G1m, G2m,
and G3 allotypes for markers of the IgG1, IgG2 and IgG2 molecules.
At present, 18 Gm allotypes are known: G1m (1, 2, 3, 17) or G1m (a,
x, f, z), G2m (23) or G2m (n), G3m (5, 6, 10, 11, 13, 14, 15, 16,
21, 24, 26, 27, 28) pr G3m (b1, c3, b5, b0, b3, b4, s, t, g, l, c5,
u, v, g5) (Lefranc, et al., The human IgG subclasses: molecular
analysis of structure, function and regulation. Pergamon, Oxford,
pp. 43-78 (1990); Lefranc, G. et al., 1979, Hum. Genet.: 50, 199-21
1, both incorporated entirely by reference).
[0170] Allelic forms of human immunoglobulins have been
well-characterized (WHO Review of the notation for the allotypic
and related markers of human immunoglobulins. J Immunogen 1976, 3:
357-362; WHO Review of the notation for the allotypic and related
markers of human immunoglobulins. 1976, Eur. J. Immunol. 6,
599-601; E. van Loghem, 1986, Allotypic markers, Monogr Allergy
19:40-51, all incorporated entirely by reference). Additionally,
other polymorphisms have been characterized (Kim et al., 2001, J.
Mol. Evol. 54:1-9, incorporated entirely by reference).
[0171] The terms "neutralizing" or "inhibits" when referring to a
targeted binding agent, such as an antibody, relates to the ability
of an antibody to eliminate, reduce, or significantly reduce, the
activity of a target antigen. Accordingly, a "neutralizing"
anti-.alpha.5.beta.1 antibody of the invention is capable of
eliminating or significantly reducing the activity of
.alpha.5.beta.1. A neutralizing .alpha.5.beta.1 antibody may, for
example, act by blocking the binding of a native
.alpha.5.beta.1-specific ligand, such as, for example, fibronectin,
to .alpha.5.beta.1. By blocking this binding, .alpha.5.beta.1
signal-mediated activity is significantly, or completely,
eliminated. Ideally, a neutralizing antibody against
.alpha.5.beta.1 inhibits cell adhesion and/or invasion and/or
angiogenesis and/or proliferation.
[0172] An "antagonist of the biological activity of
.alpha.5.beta.1" is capable of eliminating, reducing or
significantly reducing the activity of .alpha.5.beta.1. An
"antagonist of the biological activity of .alpha.5.beta.1" is
capable of eliminating, reducing or significantly reducing
.alpha.5.beta.1 signaling. An "antagonist of the biological
activity of .alpha.5.beta.1" may eliminate or significantly reduce
cell adhesion and/or invasion and/or angiogenesis and/or
proliferation.
[0173] "Reducing .alpha.5.beta.1 signaling" encompasses a reduction
of .alpha.5.beta.1 signaling by at least 5%, at least 10%, at least
15%, at least 20%, at least 25%, at least 30%, at least 35%, at
least 40%, at least 45%, at least 50%, at least 55%, at least 60%,
at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95% in comparison with the level of
signaling in the absence of a targeted binding agent, antibody or
antagonist of the invention.
[0174] The term "polypeptide" is used herein as a generic term to
refer to native protein, fragments, or analogs of a polypeptide
sequence. Hence, native protein, fragments, and analogs are species
of the polypeptide genus. Preferred polypeptides in accordance with
the invention comprise the human heavy chain immunoglobulin
molecules and the human kappa light chain immunoglobulin molecules,
as well as antibody molecules formed by combinations comprising the
heavy chain immunoglobulin molecules with light chain
immunoglobulin molecules, such as the kappa or lambda light chain
immunoglobulin molecules, and vice versa, as well as fragments and
analogs thereof. Preferred polypeptides in accordance with the
invention may also comprise solely the human heavy chain
immunoglobulin molecules or fragments thereof.
[0175] The terms "native" or "naturally-occurring" as used herein
as applied to an object refers to the fact that an object can be
found in nature. For example, a polypeptide or polynucleotide
sequence that is present in an organism (including viruses) that
can be isolated from a source in nature and which has not been
intentionally modified by man in the laboratory or otherwise is
naturally-occurring.
[0176] The term "operably linked" as used herein refers to
positions of components so described that are in a relationship
permitting them to function in their intended manner. For example,
a control sequence "operably linked" to a coding sequence is
connected in such a way that expression of the coding sequence is
achieved under conditions compatible with the control
sequences.
[0177] The term "polynucleotide" as referred to herein means a
polymeric form of nucleotides of at least 10 bases in length,
either ribonucleotides or deoxynucleotides or a modified form of
either type of nucleotide, or RNA-DNA hetero-duplexes. The term
includes single and double stranded forms of DNA.
[0178] The term "oligonucleotide" referred to herein includes
naturally occurring, and modified nucleotides linked together by
naturally occurring, and non-naturally occurring linkages.
Oligonucleotides are a polynucleotide subset generally comprising a
length of 200 bases or fewer. Preferably, oligonucleotides are 10
to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17,
18, 19, or 20 to 40 bases in length. Oligonucleotides are usually
single stranded, e.g. for probes; although oligonucleotides may be
double stranded, e.g. for use in the construction of a gene mutant.
Oligonucleotides can be either sense or antisense
oligonucleotides.
[0179] The term "naturally occurring nucleotides" referred to
herein includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" referred to herein includes nucleotides with
modified or substituted sugar groups and the like. The term
"oligonucleotide linkages" referred to herein includes
oligonucleotides linkages such as phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoraniladate, phosphoroamidate, and the
like. See e.g., LaPlanche et al. Nucl. Acids Res. 14:9081 (1986);
Stec et al. J. Am. Chem. Soc. 106:6077 (1984); Stein et al. Nucl.
Acids Res. 16:3209 (1988); Zon et al. Anti-Cancer Drug Design 6:539
(1991); Zon et al. Oligonucleotides and Analogues: A Practical
Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press,
Oxford England (1991)); Stec et al. U.S. Pat. No. 5,151,510;
Uhlmann and Peyman Chemical Reviews 90:543 (1990), the disclosures
of which are hereby incorporated by reference. An oligonucleotide
can include a label for detection, if desired.
[0180] The term "selectively hybridise" referred to herein means to
detectably and specifically bind. Polynucleotides, oligonucleotides
and fragments thereof selectively hybridise to nucleic acid strands
under hybridisation and wash conditions that minimise appreciable
amounts of detectable binding to nonspecific nucleic acids. High
stringency conditions can be used to achieve selective
hybridisation conditions as known in the art and discussed herein.
Generally, the nucleic acid sequence homology between the
polynucleotides, oligonucleotides, or antibody fragments and a
nucleic acid sequence of interest will be at least 80%, and more
typically with preferably increasing homologies of at least 85%,
90%, 95%, 99%, and 100%.
[0181] Stringent hybridization conditions include, but are not
limited to, hybridization to filter-bound DNA in 6.times. sodium
chloride/sodium citrate (SSC) (0.9 M NaCl/90 mM NaCitrate, pH 7.0)
at about 45.degree. C. followed by one or more washes in
0.2.times.SSC/0.1% SDS at about 50-65.degree. C., highly stringent
conditions such as hybridization to filter-bound DNA in 6.times.SSC
at about 45.degree. C. followed by one or more washes in
0.1.times.SSC/0.2% SDS at about 60.degree. C., or any other
stringent hybridization conditions known to those skilled in the
art (see, for example, Ausubel, F. M. et al., eds. 1989 Current
Protocols in Molecular Biology, vol. 1, Green Publishing
Associates, Inc. and John Wiley and Sons, Inc., NY at pages 6.3.1
to 6.3.6 and 2.10.3). Two amino acid sequences are "homologous" if
there is a partial or complete identity between their sequences.
For example, 85% homology means that 85% of the amino acids are
identical when the two sequences are aligned for maximum matching.
Gaps (in either of the two sequences being matched) are allowed in
maximizing matching; gap lengths of 5 or less are preferred with 2
or less being more preferred. Alternatively and preferably, two
protein sequences (or polypeptide sequences derived from them of at
least about 30 amino acids in length) are homologous, as this term
is used herein, if they have an alignment score of more than 5 (in
standard deviation units) using the program ALIGN with the mutation
data matrix and a gap penalty of 6 or greater. See Dayhoff, M. O.,
in Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5,
National Biomedical Research Foundation (1972)) and Supplement 2 to
this volume, pp. 1-10. The two sequences or parts thereof are more
preferably homologous if their amino acids are greater than or
equal to 50% identical when optimally aligned using the ALIGN
program. It should be appreciated that there can be differing
regions of homology within two orthologous sequences. For example,
the functional sites of mouse and human orthologues may have a
higher degree of homology than non-functional regions.
[0182] The term "corresponds to" is used herein to mean that a
polynucleotide sequence is homologous (i.e., is identical, not
strictly evolutionarily related) to all or a portion of a reference
polynucleotide sequence, or that a polypeptide sequence is
identical to a reference polypeptide sequence.
[0183] In contradistinction, the term "complementary to" is used
herein to mean that the complementary sequence is homologous to all
or a portion of a reference polynucleotide sequence. For
illustration, the nucleotide sequence "TATAC" corresponds to a
reference sequence "TATAC" and is complementary to a reference
sequence "GTATA".
[0184] The term "sequence identity" means that two polynucleotide
or amino acid sequences are identical (i.e., on a
nucleotide-by-nucleotide or residue-by-residue basis) over the
comparison window. The term "percentage of sequence identity" is
calculated by comparing two optimally aligned sequences over the
window of comparison, determining the number of positions at which
the identical nucleic acid base (e.g., A, T, C, G, U, or I) or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the comparison window (i.e., the
window size), and multiplying the result by 100 to yield the
percentage of sequence identity. The terms "substantial identity"
as used herein denotes a characteristic of a polynucleotide or
amino acid sequence, wherein the polynucleotide or amino acid
comprises a sequence that has at least 85 percent sequence
identity, preferably at least 90 to 95 percent sequence identity,
more preferably at least 99 percent sequence identity, as compared
to a reference sequence over a comparison window of at least 18
nucleotide (6 amino acid) positions, frequently over a window of at
least 24-48 nucleotide (8-16 amino acid) positions, wherein the
percentage of sequence identity is calculated by comparing the
reference sequence to the sequence which may include deletions or
additions which total 20 percent or less of the reference sequence
over the comparison window. The reference sequence may be a subset
of a larger sequence.
[0185] As used herein, the twenty conventional amino acids and
their abbreviations follow conventional usage. See Immunology--A
Synthesis (2.sup.nd Edition, E. S. Golub and D. R. Gren, Eds.,
Sinauer Associates, Sunderland, Mass. (1991)), which is
incorporated herein by reference. Stereoisomers (e.g., D-amino
acids) of the twenty conventional amino acids, unnatural amino
acids such as .alpha.-, .alpha.-disubstituted amino acids, N-alkyl
amino acids, lactic acid, and other unconventional amino acids may
also be suitable components for polypeptides of the present
invention. Examples of unconventional amino acids include:
4-hydroxyproline, .gamma.-carboxyglutamate,
.epsilon.-N,N,N-trimethyllysine, .epsilon.-N-acetyllysine,
O-phosphoserine, N-acetylserine, N-formylmethionine,
3-methylhistidine, 5-hydroxylysine, .sigma.-N-methylarginine, and
other similar amino acids and imino acids (e.g., 4-hydroxyproline).
In the polypeptide notation used herein, the left-hand direction is
the amino terminal direction and the right-hand direction is the
carboxy-terminal direction, in accordance with standard usage and
convention.
[0186] Similarly, unless specified otherwise, the left-hand end of
single-stranded polynucleotide sequences is the 5' end; the
left-hand direction of double-stranded polynucleotide sequences is
referred to as the 5' direction. The direction of 5' to 3' addition
of nascent RNA transcripts is referred to as the transcription
direction; sequence regions on the DNA strand having the same
sequence as the RNA and which are 5' to the 5' end of the RNA
transcript are referred to as "upstream sequences"; sequence
regions on the DNA strand having the same sequence as the RNA and
which are 3' to the 3' end of the RNA transcript are referred to as
"downstream sequences".
[0187] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, share at
least 80 percent sequence identity, preferably at least 90 percent
sequence identity, more preferably at least 95 percent sequence
identity, and most preferably at least 99 percent sequence
identity. Preferably, residue positions that are not identical
differ by conservative amino acid substitutions. Conservative amino
acid substitutions refer to the interchangeability of residues
having similar side chains. For example, a group of amino acids
having aliphatic side chains is glycine, alanine, valine, leucine,
and isoleucine; a group of amino acids having aliphatic-hydroxyl
side chains is serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side
chains is lysine, arginine, and histidine; and a group of amino
acids having sulfur-containing side chains is cysteine and
methionine. Preferred conservative amino acids substitution groups
are: valine-leucine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine, glutamic-aspartic, and
asparagine-glutamine.
[0188] As discussed herein, minor variations in the amino acid
sequences of antibodies or immunoglobulin molecules are
contemplated as being encompassed by the present invention,
providing that the variations in the amino acid sequence maintain
at least 75%, more preferably at least 80%, 90%, 95%, and most
preferably 99% sequence identity to the antibodies or
immunoglobulin molecules described herein. In particular,
conservative amino acid replacements are contemplated. Conservative
replacements are those that take place within a family of amino
acids that have related side chains. Genetically encoded amino
acids are generally divided into families: (1) acidic=aspartate,
glutamate; (2) basic=lysine, arginine, histidine; (3)
non-polar=alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan; and (4) uncharged
polar=glycine, asparagine, glutamine, cysteine, serine, threonine,
tyrosine. More preferred families are: serine and threonine are an
aliphatic-hydroxy family; asparagine and glutamine are an
amide-containing family; alanine, valine, leucine and isoleucine
are an aliphatic family; and phenylalanine, tryptophan, and
tyrosine are an aromatic family. For example, it is reasonable to
expect that an isolated replacement of a leucine with an isoleucine
or valine, an aspartate with a glutamate, a threonine with a
serine, or a similar replacement of an amino acid with a
structurally related amino acid will not have a major effect on the
binding function or properties of the resulting molecule,
especially if the replacement does not involve an amino acid within
a framework site. Whether an amino acid change results in a
functional peptide can readily be determined by assaying the
specific activity of the polypeptide derivative. Assays are
described in detail herein. Fragments or analogs of antibodies or
immunoglobulin molecules can be readily prepared by those of
ordinary skill in the art. Preferred amino- and carboxy-termini of
fragments or analogs occur near boundaries of functional domains.
Structural and functional domains can be identified by comparison
of the nucleotide and/or amino acid sequence data to public or
proprietary sequence databases. Preferably, computerized comparison
methods are used to identify sequence motifs or predicted protein
conformation domains that occur in other proteins of known
structure and/or function. Methods to identify protein sequences
that fold into a known three-dimensional structure are known. Bowie
et al. Science 253:164 (1991). Thus, the foregoing examples
demonstrate that those of skill in the art can recognize sequence
motifs and structural conformations that may be used to define
structural and functional domains in accordance with the antibodies
described herein.
[0189] Glutaminyl and asparaginyl residues are frequently
deamidated to the corresponding glutamyl and aspartyl residues,
respectively. These residues are deamidated under neutral or basic
conditions. The deamidated form of these residues falls within the
scope of this invention.
[0190] In general, cysteine residues in proteins are either engaged
in cysteine-cysteine disulfide bonds or sterically protected from
the disulfide bond formation when they are a part of folded protein
region. Disulfide bond formation in proteins is a complex process,
which is determined by the redox potential of the environment and
specialized thiol-disulfide exchanging enzymes (Creighton, Methods
Enzymol. 107, 305-329, 1984; Houee-Levin, Methods Enzymol. 353,
35-44, 2002). When a cysteine residue does not have a pair in
protein structure and is not sterically protected by folding, it
can form a disulfide bond with a free cysteine from solution in a
process known as disulfide shuffling. In another process known as
disulfide scrambling, free cysteines may also interfere with
naturally occurring disulfide bonds (such as those present in
antibody structures) and lead to low binding, low biological
activity and/or low stability.
[0191] Preferred amino acid substitutions are those which: (1)
reduce susceptibility to proteolysis, (2) reduce susceptibility to
oxidation, (3) alter binding affinity for forming protein
complexes, (4) alter binding affinities, and (4) confer or modify
other physicochemical or functional properties of such analogs.
Analogs can include various mutations of a sequence other than the
naturally-occurring peptide sequence. For example, single or
multiple amino acid substitutions (preferably conservative amino
acid substitutions) may be made in the naturally-occurring sequence
(preferably in the portion of the polypeptide outside the domain(s)
forming intermolecular contacts. A conservative amino acid
substitution should not substantially change the structural
characteristics of the parent sequence (e.g., a replacement amino
acid should not tend to break a helix that occurs in the parent
sequence, or disrupt other types of secondary structure that
characterizes the parent sequence). Examples of art-recognized
polypeptide secondary and tertiary structures are described in
Proteins, Structures and Molecular Principles (Creighton, Ed., W.
H. Freeman and Company, New York (1984)); Introduction to Protein
Structure (C. Branden and J. Tooze, eds., Garland Publishing, New
York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991),
which are each incorporated herein by reference.
[0192] Alteration may comprise replacing one or more amino acid
residue(s) with a non-naturally occurring or non-standard amino
acid, modifying one or more amino acid residue into a non-naturally
occurring or non-standard form, or inserting one or more
non-naturally occurring or non-standard amino acid into the
sequence. Naturally occurring amino acids include the 20 "standard"
L-amino acids identified as G, A, V, L, I, M, P, F, W, S, T, N, Q,
Y, C, K, R, H, D, E by their standard single-letter codes.
Non-standard amino acids include any other residue that may be
incorporated into a polypeptide backbone or result from
modification of an existing amino acid residue. Non-standard amino
acids may be naturally occurring or non-naturally occurring.
Additionally, such methods may be used to make amino acid
substitutions or deletions of one or more variable region cysteine
residues participating in an intrachain disulfide bond to generate
antibody molecules lacking one or more intrachain disulfide
bonds.
[0193] The term "CDR region" or "CDR" is intended to indicate the
hypervariable regions of the heavy and light chains of an antibody
which confer antigen-binding specificity to the antibody. CDRs may
be defined according to the Kabat system (Kabat, E. A. et al.
(1991) Sequences of Proteins of Immunological Interest, 5th
Edition. US Department of Health and Human Services, Public
Service, NIH, Washington), and later editions. An antibody
typically contains 3 heavy chain CDRs and 3 light chain CDRs. The
term CDR or CDRs is used here in order to indicate, according to
the case, one of these regions or several, or even the whole, of
these regions which contain the majority of the amino acid residues
responsible for the binding by affinity of the antibody for the
antigen or the epitope which it recognises.
[0194] The third CDR of the heavy chain (HCDR3) has a greater size
variability (greater diversity essentially due to the mechanisms of
arrangement of the genes which give rise to it). It may be as short
as 2 amino acids although the longest size known is 26. CDR length
may also vary according to the length that can be accommodated by
the particular underlying framework. Functionally, HCDR3 plays a
role in part in the determination of the specificity of the
antibody (Segal et al., PNAS, 71:4298-4302, 1974, Amit et al.,
Science, 233:747-753, 1986, Chothia et al., J. Mol. Biol.,
196:901-917, 1987, Chothia et al., Nature, 342:877-883, 1989, Caton
et al., J. Immunol., 144:1965-1968, 1990, Sharon et al., PNAS,
87:4814-4817, 1990, Sharon et al., J. Immunol., 144:4863-4869,
1990, Kabat et al., J. Immunol., 147:1709-1719, 1991).
[0195] The term a "set of CDRs" referred to herein comprises CDR1,
CDR2 and CDR3. Thus, a set of HCDRs refers to HCDR1, HCDR2 and
HCDR3, and a set of LCDRs refers to LCDR1, LCDR2 and LCDR3.
[0196] Variants of the VH and VL domains and CDRs of the present
invention, including those for which amino acid sequences are set
out herein, and which can be employed in targeting agents and
antibodies for .alpha.5.beta.1 can be obtained by means of methods
of sequence alteration or mutation and screening for antigen
targeting with desired characteristics. Examples of desired
characteristics include but are not limited to: increased binding
affinity for antigen relative to known antibodies which are
specific for the antigen; increased neutralisation of an antigen
activity relative to known antibodies which are specific for the
antigen if the activity is known; specified competitive ability
with a known antibody or ligand to the antigen at a specific molar
ratio; ability to immunoprecipitate ligand-receptor complex;
ability to bind to a specified epitope; linear epitope, e.g.
peptide sequence identified using peptide-binding scan, e.g. using
peptides screened in linear and/or constrained conformation;
conformational epitope, formed by non-continuous residues; ability
to modulate a new biological activity of .alpha.5.beta.1, or
downstream molecule; ability to bind and/or neutralise
.alpha.5.beta.1 and/or for any other desired property.
[0197] The techniques required to make substitutions within amino
acid sequences of CDRs, antibody VH or VL domains and antigen
binding sites are available in the art. Variants of antibody
molecules disclosed herein may be produced and used in the present
invention. Following the lead of computational chemistry in
applying multivariate data analysis techniques to the
structure/property-activity relationships (Wold, et al.
