U.S. patent application number 11/833486 was filed with the patent office on 2008-03-06 for antibodies directed to alphavbeta6 and uses thereof.
Invention is credited to Avril Alfred, Simon Thomas Barry, Vahe Bedian, Ian Foltz, Julie Rinkenberger.
Application Number | 20080057063 11/833486 |
Document ID | / |
Family ID | 39146822 |
Filed Date | 2008-03-06 |
United States Patent
Application |
20080057063 |
Kind Code |
A1 |
Rinkenberger; Julie ; et
al. |
March 6, 2008 |
Antibodies Directed to AlphaVBeta6 and Uses Thereof
Abstract
Targeted binding agents, such antibodies directed to the antigen
.alpha.V.beta.6 and uses of such agents are described. In
particular, fully human monoclonal antibodies directed to the
antigen .alpha.V.beta.6 are disclosed. Nucleotide sequences
encoding, and amino acid sequences comprising, heavy and light
chain immunoglobulin molecules, particularly sequences
corresponding to contiguous heavy and light chain sequences
spanning the framework regions and/or complementarity determining
regions (CDR's), specifically from FR1 through FR4 or CDR1 through
CDR3 are disclosed. Hybridomas or other cell lines expressing such
immunoglobulin molecules and monoclonal antibodies are also
disclosed.
Inventors: |
Rinkenberger; Julie;
(Moraga, CA) ; Foltz; Ian; (Burnaby, CA) ;
Alfred; Avril; (Chilliwack, CA) ; Barry; Simon
Thomas; (Macclesfield, GB) ; Bedian; Vahe;
(Waltham, MA) |
Correspondence
Address: |
COOLEY GODWARD KRONISH LLP;ATTN: Patent Group
Suite 1100
777 - 6th Street, NW
WASHINGTON
DC
20001
US
|
Family ID: |
39146822 |
Appl. No.: |
11/833486 |
Filed: |
August 3, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60835559 |
Aug 3, 2006 |
|
|
|
Current U.S.
Class: |
424/138.1 ;
424/130.1; 435/320.1; 435/325; 435/69.1; 530/387.1; 530/391.1;
536/23.5 |
Current CPC
Class: |
A61P 37/06 20180101;
C07K 2317/567 20130101; A61P 17/00 20180101; C07K 16/2839 20130101;
C12N 2501/998 20130101; C12N 5/0686 20130101; C07K 2317/565
20130101; A61P 35/00 20180101; A61P 17/06 20180101; C07K 2317/92
20130101; A61P 9/14 20180101; A61P 13/00 20180101; A61P 11/00
20180101; A61P 5/14 20180101; A61P 1/16 20180101; A61P 25/02
20180101; A61P 27/02 20180101; A61P 37/08 20180101; A61P 9/00
20180101; C07K 2317/56 20130101; A61K 2039/505 20130101; A61P 25/00
20180101; A61P 1/04 20180101; A61P 35/02 20180101; A61P 19/02
20180101; C07K 2317/21 20130101; A61P 13/12 20180101; A61P 21/00
20180101; A61P 29/00 20180101; A61P 43/00 20180101; A61P 37/02
20180101; C07K 2317/76 20130101; A61P 31/12 20180101; A61P 15/00
20180101; A61P 13/08 20180101 |
Class at
Publication: |
424/138.1 ;
424/130.1; 435/320.1; 435/325; 435/069.1; 530/387.1; 530/391.1;
536/023.5 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 43/00 20060101 A61P043/00; C07K 16/18 20060101
C07K016/18; C12N 15/00 20060101 C12N015/00; C12N 15/11 20060101
C12N015/11; C12N 5/06 20060101 C12N005/06; C12P 21/04 20060101
C12P021/04 |
Claims
1. A targeted binding agent that specifically binds to
.alpha.V.beta.6 and inhibits binding of ligands to
.alpha.V.beta.6.
2. The targeted binding agent of claim 1, wherein said targeted
binding agent inhibits greater than 99% of TGF.beta.-LAP mediated
adhesion of HT29 cells.
3. The targeted binding agent of claim 1, wherein said targeted
binding agent inhibits TGF.beta.-LAP mediated adhesion of HT29
cells with an IC.sub.50 of less than 0.070 .mu.g/ml.
4. The targeted binding agent of claim 1, wherein said targeted
binding agent binds .alpha.V.beta.6 with a Kd of less than 35
nanomolar (nM).
5. The targeted binding agent of claim 1, wherein said targeted
binding agent binds .alpha.V.beta.6 with a Kd of less than 25
nanomolar (nM).
6. The targeted binding agent of claim 1, wherein said targeted
binding agent binds .alpha.V.beta.6 with a Kd of less than 10
nanomolar (nM).
7. The targeted binding agent, of claim 1, wherein said targeted
binding agent binds .alpha.V.beta.6 with a Kd of less than 60
picomolar (pM).
8. The targeted binding agent of claim 1, wherein said targeted
binding agent is a monoclonal antibody.
9. The targeted binding agent of claim 8, wherein said targeted
binding agent is a fully human monoclonal antibody.
10. The targeted binding agent of claim 9, wherein said targeted
binding agent is sc 264 RAD, sc 264 RAD/ADY, sc 188 SDM, sc 133, sc
133 TMT, sc 133 WDS, sc 133 TMT/WDS, sc 188, sc 254, sc 264 or sc
298.
11. The targeted binding agent of claim 11, wherein said binding
agent comprises at least the VH CDR3 having amino acids 98-102 of
SEQ ID NO.: 14.
12. The targeted binding agent of claim 1, wherein said binding
agent comprises at least the VH CDR3 having amino acids 99-113 of
SEQ ID NO.: 22.
13. The targeted binding agent of claim 1, wherein said binding
agent comprises at least the VH CDR3 having amino acids 99-117 of
SEQ ID NO.: 26.
14. The targeted binding agent of claim 1, wherein said binding
agent comprises at least the VH CDR3 having amino acids 99-114 of
SEQ ID NO.: 30.
15. The targeted binding agent of claim 1, wherein said binding
agent comprises at least the VH CDR3 having amino acids 97-113 of
SEQ ID NO.: 38.
16. The targeted binding agent of claim 1, wherein said binding
agent comprises a heavy chain polypeptide having the sequence of
SEQ ID NO.: 14.
17. The targeted binding agent of claim 1, wherein said binding
agent comprises a heavy chain polypeptide having the sequence of
SEQ ID NO.: 22.
18. The targeted binding agent of claim 1, wherein said binding
agent comprises a heavy chain polypeptide having the sequence of
SEQ ID NO.: 26.
19. The targeted binding agent of claim 1, wherein said binding
agent comprises a heavy chain polypeptide having the sequence of
SEQ ID NO.: 30.
20. The targeted binding agent of claim 1, wherein said binding
agent comprises a heavy chain polypeptide having the sequence of
SEQ ID NO.: 38.
21. The targeted binding agent of claim 1, wherein said binding
agent comprises a heavy chain polypeptide having the sequence of
SEQ ID NO.: 71.
22. The targeted binding agent of claim 1, wherein said binding
agent comprises a heavy chain polypeptide having the sequence of
SEQ ID NO.: 75.