Multivariate data analysis in chemistry. Chemometrics--Mathematics
and Statistics in Chemistry (Ed.: B. Kowalski), D. Reidel
Publishing Company, Dordrecht, Holland, 1984) quantitative
activity-property relationships of antibodies can be derived using
well-known mathematical techniques, such as statistical regression,
pattern recognition and classification (Norman et al. Applied
Regression Analysis. Wiley-Interscience; 3rd edition (April 1998);
Kandel, Abraham & Backer, Eric. Computer-Assisted Reasoning in
Cluster Analysis. Prentice Hall PTR, (May 11, 1995); Krzanowski,
Wojtek. Principles of Multivariate Analysis: A User's Perspective
(Oxford Statistical Science Series, No 22 (Paper)). Oxford
University Press; (December 2000); Witten, Ian H. & Frank,
Eibe. Data Mining: Practical Machine Learning Tools and Techniques
with Java Implementations. Morgan Kaufmann; (Oct. 11, 1999);
Denison David G. T. (Editor), Christopher C. Holmes, Bani K.
Mallick, Adrian F. M. Smith. Bayesian Methods for Nonlinear
Classification and Regression (Wiley Series in Probability and
Statistics). John Wiley & Sons; (July 2002); Ghose, Arup K.
& Viswanadhan, Vellarkad N. Combinatorial Library Design and
Evaluation Principles, Software, Tools, and Applications in Drug
Discovery). In some cases the properties of antibodies can be
derived from empirical and theoretical models (for example,
analysis of likely contact residues or calculated physicochemical
property) of antibody sequence, functional and three-dimensional
structures and these properties can be considered singly and in
combination.
[0198] An antibody antigen-binding site composed of a VH domain and
a VL domain is typically formed by six loops of polypeptide: three
from the light chain variable domain (VL) and three from the heavy
chain variable domain (VH). Analysis of antibodies of known atomic
structure has elucidated relationships between the sequence and
three-dimensional structure of antibody combining sites. These
relationships imply that, except for the third region (loop) in VH
domains, binding site loops have one of a small number of
main-chain conformations: canonical structures. The canonical
structure formed in a particular loop has been shown to be
determined by its size and the presence of certain residues at key
sites in both the loop and in framework regions.
[0199] This study of sequence-structure relationship can be used
for prediction of those residues in an antibody of known sequence,
but of an unknown three-dimensional structure, which are important
in maintaining the three-dimensional structure of its CDR loops and
hence maintain binding specificity. These predictions can be backed
up by comparison of the predictions to the output from lead
optimisation experiments. In a structural approach, a model can be
created of the antibody molecule using any freely available or
commercial package, such as WAM. A protein visualisation and
analysis software package, such as Insight II (Accelrys, Inc.) or
Deep View may then be used to evaluate possible substitutions at
each position in the CDR. This information may then be used to make
substitutions likely to have a minimal or beneficial effect on
activity or confer other desirable properties.
[0200] The term "polypeptide fragment" as used herein refers to a
polypeptide that has an amino-terminal and/or carboxy-terminal
deletion, but where the remaining amino acid sequence is identical
to the corresponding positions in the naturally-occurring sequence
deduced, for example, from a full-length cDNA sequence. Fragments
typically are at least 5, 6, 8 or 10 amino acids long, preferably
at least 14 amino acids long, more preferably at least 20 amino
acids long, usually at least 50 amino acids long, and even more
preferably at least 70 amino acids long. The term "analog" as used
herein refers to polypeptides which are comprised of a segment of
at least 25 amino acids that has substantial identity to a portion
of a deduced amino acid sequence and which has at least one of the
following properties: (1) specific binding to .alpha.5.beta.1,
under suitable binding conditions, (2) ability to block appropriate
fibronectin/.alpha.5.beta.1 binding. Typically, polypeptide analogs
comprise a conservative amino acid substitution (or addition or
deletion) with respect to the naturally-occurring sequence. Analogs
typically are at least 20 amino acids long, preferably at least 50
amino acids long or longer, and can often be as long as a
full-length naturally-occurring polypeptide.
[0201] Peptide analogs are commonly used in the pharmaceutical
industry as non-peptide drugs with properties analogous to those of
the template peptide. These types of non-peptide compound are
termed "peptide mimetics" or "peptidomimetics" (Fauchere, J. Adv.
Drug Res. 15:29 (1986); Veber and Freidinger TINS p. 392 (1985);
and Evans et al. J. Med. Chem. 30:1229 (1987), which are
incorporated herein by reference). Such compounds are often
developed with the aid of computerized molecular modeling. Peptide
mimetics that are structurally similar to therapeutically useful
peptides may be used to produce an equivalent therapeutic or
prophylactic effect. Generally, peptidomimetics are structurally
similar to a paradigm polypeptide (i.e., a polypeptide that has a
biochemical property or pharmacological activity), such as human
antibody, but have one or more peptide linkages optionally replaced
by a linkage selected from the group consisting of: --CH.sub.2NH--,
--CH.sub.2S--, --CH.sub.2--CH.sub.2--, --CH.dbd.CH-- (cis and
trans), --COCH.sub.2--, --CH(OH)CH.sub.2--, and --CH.sub.2SO--, by
methods well known in the art. Systematic substitution of one or
more amino acids of a consensus sequence with a D-amino acid of the
same type (e.g., D-lysine in place of L-lysine) may be used to
generate more stable peptides. In addition, constrained peptides
comprising a consensus sequence or a substantially identical
consensus sequence variation may be generated by methods known in
the art (Rizo and Gierasch Ann. Rev. Biochem. 61:387 (1992),
incorporated herein by reference); for example, by adding internal
cysteine residues capable of forming intramolecular disulfide
bridges which cyclize the peptide.
[0202] As used herein, the terms "antibody" and "antibodies"
(immunoglobulins) encompass an oligoclonal antibody, a monoclonal
antibody (including full-length monoclonal antibody), a polyclonal
antibody, a chimeric antibody, a CDR-grafted antibody, a
multi-specific antibody, a bi-specific antibody, a catalytic
antibody, a chimeric antibody, a humanized antibody, a fully human
antibody, an anti-idiotypic antibody and antibodies that can be
labeled in soluble or bound form as well as fragments, variants or
derivatives thereof, either alone or in combination with other
amino acid sequences provided by known techniques. An antibody may
be from any species. As used herein, the term "antibody" or
"antibodies" refers to a polypeptide or group of polypeptides that
are comprised of at least one binding domain that is formed from
the folding of polypeptide chains having three-dimensional binding
spaces with internal surface shapes and charge distributions
complementary to the features of an antigenic determinant of an
antigen. chain. Native antibodies are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical
light (L) chains and two identical heavy (H) chains. Each light
chain is linked to a heavy chain by one covalent disulfide bond,
while the number of disulfide linkages varies between the heavy
chains of different immunoglobulin isotypes. Each heavy and light
chain also has regularly spaced intrachain disulfide bridges. Each
heavy chain has at one end a variable domain (VH) followed by a
number of constant domains. Each light chain has a variable domain
at one end (VL) and a constant domain at its other end; the
constant domain of the light chain is aligned with the first
constant domain of the heavy chain, and the light chain variable
domain is aligned with the variable domain of the heavy chain.
Light chains are classified as either lambda chains or kappa chains
based on the amino acid sequence of the light chain constant
region. The variable domain of a kappa light chain may also be
denoted herein as VK. The term "variable region" may also be used
to describe the variable domain of a heavy chain or light chain.
Particular amino acid residues are believed to form an interface
between the light and heavy chain variable domains. The variable
regions of each light/heavy chain pair form an antibody binding
site. Such antibodies may be derived from any mammal, including,
but not limited to, humans, monkeys, pigs, horses, rabbits, dogs,
cats, mice, etc.
[0203] The term "antibody" or "antibodies" includes binding
fragments of the antibodies of the invention, exemplary fragments
include single-chain Fvs (scFv), single-chain antibodies, single
domain antibodies, domain antibodies, Fv fragments, Fab fragments,
F(ab') fragments, F(ab')2 fragments, antibody fragments that
exhibit the desired biological activity, disulfide-stabilised
variable region (dsFv), dimeric variable region (Diabody),
anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id
antibodies to antibodies of the invention), intrabodies, linear
antibodies, single-chain antibody molecules and multispecific
antibodies formed from antibody fragments and epitope-binding
fragments of any of the above. In particular, antibodies include
immunoglobulin molecules and immunologically active fragments of
immunoglobulin molecules, i.e., molecules that contain an
antigen-binding site. Immunoglobulin molecules can be of any type
(e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2,
IgG3, IgG4, IgA1 and IgA2) or subclass.
[0204] Digestion of antibodies with the enzyme, papain, results in
two identical antigen-binding fragments, known also as "Fab"
fragments, and a "Fc" fragment, having no antigen-binding activity
but having the ability to crystallize. Digestion of antibodies with
the enzyme, pepsin, results in the a F(ab').sub.2 fragment in which
the two arms of the antibody molecule remain linked and comprise
two-antigen binding sites. The F(ab').sub.2 fragment has the
ability to crosslink antigen.
[0205] "Fv" when used herein refers to the minimum fragment of an
antibody that retains both antigen-recognition and antigen-binding
sites. This region consists of a dimer of one heavy and one light
chain variable domain in tight, non-covalent or covalent
association. It is in this configuration that the three CDRs of
each variable domain interact to define an antigen-binding site on
the surface of the VH-VL dimer. Collectively, the six CDRs confer
antigen-binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding
site.
[0206] "Fab" when used herein refers to a fragment of an antibody
that comprises the constant domain of the light chain and the CH1
domain of the heavy chain.
[0207] "dAb" when used herein refers to a fragment of an antibody
that is the smallest functional binding unit of a human antibodies.
A "dAb" is a single domain antibody and comprises either the
variable domain of an antibody heavy chain (VH domain) or the
variable domain of an antibody light chain (VL domain). Each dAb
contains three of the six naturally occurring CDRs (Ward et al.,
Binding activities of a repertoire of single immunoglobulin
variable domains secreted from Escherichia coli. Nature 341,
544-546 (1989); Holt, et al., Domain antibodies: protein for
therapy, Trends Biotechnol. 21, 484-49 (2003)). With molecular
weights ranging from 11 to 15 kDa, they are four times smaller than
a fragment antigen binding (Fab)2 and half the size of a single
chain Fv (scFv) molecule.
[0208] "Camelid" when used herein refers to antibody molecules are
composed of heavy-chain dimers which are devoid of light chains,
but nevertheless have an extensive antigen-binding repertoire
(Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers
C, Songa E B, Bendahman N, Hamers R (1993) Naturally occurring
antibodies devoid of light chains. Nature 363:446-448).
[0209] 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-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).
[0210] It has been shown that fragments of a whole antibody can
perform the function of binding antigens. Examples of binding
fragments are (Ward, E. S. et al., (1989) Nature 341, 544-546) the
Fab fragment consisting of VL, VH, CL and CH1 domains; (McCafferty
et al (1990) Nature, 348, 552-554) the Fd fragment consisting of
the VH and CH1 domains; (Holt et al (2003) Trends in Biotechnology
21, 484-490) the Fv fragment consisting of the VL and VH domains of
a single antibody; (iv) the dAb fragment (Ward, E. S. et al.,
Nature 341, 544-546 (1989), McCafferty et al (1990) Nature, 348,
552-554, Holt et al (2003) Trends in Biotechnology 21, 484-490],
which consists of a VH or a VL domain; (v) isolated CDR regions;
(vi) F(ab')2 fragments, a bivalent fragment comprising two linked
Fab fragments (vii) single chain Fv molecules (scFv), wherein a VH
domain and a VL domain are linked by a peptide linker which allows
the two domains to associate to form an antigen binding site (Bird
et al, (1988) Science, 242, 423-426, Huston et al, (1988) PNAS USA,
85, 5879-5883); (viii) bispecific single chain Fv dimers
(PCT/US92/09965) and (ix) "diabodies", multivalent or multispecific
fragments constructed by gene fusion (WO94/13804; Holliger, P.
(1993) et al, Proc. Natl. Acad. Sci. USA 90 6444-6448). Fv, scFv or
diabody molecules may be stabilised by the incorporation of
disulphide bridges linking the VH and VL domains (Reiter, Y. et al,
Nature Biotech, 14, 1239-1245, 1996). Minibodies comprising a scFv
joined to a CH3 domain may also be made (Hu, S. et al, (1996)
Cancer Res., 56, 3055-3061). Other examples of binding fragments
are Fab', which differs from Fab fragments by the addition of a few
residues at the carboxyl terminus of the heavy chain CH1 domain,
including one or more cysteines from the antibody hinge region, and
Fab'-SH, which is a Fab' fragment in is which the cysteine
residue(s) of the constant domains bear a free thiol group.
[0211] The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among
antibodies and are responsible for the binding specificity of each
particular antibody for its particular antigen. However, the
variability is not evenly distributed through the variable domains
of antibodies. It is concentrated in segments called
Complementarity Determining Regions (CDRs) both in the light chain
and the heavy chain variable domains. The more highly conserved
portions of the variable domains are called the framework regions
(FR). The variable domains of native heavy and light chains each
comprise four FR regions, largely adopting a .beta.-sheet
configuration, connected by three CDRs, which form loops
connecting, and in some cases forming part of, the .beta.-sheet
structure. The CDRs in each chain are held together in close
proximity by the FR regions and, with the CDRs from the other
chain, contribute to the formation of the antigen-binding site of
antibodies (see, Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)). The constant domains
are generally not involved directly in antigen binding, but may
influence antigen binding affinity and may exhibit various effector
functions, such as participation of the antibody in ADCC, CDC,
and/or apoptosis.
[0212] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are associated with
its binding to antigen. The hypervariable regions encompass the
amino acid residues of the "complementarity determining regions" or
"CDRs" (e.g., residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) of the
light chain variable domain and residues 31-35 (H1), 50-65 (H2) and
95-102 (H3) of the heavy chain variable domain; Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.
(1991)) and/or those residues from a "hypervariable loop" (e.g.,
residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light chain
variable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the
heavy chain variable domain; Chothia and Lesk, J. Mol. Biol.,
196:901-917 (1987)). "Framework" or "FR" residues are those
variable domain residues flanking the CDRs. FR residues are present
in chimeric, humanized, human, domain antibodies, diabodies,
vaccibodies, linear antibodies, and bispecific antibodies.
[0213] As used herein, targeted binding agent, targeted binding
protein, specific binding protein and like terms refer to an
antibody, or binding fragment thereof that preferentially binds to
a target site. In one embodiment, the targeted binding agent is
specific for only one target site. In other embodiments, the
targeted binding agent is specific for more than one target site.
In one embodiment, the targeted binding agent may be a monoclonal
antibody and the target site may be an epitope.
[0214] "Binding fragments" of an antibody are produced by
recombinant DNA techniques, or by enzymatic or chemical cleavage of
intact antibodies. Binding fragments include Fab, Fab',
F(ab').sub.2, Fv, dAb and single-chain antibodies. An antibody
other than a "bispecific" or "bifunctional" antibody is understood
to have each of its binding sites identical. An antibody
substantially inhibits adhesion of a receptor to a counter-receptor
when an excess of antibody reduces the quantity of receptor bound
to counter-receptor by at least about 20%, 40%, 60% or 80%, and
more usually greater than about 85% (as measured in an in vitro
competitive binding assay).
[0215] The term "epitope" includes any protein determinant capable
of specific binding to an immunoglobulin or T-cell receptor.
Epitopic determinants usually consist of chemically active surface
groupings of molecules such as amino acids or sugar side chains and
may, but not always, have specific three-dimensional structural
characteristics, as well as specific charge characteristics. An
antibody is said to specifically bind an antigen when the
dissociation constant is .ltoreq.1 .mu.M, preferably .ltoreq.100 nM
and most preferably .ltoreq.10 nM.
[0216] The term "Geomean" (also known as geometric mean), refers to
the average of the logarithmic values of a data set, converted back
to a base 10 number. This requires there to be at least two
measurements, e.g. at least 2, preferably at least 5, more
preferably at least 10 replicate. The person skilled in the art
will appreciate that the greater the number of replicates the more
robust the geomean value will be. The choice of replicate number
can be left to the discretion of the person skilled in the art.
[0217] The term "agent" is used herein to denote a chemical
compound, a mixture of chemical compounds, a biological
macromolecule, or an extract made from biological materials.
[0218] "Active" or "activity" in regard to an .alpha.5.beta.1
polypeptide refers to a portion of an .alpha.5.beta.1 polypeptide
that has a biological or an immunological activity of a native
.alpha.5.beta.1 polypeptide. "Biological" when used herein refers
to a biological function that results from the activity of the
native .alpha.5.beta.1 polypeptide. A preferred .alpha.5.beta.1
biological activity includes, for example, .alpha.5.beta.1 induced
cell adhesion and invasion and/or angiogenesis and/or
proliferation.
[0219] "Mammal" when used herein refers to any animal that is
considered a mammal. Preferably, the mammal is human.
[0220] "Animal" when used herein encompasses animals considered a
mammal. Preferably the animal is human.
[0221] The term "mAb" refers to monoclonal antibody.
[0222] "Liposome" when used herein refers to a small vesicle that
may be useful for delivery of drugs that may include the
.alpha.5.beta.1 polypeptide of the invention or antibodies to such
an .alpha.5.beta.1 polypeptide to a mammal.
[0223] "Label" or "labeled" as used herein refers to the addition
of a detectable moiety to a polypeptide, for example, a radiolabel,
fluorescent label, enzymatic label chemiluminescent labeled or a
biotinyl group. Radioisotopes or radionuclides may include .sup.3H,
.sup.14C, .sup.15N, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In,
.sup.125I, .sup.131I, fluorescent labels may include rhodamine,
lanthanide phosphors or FITC and enzymatic labels may include
horseradish peroxidase, .beta.-galactosidase, luciferase, alkaline
phosphatase.
[0224] Additional labels include, by way of illustration and not
limitation: enzymes, such as glucose-6-phosphate dehydrogenase
("G6PDH"), alpha-D-galactosidase, glucose oxydase, glucose amylase,
carbonic anhydrase, acetylcholinesterase, lysozyme, malate
dehydrogenase and peroxidase; dyes; additional fluorescent labels
or fluorescers include, such as fluorescein and its derivatives,
fluorochrome, GFP (GFP for "Green Fluorescent Protein"), dansyl,
umbelliferone, phycoerythrin, phycocyanin, allophycocyanin,
o-phthaldehyde, and fluorescamine; fluorophores such as lanthanide
cryptates and chelates e.g. Europium etc (Perkin Elmer and Cis
Biointernational); chemoluminescent labels or chemiluminescers,
such as isoluminol, luminol and the dioxetanes; sensitisers;
coenzymes; enzyme substrates; particles, such as latex or carbon
particles; metal sol; crystallite; liposomes; cells, etc., which
may be further labelled with a dye, catalyst or other detectable
group; molecules such as biotin, digoxygenin or
5-bromodeoxyuridine; toxin moieties, such as for example a toxin
moiety selected from a group of Pseudomonas exotoxin (PE or a
cytotoxic fragment or mutant thereof), Diptheria toxin or a
cytotoxic fragment or mutant thereof, a botulinum toxin A, B, C, D,
E or F, ricin or a cytotoxic fragment thereof e.g. ricin A, abrin
or a cytotoxic fragment thereof, saporin or a cytotoxic fragment
thereof, pokeweed antiviral toxin or a cytotoxic fragment thereof
and bryodin 1 or a cytotoxic fragment thereof.
[0225] The term "pharmaceutical agent or drug" as used herein
refers to a chemical compound or composition capable of inducing a
desired therapeutic effect when properly administered to a patient.
Other chemistry terms herein are used according to conventional
usage in the art, as exemplified by The McGraw-Hill Dictionary of
Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco
(1985)), (incorporated herein by reference).
[0226] As used herein, "substantially pure" means an object species
is the predominant species present (i.e., on a molar basis it is
more abundant than any other individual species in the
composition), and preferably a substantially purified fraction is a
composition wherein the object species comprises at least about 50
percent (on a molar basis) of all macromolecular species present.
Generally, a substantially pure composition will comprise more than
about 80 percent of all macromolecular species present in the
composition, more preferably more than about 85%, 90%, 95%, and
99%. Most preferably, the object species is purified to essential
homogeneity (contaminant species cannot be detected in the
composition by conventional detection methods) wherein the
composition consists essentially of a single macromolecular
species.
[0227] The term "patient" includes human and veterinary
subjects.
[0228] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC"
refer to a cell-mediated reaction in which non-specific cytotoxic
cells that express Ig Fc receptors (FcRs) (e.g. Natural Killer (NK)
cells, monocytes, neutrophils, and macrophages) recognise bound
antibody on a target cell and subsequently cause lysis of the
target cell. 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. FcRs expression on hematopoietic
cells is summarised in Table 3 on page 464 of Ravetch and Kinet,
Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a
molecule of interest, an in vitro ADCC assay, such as that
described in U.S. Pat. No. 5,500,362, or U.S. Pat. No. 5,821,337
can be performed. 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 can be assessed in vivo, e.g., in an animal
model such as that disclosed in Clynes et al. PNAS (USA) 95:652-656
(1988). "Complement dependent cytotoxicity" and "CDC" refer to the
mechanism by which antibodies carry out their cell-killing
function. It is initiated by the binding of C1q, a constituent of
the first component of complement, to the Fc domain of Igs, IgG or
IgM, which are in complex with antigen (Hughs-Jones, N. C., and B.