23. An isolated antibody that binds .alpha.V.beta.6, wherein the
antibody comprises: a heavy chain variable region comprising CDR1,
CDR2, and CDR3 sequences; and a light chain variable region
comprising CDR1, CDR2, and CDR3 sequences, wherein the heavy chain
variable region CDR3 sequence comprises an amino acid sequence
selected from the group consisting of SEQ ID NO.: 14, SEQ ID NO.:
22, SEQ ID NO.: 26, SEQ ID NO.: 30 or SEQ ID NO.: 38, SEQ ID NO:71,
SEQ ID NO: 76, SEQ ID NO: 79 and conservative sequence
modifications thereof, and the light chain variable region CDR3
sequence comprises an amino acid sequence selected from the group
consisting of SEQ ID NOs: SEQ ID NO.: 16, SEQ ID NO.: 24, SEQ ID
NO.: 28, SEQ ID NO.: 32 or SEQ ID NO.: 40, SEQ ID NO:85, SEQ ID NO:
93, and conservative sequence modifications thereof.
24. An isolated fully human antibody that binds .alpha.V.beta.6,
wherein the antibody comprises: a heavy chain variable region
comprising CDR1, CDR2, and CDR3 sequences; and a light chain
variable region comprising CDR1, CDR2, and CDR3 sequences, wherein
the heavy chain variable region CDR3 sequence comprises an amino
acid sequence selected from the group consisting of SEQ ID NO.: 14,
SEQ ID NO.: 22, SEQ ID NO.: 26, SEQ ID NO.: 30 or SEQ ID NO.: 38,
SEQ ID NO:71, SEQ ID NO: 76, SEQ ID NO: 79 and conservative
sequence modifications thereof, and the light chain variable region
CDR3 sequence comprises an amino acid sequence selected from the
group consisting of SEQ ID NOs: SEQ ID NO.: 16, SEQ ID NO.: 24, SEQ
ID NO.: 28, SEQ ID NO.: 32 or SEQ ID NO.: 40, SEQ ID NO:85, SEQ ID
NO: 93, and conservative sequence modifications thereof.
25. An isolated antibody fragment that binds .alpha.V.beta.6,
wherein the antibody fragment comprises: a heavy chain variable
region comprising CDR1, CDR2, and CDR3 sequences; and a light chain
variable region comprising CDR1, CDR2, and CDR3 sequences, wherein
the heavy chain variable region CDR3 sequence comprises an amino
acid sequence selected from the group consisting of SEQ ID NO.: 14,
SEQ ID NO.: 22, SEQ ID NO.: 26, SEQ ID NO.: 30 or SEQ ID NO.: 38,
SEQ ID NO:71, SEQ ID NO: 76, SEQ ID NO: 79 and conservative
sequence modifications thereof, and the light chain variable region
CDR3 sequence comprises an amino acid sequence selected from the
group consisting of SEQ ID NOs: SEQ ID NO.: 16, SEQ ID NO.: 24, SEQ
ID NO.: 28, SEQ ID NO.: 32 or SEQ ID NO.: 40, SEQ ID NO:85, SEQ ID
NO: 93, and conservative sequence modifications thereof.
26. The targeted binding agent of claim 1, in association with a
pharmaceutically acceptable carrier.
27. The targeted binding agent of claim 1, wherein said targeted
binding agent is a fully human isolated binding fragment of an
anti-.alpha.V.beta.6 antibody.
28. A nucleic acid molecule encoding the targeted binding agent of
claim 1.
29. A vector comprising the nucleic acid molecule of claim 26.
30. A host cell comprising the vector of claim 27.
31. A method of producing a targeted binding agent, comprising
culturing a host cell of claim 30.
32. A nucleic acid molecule encoding an antibody of claim 23.
33. A vector comprising the nucleic acid molecule of claim 32.
34. A host cell comprising the vector of claim 33.
35. A method of producing an antibody, comprising culturing a host
cell of claim 34.
36. A nucleic acid molecule encoding a fully human antibody of
claim 24.
37. A vector comprising the nucleic acid molecule of claim 36.
38. A host cell comprising the vector of claim 37.
39. A method of producing a fully human antibody, comprising
culturing a host cell of claim 38.
40. A nucleic acid molecule encoding an antibody fragment of claim
25.
41. A vector comprising the nucleic acid molecule of claim 40.
42. A host cell comprising the vector of claim 41.
43. A method of producing an antibody fragment, comprising
culturing a host cell of claim 42.
44. A method of treating a malignant tumor in an animal, comprising
administering to said animal in need thereof a therapeutically
effective dose of the targeted binding agent of claim 1.
45. The method of claim 29, wherein said animal is human.
46. The method of claim 29, wherein said targeted binding agent is
the monoclonal antibody sc 264 RAD, sc 264 RAD/ADY, sc 188 SDM, sc
133, sc 133 TMT, sc 133 WDS, sc 133 TMT/WDS, sc 188, sc 254, sc 264
or sc 298.
47. The method of claim 29, wherein said malignant tumor is
selected from the group consisting of: melanoma, small cell lung
cancer, non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, thyroid tumor, 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.
48. A method of treating inflammation, comprising administering to
an animal in need thereof a therapeutically effective dose of the
targeted binding agent of claim 1.
49. The method of claim 33, wherein said animal is human.
50. The method of claim 33, wherein said targeted binding agent is
a fully human monoclonal antibody.
51. The method of claim 33, wherein the targeted binding agent
binds to .alpha.V.beta.6.
52. A method of inhibiting the interaction of .alpha.V.beta.6 with
it's ligand, comprising contacting .alpha.V.beta.6 with a targeted
binding agent of claim 1.
53. A method of treating a malignant tumor in an animal, comprising
administering to said animal in need thereof a therapeutically
effective dose of an antibody of claim 23.
54. A method of treating inflammation, comprising administering to
an animal in need thereof a therapeutically effective dose of an
antibody of claim 23.
55. A method of treating a malignant tumor in an animal, comprising
administering to an animal in need thereof a therapeutically
effective dose of a fully human antibody of claim 24.
56. A method of treating inflammation, comprising administering to
an animal in need thereof a therapeutically effective dose of a
fully human antibody of claim 24.
57. A method of treating a malignant tumor in an animal, comprising
administering to an animal in need thereof a therapeutically
effective dose of an antibody fragment of claim 25.
58. A method of treating inflammation, comprising administering to
an animal in need thereof a therapeutically effective dose of an
antibody fragment of claim 25.
59. A conjugate comprising a targeted binding agent of claim 1.
60. A conjugate comprising an antibody of claim 23.
61. A conjugate comprising a fully human antibody of claim 24.
62. A conjugate comprising an antibody fragment of claim 25.
Description
[0001] This application claims priority to U.S. provisional
application 60/835,559, filed Aug. 3, 2006, which is incorporated
by reference herein in its entirety for all purposes.
FIELD
[0002] The invention relates to monoclonal antibodies against
alphaVbeta6 integrin (.alpha.V.beta.6) and uses of such antibodies.
In some embodiments, the invention relates to fully human
monoclonal antibodies directed to .alpha.V.beta.6. The described
antibodies are useful as diagnostics and for the treatment of
diseases associated with the activity and/or overproduction of
.alpha.V.beta.6.
BACKGROUND
[0003] 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 tumor
invasion and metastasis (ffrench-Constant and Colognato, (2004)
Trends Cell Biol. 14: 678-86). 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 special 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).
[0004] 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 heterodimers (Thomas et al., (2006) J. Oral Pathol.
Med. 35: 1-10). While the .alpha.V chain can pair with 5 known beta
chains, several of these beta chains can only pair with .alpha.V.
The .beta.6 chain is selective for heterodimerization to .alpha.V
and this pair binds extracellular matrix and cytokine proteins with
either high or low affinity. .alpha.V.beta.6 binds to the RGD
motifs on both TGF.beta.1LAP and TGF.beta.3LAP latent complexes and
activates them (Munger et al, (1999) Cell 96: 319-328; Annes et
al., (2002) FEBS Letters 511: 65-68). However, it does not bind to
or activate TGF.beta.2LAP, which does not have the tri-peptide
(Ludbrook et al., (2003) Biochem. J. 369: 311-18).
.alpha.V.beta.6-mediated activation of TGF.beta. requires the
latent TGF.beta. binding protein 1 (LTBP1), which tethers the
latent TGF.beta. complex to the extracellular matrix. Activation is
proposed to result from a conformation change induced as the
TGF.beta.LAP is held between the cell and the matrix by
.alpha.V.beta.6 and LTBP1, respectively (Keski-Oja et al., (2004)
Trends Cell Biol. 14: 657-659; Annes et al., (2004) J. Cell Biol.
165: 723-34). The picoMolar binding affinity of .alpha.V.beta.6 for
the TGF.beta.LAP complexes is the highest for any of its known
ligands. Other ligands for .alpha.V.beta.6 include fibronectin,
tenascin, vitronectin and osteopontin (Busk et al., (1992) J. Biol.
Chem. 267: 5790-6; Prieto et al., (1993) PNAS 90: 10154-8; Huang et
al., (1998) J. Cell. Sci. 111(Pt 15): 2189-95; Yokosaki et al.,
(2005) Matrix Biol. 24: 418-27). The binding affinity of
.alpha.V.beta.6 for these extracellular matrix proteins is low
affinity and in the nanoMolar range.
[0005] Expression of .alpha.V.beta.6 integrin is restricted to
areas of active tissue remodeling in the adult, specifically on the
epithelia of healing wounds and at the edge of invading tumors
(Breuss et al., (1995) J. Cell Sci. 108: 2241-51). Keratinocytes at
the wound edge upregulate the expression of .alpha.V.beta.6 during
their migration into the wound, but expression remains high after
the edges of the wound epithelium have joined (Breuss et al.,
(1995) J. Cell Sci. 108: 2241-51; Haapasalmi et al., (1996) J.
Invest. Dermatol. 106: 42-48). The wound extracellular matrix
contains fibronectin, tenascin and vitronectin, all of which are
ligands for .alpha.V.beta.6 (Busk et al., (1992) J. Biol. Chem.
267: 5790-6; Koivisto et al., (1999) Cell Adhes. Commun. 7: 245-57;
Hakkinen et al., (2000) J. Histochem. Cytochem. 48: 985-98). In
addition, .alpha.V.beta.6 upregulates the expression of the matrix
metalloproteinase, MMP-9, that can degrade Type IV collagen and
promote cell movement (Niu et al., (1998) Biochem. Biophys. Res.
Com. 249: 287-91; Agrez et al., (1999) Int. J. Can. 81: 90-97;
Thomas et al., (2001) Int. J. Cancer 92: 641-50; Gu et al., (2002)
Br. J. Can. 87: 348-51). Based on its expression pattern in wounds
and in vitro studies, .alpha.V.beta.6 may have dual roles to
promote keratinocyte migration during wound closure and later to
resolve the wound through the activation of TGF.beta.. The
activation of TGF.beta. by .alpha.V.beta.6 would contribute to
wound resolution through the regulation of re-epithelialization,
suppression of inflammation and promotion of connective tissue
regeneration and scar formation (Thomas et al., (2006) J. Oral
Pathol. Med. 35: 1-10). In vivo wound studies using beta 6 null
mice indicated that wounds healed but there was a markedly
increased inflammatory response in the skin. Wound closure and
keratinocyte activity was likely unaffected by the loss of
.alpha.V.beta.6 because of the expression of other integrin family
members (Huang et al., (1996) J. Cell Biol. 133: 921-8). The
inflammatory infiltrate in the beta 6 null mouse wounds resembled
those from TGF.beta.1 null mice, su.mu.gesting that there was
insufficient activity of this cytokine to suppress the immune
response in the absence of .alpha.V.beta.6 (Shull et al., (1992)
Nature 359: 693-9; Thomas et al., (2006) J. Oral Pathol. Med. 35:
1-10).
[0006] Analysis of the beta 6 null mice in lung injury and kidney
disease models has also identified a role for .alpha.V.beta.6 in
fibrosis. Lung fibrosis in the beta 6 null mice was inhibited in a
bleomycin injury model (Munger et al., (1999) Cell 96: 319-328).
These animals also were protected from an MMP12 dependent
emphasema-like phenotype (Morris et al., (2003) Nature 422:
169-73). Both disease phenotypes are dependent on the activation of
TGF.beta. (Munger et al., (1999) Cell 96: 319-328). Inhibition of
.alpha.V.beta.6 integrin-mediated TGF.beta. activation was also
hypothesized to promote pulmonary edema in the early phase response
to acute lung injury (Pittet et al., (2001) J. Clin. Invest. 107:
1537-44). Beta 6 null mice were also protected from fibrosis in a
kidney disease model, where TGF.beta. activation is essential for
the development of tubulointerstitial fibrotic lesions (Ma et al.,
(2003) Am. J. Pathol. 63: 1261-73).
[0007] In addition to its expression in wound healing, the
.alpha.V.beta.6 integrin is upregulated at the periphery of many
human tumors. .alpha.V.beta.6 expression has been reported in oral
(Breuss et al., (1995) J. Cell Sci. 108: 2241-51; Jones et al.,
(1997) J. Oral Pathol. Med. 26: 63-8; Hamidi et al., (2000) Br. J.
Cancer 82: 1433-40; Regezi et al., (2002) Oral Oncology 38: 332-6;
Impola et al., (2004) J. Pathol. 202: 14-22) and skin squamous cell
carcinomas, as well as carcinomas of the lung (Smythe et al.,
(1995) Can. Met. Rev. 14: 229-39), breast (Arhiro et al., (2000)
Breast Can. 7: 19-26), pancreas (Sipos, et al., (2004)
Histopathology 45: 226-36), stomach (Kawashima et al., (2003)
Pathol. Res. Pract. 199: 57-64), colon (Bates et al., (2005) J.
Clin. Invest. 115: 339-47), ovary (Ahmed et al., (2002)
Carcinogenesis 23: 237-44; Ahmed et al., (2002) J. Histochem.