Gardner. 1979. Mol. Immunol. 16:697). C1q is a large, structurally
complex glycoprotein of .about.410 kDa present in human serum at a
concentration of 70 .mu.g/ml (Cooper, N. R. 1985. Adv. Immunol.
37:151). Together with two serine proteases, C1r and C1s, C1q forms
the complex C1, the first component of complement. At least two of
the N-terminal globular heads of C1q must be bound to the Fc of Igs
for C1 activation, hence for initiation of the complement cascade
(Cooper, N. R. 1985. Adv. Immunol. 37:151).
[0229] The term "antibody half-life" as used herein means a
pharmacokinetic property of an antibody that is a measure of the
mean survival time of antibody molecules following their
administration. Antibody half-life can be expressed as the time
required to eliminate 50 percent of a known quantity of
immunoglobulin from the patient's body or a specific compartment
thereof, for example, as measured in serum or plasma, i.e.,
circulating half-life, or in other tissues. Half-life may vary from
one immunoglobulin or class of immunoglobulin to another. In
general, an increase in antibody half-life results in an increase
in mean residence time (MRT) in circulation for the antibody
administered.
[0230] The term "isotype" refers to the classification of an
antibody's heavy or light chain constant region. The constant
domains of antibodies are not involved in binding to antigen, but
exhibit various effector functions. Depending on the amino acid
sequence of the heavy chain constant region, a given human antibody
or immunoglobulin can be assigned to one of five major classes of
immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Several of these
classes may be further divided into subclasses (isotypes), e.g.,
IgG1 (gamma 1), IgG2 (gamma 2), IgG3 (gamma 3), and IgG4 (gamma 4),
and IgA1 and IgA2. The heavy chain constant regions that correspond
to the different classes of immunoglobulins are called .alpha.,
.delta., .epsilon., .gamma., and .mu., respectively. The structures
and three-dimensional configurations of different classes of
immunoglobulins are well-known. Of the various human immunoglobulin
classes, only human IgG1, IgG2, IgG3, IgG4, and IgM are known to
activate complement. Human IgG1 and IgG3 are known to mediate in
humans. Human light chain constant regions may be classified into
two major classes, kappa and lambda.
[0231] If desired, the isotype of an antibody that specifically
binds .alpha.5.beta.1 can be switched, for example to take
advantage of a biological property of a different isotype. For
example, in some circumstances it can be desirable in connection
with the generation of antibodies as therapeutic antibodies against
.alpha.5.beta.1 that the antibodies be capable of fixing complement
and participating in complement-dependent cytotoxicity (CDC). There
are a number of isotypes of antibodies that are capable of the
same, including, without limitation, the following: murine IgM,
murine IgG2a, murine IgG2b, murine IgG3, human IgM, human IgA,
human IgG1, and human IgG3. In other embodiments it can be
desirable in connection with the generation of antibodies as
therapeutic antibodies against .alpha.5.beta.1 that the antibodies
be capable of binding Fc receptors on effector cells and
participating in antibody-dependent cytotoxicity (ADCC). There are
a number of isotypes of antibodies that are capable of the same,
including, without limitation, the following: murine IgG2a, murine
IgG2b, murine IgG3, human IgG1, and human IgG3. It will be
appreciated that antibodies that are generated need not initially
possess such an isotype but, rather, the antibody as generated can
possess any isotype and the antibody can be isotype switched
thereafter using conventional techniques that are well known in the
art. Such techniques include the use of direct recombinant
techniques (see e.g., U.S. Pat. No. 4,816,397), cell-cell fusion
techniques (see e.g., U.S. Pat. Nos. 5,916,771 and 6,207,418),
among others.
[0232] By way of example, the anti-.alpha.5.beta.1 antibodies
discussed herein are fully human antibodies. If an antibody
possessed desired binding to .alpha.5.beta.1, it could be readily
isotype switched to generate a human IgM, human IgG1, or human IgG3
isotype, while still possessing the same variable region (which
defines the antibody's specificity and some of its affinity). Such
molecule would then be capable of fixing complement and
participating in CDC and/or be capable of binding to Fc receptors
on effector cells and participating in ADCC.
[0233] "Whole blood assays" use unfractionated blood as a source of
natural effectors. Blood contains complement in the plasma,
together with FcR-expressing cellular effectors, such as
polymorphonuclear cells (PMNs) and mononuclear cells (MNCs). Thus,
whole blood assays allow simultaneous evaluation of the synergy of
both ADCC and CDC effector mechanisms in vitro.
[0234] A "therapeutically effective" amount as used herein is an
amount that provides some improvement or benefit to the subject.
Stated in another way, a "therapeutically effective" amount is an
amount that provides some alleviation, mitigation, and/or decrease
in at least one clinical symptom. Clinical symptoms associated with
the disorders that can be treated by the methods of the invention
are well-known to those skilled in the art. Further, those skilled
in the art will appreciate that the therapeutic effects need not be
complete or curative, as long as some benefit is provided to the
subject.
[0235] The term "and/or" as used herein is to be taken as specific
disclosure of each of the two specified features or components with
or without the other. For example "A and/or B" is to be taken as
specific disclosure of each of (i) A, (ii) B and (iii) A and B,
just as if each is set out individually herein.
Antibody Structure
[0236] The basic antibody structural unit is known to comprise a
tetramer. Each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kDa) and
one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain includes a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function. Human light chains are
classified as kappa and lambda light chains. Heavy chains are
classified as mu, delta, gamma, alpha, or epsilon, and define the
antibody's isotype as IgM, IgD, IgA, and IgE, respectively. Within
light and heavy chains, the variable and constant regions are
joined by a "J" region of about 12 or more amino acids, with the
heavy chain also including a "D" region of about 10 more amino
acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed.,
2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its
entirety for all purposes). The variable regions of each
light/heavy chain pair form the antibody binding site.
[0237] Thus, an intact antibody has two binding sites. Except in
bifunctional or bispecific antibodies, the two binding sites are
the same.
[0238] The chains all exhibit the same general structure of
relatively conserved framework regions (FR) joined by three hyper
variable regions, also called CDRs. The CDRs from the two chains of
each pair are aligned by the framework regions, enabling binding to
a specific epitope. From N-terminal to C-terminal, both light and
heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3
and FR4. The assignment of amino acids to each domain is in
accordance with the definitions of Kabat Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md. (1987 and 1991)), or Chothia & Lesk J. Mol. Biol.
196:901-917 (1987); Chothia et al. Nature 342:878-883 (1989).
[0239] A bispecific or bifunctional antibody is an artificial
hybrid antibody having two different heavy/light chain pairs and
two different binding sites. Bispecific antibodies can be produced
by a variety of methods including fusion of hybridomas or linking
of Fab' fragments. See, e.g., Songsivilai & Lachmann Clin. Exp.
Immunol. 79: 315-321 (1990), Kostelny et al. J. Immunol.
148:1547-1553 (1992). Bispecific antibodies do not exist in the
form of fragments having a single binding site (e.g., Fab, Fab',
and Fv).
[0240] Typically, a VH domain is paired with a VL domain to provide
an antibody antigen-binding site, although a VH or VL domain alone
may be used to bind antigen. The VH domain (see Table 10) may be
paired with the VL domain (see Table 11), so that an antibody
antigen-binding site is formed comprising both the VH and VL
domains.
Human Antibodies and Humanization of Antibodies
[0241] Targeted binding agents of the invention include human
antibodies. Human antibodies avoid some of the problems associated
with antibodies that possess murine or rat variable and/or constant
regions. The presence of such murine or rat derived proteins can
lead to the rapid clearance of the antibodies or can lead to the
generation of an immune response against the antibody by a patient.
In order to avoid the utilization of murine or rat derived
antibodies, fully human antibodies can be generated through the
introduction of functional human antibody loci into a rodent, other
mammal or animal so that the rodent, other mammal or animal
produces fully human antibodies.
[0242] One method for generating fully human antibodies is through
the use of XenoMouse.RTM. strains of mice that have been engineered
to contain up to but less than 1000 kb-sized germline configured
fragments of the human heavy chain locus and kappa light chain
locus. See Mendez et al. Nature Genetics 15:146-156 (1997) and
Green and Jakobovits J. Exp. Med. 188:483-495 (1998). The
XenoMouse.RTM. strains are available from Amgen, Inc. (Fremont,
Calif., U.S.A).
[0243] Such mice, then, are capable of producing human
immunoglobulin molecules and antibodies and are deficient in the
production of murine immunoglobulin molecules and antibodies.
Technologies utilised for achieving the same are disclosed in U.S.
patent application Ser. No. 08/759,620, filed Dec. 3, 1996 and
International Patent Application Nos. WO 98/24893, published Jun.
11, 1998 and WO 00/76310, published Dec. 21, 2000, the disclosures
of which are hereby incorporated by reference. See also Mendez et
al. Nature Genetics 15:146-156 (1997), the disclosure of which is
hereby incorporated by reference.
[0244] The production of the XenoMouse.RTM. strains of mice is
further discussed and delineated in U.S. patent application Ser.
Nos. 07/466,008, filed Jan. 12, 1990, 07/610,515, filed Nov. 8,
1990, 07/919,297, filed Jul. 24, 1992, 07/922,649, filed Jul. 30,
1992, 08/031,801, filed Mar. 15, 1993, 08/112,848, filed Aug. 27,
1993, 08/234,145, filed Apr. 28, 1994, 08/376,279, filed Jan. 20,
1995, 08/430,938, filed Apr. 27, 1995, 08/464,584, filed Jun. 5,
1995, 08/464,582, filed Jun. 5, 1995, 08/463,191, filed Jun. 5,
1995, 08/462,837, filed Jun. 5, 1995, 08/486,853, filed Jun. 5,
1995, 08/486,857, filed Jun. 5, 1995, 08/486,859, filed Jun. 5,
1995, 08/462,513, filed Jun. 5, 1995, 08/724,752, filed Oct. 2,
1996, 08/759,620, filed Dec. 3, 1996, U.S. Publication
2003/0093820, filed Nov. 30, 2001 and U.S. Pat. Nos. 6,162,963,
6,150,584, 6,114,598, 6,075,181, and 5,939,598 and Japanese Patent
Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. See also
European Patent No., EP 0 463 151 B1, grant published Jun. 12,
1996, International Patent Application No., WO 94/02602, published
Feb. 3, 1994, International Patent Application No., WO 96/34096,
published Oct. 31, 1996, WO 98/24893, published Jun. 11, 1998, WO
00/76310, published Dec. 21, 2000. The disclosures of each of the
above-cited patents, applications, and references are hereby
incorporated by reference in their entirety.
[0245] In an alternative approach, others, including GenPharm
International, Inc., have utilised a "minilocus" approach. In the
minilocus approach, an exogenous Ig locus is mimicked through the
inclusion of pieces (individual genes) from the Ig locus. Thus, one
or more V.sub.H genes, one or more D.sub.H genes, one or more
J.sub.H genes, a mu constant region, and usually a second constant
region (preferably a gamma constant region) are formed into a
construct for insertion into an animal. This approach is described
in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat. Nos.
5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,
5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each to
Lonberg and Kay, U.S. Pat. Nos. 5,591,669 and 6,023,010 to
Krimpenfort and Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and
5,789,215 to Berns et al., and U.S. Pat. No. 5,643,763 to Choi and
Dunn, and GenPharm International U.S. patent application Ser. Nos.
07/574,748, filed Aug. 29, 1990, 07/575,962, filed Aug. 31, 1990,
07/810,279, filed Dec. 17, 1991, 07/853,408, filed Mar. 18, 1992,
07/904,068, filed Jun. 23, 1992, 07/990,860, filed Dec. 16, 1992,
08/053,131, filed Apr. 26, 1993, 08/096,762, filed Jul. 22, 1993,
08/155,301, filed Nov. 18, 1993, 08/161,739, filed Dec. 3, 1993,
08/165,699, filed Dec. 10, 1993, 08/209,741, filed Mar. 9, 1994,
the disclosures of which are hereby incorporated by reference. See
also European Patent No. 0 546 073 B1, International Patent
Application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO
92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO
97/13852, and WO 98/24884 and U.S. Pat. No. 5,981,175, the
disclosures of which are hereby incorporated by reference in their
entirety. See further Taylor et al., 1992, Chen et al., 1993,
Tuaillon et al., 1993, Choi et al., 1993, Lonberg et al., (1994),
Taylor et al., (1994), and Tuaillon et al., (1995), Fishwild et
al., (1996), the disclosures of which are hereby incorporated by
reference in their entirety.
[0246] Kirin has also demonstrated the generation of human
antibodies from mice in which, through microcell fusion, large
pieces of chromosomes, or entire chromosomes, have been introduced.
See European Patent Application Nos. 773 288 and 843 961, the
disclosures of which are hereby incorporated by reference.
Additionally, KM.TM.-mice, which are the result of cross-breeding
of Kirin's Tc mice with Medarex's minilocus (Humab) mice have been
generated. These mice possess the human IgH transchromosome of the
Kirin mice and the kappa chain transgene of the Genpharm mice
(Ishida et al., Cloning Stem Cells, (2002) 4:91-102).
[0247] Human antibodies can also be derived by in vitro methods.
Suitable examples include but are not limited to phage display
(Medimmune, Morphosys, Dyax, Biosite/Medarex, Xoma, Symphogen,
Alexion (formerly Proliferon), Affimed) ribosome display
(Medimmune), yeast display, and the like.
Preparation of Antibodies
[0248] Antibodies, as described herein, were prepared through the
utilization of the XenoMouse.RTM. technology, as described below.
Such mice are capable of producing human immunoglobulin molecules
and antibodies and are deficient in the production of murine
immunoglobulin molecules and antibodies. Technologies utilised for
achieving the same are disclosed in the patents, applications, and
references disclosed in the background section herein. In
particular, however, a preferred embodiment of transgenic
production of mice and antibodies therefrom is disclosed in U.S.
patent application Ser. No. 08/759,620, filed Dec. 3, 1996 and
International Patent Application Nos. WO 98/24893, published Jun.
11, 1998 and WO 00/76310, published Dec. 21, 2000, the disclosures
of which are hereby incorporated by reference. See also Mendez et
al. Nature Genetics 15:146-156 (1997), the disclosure of which is
hereby incorporated by reference.
[0249] Through the use of such technology, fully human monoclonal
antibodies to a variety of antigens have been produced.
Essentially, XenoMouse.RTM. lines of mice are immunised with an
antigen of interest (e.g. .alpha.5.beta.1), lymphatic cells (such
as B-cells) are recovered from the hyper-immunised mice, and the
recovered lymphocytes are fused with a myeloid-type cell line to
prepare immortal hybridoma cell lines. These hybridoma cell lines
are screened and selected to identify hybridoma cell lines that
produced antibodies specific to the antigen of interest. Provided
herein are methods for the production of multiple hybridoma cell
lines that produce antibodies specific to .alpha.5.beta.1. Further,
provided herein are characterisation of the antibodies produced by
such cell lines, including nucleotide and amino acid sequence
analyses of the heavy and light chains of such antibodies.
[0250] Alternatively, instead of being fused to myeloma cells to
generate hybridomas, B cells can be directly assayed. For example,
CD19+ B cells can be isolated from hyperimmune XenoMouse.RTM. mice
and allowed to proliferate and differentiate into
antibody-secreting plasma cells. Antibodies from the cell
supernatants are then screened by ELISA for reactivity against the
.alpha.5.beta.1 immunogen. The supernatants might also be screened
for immunoreactivity against fragments of .alpha.5.beta.1 to
further map the different antibodies for binding to domains of
functional interest on .alpha.5.beta.1. The antibodies may also be
screened other related human endoglycosidases and against the rat,
the mouse, and non-human primate, such as Cynomolgus monkey,
orthologues of .alpha.5.beta.1, the last to determine species
cross-reactivity. B cells from wells containing antibodies of
interest may be immortalised by various methods including fusion to
make hybridomas either from individual or from pooled wells, or by
infection with EBV or transfection by known immortalising genes and
then plating in suitable medium. Alternatively, single plasma cells
secreting antibodies with the desired specificities are then
isolated using an .alpha.5.beta.1-specific hemolytic plaque assay
(see for example Babcook et al., Proc. Natl. Acad. Sci. USA
93:7843-48 (1996)). Cells targeted for lysis are preferably sheep
red blood cells (SRBCs) coated with the .alpha.5.beta.1
antigen.
[0251] In the presence of a B-cell culture containing plasma cells
secreting the immunoglobulin of interest and complement, the
formation of a plaque indicates specific .alpha.5.beta.1-mediated
lysis of the sheep red blood cells surrounding the plasma cell of
interest. The single antigen-specific plasma cell in the center of
the plaque can be isolated and the genetic information that encodes
the specificity of the antibody is isolated from the single plasma
cell. Using reverse-transcription followed by PCR (RT-PCR), the DNA
encoding the heavy and light chain variable regions of the antibody
can be cloned. Such cloned DNA can then be further inserted into a
suitable expression vector, preferably a vector cassette such as a
pcDNA, more preferably such a pcDNA vector containing the constant
domains of immunglobulin heavy and light chain. The generated
vector can then be transfected into host cells, e.g., HEK293 cells,
CHO cells, and cultured in conventional nutrient media modified as
appropriate for inducing transcription, selecting transformants, or
amplifying the genes encoding the desired sequences.
[0252] As will be appreciated, antibodies that specifically bind
.alpha.5.beta.1 can be expressed in cell lines other than hybridoma
cell lines. Sequences encoding particular antibodies can be used to
transform a suitable mammalian host cell. Transformation can be by
any known method for introducing polynucleotides into a host cell,
including, for example packaging the polynucleotide in a virus (or
into a viral vector) and transducing a host cell with the virus (or
vector) or by transfection procedures known in the art, as
exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and
4,959,455 (which patents are hereby incorporated herein by
reference). The transformation procedure used depends upon the host
to be transformed. Methods for introducing heterologous
polynucleotides into mammalian cells are well known in the art and
include dextran-mediated transfection, calcium phosphate
precipitation, polybrene mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, and direct microinjection of the DNA into nuclei.
[0253] Mammalian cell lines available as hosts for expression are
well known in the art and include many immortalized cell lines
available from the American Type Culture Collection (ATCC),
including but not limited to Chinese hamster ovary (CHO) cells,
HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells
(COS), human hepatocellular carcinoma cells (e.g., Hep G2), human
epithelial kidney 293 cells, and a number of other cell lines
(Chadd, H. E. and Chamow, S. M., (2001) Curr Opin in Biotech. 12:
188-194; Andersen, D. C. and Krummen, L, (2002) Curr Opin in
Biotech. 13:117; Larrick, J. W., and Thomas, D. W., (2001) Curr
Opin in Biotech. 12: 411-418). Cell lines of particular preference
are selected through determining which cell lines have high
expression levels and produce antibodies with constitutive
.alpha.5.beta.1 binding properties.
[0254] In the cell-cell fusion technique, a myeloma, CHO cell or
other cell line is prepared that possesses a heavy chain with any
desired isotype and another myeloma, CHO cell or other cell line is
prepared that possesses the light chain. Such cells can,
thereafter, be fused and a cell line expressing an intact antibody
can be isolated.
[0255] Accordingly, as antibody candidates are generated that meet
desired "structural" attributes as discussed above, they can
generally be provided with at least certain of the desired
"functional" attributes through isotype switching.
Therapeutic Administration and Formulations
[0256] Embodiments of the invention include sterile pharmaceutical
formulations of anti-.alpha.5.beta.1 antibodies that are useful as
treatments for diseases. Such formulations would inhibit the
binding of a native .alpha.5.beta.1-specific ligand such as, for
example, fibronectin, to .alpha.5.beta.1, thereby effectively
treating pathological conditions where, for example, serum or
tissue .alpha.5.beta.1 expression is abnormally elevated.
Anti-.alpha.5.beta.1 antibodies preferably possess adequate
affinity to potently inhibit native .alpha.5.beta.1-specific
ligands such as, for example, fibronectin, and preferably have an
adequate duration of action to allow for infrequent dosing in
humans. A prolonged duration of action will allow for less frequent
and more convenient dosing schedules by alternate parenteral routes
such as subcutaneous or intramuscular injection.
[0257] Sterile formulations can be created, for example, by
filtration through sterile filtration membranes, prior to or
following lyophilization and reconstitution of the antibody. The
antibody ordinarily will be stored in lyophilized form or in
solution. Therapeutic antibody compositions generally are placed
into a container having a sterile access port, for example, an
intravenous solution bag or vial having an adapter that allows
retrieval of the formulation, such as a stopper pierceable by a
hypodermic injection needle.
[0258] The route of antibody administration is in accord with known
methods, e.g., injection or infusion by intravenous,
intraperitoneal, intracerebral, intramuscular, intraocular,
intraarterial, intrathecal, inhalation or intralesional routes,
direct injection to a tumour site, or by sustained release systems
as noted below. The antibody is preferably administered
continuously by infusion or by bolus injection.