Cytochem. 50: 1371-79), and salivary gland (Westernoff et al.,
(2005) Oral Oncology 41: 170-74). In many of these reports the
expression of .alpha.V.beta.6 correlated with increasing tumor
grade (Ahmed et al., (2002) J. Histochem. Cytochem. 50: 1371-79;
Arihiro et al., (2000) Breast Can. 7: 19-26), eventual metastases
to lymph nodes (Kawashima et al., (2003) Pathol. Res. Pract. 199:
57-64; Bates et al., (2005) J. Clin. Invest. 115: 339-47), or poor
prognosis (Bates et al., (2005) J. Clin. Invest. 115: 339-47). The
most well studied tumor type is oral squamous cell carcinoma, where
investigators have also examined .alpha.V.beta.6 in pre-cancerous
lesions and correlated its expression with progression to
malignancy (Hamidi et al., (2000) Br. J. Cancer 82: 1433-40). The
link between .alpha.V.beta.6 expression and tumor progression has
also been investigated in colon carcinoma where the presence of
integrin correlated with the epithelial-to-mesenchymal transition
(EMT) of colon cells in an in vitro model (Brunton et al., (2001)
Neoplasia 3: 215-26; Bates et al., (2005) J. Clin. Invest. 115:
339-47). The EMT is a normal developmental process that enables
epithelial cells to leave their home tissue and migrate out to new
areas (Thiery and Sleeman, (2006) Nat. Rev. Mol. Cell. Biol. 7:
131-42). It is marked by an increase in the expression of proteins
that promote the migration and invasion of cells, such as matrix
proteases, cytokines like TGF.beta. and a variety of cellular
adhesion molecules, including integrins (Zavadil and Bottinger,
(2005) Oncogene 24: 5764-5774). The expression of EMT markers has
also been identified in tumors, particularly in a.mu.gressively
invasive and metastatic carcinomas. The ability of .alpha.V.beta.6
to promote adhesion to interstitial fibronectin, upregulate the
expression of MMP-9 and other matrix proteases and to activate
TGF.beta. indicates it may facilitate the EMT of malignant cells
and tumor progression (Bates and Mercurio, (2005) Cancer Bio. &
Ther. 4: 365-70).
[0008] Animal and in vitro models of human cancer have implicated
.alpha.V.beta.6 mediated signal transduction in the promotion of
cell proliferation and inhibition of apoptosis. The residues within
the C-terminus of the beta 6 chain that promote proliferation of
.alpha.V.beta.6-transfected SW480 colon tumor cells in a collagen
gel matrix in vitro were identified. Compared to the full-length
.beta.6 transfected SW480 cells, the .beta.6 deletion mutant had
markedly reduced ability to grow sub-cutaneously in Nude mice
(Agrez et al., (1994) J. Cell. Biol. 127: 547-56). In an oral
cancer cell line that stably expressed .alpha.V.beta.6, binding to
fibronectin resulted in the recruitment and activation of the Fyn
kinase by the beta 6 subunit. Downstream signal transduction
resulted in the production of MMP-3, promoted cell proliferation in
vitro, tumor invasion in an orthotopic model, and metastasis in a
tail vein injection model (Li et al., (2003) J. Biol. Chem. 278:
41646-53).
[0009] Suppression of apoptosis, like cell proliferation, is
another way that .alpha.V.beta.6 may promote tumor growth. Normal
stratified squamous epithelia express the .alpha.V.beta.5 integrin
but down-regulate it and upregulate .alpha.V.beta.6 expression upon
transformation to carcinomas. Using carcinoma cell lines that
over-expressed .alpha.V.beta.5, .alpha.V.beta.6 expression was
shown to prevent suspension-induced cell death (anoikis) in vitro
(Janes and Watt, (2004) J. Cell Biol. 166: 419-31). Apoptosis
inhibition has also been observed in vitro in ovarian cancer cell
lines treated with cisplatin, which may represent a mechanism for
drug resistance of these tumors in vivo (Wu et al., (2004) Zhonghua
Fu Chan Ke Za Zhi 39: 112-14).
[0010] A number of investigators have developed therapeutics to
target .alpha.V.beta.6 activity in fibrosis and cancer. A murine
antibody with specificity for the .alpha.V.beta.6, .alpha.V.beta.3
and .alpha.V.beta.5 integrins was shown to prevent adhesion of HT29
colon carcinoma cells to vitronectin and fibronectin in vitro
(Lehmann et al., (1994) Can. Res. 54: 2102-07). Another murine
antibody therapeutic specific for the human .alpha.V.beta.6 protein
was demonstrated to inhibit the invasive growth of HSC-3 oral
carcinoma cells in a transoral xenograft tumor model in mice (Xue
et al., (2001) Biochem. Biophys. Res. Com. 288: 610-18). A series
of human .alpha.V.beta.6 specific antibodies were raised using the
beta 6 null mouse model as the host. These antibodies were able to
block both TGF.beta.LAP and fibronectin binding to integrin in
vitro (Weinreb et al., (2004) J. Biol. Chem. 279: 17875-87). They
also demonstrated significant tumor growth inhibition in a human
pharyngeal cancer xenograft model (Leone et al., (2003) Proc. of
the Am. Assoc. Can. Res. 44, Abstract #4069).
[0011] In addition to function blocking antibodies the creation of
a peptidomimetic inhibitor of the human .alpha.V.beta.6 integrin
has been reported. This compound was shown to inhibit UCLAP-3 cell
binding to fibronectin with an IC50 in the 200 nM range with
additional activity to block .alpha.V.beta.5 and .alpha.V.beta.3
integrin-mediated cell binding to vitronectin in the 3-20 uM range,
respectively (Goodman et al., (2002) J. Med. Chem. 45:
1045-51).
[0012] Another recently described role for .alpha.V.beta.6 is as a
cellular receptor for viral pathogens. It mediates the binding of
the viral capsid for foot-and-mouth disease virus and the
Coxsackievirus 9 to enable viral entry in vitro (Miller et al.,
(2001) J. Virol. 75: 4158-64; Williams et al., (2004) J. Virol. 78:
6967-73). Both foot-and-mouth disease virus and Coxsackievirus 9
capsid proteins contain an RGD sequence that is recognized by
multiple integrin family members. Viral entry of both pathogens is
blocked by antibody to .alpha.V.beta.6 integrin (Williams et al.,
(2004) J. Virol. 78: 6967-73).
SUMMARY
[0013] The invention is generally directed to targeted binding
agents that bind to .alpha.V.beta.6. Embodiments of the invention
relate to fully human targeted binding agents that specifically
bind to .alpha.V.beta.6 and thereby inhibit binding of ligands to
.alpha.V.beta.6. The targeted binding agents also inhibit tumor
cell adhesion. In addition, the targeted binding agents are useful
for reducing tumor growth. Mechanisms by which this can be achieved
can include and are not limited to either inhibiting binding of a
ligand to its receptor .alpha.V.beta.6, abrogation of intereactions
with ligands such as TGF.beta.-LAP, thereby reducing the effective
concentration of .alpha.V.beta.6.
[0014] In one embodiment of the invention, the targeted binding
agent is a fully human antibody that binds to .alpha.V.beta.6 and
prevents .alpha.V.beta.6 binding to ligands of .alpha.V.beta.6.
Examples of ligands of .alpha.V.beta.6 include TGF.beta.LAP,
fibronectin, tenascin, vitronectin and osteopontin. The antibody
may bind .alpha.V.beta.6 with a K.sub.d of less than 35 nM, 25 nM,
10 nM, or 60 pM.