[0259] An effective amount of antibody to be employed
therapeutically will depend, for example, upon the therapeutic
objectives, the route of administration, and the condition of the
patient. Accordingly, it is preferred that the therapist titer the
dosage and modify the route of administration as required to obtain
the optimal therapeutic effect. Typically, the clinician will
administer antibody until a dosage is reached that achieves the
desired effect. The progress of this therapy is easily monitored by
conventional assays or by the assays described herein.
[0260] Antibodies, as described herein, can be prepared in a
mixture with a pharmaceutically acceptable carrier. This
therapeutic composition can be administered intravenously or
through the nose or lung, preferably as a liquid or powder aerosol
(lyophilized). The composition may also be administered
parenterally or subcutaneously as desired. When administered
systemically, the therapeutic composition should be sterile,
pyrogen-free and in a parenterally acceptable solution having due
regard for pH, isotonicity, and stability. These conditions are
known to those skilled in the art. Briefly, dosage formulations of
the compounds described herein are prepared for storage or
administration by mixing the compound having the desired degree of
purity with pharmaceutically acceptable carriers, excipients, or
stabilizers. Such materials are non-toxic to the recipients at the
dosages and concentrations employed, and include buffers such as
TRIS HCl, phosphate, citrate, acetate and other organic acid salts;
antioxidants such as ascorbic acid; low molecular weight (less than
about ten residues) peptides such as polyarginine, proteins, such
as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers
such as polyvinylpyrrolidinone; amino acids such as glycine,
glutamic acid, aspartic acid, or arginine; monosaccharides,
disaccharides, and other carbohydrates including cellulose or its
derivatives, glucose, mannose, or dextrins; chelating agents such
as EDTA; sugar alcohols such as mannitol or sorbitol; counterions
such as sodium and/or nonionic surfactants such as TWEEN, PLURONICS
or polyethyleneglycol.
[0261] Sterile compositions for injection can be formulated
according to conventional pharmaceutical practice as described in
Remington: The Science and Practice of Pharmacy (20.sup.th ed,
Lippincott Williams & Wilkens Publishers (2003)). For example,
dissolution or suspension of the active compound in a
pharmaceutically acceptable carrier such as water or naturally
occurring vegetable oil like sesame, peanut, or cottonseed oil or a
synthetic fatty vehicle like ethyl oleate or the like may be
desired. Buffers, preservatives, antioxidants and the like can be
incorporated according to accepted pharmaceutical practice.
[0262] Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
polypeptide, which matrices are in the form of shaped articles,
films or microcapsules. Examples of sustained-release matrices
include polyesters, hydrogels (e.g.,
poly(2-hydroxyethyl-methacrylate) as described by Langer et al., J.
Biomed Mater. Res., (1981) 15:167-277 and Langer, Chem. Tech.,
(1982) 12:98-105, or poly(vinylalcohol)), polylactides (U.S. Pat.
No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma
ethyl-L-glutamate (Sidman et al., Biopolymers, (1983) 22:547-556),
non-degradable ethylene-vinyl acetate (Langer et al., supra),
degradable lactic acid-glycolic acid copolymers such as the LUPRON
Depot.TM. (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
[0263] While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels release proteins for shorter time periods. When
encapsulated proteins remain in the body for a long time, they may
denature or aggregate as a result of exposure to moisture at
37.degree. C., resulting in a loss of biological activity and
possible changes in immunogenicity. Rational strategies can be
devised for protein stabilization depending on the mechanism
involved. For example, if the aggregation mechanism is discovered
to be intermolecular S--S bond formation through disulfide
interchange, stabilization may be achieved by modifying sulfhydryl
residues, lyophilizing from acidic solutions, controlling moisture
content, using appropriate additives, and developing specific
polymer matrix compositions.
[0264] Sustained-released compositions also include preparations of
crystals of the antibody suspended in suitable formulations capable
of maintaining crystals in suspension. These preparations when
injected subcutaneously or intraperitonealy can produce a sustained
release effect. Other compositions also include liposomally
entrapped antibodies. Liposomes containing such antibodies are
prepared by methods known per se: U.S. Pat. No. DE 3,218,121;
Epstein et al., Proc. Natl. Acad. Sci. USA, (1985) 82:3688-3692;
Hwang et al., Proc. Natl. Acad. Sci. USA, (1980) 77:4030-4034; EP
52,322; EP 36,676; EP 88,046; EP 143,949; 142,641; Japanese patent
application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and
EP 102,324.
[0265] The dosage of the antibody formulation for a given patient
will be determined by the attending physician taking into
consideration various factors known to modify the action of drugs
including severity and type of disease, body weight, sex, diet,
time and route of administration, other medications and other
relevant clinical factors. Therapeutically effective dosages may be
determined by either in vitro or in vivo methods.
[0266] An effective amount of the antibodies, described herein, to
be employed therapeutically will depend, for example, upon the
therapeutic objectives, the route of administration, and the
condition of the patient. Accordingly, it is preferred for the
therapist to titer the dosage and modify the route of
administration as required to obtain the optimal therapeutic
effect. A typical daily dosage might range from about 0.0001 mg/kg,
0.001 mg/kg, 0.01 mg/kg, 0.1 mg/kg, 1 mg/kg, 10 mg/kg to up to 100
mg/kg, 1000 mg/kg, 10000 mg/kg or more, of the patient's body
weight depending on the factors mentioned above. The dosage may be
between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg,
0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg,
0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001
mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg,
0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the
patient's body weight depending on the factors mentioned above.
Typically, the clinician will administer the therapeutic antibody
until a dosage is reached that achieves the desired effect. The
progress of this therapy is easily monitored by conventional assays
or as described herein.
[0267] Doses of antibodies of the invention may be repeated and the
administrations may be separated by at least 1 day, 2 days, 3 days,
5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3
months, or at least 6 months.
[0268] It will be appreciated that administration of therapeutic
entities in accordance with the compositions and methods herein
will be administered with suitable carriers, excipients, and other
agents that are incorporated into formulations to provide improved
transfer, delivery, tolerance, and the like. These formulations
include, for example, powders, pastes, ointments, jellies, waxes,
oils, lipids, lipid (cationic or anionic) containing vesicles (such
as Lipofectin.TM.), DNA conjugates, anhydrous absorption pastes,
oil-in-water and water-in-oil emulsions, emulsions carbowax
(polyethylene glycols of various molecular weights), semi-solid
gels, and semi-solid mixtures containing carbowax. Any of the
foregoing mixtures may be appropriate in treatments and therapies
in accordance with the present invention, provided that the active
ingredient in the formulation is not inactivated by the formulation
and the formulation is physiologically compatible and tolerable
with the route of administration. See also Baldrick P.
"Pharmaceutical excipient development: the need for preclinical
guidance." Regul. Toxicol. Pharmacol. 32(2):210-8 (2000), Wang W.
"Lyophilization and development of solid protein pharmaceuticals."
Int. J. Pharm. 203(1-2):1-60 (2000), Charman W N "Lipids,
lipophilic drugs, and oral drug delivery-some emerging concepts." J
Pharm Sci 0.89(8):967-78 (2000), Powell et al. "Compendium of
excipients for parenteral formulations" PDA J Pharm Sci Technol.
52:238-311 (1998) and the citations therein for additional
information related to formulations, excipients and carriers well
known to pharmaceutical chemists.
Design and Generation of Other Therapeutics
[0269] In accordance with the present invention and based on the
activity of the antibodies that are produced and characterized
herein with respect to .alpha.5.beta.1, the design of other
therapeutic modalities beyond antibody moieties is facilitated.
Such modalities include, without limitation, advanced antibody
therapeutics, such as bispecific antibodies, immunotoxins, and
radiolabeled therapeutics, single domain antibodies, antibody
fragments, such as a Fab, Fab', F(ab').sub.2, Fv or dAb, generation
of peptide therapeutics, .alpha.5.beta.1 binding domains in novel
scaffolds, gene therapies, particularly intrabodies, antisense
therapeutics, and small molecules.
[0270] An antigen binding site may be provided by means of
arrangement of CDRs on non-antibody protein scaffolds, such as
fibronectin or cytochrome B etc. (Haan & Maggos (2004)
BioCentury, 12(5): A1-A6; Koide et al. (1998) Journal of Molecular
Biology, 284: 1141-1151; Nygren et al. (1997) Current Opinion in
Structural Biology, 7: 463-469) or by randomising or mutating amino
acid residues of a loop within a protein scaffold to confer binding
specificity for a desired target. Scaffolds for engineering novel
binding sites in proteins have been reviewed in detail by Nygren et
al. (Nygren et al. (1997) Current Opinion in Structural Biology, 7:
463-469). Protein scaffolds for antibody mimics are disclosed in
WO/0034784, which is herein incorporated by reference in its
entirety, in which the inventors describe proteins (antibody
mimics) that include a fibronectin type III domain having at least
one randomised loop. A suitable scaffold into which to graft one or
more CDRs, e.g. a set of HCDRs, may be provided by any domain
member of the immunoglobulin gene superfamily. The scaffold may be
a human or non-human protein. An advantage of a non-antibody
protein scaffold is that it may provide an antigen-binding site in
a scaffold molecule that is smaller and/or easier to manufacture
than at least some antibody molecules. Small size of a binding
member may confer useful physiological properties, such as an
ability to enter cells, penetrate deep into tissues or reach
targets within other structures, or to bind within protein cavities
of the target antigen. Use of antigen binding sites in non-antibody
protein scaffolds is reviewed in Wess, 2004 (Wess, L. In:
BioCentury, The Bernstein Report on BioBusiness, 12(42), A1-A7,
2004). Typical are proteins having a stable backbone and one or
more variable loops, in which the amino acid sequence of the loop
or loops is specifically or randomly mutated to create an
antigen-binding site that binds the target antigen. Such proteins
include the IgG-binding domains of protein A from S. aureus,
transferrin, albumin, tetranectin, fibronectin (e.g. 10th
fibronectin type III domain), lipocalins as well as
gamma-crystalline and other Affilin.TM. scaffolds (Scil Proteins).
Examples of other approaches include synthetic "Microbodies" based
on cyclotides--small proteins having intra-molecular disulphide
bonds, Microproteins (Versabodies.TM., Amunix) and ankyrin repeat
proteins (DARPins, Molecular Partners).
[0271] In addition to antibody sequences and/or an antigen-binding
site, a targeted binding agent according to the present invention
may comprise other amino acids, e.g. forming a peptide or
polypeptide, such as a folded domain, or to impart to the molecule
another functional characteristic in addition to ability to bind
antigen. Targeted binding agents of the invention may carry a
detectable label, or may be conjugated to a toxin or a targeting
moiety or enzyme (e.g. via a peptidyl bond or linker). For example,
a targeted binding agent may comprise a catalytic site (e.g. in an
enzyme domain) as well as an antigen binding site, wherein the
antigen binding site binds to the antigen and thus targets the
catalytic site to the antigen. The catalytic site may inhibit
biological function of the antigen, e.g. by cleavage.
[0272] In connection with the generation of advanced antibody
therapeutics, where complement fixation is a desirable attribute,
it may be possible to sidestep the dependence on complement for
cell killing through the use of bispecific antibodies,
immunotoxins, or radiolabels, for example.
[0273] Antibodies can also be modified to act as immunotoxins,
utilizing techniques that are well known in the art. See e.g.,
Vitetta Immunol Today 14:252 (1993). See also U.S. Pat. No.
5,194,594. In connection with the preparation of radiolabeled
antibodies, such modified antibodies can also be readily prepared
utilizing techniques that are well known in the art. See e.g.,
Junghans et al. in Cancer Chemotherapy and Biotherapy 655-686 (2d
edition, Chafner and Longo, eds., Lippincott Raven (1996)). See
also U.S. Pat. Nos. 4,681,581, 4,735,210, 5,101,827, 5,102,990 (RE
35,500), 5,648,471, and 5,697,902. Each immunotoxin or radiolabeled
molecule would be likely to kill cells expressing the desired
multimeric enzyme subunit oligomerisation domain.
[0274] When an antibody is linked to an agent (e.g., radioisotope,
pharmaceutical composition, or a toxin), it is contemplated that
the agent possess a pharmaceutical property selected from the group
of antimitotic, alkylating, antimetabolite, antiangiogenic,
apoptotic, alkaloid, COX-2, and antibiotic agents and combinations
thereof. The drug can be selected from the group of nitrogen
mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas,
triazenes, folic acid analogs, anthracyclines, taxanes, COX-2
inhibitors, pyrimidine analogs, purine analogs, antimetabolites,
antibiotics, enzymes, epipodophyllotoxins, platinum coordination
complexes, vinca alkaloids, substituted ureas, methyl hydrazine
derivatives, adrenocortical suppressants, antagonists, endostatin,
taxols, camptothecins, oxaliplatin, doxorubicins and their analogs,
and a combination thereof.
[0275] Examples of toxins further include gelonin, Pseudomonas
exotoxin (PE), PE40, PE38, diphtheria toxin, ricin, abrin, alpha
toxin, saporin, ribonuclease (RNase), DNase I, Staphylococcal
enterotoxin-A, pokeweed antiviral protein, gelonin, Pseudomonas
endotoxin, members of the enediyne family of molecules, such as
calicheamicin and esperamicin, as well as derivatives, combinations
and modifications thereof. Chemical toxins can also be taken from
the group consisting of duocarmycin (see, e.g., U.S. Pat. No.
5,703,080 and U.S. Pat. No. 4,923,990), methotrexate, doxorubicin,
melphalan, chlorambucil, ARA-C, vindesine, mitomycin C,
cis-platinum, etoposide, bleomycin and 5-fluorouracil. Examples of
chemotherapeutic agents also include Adriamycin, Doxorubicin,
5-Fluorouracil, Cytosine arabinoside (Ara-C), Cyclophosphamide,
Thiotepa, Taxotere (docetaxel), Busulfan, Cytoxin, Taxol,
Methotrexate, Cisplatin, Melphalan, Vinblastine, Bleomycin,
Etoposide, Ifosfamide, Mitomycin C, Mitoxantrone, Vincreistine,
Vinorelbine, Carboplatin, Teniposide, Daunomycin, Caminomycin,
Aminopterin, Dactinomycin, Mitomycins, Esperamicins (see, U.S. Pat.
No. 4,675,187), Melphalan, and other related nitrogen mustards.
Suitable toxins and chemotherapeutic agents are described in
Remington's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co.
1995), and in Goodman And Gilman's The Pharmacological Basis of
Therapeutics, 7th Ed. (MacMillan Publishing Co. 1985). Other
suitable toxins and/or chemotherapeutic agents are known to those
of skill in the art.
[0276] Examples of radioisotopes include gamma-emitters,
positron-emitters, and x-ray emitters that can be used for
localisation and/or therapy, and beta-emitters and alpha-emitters
that can be used for therapy. The radioisotopes described
previously as useful for diagnostics, prognostics and staging are
also useful for therapeutics.
[0277] Non-limiting examples of anti-cancer or anti-leukemia agents
include anthracyclines such as doxorubicin (adriamycin),
daunorubicin (daunomycin), idarubicin, detorubicin, caminomycin,
epirubicin, esorubicin, and morpholino and substituted derivatives,
combinations and modifications thereof. Exemplary pharmaceutical
agents include cis-platinum, taxol, calicheamicin, vincristine,
cytarabine (Ara-C), cyclophosphamide, prednisone, daunorubicin,
idarubicin, fludarabine, chlorambucil, interferon alpha,
hydroxyurea, temozolomide, thalidomide, and bleomycin, and
derivatives, combinations and modifications thereof. Preferably,
the anti-cancer or anti-leukemia is doxorubicin,
morpholinodoxorubicin, or morpholinodaunorubicin.
[0278] The antibodies of the invention also encompass antibodies
that have half-lives (e.g., serum half-lives) in a mammal,
preferably a human, of greater than that of an unmodified antibody.
Said antibody half life may be greater than about 15 days, greater
than about 20 days, greater than about 25 days, greater than about
30 days, greater than about 35 days, greater than about 40 days,
greater than about 45 days, greater than about 2 months, greater
than about 3 months, greater than about 4 months, or greater than
about 5 months. The increased half-lives of the antibodies of the
present invention or fragments thereof in a mammal, preferably a
human, result in a higher serum titer of said antibodies or
antibody fragments in the mammal, and thus, reduce the frequency of
the administration of said antibodies or antibody fragments and/or
reduces the concentration of said antibodies or antibody fragments
to be administered. Antibodies or fragments thereof having
increased in vivo half-lives can be generated by techniques known
to those of skill in the art. For example, antibodies or fragments
thereof with increased in vivo half-lives can be generated by
modifying (e.g., substituting, deleting or adding) amino acid
residues identified as involved in the interaction between the Fc
domain and the FcRn receptor (see, e.g., International Publication
Nos. WO 97/34631 and WO 02/060919, which are incorporated herein by
reference in their entireties). Antibodies or fragments thereof
with increased in vivo half-lives can be generated by attaching to
said antibodies or antibody fragments polymer molecules such as
high molecular weight polyethyleneglycol (PEG). PEG can be attached
to said antibodies or antibody fragments with or without a
multifunctional linker either through site-specific conjugation of
the PEG to the N- or C-terminus of said antibodies or antibody
fragments or via epsilon-amino groups present on lysine residues.
Linear or branched polymer derivatisation that results in minimal
loss of biological activity will be used. The degree of conjugation
will be closely monitored by SDS-PAGE and mass spectrometry to
ensure proper conjugation of PEG molecules to the antibodies.
Unreacted PEG can be separated from antibody-PEG conjugates by,
e.g., size exclusion or ion-exchange chromatography.
[0279] As will be appreciated by one of skill in the art, in the
above embodiments, while affinity values can be important, other
factors can be as important or more so, depending upon the
particular function of the antibody. For example, for an
immunotoxin (toxin associated with an antibody), the act of binding
of the antibody to the target can be useful; however, in some
embodiments, it is the internalisation of the toxin into the cell
that is the desired end result. As such, antibodies with a high
percent internalisation can be desirable in these situations. Thus,
in one embodiment, antibodies with a high efficiency in
internalisation are contemplated. A high efficiency of
internalisation can be measured as a percent internalised antibody,
and can be from a low value to 100%. For example, in varying
embodiments, 0.1-5, 5-10, 10-20, 20-30, 30-40, 40-45, 45-50, 50-60,
60-70, 70-80, 80-90, 90-99, and 99-100% can be a high efficiency.
As will be appreciated by one of skill in the art, the desirable
efficiency can be different in different embodiments, depending
upon, for example, the associated agent, the amount of antibody
that can be administered to an area, the side effects of the
antibody-agent complex, the type (e.g., cancer type) and severity
of the problem to be treated.
[0280] In other embodiments, the antibodies disclosed herein
provide an assay kit for the detection of .alpha.5.beta.1
expression in mammalian tissues or cells in order to screen for a
disease or disorder associated with changes in expression of
.alpha.5.beta.1. The kit comprises an antibody that binds
.alpha.5.beta.1 and means for indicating the reaction of the
antibody with the antigen, if present.
Combinations
[0281] The targeted binding agent or antibody defined herein may be
applied as a sole therapy or may involve, in addition to the
compounds of the invention, conventional surgery or radiotherapy or
chemotherapy. Such chemotherapy may include one or more of the
following categories of anti tumour agents:
[0282] (i) other antiproliferative/antineoplastic drugs and
combinations thereof, as used in medical oncology, such as
alkylating agents (for example cis-platin, oxaliplatin,
carboplatin, cyclophosphamide, nitrogen mustard, melphalan,
chlorambucil, busulphan, temozolamide and nitrosoureas);
antimetabolites (for example gemcitabine and antifolates such as
fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed,
methotrexate, cytosine arabinoside, and hydroxyurea); antitumour
antibiotics (for example anthracyclines like adriamycin, bleomycin,
doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,
dactinomycin and mithramycin); antimitotic agents (for example
vinca alkaloids like vincristine, vinblastine, vindesine and
vinorelbine and taxoids like taxol and taxotere and polokinase
inhibitors); and topoisomerase inhibitors (for example
epipodophyllotoxins like etoposide and teniposide, amsacrine,
topotecan and camptothecin);
[0283] (ii) cytostatic agents such as antioestrogens (for example
tamoxifen, fulvestrant, toremifene, raloxifene, droloxifene and
iodoxyfene), antiandrogens (for example bicalutamide, flutamide,
nilutamide and cyproterone acetate), LHRH antagonists or LHRH
agonists (for example goserelin, leuprorelin and buserelin),
progestogens (for example megestrol acetate), aromatase inhibitors
(for example as anastrozole, letrozole, vorazole and exemestane)
and inhibitors of 5.alpha.-reductase such as finasteride;
[0284] (iii) anti-invasion agents (for example c-Src kinase family
inhibitors like
4-(6-chloro-2,3-methylenedioxyanilino)-7-[2-(4-methylpiperazin-1-yl)ethox-
y]-5-tetrahydropyran-4-yloxyquinazoline (AZD0530; International
Patent Application WO 01/94341) and
N-(2-chloro-6-methylphenyl)-2-{6-[4-(2-hydroxyethyl)piperazin-1-yl]-2-met-
hylpyrimidin-4-ylamino}thiazole-5-carboxamide (dasatinib,
BMS-354825; J. Med. Chem., 2004, 47, 6658-6661), and
metalloproteinase inhibitors like marimastat, inhibitors of
urokinase plasminogen activator receptor function or, inhibitors of
cathepsins, inhibitors of serine proteases for example matriptase,
hepsin, urokinase, inhibitors of heparanase);
[0285] (iv) cytotoxic agents such as fludarabine,
2-chlorodeoxyadenosine, chlorambucil or doxorubicin and combination
thereof such as Fludarabine+cyclophosphamide, CVP:
cyclophosphamide+vincristine+prednisone, ACVBP:
doxorubicin+cyclophosphamide+vindesine+bleomycin+prednisone, CHOP:
cyclophosphamide+doxorubicin+vincristine+prednisone, CNOP:
cyclophosphamide+mitoxantrone+vincristine+prednisone, m-BACOD:
methotrexate+bleomycin+doxorubicin+cyclophosphamide+vincristine+dexametha-
sone+leucovorin, MACOP-B:
methotrexate+doxorubicin+cyclophosphamide+vincristine+prednisone
fixed dose+bleomycin+leucovorin, or ProMACE CytaBOM:
prednisone+doxorubicin+cyclophosphamide+etoposide+cytarabine+bleomycin+vi-
ncristine+methotrexate+leucovorin.