[0015] Yet another embodiment is a fully human antibody that binds
to .alpha.V.beta.6 and inhibits greater than 80%, 85%, 90% or 99%
of TGF.beta.-LAP mediated adhesion of HT29 cells at antibody
concentrations as low as 1 .mu.g/ml or less.
[0016] Yet another embodiment is a fully human antibody that binds
to .alpha.V.beta.6 and inhibits TGF.beta.-LAP mediated adhesion of
HT29 cells with an IC.sub.50 of less than 0.070 .mu.g/ml.
[0017] The targeted binding agent (i.e. an antibody) may comprise a
heavy chain amino acid sequence having a complementarity
determining region (CDR) with one of the sequences shown in Table 8
or Table 29. In one embodiment the targeted binding agent may
comprise a sequence comprising any one of a CDR1, CDR2 or CDR3
sequence as shown in Table 8 or Table 29. In another embodiment the
targeted binding agent may comprise a sequence comprising any two
of a CDR1, CDR2 or CDR3 sequence as shown in Table 8 or Table 29
(that is either a CDR1 and CDR2, a CDR1 and CDR3 or a CDR2 and
CDR3). In another embodiment the targeted binding agent may
comprise a sequence comprising a CDR1, CDR2 and CDR3 sequence as
shown in Table 8 or Table 29. 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.
[0018] In another embodiment the targeted binding agent (i.e. an
antibody) may comprise a light chain amino acid sequence having a
complementarity determining region (CDR), CDR1, CDR2 or CDR3
sequences as shown in Table 9 or Table 30. In another embodiment
the targeted binding agent may comprise a sequence comprising any
two of a CDR1, CDR2 or CDR3 sequence as shown in Table 9 or Table
30 (that is either a CDR1 and CDR2, a CDR1 and CDR3, or a CDR2 and
CDR3). In another embodiment the targeted binding agent may
comprise a sequence comprising a CDR1, CDR2 and CDR3 sequence as
shown in Table 9 or Table 30.
[0019] The targeted binding agent of the invention may comprise an
antigen-binding site within a non-antibody molecule, normally
provided by one or more CDRs e.g. a set of CDRs in a non-antibody
protein scaffold, as discussed further below.
[0020] In another embodiment the targeted binding agent comprises a
sequence comprising any one of a CDR1, CDR2 or CDR3 sequence of
fully human monoclonal antibodies sc 264 RAD, sc 264 RAD/ADY, sc
188 SDM, sc 133, sc 133 TMT, sc 133 WDS, sc 133 TMT/WDS, sc 188, sc
254, sc 264 or sc 298.
[0021] In another embodiment the targeted binding agent comprises
any two of a CDR1, CDR2 or CDR3 sequence of fully human monoclonal
antibodies sc 264 RAD, sc 264 RAD/ADY, sc 188 SDM, sc 133, sc 133
TMT, sc 133 WDS, sc 133 TMT/WDS, sc 188, sc 254, sc 264 or sc 298
(that is either a CDR1 and CDR2, a CDR1 and CDR3 or a CDR2 and
CDR3).
[0022] In another embodiment the targeted binding agent comprises a
CDR1, CDR2 and CDR3 sequence of fully human monoclonal antibodies
sc 264 RAD, sc 264 RAD/ADY, sc 188 SDM, sc 133, sc 133 TMT, sc 133
WDS, sc 133 TMT/WDS, sc 188, sc 254, sc 264 or sc 298.
[0023] 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.
[0024] Another embodiment of the invention comprises an antibody
that binds to .alpha.V.beta.6 and comprises a light chain amino
acid sequence comprising any one of a CDR1, CDR2 or CDR3 sequence
as shown in Table 9 or Table 30. Another embodiment of the
invention comprises an antibody that binds to .alpha.V.beta.6 and
comprises a light chain amino acid sequence comprising any two of a
CDR1, CDR2 or CDR3 sequence as shown in Table 9 or Table 30 (that
is either a CDR1 and CDR2, a CDR1 and CDR3 or a CDR2 and CDR3).
Another embodiment of the invention comprises an antibody that
binds to .alpha.V.beta.6 and comprises a light chain amino acid
sequence comprising a CDR1, a CDR2 and a CDR3 sequence as shown in
Table 9 or Table 30. In certain embodiments the antibody is a fully
human monoclonal antibody.
[0025] Yet another embodiment of the invention comprises an
antibody that binds to .alpha.V.beta.6 and comprises a heavy chain
amino acid sequence comprising any one of a CDR1, CDR2 or CDR3
sequence as shown in Table 8 or Table 29. Another embodiment of the
invention comprises an antibody that binds to .alpha.V.beta.6 and
comprises a heavy chain amino acid sequence comprising any two of a
CDR1, CDR2 or CDR3 sequence as shown in Table 8 or Table 29 (that
is either a CDR1 and CDR2, a CDR1 and CDR3 or a CDR2 and CDR3).
Another embodiment of the invention comprises an antibody that
binds to .alpha.V.beta.6 and comprises a heavy chain amino acid
sequence comprising a CDR1, a CDR2 and a CDR3 sequence as shown in
Table 8 or Table 29. In certain embodiments the antibody is a fully
human monoclonal antibody.
[0026] One embodiment of the invention comprises one or more of
fully human monoclonal antibodies sc 264 RAD, sc 264 RAD/ADY, sc
188 SDM, sc 133, sc 133 TMT, sc 133 WDS, sc 133 TMT/WDS, sc 188, sc
254, sc 264 or sc 298 which specifically bind to .alpha.V.beta.6,
as discussed in more detail below.
[0027] Yet another embodiment is an antibody that binds to
.alpha.V.beta.6 and comprises a light chain amino acid sequence
having a CDR comprising one of the sequences shown in Table 9 or
Table 30. Another embodiment is an antibody that binds to
.alpha.V.beta.6 and comprises a heavy chain amino acid sequence
having a CDR comprising one of the sequences shown in Table 8 or
Table 29. In certain embodiments the antibody is a fully human
monoclonal antibody.
[0028] A further embodiment is an antibody that binds to
.alpha.V.beta.6 and comprises a heavy chain amino acid sequence
having one of the CDR sequences shown in Table 8 or Table 29 and a
light chain amino acid sequence having one of the CDR sequences
shown in Table 9 or Table 30. In certain embodiments the antibody
is a fully human monoclonal antibody.
[0029] A further embodiment of the invention is a targeted binding
agent (i.e. an antibody) that competes for binding to
.alpha.V.beta.6 with the antibodies of the invention. In one
embodiment, said targeted binding agent comprises a heavy chain
amino acid sequence having at least one of the CDR sequences shown
in Table 8 or Table 29 and a light chain amino acid sequence having
at least one of the CDR sequences shown in Table 9 or Table 30.
[0030] A further embodiment of the invention is a targeted binding
agent that binds to the same epitope on .alpha.V.beta.6 as the
antibodies of the invention. In one embodiment, said targeted
binding agent comprised a heavy chain amino acid sequence having at
least one of the CDR sequences shown in Table 8 or Table 29 and a
light chain amino acid sequence having at least one of the CDR
sequences shown in Table 9 or Table 30.
[0031] In another embodiment the targeted binding agent comprises a
sequence comprising any one of a CDR1, CDR2 or CDR3 sequence as
shown in Table 8 or Table 29 and any one of a CDR1, CDR2 or CDR3
sequence as shown in Table 9 or Table 30. In another embodiment the
targeted binding agent comprises any two of a CDR1, CDR2 or CDR3
sequence shown in Table 8 or Table 29 and any two of a CDR1, CDR2
or CDR3 sequence as shown in Table 9 or Table 30 (that is either a
CDR1 and CDR2, a CDR1 and CDR3 or a CDR2 and CDR3). In another
embodiment the targeted binding agent comprises a CDR1, CDR2 and
CDR3 sequence as shown in Table 8 or Table 29 and a CDR1, CDR2 and
CDR3 sequence as shown in Table 9 or Table 30.
[0032] In some embodiments, a binding agent of the invention may
comprise an antigen-binding site within a non-antibody molecule,
normally provided by one or more CDRs e.g. a set of CDRs in a
non-antibody protein scaffold, as discussed further below.
[0033] A still further embodiment is an antibody that binds to
.alpha.V.beta.6 and comprises an amino acid sequence having one or
more corrective mutations where the antibody sequence is mutated
back to its respective germline sequence. For example, the antibody
can have a sequence as shown in any of Tables 10-13.
[0034] The invention further provides methods for assaying the
level of .alpha.V.beta.6 in a patient or patient sample, comprising
contacting an anti-.alpha.V.beta.6 antibody with a biological
sample from a patient, and detecting the level of binding between
said antibody and .alpha.V.beta.6 in said sample. In more specific
embodiments, the biological sample is blood, or plasma.
[0035] In other embodiments the invention provides compositions
comprising targeted binding agent, including an antibody or
functional fragment thereof, and a pharmaceutically acceptable
carrier.
[0036] Still further embodiments of the invention include methods
of effectively treating an animal suffering from an
.alpha.V.beta.6-related disease or disorder, including selecting an
animal in need of treatment for a neoplastic or non-neoplastic
disease, and administering to the animal a therapeutically
effective dose of a fully human monoclonal antibody that
specifically binds to .alpha.V.beta.6.
[0037] The antibodies of the invention can be used to treat an
.alpha.V.beta.6-related disease or disorder. An
.alpha.V.beta.6-related disease or disorder can be any condition
arising due to the aberrant activation or expression of
.alpha.V.beta.6. Examples of such diseases include where
.alpha.V.beta.6 aberrantly interacts with its ligands thereby
altering cell-adhesion or cell signaling properties. This
alteration in cell adhesion or cell signaling properties can result
in neoplastic diseases. Other .alpha.V.beta.6-related diseases or
disorders include inflammatory disorders, lung disease, diseases
associated with fibrosis and any disease associated with
dysregulated TGF-.beta..
[0038] In one example, the .alpha.V.beta.6-related disease is a
neoplastic disease such as melanoma, small cell lung cancer,
non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, thyroid tumor, 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.
[0039] In another example, the .alpha.V.beta.6-related disease is
an inflammatory disorder such as inflammatory bowel disease;
systemic lupus erythematosus; rheumatoid arthritis; juvenile
chronic arthritis; spondyloarthropathies; systemic sclerosis, for
example, scleroderma; idiopathic inflammatory myopathies for
example, dermatomyositis, polymyositis; Sjogren's syndrome;
systemic vaculitis; sarcoidosis; thyroiditis, for example, Grave's
disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis,
atrophic thyroiditis; immune-mediated renal disease, for example,
glomerulonephritis, tubulointerstitial nephritis; demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic polyneuropathy; hepatobiliary
diseases such as infectious hepatitis such as hepatitis A, B, C, D,
E and other nonhepatotropic viruses; autoimmune chronic active
hepatitis; primary biliary cirrhosis; granulomatous hepatitis; and
sclerosing cholangitis; inflammatory and fibrotic lung diseases
(e.g., cystic fibrosis); gluten-sensitive enteropathy; autoimmune
or immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases of the lung such as eosinophilic pneumonia, idiopathic
pulmonary fibrosis, allergic conjunctivitis and hypersensitivity
pneumonitis, transplantation associated diseases including graft
rejection and graft-versus host disease.
[0040] In yet another example, the .alpha.V.beta.6-related disease
is fibrosis such as kidney or lung fibrosis.
[0041] In yet another example, the .alpha.V.beta.6-related disease
is associated with dysregulated TGF-.beta. include cancer and
connective tissue (fibrotic) disorders.
[0042] Additional embodiments of the invention include methods of
inhibiting .alpha.V.beta.6 induced cell adhesion in an animal.
These methods include selecting an animal in need of treatment for
.alpha.V.beta.6 induced cell adhesion, and administering to said
animal a therapeutically effective dose of a fully human monoclonal
antibody wherein said antibody specifically binds to
.alpha.V.beta.6.
[0043] Further embodiments of the invention include the use of an
antibody in the preparation of medicament for the treatment of an
.alpha.V.beta.6 related disease or disorder in an animal, wherein
said monoclonal antibody specifically binds to .alpha.V.beta.6.
[0044] In still further embodiments, the targeted binding agents
described herein can be used for the preparation of a medicament
for the effective treatment of .alpha.V.beta.6 induced cell
adhesion in an animal, wherein said monoclonal antibody
specifically binds to .alpha.V.beta.6.
[0045] Embodiments of the invention described herein relate to
monoclonal antibodies that bind .alpha.V.beta.6 and affect
.alpha.V.beta.6 function. Other embodiments relate to fully human
anti-.alpha.V.beta.6 antibodies and anti-.alpha.V.beta.6 antibody
preparations with desirable properties from a therapeutic
perspective, including high binding affinity for .alpha.V.beta.6,
the ability to neutralize .alpha.V.beta.6 in vitro and in vivo, and
the ability to inhibit .alpha.V.beta.6 induced cell adhesion and
tumor growth.
[0046] In one embodiment, the invention includes antibodies that
bind to .alpha.V.beta.6 with very high affinities (KD). For example
a human, rabbit, mouse, chimeric or humanized antibody that is
capable of binding .alpha.V.beta.6 with a Kd less than, but not
limited to, about 10.sup.-5, 10.sup.-6, 10.sup.-7, 10.sup.-8,
10.sup.-9, 10.sup.-10 or about 10.sup.-11M, or any range or value
therein. Affinity and/or avidity measurements can be measured by
KinExA.RTM. and/or BIACORE.RTM., as described herein.
[0047] One embodiment of the invention includes isolated
antibodies, or fragments of those antibodies, that bind to
.alpha.V.beta.6. As known in the art, the antibodies can be, for
example, polyclonal, oligoclonal, monoclonal, chimeric, humanized,
and/or fully human antibodies. Embodiments of the invention
described herein also provide cells for producing these
antibodies.
[0048] It will be appreciated that embodiments of the invention are
not limited to any particular form of an antibody or method of
generation or production. For example, the anti-.alpha.V.beta.6
antibody may be a full-length antibody (e.g., having an intact
human Fc region) or an antibody fragment (e.g., a Fab, Fab' or
F(ab').sub.2, FV or Dab (Dabs are the smallest functional binding
units of human antibodies). In addition, the antibody may be
manufactured from a hybridoma that secretes the antibody, or from a
recombinantly produced cell that has been transformed or
transfected with a gene or genes encoding the antibody.