[0286] (v) inhibitors of growth factor function, for example such
inhibitors include growth factor antibodies and growth factor
receptor antibodies (for example the anti-erbB2 antibody
trastuzumab [Herceptin.TM.], the anti-EGFR antibody panitumumab,
the anti-erbB1 antibody cetuximab [Erbitux, C225] and any growth
factor or growth factor receptor antibodies disclosed by Stern et
al. Critical reviews in oncology/haematology, 2005, Vol. 54, pp
11-29); such inhibitors also include tyrosine kinase inhibitors,
for example inhibitors of the epidermal growth factor family (for
example EGFR family tyrosine kinase inhibitors such as
N-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-
-amine (gefitinib, ZD1839),
N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine
(erlotinib, OSI-774) and
6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)-quinazol-
in-4-amine (CI-1033), 1033), erbB2 tyrosine kinase inhibitors such
as lapatinib, inhibitors of the hepatocyte growth factor family,
inhibitors of the platelet-derived growth factor family such as
imatinib, inhibitors of serine/threonine kinases (for example
Ras/Raf signalling inhibitors such as farnesyl transferase
inhibitors, for example sorafenib (BAY 43-9006)), inhibitors of
cell signalling through MEK and/or AKT kinases, inhibitors of the
hepatocyte growth factor family, c-kit inhibitors, abl kinase
inhibitors, IGF receptor (insulin-like growth factor) kinase
inhibitors, aurora kinase inhibitors (for example AZD1152,
PH739358, VX-680, MLN8054, R763, MP235, MP529, VX-528 and AX39459),
cyclin dependent kinase inhibitors such as CDK2 and/or CDK4
inhibitors, and inhibitors of survival signaling proteins such as
Bcl-2, Bcl-XL for example ABT-737;
[0287] (vi) antiangiogenic agents such as those which inhibit the
effects of vascular endothelial growth factor, [for example the
anti-vascular endothelial cell growth factor antibody bevacizumab
(Avastin.TM.) and VEGF receptor tyrosine kinase inhibitors such as
4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)qu-
inazoline (ZD6474; Example 2 within WO 01/32651),
4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)-
quinazoline (AZD2171; Example 240 within WO 00/47212), vatalanib
(PTK787; WO 98/35985) and SU11248 (sunitinib; WO 01/60814),
compounds such as those disclosed in International Patent
Applications WO97/22596, WO 97/30035, WO 97/32856, WO 98/13354,
WO00/47212 and WO01/32651 and compounds that work by other
mechanisms (for example linomide, inhibitors of integrin
.alpha.v.beta.3 function and angiostatin)] or colony stimulating
factor 1 (CSF1) or CSF1 receptor;
[0288] (vii) vascular damaging agents such as Combretastatin A4 and
compounds disclosed in International Patent Applications WO
99/02166, WO 00/40529, WO 00/41669, WO 01/92224, WO 02/04434 and WO
02/08213;
[0289] (viii) antisense therapies, for example those which are
directed to the targets listed above, such as G-3139 (Genasense),
an anti bcl2 antisense;
[0290] (ix) gene therapy approaches, including for example
approaches to replace aberrant genes such as aberrant p53 or
aberrant BRCA1 or BRCA2, GDEPT (gene directed enzyme pro drug
therapy) approaches such as those using cytosine deaminase,
thymidine kinase or a bacterial nitroreductase enzyme and
approaches to increase patient tolerance to chemotherapy or
radiotherapy such as multi drug resistance gene therapy; and
[0291] (x) immunotherapy approaches, including for example
treatment with Alemtuzumab (campath-1H.TM.), a monoclonal antibody
directed at CD52, or treatment with antibodies directed at CD22, ex
vivo and in vivo approaches to increase the immunogenicity of
patient tumour cells, transfection with cytokines such as
interleukin 2, interleukin 4 or granulocyte macrophage colony
stimulating factor, approaches to decrease T cell anergy such as
treatment with monoclonal antibodies inhibiting CTLA-4 function,
approaches using transfected immune cells such as cytokine
transfected dendritic cells, approaches using cytokine transfected
tumour cell lines and approaches using anti idiotypic
antibodies.
[0292] (xi) inhibitors of protein degradation such as proteasome
inhibitor such as Velcade (bortezomid).
[0293] (xii) biotherapeutic therapeutic approaches for example
those which use peptides or proteins (such as antibodies or soluble
external receptor domain constructions) which either sequester
receptor ligands, block ligand binding to receptor or decrease
receptor signalling (e.g. due to enhanced receptor degradation or
lowered expression levels).
[0294] In one embodiment the anti-tumour treatment defined herein
may involve, in addition to the compounds of the invention,
treatment with other antiproliferative/antineoplastic drugs and
combinations thereof, as used in medical oncology, such as
alkylating agents (for example cis-platin, oxaliplatin,
carboplatin, cyclophosphamide, nitrogen mustard, melphalan,
chlorambucil, busulphan, temozolamide and nitrosoureas);
antimetabolites (for example gemcitabine and antifolates such as
fluoropyrimidines like 5-fluorouracil and tegafur, raltitrexed,
methotrexate, cytosine arabinoside, and hydroxyurea); antitumour
antibiotics (for example anthracyclines like adriamycin, bleomycin,
doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,
dactinomycin and mithramycin); antimitotic agents (for example
vinca alkaloids like vincristine, vinblastine, vindesine and
vinorelbine and taxoids like taxol and taxotere and polokinase
inhibitors); and topoisomerase inhibitors (for example
epipodophyllotoxins like etoposide and teniposide, amsacrine,
topotecan and camptothecin).
[0295] In one embodiment the anti-tumour treatment defined herein
may involve, in addition to the compounds of the invention,
treatment with gemcitabine.
[0296] Such conjoint treatment may be achieved by way of the
simultaneous, sequential or separate dosing of the individual
components of the treatment. Such combination products employ the
compounds of this invention, or pharmaceutically acceptable salts
thereof, within the dosage range described hereinbefore and the
other pharmaceutically active agent within its approved dosage
range.
[0297] For treatment of an inflammatory disease, e.g. rheumatoid
arthritis, osteoarthritis, asthma, allergic thinitis, chronic
obstructive pulmonary disease (COPD), or psoriasis, a targeted
binding agent of the invention may be combined with one or agents,
such as non-steroidal anti-inflammatory agents (hereinafter NSAIDs)
including non-selective cyclo-oxygenase (COX)--1/COX-2 inhibitors
whether applied topically or systemically, such as piroxicam,
diclofenac, propionic acids, such as naproxen, flurbiprofen,
fenoprofen, ketoprofen and ibuprofen, fenamates, such as mefenamic
acid, indomethacin, sulindac, azapropazone, pyrazolones, such as
phenylbutazone, salicylates, such as aspirin); selective COX-2
inhibitors (such as meloxicam, celecoxib, rofecoxib, valdecoxib,
lumarocoxib, parecoxib and etoricoxib); cyclo-oxygenase inhibiting
nitric oxide donors (CINODs); glucocorticosteroids (whether
administered by topical, oral, intra-muscular, intra-venous or
intra-articular routes); methotrexate, leflunomide;
hydroxychloroquine, d-penicillamine, auranofin or other parenteral
or oral gold preparations; analgesics; diacerein; intra-articular
therapies, such as hylauronic acid derivatives; and nutritional
supplements, such as glucosamine.
[0298] The targeted binding agent or antibody defined herein may be
applied in combination with an antagonist of VEGF. The targeted
binding agent or antibody defined herein and the antagonist of VEGF
can be administered in concurrent or sequential treatment cycles.
Such combination treatments are useful for treating diseases having
abnormal angiogenesis and/or vascular permeability. In one
embodiment, the antagonist of VEGF is Avastin.TM., ZD6474
(4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)q-
uinazoline) or AZD2171
(4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-(pyrrolidin-1-yl)propo-
xy)quinazoline).
[0299] All references cited herein, including patents, patent
applications, papers, text books, and the like, and the references
cited therein, to the extent that they are not already, are hereby
incorporated herein by reference in their entirety.
[0300] This application claims the benefit of priority of U.S.
Provisional Application Ser. No. 61/140,336 filed Dec. 23, 2008,
herein incorporated by reference for all purposes.
EXAMPLES
[0301] The following examples, including the experiments conducted
and results achieved are provided for illustrative purposes only
and are not to be construed as limiting upon the teachings
herein.
Example 1
Immunization and Titering
Immunization
[0302] Immunizations were conducted using soluble .alpha.5.beta.1
and cell-bound .alpha.5.beta.1 (CHO transfectants expressing human
.alpha.5.beta.1 at the cell surface), respectively. For the
generation of CHO transfectants, human full-length .alpha.5.beta.1
cDNA was inserted into the pcDNA 3 expression vector. CHO cells
were transiently transfected via electroporation. Expression of
human .alpha.5.beta.1 on the cell surface at the level suitable for
immunogen purpose was confirmed by Fluorescence-Activated Cell
Sorter (FACS) analysis. For the campaign, an initial injection of
2.times.10.sup.6 cells/mouse of transfected CHO cells (group 1 and
2) and 10 .mu.g/mouse of soluble protein (Groups 3 and 4) were used
for immunization in XenoMouse.TM.. The immunization was carried out
according to the methods disclosed in U.S. patent application Ser.
No. 08/759,620, filed Dec. 3, 1996 and International Patent
Application Nos. WO 98/24893, published Jun. 11, 1998 and WO
00/76310, published Dec. 21, 2000, the disclosures of which are
hereby incorporated by reference. Following the initial
immunization, 11 subsequent boost immunizations of 1.times.10.sup.6
cells/mouse were administered for groups 1 and 2 (cell-bound
antigen), and thirteen subsequent boost immunizations of 5
.mu.g/mouse were administered for groups 3 and 4 (soluble antigen).
The immunization programs are summarized in Table 2.
Selection of Animals for Harvest by Titer
[0303] Titers of the antibody against native (cell-bound) antigen
were tested by FACS staining for native antigen binding using
untransfected 300.19 cells (Amgen, Vancouver) or human
.alpha.5.beta.1-transfected 300.19 cells. At the end of the
immunization program, fusions were performed using mouse myeloma
cells and lymphocytes isolated from the spleens and lymph nodes of
the immunized mice by means of electroporation, as described in
Example 2.
TABLE-US-00002 TABLE 2 Summary of Immunization Programs No of Group
Immunogen Strain mice Immunization routes 1 Cell-bound IgG2 10
IP/Tail/BIP, twice/wk, x .alpha.5.beta.1 (CHO 5 wks, transfectants)
2 Cell-bound IgG4 10 IP/Tail/BIP, twice/wk, x .alpha.5.beta.1 (CHO
5 wks transfectants) 3 Soluble .alpha.5.beta.1 IgG2 10 IP/Tail/BIP,
twice/wk, x 6 wks 4 Soluble .alpha.5.beta.1 IgG4 10 IP/Tail/BIP,
twice/wk, x 6 wks "IP" refers to "intraperitoneal" "BIP" refers to
"Base of Tail/Intraperitoneal"
Example 2
Recovery of Lymphocytes, B-Cell Isolations, Fusions and Generation
of Hybridomas
[0304] Immunized mice were sacrificed by cervical dislocation, and
the draining lymph nodes harvested and pooled from each cohort.
Three independent harvests were conducted. Harvest 1 used five mice
from group 1 with ID numbers 150927, 150928, 150929, 150930,
150031. Harvest 2 used six mice from group 2 with ID numbers
151037, 151038, 151039, 151040, 150588, 150589. The third harvest
used five mice from group 1 with ID numbers 150932, 150919, 150920,
150921, 150926.
[0305] The lymphoid cells were dissociated by grinding in DMEM to
release the cells from the tissues and the cells were suspended in
DMEM. The cells were counted, and 0.9 ml DMEM per 100 million
lymphocytes added to the cell pellet to resuspend the cells gently
but completely. Using 100 .mu.l of CD90+ magnetic beads per 100
million cells, the cells were labeled by incubating the cells with
the magnetic beads at 4.degree. C. for 15 minutes. The magnetically
labeled cell suspension containing up to 10.sup.8 positive cells
(or up to 2.times.10.sup.9 total cells) was loaded onto a LS+
column and the column washed with DMEM. The total effluent was
collected as the CD90- negative fraction (most of these cells were
expected to be B cells).
[0306] The fusion was performed by mixing washed enriched Day 6 B
cells with nonsecretory myeloma P3X63Ag8.653 cells purchased from
ATCC, cat.# CRL 1580 (Kearney et al, J. Immunol. 123, 1979,
1548-1550) at a ratio of 1:4. The cell mixture was gently pelleted
by centrifugation at 400.times.g for 4 minutes. After decanting of
the supernatant, the cells were gently mixed using a 1 ml pipette.
Preheated PEG (1 ml per 10.sup.6 B-cells) was slowly added with
gentle agitation over 1 minute followed by 1 minute of mixing.
Preheated IDMEM (2 ml per 10.sup.6 B-cells) was then added over 2
minutes with gentle agitation. Finally preheated IDMEM (8 ml per
10.sup.6 B-cells) was added over 3 minutes.
[0307] The fused cells were spun down at 400.times.g for 6 minutes
and resuspended in 20 ml of Selection media (DMEM (Invitrogen), 15%
FBS (Hyclone), supplemented with L-glutamine, pen/strep, MEM
Non-essential amino acids, Sodium Pyruvate, 2-Mercaptoethanol (all
from Invitrogen), HA-Azaserine Hypoxanthine and OPI (oxaloacetate,
pyruvate, bovine insulin) (both from Sigma) and IL-6 (Boehringer
Mannheim)) per 10.sup.6 B-cells. Cells were incubated for 20-30
minutes at 37.degree. C. and then resuspended in 200 ml Selection
media and cultured for 3-4 days in a T175 flask.
[0308] Day 3 post fusion the cells were collected, spun for 8
minutes at 400.times.g and resuspended in 10 ml Selection media per
10.sup.6 fused B-cells. FACS analysis of hybridoma population was
performed, and cells were subsequently frozen down.
[0309] Hybridomas were grown as routine in the selective medium.
Exhaustive supernatants collected from the hybridomas that
potentially produce anti-human .alpha.5.beta.1 antibodies were
subjected to subsequent screening assays.
Example 3
Antibody Titer Measurement: Native Antigen Binding of 300.19
Cells
[0310] FACS analysis was performed on 300.19 cells to measure the
titers of antibody against .alpha.5.beta.1 expressed on B300.19
cells. 300.19 cells (control and transfected with human
.alpha.5.beta.1) were seeded at 50,000 cells/well and incubated
with 90 .mu.L of sample supernatant (at 1:50 dilution) for one hour
at 4.degree. C. The wells were then washed and incubated with
Cy5-conjugated goat anti-human antibody (Jackson Laboratories) at
5n/mL and 7-Amino-Actinomycin (7AAD) at 5 .mu.g/mL for 15 minutes
at 4.degree. C. Bound .alpha.5.beta.1 was detected using FACS
analysis. The positive control was mouse anti-.alpha.5.beta.1
antibody (R&D Systems, Inc.), and negative controls included
goat anti-human and anti-mouse Fc Cy5 coupled antibody (Jackson
Laboratories) alone, as well binding or irrelevant mouse IgG1 and
human IgG2, irrelevant supernatants as indicated. Animals with the
greatest FACS Geometric Mean Fluorescence were selected for
subsequent hybridoma generation. Table 3 lists the FACS data
obtained from analysis of the 300.19 cells.
TABLE-US-00003 TABLE 3 Titers of antibody against human
.alpha.5.beta.1 as measured by FACS analysis of 300.19 cells
.alpha.5.beta.1 expressing B300.19 B300.19 cells cells X Geo % X
Geo ID's Events % Total Mean Events Total Mean Ratio cells 1397
13.97 3.0 2795 28 4.2 1.4 Cells + Mo 2' alone 1777 17.77 18.1 1548
15 15.4 0.9 Cells + Hu 2' alone 1730 17.3 15.1 1450 15 14.0 0.9 Mo
IgG1 2.0 ug/mL + 2' 1453 14.53 17.3 1229 12 14.9 0.9 Hu IgG2 @ 2.0
ug/mL + 2' 1372 13.72 14.5 1144 11 12.0 0.8 Irrel. Sera1 1907 19.07
25.1 1494 15 20.3 0.8 Irrel. Sera2 2041 20.41 44.4 1761 18 24.5 0.6
150919 1912 19.12 23.8 1505 15 95.6 4.0 150920 2185 21.85 25.1 1669
17 92.6 3.7 150921 2012 20.12 69.4 1636 16 142.5 2.1 150926 2031
20.31 23.7 1627 16 77.0 3.3 150927 1535 15.35 35.1 1340 13 156.2
4.5 150928 2003 20.03 28.4 1760 18 251.6 8.9 150929 1862 18.62 22.4
1614 16 102.0 4.5 150930 2118 21.18 23.9 1852 19 154.1 6.4 150931
2006 20.06 20.3 1659 17 117.5 5.8 150932 2228 22.28 30.3 1796 18
107.2 3.5 151037 1978 19.78 18.5 1680 17 77.1 4.2 151038 2218 22.18
22.6 1882 19 27.0 1.2 151039 2099 20.99 29.5 1698 17 88.7 3.0
151040 2013 20.13 24.4 1627 16 75.6 3.1 150588 2323 23.23 21.1 1865
19 81.6 3.9 150589 2448 24.48 17.1 2016 20 62.1 3.6 mAb 1969 @ 2.0
ug/mL 2048 20.48 20.4 1804 18 194.1 9.5 mAb 1969 @ 0.2 ug/mL 2062
20.62 22.1 1796 18 151.8 6.9 mAb 1969 @ 0.02 ug/mL 2109 21.09 18.1
1769 18 45.1 2.5
[0311] Only fusions derived from animals receiving cell bound
immunogen were progressed to further screening.
Example 4
[0312] Hybridoma Supernatant Screening by Binding Assay--Binding to
.alpha.5.beta.1 Expressed on HEK 293T Cells
[0313] Hybridoma supernatants containing antibody, produced as
described in Examples 1 and 2, were screened by assays that measure
binding to immobilized native .alpha.5.beta.1. Supernatants
collected from harvested cells were tested to assess the binding of
secreted antibodies to HEK 293T (ATCC, cat.# CRL 11268) cells.
Cells in FACS buffer were seeded into 384-well FMAT plates in a
volume of 40 .mu.L/well at a density of 7500 cells/well. Then, 10
.mu.L/well of supernatant was added, and plates were incubated for
approximately 1.5 hour at room temperature, after which 10
.mu.L/well of anti-human IgG-Cy5 secondary antibody (Jackson
Laboratories) was added to a final concentration of 750 ng/ml.
Plates were then incubated for one hour at 4.degree. C., and
fluorescence was read using an FMAT macroconfocal scanner (Applied
Biosystems). A total of 1790 antigen-specific wells were identified
across the three harvests. The breakdown of these hits were as
follows; harvest 1--459 native binding wells identified, harvest
2--860 native binding wells identified and harvest 3--471 native
binding wells identified.
Example 5
Determination of Relative Potency of Antibody-Containing
Supernatants: Ability to Inhibit .alpha.5.beta.1 Mediated Binding
to Fibronectin
[0314] The relative potency of the different antibody-containing
supernatants was assayed by how well the antibodies blocked
adhesion of K562 cells (ATCC, cat.# CCL 243) to fibronectin. Plates
were coated overnight with 3-5 .mu.g/ml Fibronectin or
GST-Fibronectin type III domains 9-10, and pre-blocked with 3%
BSA/PBS for 1 hour prior to the assay. Cells were then pelleted and
washed twice in HBSS, after which the cells were then resuspended
in HBSS at 1.times.10.sup.6 cells/ml. To select the best antibodies
the cells were incubated in the presence of appropriate antibodies
at 4.degree. C. for 60 minutes in a V-bottom plate. To increase the
stringency of the assay cells the pre-incubation step was removed,
and the cation conditions modified to increase binding affinity.