[0049] Other embodiments of the invention include isolated nucleic
acid molecules encoding any of the antibodies described herein,
vectors having isolated nucleic acid molecules encoding
anti-.alpha.V.beta.6 antibodies or a host cell transformed with any
of such nucleic acid molecules. In addition, one embodiment of the
invention is a method of producing an anti-.alpha.V.beta.6 antibody
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 realized 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 optimized for increasing yields of
antibodies or fragments thereof when transfected into host cells
for antibody production.
[0050] A further embodiment includes a method of producing high
affinity antibodies to .alpha.V.beta.6 by immunizing a mammal with
human .alpha.V.beta.6, or a fragment thereof, and one or more
orthologous sequences or fragments thereof.
[0051] Other embodiments are based upon the generation and
identification of isolated antibodies that bind specifically to
.alpha.V.beta.6. Inhibition of the biological activity of
.alpha.V.beta.6 can prevent .alpha.V.beta.6 induced cell adhesion
and other desired effects.
[0052] Another embodiment of the invention includes a method of
diagnosing diseases or conditions in which an antibody prepared as
described herein is utilized to detect the level of .alpha.V.beta.6
in a patient sample. In one embodiment, the patient sample is blood
or blood serum. 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
overexpression of .alpha.V.beta.6 using anti-.alpha.V.beta.6
antibodies.
[0053] Another embodiment of the invention includes a method for
diagnosing a condition associated with the expression of
.alpha.V.beta.6 in a cell by contacting the serum or a cell with an
anti-.alpha.V.beta.6 antibody, and thereafter detecting the
presence of .alpha.V.beta.6. Preferred conditions include an
.alpha.V.beta.6 related disease or disorder including, but not
limited to, neoplastic diseases, such as, melanoma, small cell lung
cancer, non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, glioblastoma, and carcinoma of the thyroid, stomach,
prostate, breast, ovary, bladder, lung, uterus, kidney, colon, and
pancreas, salivary gland, and colorectum.
[0054] In another embodiment, the invention includes an assay kit
for detecting .alpha.V.beta.6 in mammalian tissues, cells, or body
fluids to screen for .alpha.V.beta.6-related diseases. The kit
includes an antibody that binds to .alpha.V.beta.6 and a means for
indicating the reaction of the antibody with .alpha.V.beta.6, if
present. In one embodiment, the antibody is a monoclonal antibody.
In another embodiment, the antibody that binds .alpha.V.beta.6 is
labeled. In still 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 for detecting
includes a labeled second antibody that is an anti-immunoglobulin.
The antibody may be labeled with a marker selected from the group
consisting of a fluorochrome, an enzyme, a radionuclide and a
radiopaque material.
[0055] Other embodiments of the invention include pharmaceutical
compositions having an effective amount of an anti-.alpha.V.beta.6
antibody in admixture with a pharmaceutically acceptable carrier or
diluent. In yet other embodiments, the anti-.alpha.V.beta.6
antibody, or a fragment thereof, is conjugated to a therapeutic
agent. The therapeutic agent can be, for example, a toxin or a
radioisotope.
[0056] Yet another embodiment includes methods for treating
diseases or conditions associated with the expression of
.alpha.V.beta.6 in a patient, by administering to the patient an
effective amount of an anti-.alpha.V.beta.6 antibody. The
anti-.alpha.V.beta.6 antibody can be administered alone, or can be
administered in combination with additional antibodies or
chemotherapeutic drug or radiation therapy. For example, a
monoclonal, oligoclonal or polyclonal mixture of .alpha.V.beta.6
antibodies that block cell adhesion can be administered in
combination with a drug shown to inhibit tumor cell proliferation
directly. The method can be performed in vivo and the patient is
preferably a human patient. In a preferred embodiment, the method
concerns the treatment of an .alpha.V.beta.6 related disease or
disorder including, but not limited to, neoplastic diseases, such
as, melanoma, small cell lung cancer, non-small cell lung cancer,
glioma, hepatocellular (liver) carcinoma, glioblastoma, and
carcinoma of the thyroid, stomach, prostate, breast, ovary,
bladder, lung, uterus, kidney, colon, and pancreas, salivary gland,
and colorectum.
[0057] In another embodiment, the invention provides an article of
manufacture including a container. The container includes a
composition containing an anti-.alpha.V.beta.6 antibody, and a
package insert or label indicating that the composition can be used
to treat an .alpha.V.beta.6 related disease or disorder
characterized by the overexpression of .alpha.V.beta.6.
[0058] In some embodiments, the anti-.alpha.V.beta.6 antibody is
administered to a patient, followed by administration of a clearing
agent to remove excess circulating antibody from the blood.
[0059] Yet another embodiment is the use of an anti-.alpha.V.beta.6
antibody in the preparation of a medicament for the treatment of
.alpha.V.beta.6-related diseases or disorders such as neoplastic
diseases, inflammatory disorders, fibrosis, lung disease or
diseases associated with dysregulated TGF-.beta.. In one
embodiment, the neoplastic diseases include carcinoma, such as
breast, ovarian, stomach, endometrial, salivary gland, lung,
kidney, colon, colorectum, esophageal, thyroid, pancreatic,
prostate and bladder cancer. In another embodiment, the
.alpha.V.beta.6 related diseases or disorders include, but are not
limited to, neoplastic diseases, such as, melanoma, small cell lung
cancer, non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, sarcoma, head and neck cancers, mesothelioma, biliary
(cholangiocarcinoma), small bowel adenocarcinoma, pediatric
malignancies and glioblastoma.
[0060] Yet another embodiment of the invention is the use of an
anti-.alpha.V.beta.6 antibody in the preparation of a medicament
for the treatment of inflammatory, or hyperprolifearative diseases
including but not limited to arthritis, atherosclerosis, allergic
conjunctivitis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] FIG. 1 is a line graph showing the ability of the purified
monoclonal antibodies to bind to .alpha.V.beta.6 and block its
binding to a GST-LAP peptide.
[0062] FIGS. 2A and 2B are line graphs showing a plot of the
averaged Geometric Mean Fluorescence (GMF) as a function of
molecular mAb concentration, which was used to estimate the binding
affinity of one of the antibodies to K562 cells that stably express
the human .alpha.V.beta.6 antigen. Shown in FIG. 2A is affinity
data for mAb 188.
[0063] FIG. 2B shows affinity data for mAb 264 RAD.
[0064] FIGS. 3A-3E are bar graphs showing the ability of the
purified monoclonal antibodies to mediate complement-dependent
cytotoxicity in 293 cells stably expressing .alpha.V.beta.6
integrin.
[0065] FIG. 4 is a bar graph showing the ability of antibodies
264RAD, 133 and 188 SDM to inhibit tumour growth using the
Detroit-562 nasophayngeal cell line.
[0066] FIG. 5 is a bar chart showing comparison of the activity of
264 RAD with 264 RAD/ADY.
DETAILED DESCRIPTION
[0067] Embodiments of the invention relate to targeted binding
agents that bind to .alpha.V.beta.6 integrin (.alpha.V.beta.6). In
some embodiments, the binding agents bind to .alpha.V.beta.6 and
inhibit the binding of ligands to .alpha.V.beta.6. In one
embodiment, the targeted binding agents are monoclonal antibodies,
or binding fragments thereof. In another embodiment, the antibodies
bind only to the .beta.6 chain yet are able to inhibit binding of
ligands to .alpha.V.beta.6.