The 3% BSA/PBS was removed from the assay plates and the plates
washed twice with PBS or HBSS, and the cell-antibody mixtures were
transferred to the coated plate and the plate was incubated at
37.degree. C. for 60 minutes in the presence of either 1 mM or 0.2
mM MnCl.sub.2. The cells on the coated plates were then washed four
times in warm HBSS, and the cells were thereafter frozen at
-80.degree. C. for one hour. The cells were allowed to thaw at room
temperature for one hour, and then 100 .mu.L of CyQuant dye/lysis
buffer (Molecular Probes) was added to each well according to the
manufacturer's instructions. Fluorescence was read at an excitation
wavelength of 485 nm and an emission wavelength of 530 nm.
[0315] To identify anti-functional antibodies two adhesion assays
(n=1 and n=2 in Table 3) were run using supernatant diluted 1 in 4
and with pre-incubation of the antibodies on cells prior to
addition to plates coated with full length FN and MnCl.sub.2 was
included at 1 mM final. Many neutralizing antibodies were
identified and a cut-off of 90% inhibition of adhesion was applied
for these screens. A total of 188 supernatants were advanced for
further screening based on this data (Harvest 1--16 Abs; Harvest
2--116 Abs; Harvest 3--56 Abs). To identify the best antibodies
within this group the K562 adhesion assay was run a third time (n=3
Table 3) without pre-incubation on cells to attempt to identify
antibodies with higher affinity (or good on-rates). The supernatant
was still used at 1 in 4 dilution. The antibodies and cells (in
media containing 0.2 mM MnCl.sub.2) were added to a plates coated
with GST-FNIII(9-10) at 3 ug/mL overnight. Most of the antibodies
still showed nearly complete inhibition in this assay. In an
attempt to increase the assay stringency, two more adhesion assays
(n=4 and N=5 Table 3) were run with K562 cells on GST-FNIII(9-10)
(in media containing 1 mM MnCl.sub.2) with a larger dilution of
supernatant (1 in 10). The antibodies were found to have
differences in their ability to block adhesion at this dilution,
and allowed the selection of a lead panel of antibodies for
sub-cloning. Table 4 provides a summary of the results for the
assay.
TABLE-US-00004 TABLE 4 Inhibition of adhesion of K562 cell binding
to fibronectin by selected lead antibodies % Inhibition of cell
adhesion MAb ID Isotype n = 1 n = 2 n = 3 n = 4 n = 5 4C3 IgG4/k 97
99 94 97 90 3A7 IgG4/k 98 98 92 97 91 4E9 IgG4/k 96 99 94 93 85
7B1.3A1 IgG4/k 98 100 91 93 84 7F9 IgG4/k 99 101 93 93 88 1F2.2B7
IgG4/k 93 102 100 98 77 2G2.2B5 IgG4/k 100 102 97 82 78 4F9 IgG4/k
97 101 100 82 63 2H12 IgG4/k 89 100 96 92 89 2A5 IgG4/k 89 100 96
92 89 4G3.3D11 IgG4/k 96 99 102 100 81 3F12.4A1 IgG2/k 103 100 97
93 91
Example 6
Screening for Functional Selectivity Over .alpha.5.beta.1
Integrin--Antibodies Do not Inhibit J6 Cell Adhesion to the CS-1
Fragment of Fibronectin
[0316] To confirm that the antibodies were specific to .alpha.5 or
.alpha.5.beta.1 (and not binding to .beta.1, the antibodies were
also screened in an alpha4beta dependent adhesion assay. For this,
the ability of our antibodies to block the binding of J6.77 Jurkat
cells (Amgen, Vancouver) to the CS-1 fragment of fibronectin was
tested. Plates were coated overnight at 4.degree. C. with 2.5 ug/ml
GST-CS-1 fragment of fibronectin in PBS, washed twice in PBS and
then blocked with 3% BSA/PBS for 1 hour. Cells were then pelleted
and washed 3 times with 1% BSA/HBSS and resuspended in HBSS at a
concentration of 9.times.10.sup.5/ml. Cells were dispensed into V
bottom pates (37.5 ul per well), 12.5 ul of supernatant or a
control of HBSS added to each well, and then incubated for 1 hour
at 4.degree. C. Assay plates were then washed 3 times with PBS. The
mix of cells and antibody was then transferred to the assay plate
and incubated for 40 minutes at 37.degree. C. in the presence of is
0.2 mM MnCl.sub.2. The cells on the coated plates were then washed
four times in warm HBSS, and the cells were thereafter frozen at
-80.degree. C. for one hour. The cells were allowed to thaw at room
temperature for one hour, and then 100 .mu.L of CyQuant dye/lysis
buffer (Molecular Probes) was added to each well according to the
manufacturer's instructions. Fluorescence was read at an excitation
wavelength of 485 nm and an emission wavelength of 530 nm. The
majority of the antibodies showed little to no blockade in this
assay, suggesting that their specificity is primarily against
.alpha.5 or .alpha.5.beta.1 (Table 5). Table 5 provides a summary
of the results for the assay.
TABLE-US-00005 TABLE 5 Inhibition of .alpha.4.beta.1 mediated
adhesion of J6.77 Jurkat cells to the CS-1 fragment of fibronectin
% Inhibition of adhesion MAb ID Isotype CS1 n = 1 CS1 n = 2 4C3
IgG4/k 2 31 3A7 IgG4/k 24 33 4E9 IgG4/k 17 24 1F2.2B7 IgG4/k 29 22
7F9 IgG4/k 15 -3 7B1.3A1 IgG4/k 33 12 2H12 IgG4/k 39 37 2A5 IgG4/k
39 37 4F9 IgG4/k 28 -5 2G2.2B5 IgG4/k 28 -5
Example 7
Specific Binding to .alpha.5.beta.1--Lead Antibodies Show No Cross
Reactivity to The A5 Null Line HT29 when Analysed by FACS
[0317] To confirm the antibodies bind to the .alpha.5 chain or to
the .alpha.5.beta.1 heterodimer, binding to Human colon
adenocarcinoma grade II cells (HT29 cells) was assessed by FACS.
HT29 (ATCC, cat #HTB-38) cells do not express the .alpha.5 chain,
but do express the .beta.1 chain. HT-29 cells were suspended in
HBSS with 1% BSA and 1 mM final MnCL.sub.2 at a concentration of
6.times.10.sup.5 cells/ml. 12.5 uL of the primary antibody was
added to 37.5 ul of cells and the plate incubated on ice for 60
minutes. A range of negative controls were included as described
previously (indicated as below). In addition as a positive control
antibodies to .alpha.v.beta.6 (Mab2077, Chemicon), .alpha.5.beta.1
(Mab1909, Chemicon) and .alpha.v (L230, Chemicon) were included.
100 uL of HBSS buffer was added to dilute primary antibody, the
cells pelleted by centrifuging at 1500 rpm for 3 minutes and
resuspended in 50 ul Goat anti-human IgG Fc Cy5 or Goat anti Mouse
IgG Fc Cy5 secondary at 2 ug/mL. They were then incubated on ice
for a further 7 minutes, and 100 uL of HBSS buffer was added to
dilute secondary antibody. Finally cells were pelleted by
centrifuging at 1500 rpm for 3 minutes, HBSS Buffer/secondary
supernatant removed, washed twice in 100 ul of FACS buffer was
added and the cells were resuspended and then read on a FACS
Calibur HTS. Samples were analyzed using Cell Quest Pro software.
The data is represented in Table 6 and confirm that the antibodies
are selective for .alpha.5.beta.1.
TABLE-US-00006 TABLE 6 Binding of lead antibodies to HT29 cells by
FACS MAb ID Binding Geomean 7B1.3A1 3.17 7F9 3.06 2G2.2B5 3.03 2A5
2.96 3A7 3.16 1F2.2B7 2.75 2H12 2.96 4C3 3.26 4E9 3 4G3.3D11 3.06
4F9 2.97 3F12.4A1 3.61 Cells Alone 2.07 Gt anti Mouse 2.52 Mouse
IgG2a 10.21 Mouse IgG1 2.55 MAB2077Z avb6 39.23 MAB1969 a5b1 2.69
4b4 anti beta1 361.61 L230 anti alphaV 294.02 Human Secondary 2.8
Human IgG2 2.84 Human IgG4 3.2
Example 8
Lead Antibodies Show No Cross-Reactivity to Macaque
.alpha.5.beta.1
[0318] To determine whether the antibodies cross reacted to monkey
integrin, binding to purified Macaque T-cells was assessed. Macaque
PBMCs were previously purified from whole blood, and stored frozen.
Macaque PBMCs were suspended in adhesion buffer with HBSS 1% BSA
and 1 mM Mn2+ ("FACS buffer"), at a concentration of
4.8.times.10.sup.5 live cells per ml. 12.5 uL of the primary
antibody was added to 37.5 ul of cells, and incubated at 4.degree.
C. for 1 hour. Positive and negative controls were included as
indicated. 100 uL of FACs buffer is added to dilute out primary
antibody, the cells washed and resuspended in the appropriate
secondary at 2 ug/mL (50 uL) with 10 ug/mL 7AAD, and stained on ice
for 7 minutes. 100 uL of FACs buffer was added and cells were
washed twice in FACS buffer, finally the supernatant removed and
the cells were resuspended in 100 uL of buffer.
[0319] Samples were read on HTS FACS machine and analyzed using
Cell Quest Pro software. The data shown in Table 7 confirm that the
antibodies cross react with monkey .alpha.5.beta.1.
TABLE-US-00007 TABLE 7 Cross-Reactivity Assay Results Against
Macaque .alpha.5.beta.1 Geo Mean on Macaque MAb ID T-cells IgG
detection 1F2.2B7 9 4C3 8 3A7 8 4E9 9 7B1.3A1 10 7F9 10 2A5 10 2H12
10 2G2.2B5 11 4F9 10 3F12.4A1 13 Cells alone 2 Goat anti Mouse 2
IgGFc Cy5 Mouse IgG1 2 4b4 anti beta1 141 IIA1 anti alpha5 10 Goat
anti Human IgG 3 Fc Cy5 Human IgG2/4 3 L230 anti-alphaV 3
Example 9
Structural Analysis of .alpha.5.beta.1 Antibodies
[0320] The variable heavy chains and the variable light chains of
the antibodies were sequenced to determine their DNA sequences. The
complete sequence information for the anti-.alpha.5.beta.1
antibodies is provided in the sequence listing with nucleotide and
amino acid sequences for each gamma and kappa chain combination.
The heavy and light chain variable domain cDNA sequences were
analyzed to determine the VH, D, JH, Vkappa and Jkappa gene
segments used. The sequences were then translated to determine the
primary amino acid sequence and compared to the germline
VH-D-J-region or Vk-Jk sequences to assess mutations of lead
antibody sequences from germ line.
[0321] Table 10 is a table comparing the antibody heavy chain
regions to their cognate germ line heavy chain region. Table 11 is
a table comparing the antibody kappa light chain regions to their
cognate germ line light chain region.
[0322] The variable (V) regions of immunoglobulin chains are
encoded by multiple germ line DNA segments, which are joined into
functional variable regions (V.sub.HDJ.sub.H or V.sub.KJ.sub.K)
during B-cell ontogeny. The molecular and genetic diversity of the
antibody response to .alpha.5.beta.1 was studied in detail. Based
on the VH-D-JH and Vk-Jk gene segment usage, all the antibodies
described below belong to the same family.
[0323] It should also be appreciated that where a particular
antibody differs from its respective germline sequence at the amino
acid level, it may be possible to mutate the antibody sequence back
to the germline sequence without significant loss of affinity or
potency. When such back mutations to germline do not adversely
affect affinity or potency of the antibody, they will reduce the
risk of immunogenicity of the antibody in human subject. Such
corrective mutations can occur at one, two, three or more
positions, or a combination of any of the mutated positions, using
standard molecular biological techniques. By way of non-limiting
example, Table 11 shows that the light chain sequence of mAb
1F2.2B7 (SEQ ID NO.: 8) differs from the corresponding germline
sequence (SEQ ID NO.: 36) through a His to Gln mutation (mutation
1) in the CDR1 region and a Asn to Thr mutation (mutation 2) in the
CDR1 region. on. Thus, the amino acid or nucleotide sequence
encoding the light chain of mAb 1F2.2B7 can be modified to change
mutation 1 to yield the germline sequence at the site of mutation
1. Further, the amino acid or nucleotide sequence encoding the
light chain of mAb 1F2.2B7 can be modified to change mutation 2 to
yield the germline sequence at the site of mutation 2. Still
further, the amino acid or nucleotide sequence encoding the light
chain of mAb 3C2.2A8 can be modified to change both mutation 1 and
mutation 2 to yield the germline sequence at those particular
sites. Tables 8-9 below illustrate the positions of such variations
from the germline for mAb 2H12. Each row represents a unique
combination of germline and non-germline residues at the position
indicated by bold type.
[0324] The heavy chain of the 2H12CDR3 contains an RGD sequence.
Note that in this sequence (SEQ ID NO:15) DG comes from insertional
events at the recombination junction between VH and D, AAR comes
from the D6-6 gene, RGD comes from further nucleotide additions at
the D-JH junction, and YYYYHGMDV is derived from the JH6 gene
segment (YYYYYGMDV with the 5th germline Y mutated to H). Thus, the
RGD is an arbitrary nucleotide addition segment, selected for its
target binding properties, leading to expansion of the B-cell
lineage, and further diversification (elsewhere, while maintaining
the RGD in CDR3).
TABLE-US-00008 TABLE 8 Exemplary Mutations of mAB 2H12 Light Chain
(SEQ ID NO: 12) to Germline at the Indicated Residue Number 31 32
33 36 H S N N R S N N H G N N R G N N H S H N R S H N H G H N R G H
N H S N S R S N S H G N S R G N S H S H S R S H S H G H S R G H
S
TABLE-US-00009 TABLE 9 Exemplary Mutations of mAB 2H12 Heavy Chain
(SEQ ID NO: 10) to Germline at the Indicated Residue Number 56 77
99 100 104 105 106 S N -- -- -- -- -- N N -- -- -- -- -- S T -- --
-- -- -- N T -- -- -- -- -- S N D -- -- -- -- N N D -- -- -- -- S T
D -- -- -- -- N T D -- -- -- -- S N -- G -- -- -- N N -- G -- -- --
S T -- G -- -- -- N T -- G -- -- -- S N D G -- -- -- N N D G -- --
-- S T D G -- -- -- N T D G -- -- -- S N -- -- R -- -- N N -- -- R
-- -- S T -- -- R -- -- N T -- -- R -- -- S N D -- R -- -- N N D --
R -- -- S T D -- R -- -- N T D -- R -- -- S N -- G R -- -- N N -- G
R -- -- S T -- G R -- -- N T -- G R -- -- S N D G R -- -- N N D G R
-- -- S T D G R -- -- N T D G R -- -- S N -- -- -- G -- N N -- --
-- G -- S T -- -- -- G -- N T -- -- -- G -- S N D -- -- G -- N N D
-- -- G -- S T D -- -- G -- N T D -- -- G -- S N -- G -- G -- N N
-- G -- G -- S T -- G -- G -- N T -- G -- G -- S N D G -- G -- N N
D G -- G -- S T D G -- G -- N T D G -- G -- S N -- -- R G -- N N --
-- R G -- S T -- -- R G -- N T -- -- R G -- S N D -- R G -- N N D
-- R G -- S T D -- R G -- N T D -- R G -- S N -- G R G -- N N -- G
R G -- S T -- G R G -- N T -- G R G -- S N D G R G -- N N D G R G
-- S T D G R G -- N T D G R G -- S N -- -- -- -- D N N -- -- -- --
D S T -- -- -- -- D N T -- -- -- -- D S N D -- -- -- D N N D -- --
-- D S T D -- -- -- D N T D -- -- -- D S N -- G -- -- D N N -- G --
-- D S T -- G -- -- D N T -- G -- -- D S N D G -- -- D N N D G --
-- D S T D G -- -- D N T D G -- -- D S N -- -- R -- D N N -- -- R
-- D S T -- -- R -- D N T -- -- R -- D S N D -- R -- D N N D -- R
-- D S T D -- R -- D N T D -- R -- D S N -- G R -- D N N -- G R --
D S T -- G R -- D N T -- G R -- D S N D G R -- D N N D G R -- D S T
D G R -- D N T D G R -- D S N -- -- -- G D N N -- -- -- G D S T --
-- -- G D N T -- -- -- G D S N D -- -- G D N N D -- -- G D S T D --
-- G D N T D -- -- G D S N -- G -- G D N N -- G -- G D S T -- G --
G D N T -- G -- G D S N D G -- G D N N D G -- G D S T D G -- G D N
T D G -- G D S N -- -- R G D N N -- -- R G D S T -- -- R G D N T --
-- R G D S N D -- R G D N N D -- R G D S T D -- R G D N T D -- R G
D S N -- G R G D N N -- G R G D S T -- G R G D N T -- G R G D S N D
G R G D N N D G R G D S T D G R G D N T D G R G D "--" indicates
the absence of a residue at that position with reference to SEQ ID
NO: 10
TABLE-US-00010 TABLE 10 Heavy chain analysis SEQ Chain ID Name NO:
V D J FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 35 Germline QVQLVESGGGVVQPGR
SYGMH WVRQAPG VIWYDGS RFTISRDNSKNT -- WGQGT SLRLSCAASGFTFS KGLEWVA
NKYYADS LYLQMNSLRAED AAR-- TVTVS VKG TAVYYCAR - S YYYYY GMDV
7B1.3A1 2 VH3-33 D6-6 JH6B QVQLVESGGGVVQPGR SYGMH WVRQAPG VLWYDGS
RFTISRDNSKNT DGAAR WGQGT SLRLSCAASGFTFR KGLEWVA NKNYADS
LYLQMNSLRAED RGDYY TVTVS VKG TAMYYCAR YYHGM S DV 1F2.2B7 6 VH3-33
D6-6 JH6B QVQLVESGGGVVQPGR SYGMH WVRQAPG VIWYDGS RFTISRDNSKNT DGAAR
WGQGT SLRLSCAASGFTFR KGLEWVA NKYYADS LYLQMNSLRAED RGDYY TVTVS VKG
TAVMYCAR YYHGM S DV 2H12 10 VH3-33 D6-6 JH6B QVQLVESGGGVVQPGR SYGMH
WVRQAPG VIWYDGT RFTISRDNSKTT DGAAR WGQGT SLRLSCAASGFTFS KGLEWVA
NKYYADS LYLQMNSLRAED RGDYY TVTVS VKG TAVYYCAR YYHGM S DV 2H12 20
VH3-33 D6-6 JH6B QVQLVESGGGVVQPGR SYGMH WVRQAPG VIWYDGT
RFTISRDNSKNT DGAAR WGQGT Variant SLRLSCAASGFTFS KGLEWVA NKYYADS
LYLQMNSLRAED RGDYY TVTVS 1 VKG TAVYYCAR YYHGM S DV 2G2.2B5 14
VH3-33 D6-6 JH6B QVQLVESGGGVVQPGR SYGMH WVRQAPG VIWYDGS
RFTISRDNSKNT DGAAR WGQGT SLRLSCAASGFTFS KGLEWVA NKYYADS
LYLQMNSLRAED RGDYY TVTVS VKG TAVYYCAR YYHGM S DV 3F12.4A1 18 VH3-33
D6-6 JH6B QVQLVESGGGVVQPGR SYGMH WVRQAPG VIWYDGT RFTISRDNSKNT DMAAR
WGPGT SLRLSCAASGFTFS KGLEWVA NKYYADS LYLQMNSLTAED RGDYY TVTVS VKG
TAVYYCAR YYHGM S DV 4G3.3D11 22 VH3-33 D6-6 JH6B QVQLVESGGGVVQPGR
SYGMH WVRQAPG VIWYDGT RFTISRDNSKNT DGAAR WGQGT SLRLSCAASGFIFS
KGLEWVA NKYYADS LYLQMNSLRAED RGDYY TVTVS VKG TAVYYCAR YYHGM S
DV
TABLE-US-00011 TABLE 11 Light chain analysis SEQ Chain ID Name NO:
V J FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4 36 Germline DIVMTQSPLSLPVT
RSSQSLLHSNGYN WYLQKPGQSPQL LGSNRAS GVPDRFSGSGSGTDFTL MQALQT FGPGT
PGEPASISC YLD LIY KISRVEAEDVGVYYC PFT KVDIK 7B1.3A1 4 A3 JK3
DIVMTQSPLSLPVT RSSQSLLQSTGYN WYLQKPGQSPQL LGSNRAS GVPDRFSGSGSGTDFTL
MQALQT FGPGT PGEPASISC YLD LIY KISRVEAEDVGVYYC PFT KVDIK 1F2.2B7 8
A3 JK3 DIVMTQSPLSLPVT RSSQSLLQSTGYN WYLQKPGQSPQL LGSNRAS
GVPDRFSGSGSGTDFTL MQALQT FGPGT PGEPASISC YLD LIY KISRVEAEDVGVYYC
PFT KVDIK 2H12 12 A3 JK3 DIVMTQSPLSLPVT RSSQSLLRGHGYS WYLQKPGQSPQL
LGSNRAS GVPDRFSGSGSGTDFTL MQALQT FGPGT PGEPASISC YLD LIY
KISRVEAEDVGVYYC PFT KVDIK 2H12 22 A3 JK3 DIVMTQSPLSLPVT
RSSQSLLRGHGYS WYLQKPGQSPQL LGSNRAS GVPDRFSGSGSGTDFTL MQALQT FGPGT
Variant PGEPASISC YLD LIY KISRVEAEDVGVYYC PFT KVDIK 1 2G2.2B5 16 A3
JK3 EIVMTQSPLSLPVT RSGQSLLQSTGSN WYLQKPGQSPQL LGSNRAS
GVPDRFSGSGSGTDFTL MQALQT FGPGT PGEPASISC YLA LIY KISRVEAEDVGVYYC
PFT KVDIK 3F12.4A1 20 A3 JK3 DIVMTQSPLSLPVT RSSQSLLNGIGYN
WYLQKPGQSPQL LGSNRAS GVPDRFSGSGSGTDFTL MHALQT FGPGT PGEPASISC FLD
LIY TISRVEAEDVGVYYC PFT KVDIK 4G3.3D11 24 A3 JK3 DIVMTQSPLSLPVT
RSSQSLLHGTGYS WYLQKPGQSPQL LGSNRAS GVPDRFSGSGSGTDFTL MQALQT FGPGT
PGEPASISC SLD LIY KISRVEAEDVGVYFC PFT KVDIK
Example 10
Potency (IC50) Determination of .alpha.5.beta.1 Antibodies
Inhibition of .alpha.5.beta.1-Mediated K563 Binding to
Fibronectin
[0325] In order to confirm activity and determine the relative
potency of the different cloned purified antibodies, activity in
the K562-fibronectin adhesion assay was determined. Plates were
coated overnight with 3-5 .mu.g/ml GST-Fibronectin type III domains
9-10, and pre-blocked with 3% BSA/PBS for 1 hour prior to the
assay. Cells were then pelleted and washed twice in HBSS, after
which the cells were then resuspended in HBSS at 1.times.10.sup.6
cells/ml. To select the best antibodies the cells were incubated in
the presence of appropriate antibodies at 4.degree. C. for 60
minutes in a V-bottom plate. To increase the stringency of the
assay cells the pre-incubation step was removed. The 3% BSA/PBS was
removed from the assay plates and the plates washed twice with PBS
or HBSS, and the cell-antibody mixtures were transferred to the
coated plate and the plate was incubated at 37.degree. C. for 60
minute in the presence of 1 mM MnCl.sub.2. The cells on the coated
plates were then washed four times in warm HBSS, and the cells were
thereafter frozen at -80.degree. C. for one hour. The cells were
allowed to thaw at room temperature for one hour, and then 100
.mu.L of CyQuant dye/lysis buffer (Molecular Probes) was added to
each well according to the manufacturer's instructions.
Fluorescence was read at an excitation wavelength of 485 nm and an
emission wavelength of 530 nm. As a standard control the commercial
.alpha.5.beta.1 neutralising antibody IIA1 (R&D Systems) was
included.
TABLE-US-00012 TABLE 12 Inhibition of .alpha.5.beta.1 mediated
adhesion of K562 cells to fibronectin. MAb ID IC50 ng/mL 7B1.3A1
49.8 2H12 43.2 4E9 61.5 4G3.3D11 60.2 IIA1 16.68
Example 11
Cloned Purified Antibodies Exhibit Selective Inhibition of .alpha.5
Integrin but not .alpha.4 Integrin in J6 Cells
[0326] To confirm that the antibodies were specific to .alpha.5 or
.alpha.5.beta.1 (and not binding to .beta.1), the antibodies were
also screened in an alpha4beta dependent adhesion assay. For this,
the ability of our antibodies to block the binding of J6.77 Jurkat
cells to the CS-1 fragment of fibronectin was tested. Plates were
coated overnight at 4.degree. C. with 2.5 ug/ml GST-CS-1 fragment
of fibronectin in PBS, washed twice in PBS and then and blocked
with 3% BSA/PBS for 1 hour. Cells were then pelleted and washed 3
times with 1% BSA/HBSS and resuspended in HBSS at a concentration
of 9.times.10.sup.5/ml. Cells were dispensed into V bottom pates
(35 ul per well), antibody was added at a final concentration of 5
ug/ml in 35 ul HBSS added to each well, and then incubated for 1
hour at 4.degree. C. Assay plates were then washed 3 times with
PBS. The mix of cells and antibody was then transferred to the
assay plate and incubated for 40 minutes at 37.degree. C. in the
presence of 0.2 mM MnCl.sub.2. The cells on the coated plates were
then washed four times in warm HBSS, and the cells were thereafter
frozen at -80.degree. C. for one hour. The cells were allowed to
thaw at room temperature for one hour, and then 100 .mu.L of
CyQuant dye/lysis buffer (Molecular Probes) was added to each well
according to the manufacturer's instructions. Fluorescence was read
at an excitation wavelength of 485 nm and an emission wavelength of
530 nm. The majority of the antibodies showed little to no blockade
in this assay, suggesting that their specificity is primarily
against .alpha.5 or .alpha.5.beta.1.
TABLE-US-00013 TABLE 13 Inhibition of .alpha.4.beta.1 mediated J6
cell adhesion to GST-CS-1. MAb ID Average % inhibition 7B1.3A1 -7
4E9 -32 2H12 -12 2G2.2B5 -11 3F12.4A1 -2 Inhibition at 5 ug/ml
expressed as a percentage.
Example 12
Lead Antibodies do not Inhibit of avb3 and avb5 Mediated Adhesion
to Osteopontin (OPN) and Vitronectin (VN)
[0327] To confirm the purified antibodies did not cross-react or
inhibit av integrins the ability of a smaller subset of clones to
block A375M (ATCC CRL 1619) adhesion to vitronectin (VN) and
osteopontin (OPN) was assessed. Plates were coated with either 1.25
ug/mL purified human VN (Becton Dickinson) or 313 ng/mL GST OPN
aa17-168 in phosphate buffer pH9.0, overnight at 4.degree. C.
Plates were then washed and pre-blocked with 3% BSA/PBS for 1 hour
prior to the assay.
[0328] A375M cells were cultured in DMEM (Hepes modification) with
L-Glutamine, sodium pyruvate, and 10% FCS. Cells were trypsinised,
pelleted and washed 3.times. in HBSS, then resuspended in HBSS at
appropriate concentration (30000 cells in 35 uL HBSS) and 35 uL of
2.times. antibody, each antibody was at a final of 5 ug/ml. Cells
and antibody were co-incubated for 40 min at 4.degree. C. Assay
plates were then washed 3 times with HBSS, the cells and antibody
transferred to the assay plate and incubated for 40 minutes at
37.degree. C. The plates were then washed 3 times in warm HBSS. To
determine the number of cells bound, plates were placed at
-80.degree. C. for 20 minutes, thawed at room temperature and 100
uL of CyQuant dye/lysis buffer to each well as per Molecular Probes
procedure. Plates were read Flourescence at 485 nm excitation and
530 emission. As a positive control the commercial av integrin
neutralising antibody L230 (Chemicon) was included. Collectively
the data confirm that these antibodies are specific for
.alpha.5.beta.1 integrin over avb3 and avb5 integrins.
TABLE-US-00014 TABLE 14 Inhibition of A375M adhesion to vitronectin
and osteopontin. Vitronectin Osteopontin Adhesion Adhesion MAb ID
(% inhibition) (% inhibition) 7B1.3A1 21 14 2H12 12 11 L230 96 97
Activity is expressed as % inhibition at 5 ug/ml.
Example 13
Inhibition of .alpha.5.beta.1 Mediated Adhesion of HUVEC Cells to
Fibronectin
[0329] To determine whether the .alpha.5.beta.1 blocking antibodies
were able to block .alpha.5.beta.1, function on HUVECs and adhesion
assay was performed. Black clear-bottomed plates were incubated
overnight at 4.degree. C. with 100 ul per well of a GST fusion
protein fibronectin fragment 9-10 at 10 ug/ml in PBS. The following
day plates were washed and blocked with 1% BSA in PBS. The adhesion
assay was carried out in MCDB131 media using 25000 HUVEC cells per
well. To block av integrins (which also bind to fibronectin) cells
were also pre-incubated with the av integrin blocking antibody L230
at 10 ug/ml. The cells were incubated at 37 for 45 minutes, unbound
cell were washed away, the adhered cells were fixed and stained
with Hoescht. The number of cells adhered were counted on the
Arrayscan.
[0330] The data in FIG. 1 shows that 2H12 is an effective inhibitor
of .alpha.5.beta.1 integrin on HUVEC cells.
Example 14
2H12 Exhibits as Different Cellular Binding Profile from an
.alpha.5 Integrin Binding Antibody
[0331] To further explore the cellular binding profile of 2H12
adhesion of a range of cell lines to each antibody was assessed.
Briefly 1 ug of each antibody indicated was coated onto a 96 well
plate in PBS overnight at 4.degree. C. then blocked with PBS/3% BSA
for 1 hour at 37.degree. C. Plates were washed and the cell lines
indicated were added to the wells in RPMI 10% FCS (c.
5.times.10.sup.4 per well). Plates were incubated for 45 minutes at
37.degree. C., then washed twice in PBS, fixed with 100% ethanol
and stained with Hoescht. The number of cells adhered were counted
on an a Cellomics Arrayscan.
[0332] The data demonstrate that 2H12 shows a differentiated
binding profile, although .alpha.5.beta.1 is a key binding partner
it is clear that it binds another cellular integrin. As such it is
differentiated from classic integrin antibodies such as IIA1.
TABLE-US-00015 TABLE 15 Binding of cells to a range of integrin
specific antibodies including 2H12. avb6 a5 av avb3 avb5 a4 b1
.alpha.5.beta.1 Cell line 10D5 IIA1 L230 LM609 P5H9 7.2R 4B4 2H12
A549 434 6431 5408 2700 6823 0 5898 6963 Calu3 3293 0 0 0 30 0 4390
1506 Calu 6 0 5243 1708 0 3066 0 5536 5030 Colo205 suspension 0 0
49 0 0 0 5276 534 Colo205 adhered 1917 3 2629 28 0 0 7283 6264
Detroit562 5209 2467 4092 3377 4106 0 5193 3279 H358 3496 8 2367 11
239 0 5241 1977 H1299 7 1548 1346 73 0 1078 1668 874 H1437 6170
6984 4801 0 6915 0 7089 6553 H1793 952 3861 3474 3850 2288 389 3800
4345 H1975 1495 4535 2756 4246 4124 11 3773 3242 H2085 234 137 2818
3226 2905 33 4095 3246 H2122 4553 139 705 0 3014 22 5353 3302 H2291
234 0 8 0 0 0 979 13 HCT-8 0 8176 3284 0 6732 0 7323 9045 HCT116 0
8297 3776 0 4887 0 7883 8312 HT-29 2491 0 1305 9 5116 0 7327 21
HUVEC 24 1477 267 2002 0 0 1822 1720 HX147 0 3158 755 2359 2037 0
3088 2718 LOVO 0 0 1171 0 0 0 8813 0 MB231 0 4431 2953 5579 3280 0
4555 4056 MB468 4142 0 1699 0 1415 0 7158 5577 PE/CA-PJ15 3605 2970
2336 1644 2449 0 3690 2968 PC9 304 0 11 0 1590 0 4899 0 SHP77 0 0
360 0 0 0 7089 0 SKCO-1 0 0 0 0 0 0 7355 0 SW403 BOD 0 0 719 0 51 0
7015 0 SW620 16 119 196 0 94 0 11330 5660 SW948 BOD 20 0 0 0 14 0
1699 0
Example 15
Determination of Binding Affinity of Purified Antibodies
[0333] To assess the affinity of the antibodies for
.alpha.5.beta.1, a FACS based KD analysis was employed. As the
antibodies do not interact well with recombinant integrin classical
Biacore analysis was not considered relevant. K562 cells expressing
.alpha.5.beta.1 were resuspended in filtered HBS buffer containing
1 mM MgCl.sub.2 and 1 mM CaCl.sub.2 at a concentration of
approximately 2.5-6 million cells/mL. The cells were kept on ice.
Serially diluted (2.times.) mAbs across 11 wells in a 96-well
plate. All mAbs were diluted in HBS described above. Additional HBS
and cells were added to each well so that the final volume was 300
.mu.L/well and each well contained approximately 140,000-150,000
cells. The final molecular concentration range for each mAb was
2H12: 7.5 nM-14.7 pM. Cells were incubated on a plate shaker for 5
hours at 4.degree. C. and then were spun/washed 3.times. at
4.degree. C. with HBS. 250 .mu.L of 99 nM Cy5 goat .alpha.-human
polyclonal antibody (Jackson ImmunoResearch Laboratories,
#109-175-008) was added to each well, and incubated with shaking
for 40 minutes at 4.degree. C. The cells were again spun/washed
3.times. at 4.degree. C. with HBS. The Mean Fluorescence (F) of
7000 "events" was determined using FACS Canto II HTS flow cytometry
instrumentation. To calculate affinity, data was fitted nonlinearly
to a plot of the Mean Fluorescence as a function of molecular mAb
concentration with Scientist 3.0 software using the equation:
F = P ' ( K D + L T + n M ) - ( K D + L T + n M ) 2 - 4 n M L T 2 +
B ##EQU00001##
In this equation, F=mean fluorescence, L.sub.T=total molecular mAb
concentration, P'=proportionality constant that relates arbitrary
fluorescence units to bound mAb, M=cellular concentration in
molarity, n=number of receptors per cell, B=background signal, and
K.sub.D=equilibrium dissociation constant. For each mAb titration
curve an estimate for K.sub.D is obtained as P', n, B, and K.sub.D
are allowed to float freely in the nonlinear analysis. (A. W. Drake
and S. L. Klakamp. A rigorous multiple independent binding site
model for determining cell-based equilibrium dissociation
constants. J. Immunol. Methods, 2007, Vol. 318, 157-162.)
[0334] Each plot with the nonlinear fit (green line) is shown
below. The table lists the resulting K.sub.D for each mAb in order
of decreasing affinity along with the 95% confidence interval of
the fit. The nonlinear fit for each titration curve with the
4-parameter model was able to return reasonable 95% confidence
intervals for K.sub.D. This implies each curve possesses some
K.sub.D influence and hence the returned values for K.sub.D are
most likely acceptable estimates of either the stoichiometric or
site-binding dissociation constant.
TABLE-US-00016 TABLE 16 Affinity of 2H12 for cellular
.alpha.5.beta.1 MAb ID K.sub.D (pM) 95% CI (pM) 2H12 162
.+-.61.4
Example 16
2H12 Inhibits Angiogenesis in an In Vitro Co-Culture Model of
Endothelial Tube Formation
[0335] .alpha.5.beta.1 integrin is thought to play a role in
regulating the formation of new blood vessels. To understand
whether the antibodies have the potential to modulate endothelial
cell function, the ability of the antibodies to impact endothelial
tube formation were assessed using an in vitro co-culture assay and
an in vivo angiogenesis assay.
In Vitro Co-Culture Assay:
[0336] Endothelial cells are cultured on a monolayer or feeder
layer of dermal fibroblast, and over a period of 11 days network of
endothelial cell tubes is established. The co-culture kit was
purchased from TCS Biologicals (UK) and performed as per
manufacturers instructions. Antibodies were dosed at the
concentrations indication and the media changed every 2-3 days.
Tubes were visualised by staining for PECAM/CD31 as per manufactors
instructions, and quantitated using the neurite outgrowth algorithm
on a KS400.
Example 17
Determination of In Vitro Potency of 2H12 Variants
[0337] 2H12 was identified as an IgG4 antibody. To assess the
potential of other formats of the antibody 2H12 was isotype
switched to an IgG2 format. In addition a key mutation from
germline in the framework region, a threonine at position 77 was
mutated to Asparagine. This antibody will be referred to as 2H12
Variant 1. Both forms of the antibody were assayed versus 2H12 WT
as IgG4 for equivalient potency. As outlined previously K562 cells
were allowed to adhere to fibronectin in the presence and absence
of the 2H12 variants. 96 well high protein binding plates were
coated overnight at 4.degree. C. with 1 .mu.g GST-fibronectin
repeats 8-10. The following day the plates were washed 3.times.
with PBS and blocked with PBS/3% BSA for 1 hour at 37.degree. C.
Antibodies were prepared at 10.times. the final assay concentration
in HBSS, 50 mM HEPES, 0.2 mM MnCl2. The K562 cells were resuspended
in HBSS, 50 mM HEPES, 0.2 mM MnCl2 and plated at 200,000 cells per
well, and allowed to adhere at 37.degree. C., 5% CO.sub.2 for 45
minutes. Non-adhered cells were flicked from the plate and the
wells washed 3.times. with PBS and fixed with 100% ethanol for 30
minutes at RT. Cells were then stained with 0.1% crystal violet,
washed then solublised with 0.1% Triton X100. The OD at 570 nm was
read and the data plotted. Both IgG2 variants showed equivalent
potency to the IgG4 version of 2H12. Given the potential issue
associated with having a threonine at position 77 of the heavy
chain (deviation of germiline resulting in loss of glycosylation
site), 2H12 Variant 1 was used for large scale production and
subsequent in vivo experiments.
[0338] As illustrated in FIG. 3, 2H12 IgG4, 2H12 IgG2 WT and 2H12
IgG2 Variant 1 imhibit binding of K562 cells to fibronectin with
similar potencies.
Example 18
Determination of In Vivo Efficacy of Purified Antibodies:
Evaluation of the Antiangiogenic Efficacy on a Spheroid-Based In
Vivo Angiogenesis Assay
[0339] As the antibodies do not cross react with mouse integrin,
the impact of the antibodies targeting .alpha.5.beta.1 on vessel
formation was assessed in a matrigel plug seeded with human
endothelial cells. In this model the human endothelial cells form
functional angiogenic vessels allowing the therapeutic impact of
the antibodies to be studied. Human umbilical vein endothelial cell
(HUVEC) spheroids were prepared as described earlier (Korff and
Augustin: J Cell Biol 143: 1341-52, 1998) by pipetting 100
endothelial cells (EC) in a hanging drop on plastic dishes to allow
overnight spheroid formation. The following day, using the method
previously described (Alajati et al: Nature Methods 5:439-445,
2008), EC spheroids were harvested and mixed in a Matrigel/fibrin
solution with single HUVECs to reach a final number of 100,000 ECs
as spheroids and 200,000 single ECs per injected plug. VEGF-A and
FGF-2 were added at a final concentration of 1000 ng/ml. Human
umbilical vein endothelial cell (HUVEC) spheroids (1000 spheroids;
100 cells/spheroid) and HUVECs in suspension (200,000 cells) were
injected subcutaneously into the flank of a SCID mouse in a
Matrigel/fibrin matrix containing VEGF-A/FGF-2 (each 1000 ng/ml).
The following day (day 1) treatment commenced. At day 21 the study
was terminated. The matrix plugs were removed and fixed in 4% PFA.
All matrix plugs were paraffin embedded and cut to a thickness of
8-10 .mu.m section for histological examination. Blood vessels were
visualized by staining for human CD34 and smooth muscle actin (SMA)
and the microvessel density (MVD) and pericyte coverage was
determined.
[0340] As illustrated in FIG. 4, MAb 2H12 Variant 1 is effective in
inhibiting vessel formation in vivo.
Example 19
2H12 Variant 1 Inhibits Growth of U87Mg and A549 Tumours by
Targeting Both the Tumour Cells and Host Cells
[0341] .alpha.5.beta.1 plays a role in regulating the function of a
number of different cell types including endothelial cells and
tumour cells. To explore the direct effects of targeting
.alpha.5.beta.1 inhibition in the tumour cell compartment in vivo
we exploited the fact that 2H12 Variant 1 does not cross react with
murine integrin, and assessed impact of growth on human xenografts.
U87-MG and MDA-MB-231 tumours were established by s.c injection of
2.5.times.10.sup.6 cells alone and 1.times.10.sup.7 cells in 50%
matrigel respectively, into the hind flank of nude (nu/nu genotype)
mice. Dosing, 20 mg/kg i.p. twice weekly, commenced when tumours
were established.
[0342] In order to model the impact of inhibiting .alpha.5.beta.1
simultaneously in both the host and tumour a strain of SCID mice
were bred in which the murine alpha5 chain was replaced with the
human alpha5 chain. The impact of 2H12 Variant 1 on the growth of
A549 in the human a5 expressing SCID animals was assessed.
Transgenic mice expressing human .alpha.5 were implanted with A549
tumours, established by s.c. injection of 2.times.10.sup.6 cells
(without matrigel) into the hind flank of SCID (.alpha.5.beta.1
ko/ki/SCID (h.alpha.5.beta.1-SCID)) mice. Dosing, 20 mg/kg i.p.
twice weekly, commenced to when tumours were established.
[0343] The data (Table 17) demonstrate that 2H12 Variant 1 impacts
tumour growth either directly by targeting the tumour, or
indirectly by targeting the host.
TABLE-US-00017 TABLE 17 Inhibition of tumour growth by 2H12 Variant
1 % Inhibition Geometric Mean MAb ID Model Delta Volume 2H12 U87-MG
(nude) 61*** Variant 1 A549 (ha5 SCID) 63** ***p < 0.001; **p
< 0.01; *p < 0.05; NS--not significant
Example 20
2H12 Variant 1 Shows Reduced Total Binding to Whole Blood Binding
Compared to a Conventional A5B1 Blocking Antibody Assay
[0344] The Heavy chain of the 2H12CDR3 contains an RGD sequence and
shows cation dependent binding, this mode of binding is different
from other antibodies and could influence binding of the antibody
to different cell types. Human blood was taken into heparin
containing tubes. To determine the binding profile of the antibody
in whole human blood the antibodies were biotinylated using the
Zenon antibody biotinylation labeling kit. 18 ug of each
.alpha.5.beta.1, and IgG2 antibody was labelled with biotin using
the Zenon labelling kit as per the manufacturers instructions.
Aliquots of blood were incubated with either IgG2, 3C5 (a non RGD
containing antibody) or 2H12 for 1 hour at room temperature with
agitation. Each antibody was incubated with 1 ml of whole blood to
give final antibody concentrations of 7.5. 5, 2.5, 1, 0.5, 0.25,
0.1 and 0 ug/ml. The washed WBC cell pellet was resuspended in 200
.mu.l of PBS/1% BSA/ and the primary antibody added and incubated
with the primary antibody for 45 minutes on ice. The samples were
then washed in 5 ml of PBS/1% BSA. To detect bound antibody the
cells were incubated in 200 .mu.l of PBS/1% BSA with
streptavidin-PE. The samples were washed three times as before in
PBS/1% BSA. Each pellet was resuspended in 300 .mu.l of PBS and
transferred to a FACS tube. Binding to the monocyte population was
determined by sorting on a FACS Aria (Becton Dickinson).
Surprisingly the two antibodies exhibited different binding
profiles to monocytes in whole human blood. The binding of 2H12 was
far lower than 3C5, a non-RGD containing integrin targeting
antibody. Clearly 2H12 has a differentiated binding profile to
human peripheral blood monocytes. This could indicate that 2H12
only binds activated integrin, and as such could confer a different
pharmacokinetic (PK) profile in man
[0345] As illustrated in FIG. 5, the data shows the binding profile
of biotinylated .alpha.5.beta.1 binding antibodies 3C5 (IgG2) and
2H12 Variant 1 (IgG2), versus IgG2 control, to monocytes in the
whole human blood is different.
Example 21
Inhibition of Tumour Cell Growth in Human Patients
[0346] A group of human cancer patients diagnosed with pancreatic
cancer is randomized into treatment groups. Each patient group is
treated with weekly intravenous injections of fully human
monoclonal antibodies against .alpha.5.beta.1 as described herein.
Each patient is dosed with an effective amount of the antibody
ranging from 5 mg/kg/week to 20 mg/kg/week for 4-8 months. A
control group is given only the standard chemotherapeutic
regimen.
[0347] At periodic times during and after the treatment regimen,
tumour burden is assessed by magnetic resonance imaging (MRI). It
can be expected that the patients who have received weekly antibody
treatments will show significant reductions in tumour size, time
delay to progression or prolonged survival compared to patients
that do not receive antibody treatment. In some treated patients,
it can be expected that the tumours are no longer detectable. In
contrast, it can be expected that tumour size increases or remains
substantially the same in the control group.
Example 22
Inhibition of Colon Cancer in a Human Patient
[0348] A group of human cancer patients diagnosed with colon cancer
is randomized into treatment groups. Each patient group is treated
3-weekly with intravenous injections of fully human monoclonal
antibodies against .alpha.5.beta.1 as described herein. Each
patient is dosed with an effective amount of the antibody ranging
from 5 mg/kg/week to 20 mg/kg/week for 4-8 months. A control group
is given only the standard chemotherapeutic regimen. At periodic
times during and after the treatment regimen, tumour burden is
assessed by magnetic resonance imaging (MRI). It it can be expected
that the patients who have received 3-weekly antibody treatments
show significant reductions in tumour size, time delay to
progression or prolonged survival compared to patients that do not
receive the antibody treatment. In some treated patients, it can be
expected that the tumours are no longer detectable. In contrast, it
can be expected that tumour size increases or remains substantially
the same in the control group.
Example 23
Inhibition of Melanoma in a Human Patient
[0349] A group of human cancer patients diagnosed with melanoma is
randomized into treatment groups. Each patient group is treated
3-weekly with intravenous injections of fully human monoclonal
antibodies against .alpha.5.beta.1 as described herein. Each
patient is dosed with an effective amount of the antibody ranging
from 5 mg/kg/week to 20 mg/kg/week for 4-8 months. A control group
is given only the standard chemotherapeutic regimen. At periodic
times during and after the treatment regimen, tumour burden is
assessed by magnetic resonance imaging (MRI). It it can be expected
that the patients who have received 3-weekly antibody treatments
with antibodies against .alpha.5.beta.1 show significant reductions
in melanoma, time delay to progression or prolonged survival
compared to patients that do not receive the antibody treatment. In
some treated patients, it can be expected that the melanoma lesions
are no longer detectable. In contrast, it can be expected that
melanoma increases or remains substantially the same in the control
group.
Example 24
Inhibition of Chronic Myelogenous Leukemia (CML) in a Human
Patient
[0350] A group of human cancer patients diagnosed with CML is
randomized into treatment groups. Each patient group is treated
3-weekly with intravenous injections of fully human monoclonal
antibodies against .alpha.5.beta.1 as described herein. Each
patient is dosed with an effective amount of the antibody ranging
from 5 mg/kg/week to 20 mg/kg/week for 4-8 months. A control group
is given only the standard chemotherapeutic regimen. At periodic
times during and after the treatment regimen, tumour burden is
assessed by magnetic resonance imaging (MRI). It it can be expected
that the patients who have received 3-weekly antibody treatments
show significant reductions in CML, time delay to progression or
prolonged survival compared to patients that do not receive the
antibody treatment. In some treated patients, it can be expected
that the CML is no longer detectable. In contrast, it can be
expected that CML increases or remains substantially the same in
the control group.
Example 25
Inhibition of Tumour Cell Growth in a Human Patient
[0351] A human patient is diagnosed with a malignant tumour. The
patient is treated with weekly intravenous injections of fully
human monoclonal antibodies against .alpha.5.beta.1 as described
herein for 8 weeks. At periodic times during and after the
treatment regimen, tumour burden is assessed by magnetic resonance
imaging (MRI). It can be expected that significant reductions in
tumour size are found.
Example 26
2H12IgG1TM Inhibits Tumor Growth in Nude Mice
[0352] For a target with potentially broad tissue expression, like
.alpha.5.beta.1, antibody dependent cell cytotoxicity can cause
toxicity issues when dosed chronically in the general population.
For that reason the ability of 2H12 Variant 1 to inhibit tumour
cell growth in an IgG1TM format was assessed. As described earlier
(see EXAMPLE 19) 2H12 Variant 1 was dosed twice weekly at 20 mg/kg
to mice bearing U87MG tumour xenografts. U87-MG tumours were
established by s.c injection of 2.5.times.10.sup.6 cells alone into
the hind flank of nude (nu/nu genotype) mice. Dosing, 20 mg/kg i.p.
twice weekly, commenced when tumours were established. 2H12IgG1TM
inhibited growth of the tumours. These data demonstrate that 2H12
Variant 1 IgG1 TM is effective at inhibiting tumour growth in vivo,
and supports the conclusion that the antibody has potential as a
therapeutic in this format.
Example 27
2H12IgG1TM is as Effective as 2H12IgG2 at Inhibiting Adhesion of
K562 Cel to Fibronectin
[0353] To assess the in vitro activity of 2H12 as an IgG1TM format,
inhibition of adhesion of K562 cells to fibronectin was compared
for 2H12IgG1TM and 2H12IgG2. Both 2H12IgG1TM and 2H12IgG2 Abs were
Variant 1 Abs. As outlined previously K562 cells were allowed to
adhere to fibronectin in the presence and absence of the 2H12
variants. 96 well high protein binding plates were coated overnight
at 4.degree. C. with 1 .mu.g GST-fibronectin repeats 8-10. The
following day the plates were washed 3.times. with PBS and blocked
with PBS/3% BSA for 1 hour at 37.degree. C. Antibodies were
prepared at 10.times. the final assay concentration in HBSS, 50 mM
HEPES, 0.2 mM MnCl2. The K562 cells were resuspended in HBSS, 50 mM
HEPES, 0.2 mM MnCl2 and plated at 200,000 cells per well, and
allowed to adhere at 37.degree. C., 5% CO2 for 45 minutes.
Non-adhered cells were flicked from the plate and the wells washed
3.times. with PBS and fixed with 100% ethanol for 30 minutes at RT.
Cells were then stained with 0.1% crystal violet, washed then
solublised with 0.1% Triton X100. The OD at 570 nm was read and the
data plotted. Both formats of the antibody were as effective at
blocking binding of K562 cells to fibronectin demonstrating that
the TM format does not impact the activity of 2H12 variants.
DEPOSIT OF BIOLOGICAL MATERIAL
[0354] A deposit of E. coli Top 10 containing a plasmid which
encodes the 2H12 antibody light chain was made under the terms of
the Budapest Treaty on Nov. 26, 2009 at the National Collections of
Industrial and Marine Bacteria (NCIMB) Ltd., Ferguson Building,
Craibstone Estate, Bucksburn, Aberdeen, Scotland, AB21 9YA, UK and
has been assigned Accession No. 41666.
[0355] A deposit of E. coli Top 10 containing a plasmid which
encodes the 2H12 antibody heavy chain variant 1 was made under the
terms of the Budapest Treaty on Nov. 26, 2009 at the NCIMB Ltd.,
Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen,
Scotland, AB21 9YA, UK and has been assigned Accession No.
41667.
EQUIVALENTS
[0356] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The foregoing description and Examples detail certain
preferred embodiments of the invention and describes the best mode
contemplated by the inventors. It will be appreciated, however,
that no matter how detailed the foregoing may appear in text, the
invention may be practiced in many ways and the invention should be
construed in accordance with the appended claims and any
equivalents thereof.
Sequence CWU 1
1
361378DNAHomo sapiens 1caggtgcagc tggtggagtc ggggggaggc gtggtccagc
ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagg agctatggca
tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg ggtggcagtt
ctatggtatg atggaagtaa taaaaactat 180gcagactccg tgaagggccg
attcaccatc tccagagaca attccaagaa cacgttgtat 240ctgcaaatga
acagcctgag agccgaggac acggctatgt attactgtgc gagagatgga
300gcagctcgtc gcggagatta ctattactac cacggtatgg acgtctgggg
ccaagggacc 360acggtcaccg tctcctct 3782126PRTHomo sapiens 2Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 25
30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Val Leu Trp Tyr Asp Gly Ser Asn Lys Asn Tyr Ala Asp Ser
Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Met Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ala Ala Arg Arg Gly Asp Tyr
Tyr Tyr Tyr His Gly 100 105 110Met Asp Val Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser 115 120 1253336DNAHomo sapiens 3gatattgtga
tgactcagtc tccactctcc ctgcccgtca cccctggaga gccggcctcc 60atctcctgca
ggtctagtca gagcctcctg caaagtactg gatacaacta tttggattgg
120tacctgcaga agccagggca gtctccacaa ctcctgatct atttgggttc
taatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca
cagattttac actgaaaatc 240agcagagtgg aggctgagga tgttggggtt
tattactgca tgcaagctct acaaactcca 300ttcactttcg gccctgggac
caaagtggat atcaaa 3364112PRTHomo sapiens 4Asp Ile Val Met Thr Gln
Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile
Ser Cys Arg Ser Ser Gln Ser Leu Leu Gln Ser 20 25 30Thr Gly Tyr Asn
Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu
Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95Leu Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile
Lys 100 105 1105378DNAHomo sapiens 5caggtgcagt tggtggagtc
ggggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagg agctatggca tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcagtt atatggtatg atggaagtaa taaatactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctatgt
attactgtgc gagagatgga 300gcagctcgtc gcggagatta ctactactac
cacggtatgg acgtctgggg ccaagggacc 360acggtcaccg tctcctca
3786126PRTHomo sapiens 6Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
Phe Thr Phe Arg Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asp Gly Ser
Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95Ala Arg Asp Gly
Ala Ala Arg Arg Gly Asp Tyr Tyr Tyr Tyr His Gly 100 105 110Met Asp
Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
1257336DNAHomo sapiens 7gatattgtga tgactcagtc tccactctcc ctgcccgtca
cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcctg caaagtactg
gatacaacta tttggattgg 120tacctgcaga agccagggca gtctccacaa
ctcctgatct atttgggttc taatcgggcc 180tccggggtcc ctgacaggtt
cagtggcagt ggatcaggca cagattttac actgaaaatc 240agcagagtgg
aggctgagga tgttggggtt tattactgca tgcaagctct acaaactcca
300ttcactttcg gccctgggac caaagtggat atcaaa 3368112PRTHomo sapiens
8Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5
10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Gln
Ser 20 25 30Thr Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser
Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Met Gln Ala 85 90 95Leu Gln Thr Pro Phe Thr Phe Gly Pro
Gly Thr Lys Val Asp Ile Lys 100 105 1109379DNAHomo sapiens
9caggtccagc tggtcgagag cggcggaggg gtggtgcagc ccggcagaag cctgaggctg
60tcctgcgccg ccagcggctt caccttcagc agctacggca tgcactgggt gcggcaggcc
120ccaggcaagg gcctggaatg ggtggccgtg atctggtacg acggcaccaa
caagtactac 180gccgacagcg tgaagggcag gttcaccatc agcagggaca
acagcaagaa caccctgtac 240ctgcagatga acagcctgag ggccgaggac
accgccgtgt actactgcgc cagggacgga 300gccgccagaa ggggcgacta
ctactactat tacggcatgg acgtgtgggg ccagggcacc 360accgtgaccg tgagcagcg
37910126PRTHomo sapiens 10Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asp Gly
Thr Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Gly Ala Ala Arg Arg Gly Asp Tyr Tyr Tyr Tyr Tyr Gly 100 105 110Met
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
12511336DNAHomo sapiens 11gatattgtga tgactcagtc tccactctcc
ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcctg
cgtggtcatg gatacagcta tttggattgg 120tacctgcaga agccagggca
gtctccacag ctcctgatct atttgggttc taatcgggcc 180tccggggtcc
ctgacaggtt cagtggcagt ggatcaggca cagattttac actgaaaatc
240agcagagtgg aggctgagga tgttggggtt tattactgca tgcaagctct
acaaactcca 300ttcactttcg gccctgggac caaagtggat atcaaa
33612112PRTHomo sapiens 12Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu Arg Gly 20 25 30His Gly Tyr Ser Tyr Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu
Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95Leu Gln Thr
Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105
110135PRTHomo sapiens 13Ser Tyr Gly Met His1 51417PRTHomo sapiens
14Val Ile Trp Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp Ser Val Lys1
5 10 15Gly1517PRTHomo sapiens 15Asp Gly Ala Ala Arg Arg Gly Asp Tyr
Tyr Tyr Tyr His Gly Met Asp1 5 10 15Val1616PRTHomo sapiens 16Arg
Ser Ser Gln Ser Leu Leu Arg Gly His Gly Tyr Ser Tyr Leu Asp1 5 10
15177PRTHomo sapiens 17Leu Gly Ser Asn Arg Ala Ser1 5189PRTHomo
sapiens 18Met Gln Ala Leu Gln Thr Pro Phe Thr1 519378DNAHomo
sapiens 19caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc
cctgagactc 60tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt
ccgccaggct 120ccaggcaagg gactggagtg ggtggcagtt atatggtatg
atggaactaa taaatactat 180gcagactccg tgaagggccg attcaccatc
tccagagaca attccaagac cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagagatgga 300gcagctcgtc
gcggggatta ctactactac tacggtatgg acgtctgggg ccaagggacc
360acggtcaccg tctcctca 37820126PRTHomo sapiens 20Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala
Val Ile Trp Tyr Asp Gly Thr Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Thr Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Asp Gly Ala Ala Arg Arg Gly Asp Tyr Tyr Tyr Tyr
Tyr Gly 100 105 110Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser 115 120 12521336DNAHomo sapiens 21gatattgtga tgactcagtc
tccactctcc ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctagtca
gagcctcctg cgtggtcatg gatacagcta tttggattgg 120tacctgcaga
agccagggca gtctccacag ctcctgatct atttgggttc taatcgggcc
180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca cagattttac
actgaaaatc 240agcagagtgg aggctgagga tgttggggtt tattactgca
tgcaagctct acaaactcca 300ttcactttcg gccctgggac caaagtggat atcaaa
33622112PRTHomo sapiens 22Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu Arg Gly 20 25 30His Gly Tyr Ser Tyr Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu
Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95Leu Gln Thr
Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105
11023378DNAHomo sapiens 23caggtgcagc tggtggagtc tgggggaggc
gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt caccttcagt
agctatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcagtt atatggtatg atggaagtaa taaatactat 180gcagactccg
tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc
gagagatggg 300gcagcccgtc ggggagatta ctactactac tacggtatgg
acgtctgggg ccaagggacc 360acggtcaccg tctcctct 37824126PRTHomo
sapiens 24Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr
Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ala Ala Arg
Arg Gly Asp Tyr Tyr Tyr Tyr Tyr Gly 100 105 110Met Asp Val Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 12525336DNAHomo sapiens
25gagattgtga tgactcagtc tccactctcc ctgcccgtca ctcctggaga gccggcctcc
60atctcctgca ggtctggtca gagcctcctg caaagtactg gatccaacta tttggcttgg
120tacctgcaga agccagggca gtctccacag ctcctgatct atttgggttc
taatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt ggatcaggca
cagattttac actgaaaatc 240agcagagtgg aggctgagga tgttggggtt
tattactgca tgcaagctct acaaactcca 300ttcactttcg gccctgggac
caaagtggat atcaaa 33626112PRTHomo sapiens 26Glu Ile Val Met Thr Gln
Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile
Ser Cys Arg Ser Gly Gln Ser Leu Leu Gln Ser 20 25 30Thr Gly Ser Asn
Tyr Leu Ala Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu
Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg
Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75
80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala
85 90 95Leu Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile
Lys 100 105 11027378DNAHomo sapiens 27caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcagt agctatggca tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcagtt atatggtatg atggaactaa taaatactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgac agccgaggac acggctgtgt
attactgtgc gagagatatg 300gcagctcgtc ggggggatta ctactactac
tacggtatgg acgtctgggg cccagggacc 360acggtcaccg tctcctca
37828126PRTHomo sapiens 28Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asp Gly
Thr Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp
Met Ala Ala Arg Arg Gly Asp Tyr Tyr Tyr Tyr Tyr Gly 100 105 110Met
Asp Val Trp Gly Pro Gly Thr Thr Val Thr Val Ser Ser 115 120
12529336DNAHomo sapiens 29gatattgtga tgactcagtc tccactctcc
ctgcccgtca cccctggaga gccggcctcc 60atctcctgca ggtctagtca gagcctcctg
aatggtattg gatacaactt tttggattgg 120tacctgcaga agccagggca
gtctccacag ctcctgatct atttgggttc taatcgggcc 180tccggggtcc
ctgacaggtt cagtggcagt ggatcaggca cagattttac actgacaatc
240agcagagtgg aggctgagga tgttggggtt tattactgca tgcacgctct
acaaactcca 300ttcactttcg gccctgggac caaagtggat atcaaa
33630112PRTHomo sapiens 30Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu Asn Gly 20 25 30Ile Gly Tyr Asn Phe Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu
Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile65 70 75 80Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met His Ala 85 90 95Leu Gln Thr
Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105
11031378DNAHomo sapiens 31caggtgcagc tggtggagtc tgggggaggc
gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt catcttcagt
agctatggca tgcactgggt ccgccaggct 120ccaggcaagg ggctggagtg
ggtggcagtt atatggtatg atggaactaa taaatactat 180gcagactccg
tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat
240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc
gagagatggg 300gcagcccgtc ggggagatta ctactactac tacggtatgg
acgtctgggg ccaagggacc 360acggtcaccg tctcctca 37832126PRTHomo
sapiens 32Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro
Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe
Ser Ser Tyr 20 25 30Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asp Gly Thr Asn
Lys Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu
Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Asp Gly Ala
Ala Arg Arg Gly Asp Tyr Tyr Tyr Tyr Tyr Gly 100 105 110Met Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 12533336DNAHomo
sapiens 33gatattgtga tgactcagtc tccactctcc ctgcccgtca cccctggaga
gccggcctcc 60atctcctgca ggtctagtca gagcctcctg catggtactg gatacagctc
tttggattgg 120tacctgcaga agccagggca gtctccacag ctcctgatct
atttgggttc taatcgggcc 180tccggggtcc ctgacaggtt cagtggcagt
ggatcaggca cagattttac actgaaaatc 240agcagagtgg aggctgagga
tgttggggtt tatttctgca tgcaagctct acaaactcca 300ttcactttcg
gccctgggac caaagtggat atcaaa 33634112PRTHomo sapiens 34Asp Ile Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Gly 20 25 30Thr
Gly Tyr Ser Ser Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro
50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys
Ile65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Phe Cys
Met Gln Ala 85 90 95Leu Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys
Val Asp Ile Lys 100 105 11035121PRTHomo sapiens 35Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala
Val Ile Trp Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55
60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65
70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Ala Ala Arg Tyr Tyr Tyr Tyr Tyr Gly Met Asp Val
Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser 115
12036112PRTHomo sapiens 36Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro Gly1 5 10 15Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu His Ser 20 25 30Asn Gly Tyr Asn Tyr Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr Leu
Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95Leu Gln Thr
Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100 105 110
* * * * *