[0068] Other embodiments of the invention include fully human
anti-.alpha.V.beta.6 antibodies, and antibody preparations that are
therapeutically useful. In one embodiment, the anti-.alpha.V.beta.6
antibody preparations have desirable therapeutic properties,
including strong binding affinity for .alpha.V.beta.6, and the
ability to inhibit TGF.beta.LAP mediated cell adhesion in
vitro.
[0069] Embodiments of the invention also include fully human
isolated binding fragments of anti-.alpha.V.beta.6 antibodies. In
one embodiment the binding fragments are derived from fully human
anti-.alpha.V.beta.6 antibodies. Exemplary fragments include Fv,
Fab' or other well-known antibody fragments, as described in more
detail below. Embodiments of the invention also include cells that
express fully human antibodies against .alpha.V.beta.6. Examples of
cells include hybridomas, or recombinantly created cells, such as
Chinese hamster ovary (CHO) cells, variants of CHO cells (for
example DG44) and NSO cells that produce antibodies against
.alpha.V.beta.6. 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.
[0070] In addition, embodiments of the invention include methods of
using these antibodies for treating an .alpha.V.beta.6-related
disease or disorder. An .alpha.V.beta.6-related disease or disorder
can be any condition arising due to the aberrant activation or
expression of .alpha.V.beta.6. Examples of such diseases include
where .alpha.V.beta.6 aberrantly interacts with its ligands thereby
altering cell-adhesion or cell signaling properties. This
alteration in cell adhesion or cell signaling properties can result
in neoplastic diseases. Other .alpha.V.beta.6-related diseases or
disorders include inflammatory disorders, lung disease, diseases
associated with fibrosis and any disease associated with
dysregulated TGF-.beta..
[0071] In one example, the .alpha.V.beta.6-related disease is a
neoplastic disease such as melanoma, small cell lung cancer,
non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, thyroid tumor, 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.
[0072] In another example, the .alpha.V.beta.6-related disease is
an inflammatory disorder such as inflammatory bowel disease;
systemic lupus erythematosus; rheumatoid arthritis; juvenile
chronic arthritis; spondyloarthropathies; systemic sclerosis, for
example, scleroderma; idiopathic inflammatory myopathies for
example, dermatomyositis, polymyositis; Sjogren's syndrome;
systemic vaculitis; sarcoidosis; thyroiditis, for example, Grave's
disease, Hashimoto's thyroiditis, juvenile lymphocytic thyroiditis,
atrophic thyroiditis; immune-mediated renal disease, for example,
glomerulonephritis, tubulointerstitial nephritis; demyelinating
diseases of the central and peripheral nervous systems such as
multiple sclerosis, idiopathic polyneuropathy; hepatobiliary
diseases such as infectious hepatitis such as hepatitis A, B, C, D,
E and other nonhepatotropic viruses; autoimmune chronic active
hepatitis; primary biliary cirrhosis; granulomatous hepatitis; and
sclerosing cholangitis; inflammatory and fibrotic lung diseases
(e.g., cystic fibrosis); gluten-sensitive enteropathy; autoimmune
or immune-mediated skin diseases including bullous skin diseases,
erythema multiforme and contact dermatitis, psoriasis; allergic
diseases of the lung such as eosinophilic pneumonia, idiopathic
pulmonary fibrosis, allergic conjunctivitis and hypersensitivity
pneumonitis, transplantation associated diseases including graft
rejection and graft-versus host disease.
[0073] In yet another example, the .alpha.V.beta.6-related disease
is fibrosis such as kidney or lung fibrosis.
[0074] In yet another example, the .alpha.V.beta.6-related disease
is associated with dysregulated TGF-.beta. include cancer and
connective tissue (fibrotic) disorders.
[0075] Other embodiments of the invention include diagnostic assays
for specifically determining the quantity of .alpha.V.beta.6 in a
biological sample. The assay kit can include anti-.alpha.V.beta.6
antibodies along with the necessary labels for detecting such
antibodies. These diagnostic assays are useful to screen for
.alpha.V related diseases or .beta.6 disorders including, but not
limited to, neoplastic diseases, such as, melanoma, small cell lung
cancer, non-small cell lung cancer, glioma, hepatocellular (liver)
carcinoma, glioblastoma, and carcinoma of the thyroid, stomach,
prostate, breast, ovary, bladder, lung, uterus, kidney, colon, and
pancreas, salivary gland, and colorectum.
[0076] Another aspect of the invention is an antagonist of the
biological activity of .alpha.V.beta.6 wherein the antagonist binds
to .alpha.V.beta.6. In one embodiment, the antagonist is a targeted
binding agent, such as an antibody. The antagonist may bind to:
[0077] i) .beta.6 alone; [0078] ii) .alpha.V.beta.6; or [0079] iii)
the .alpha.V.beta.6/ligand complex, or a combination of these. In
one embodiment the antibody is able to antagonize the biological
activity of .alpha.V.beta.6 in vitro and in vivo. The antibody may
be selected from fully human monoclonal antibody e.g., sc 264 RAD,
sc 264 RAD/ADY, sc 188 SDM, sc 133, sc 133 TMT, sc 133 WDS, sc 133
TMT/WDS, sc 188, sc 254, sc 264 or sc 298 or variants thereof.
[0080] In one embodiment the antagonist of the biological activity
of .alpha.V.beta.6 may bind to .alpha.V.beta.6 and thereby prevent
TGF.beta.LAP mediated cell adhesion.
[0081] One embodiment is an antibody which binds to the same
epitope or epitopes as fully human monoclonal antibody c 264 RAD,
sc 264 RAD/ADY, sc 188 SDM, sc 133, sc 133 TMT, sc 133 WDS, sc 133
TMT/WDS, sc 188, sc 254, sc 264 or sc 298.
[0082] In one embodiment, the targeted binding agent binds
.alpha.V.beta.6 with a K.sub.d of less than 100 nanomolar (nM). The
targeted binding agent may bind with a K.sub.d less than about 35
nanomolar (nM). The targeted binding agent may bind with a K.sub.d
less than about 25 nanomolar (nM). The targeted binding agent may
bind with a K.sub.d less than about 10 nanomolar (nM). In another
embodiment, the targeted binding agent binds with a K.sub.d of less
than about 60 picomolar (pM).
[0083] One embodiment is an antibody-secreting plasma cell that
produces the light chain and/or the heavy chain of antibody as
described hereinabove. In one embodiment the plasma cell produces
the light chain and/or the heavy chain of a fully human monoclonal
antibody. In another embodiment the plasma cell produces the light
chain and/or the heavy chain of the fully human monoclonal antibody
c 264 RAD, sc 264 RAD/ADY, sc 188 SDM, sc 133, sc 133 TMT, sc 133
WDS, sc 133 TMT/WDS, sc 188, sc 254, sc 264 or sc 298.
Alternatively the plasma cell may produce an antibody which binds
to the same epitope or epitopes as fully human monoclonal antibody
sc c 264 RAD, sc 264 RAD/ADY, sc 188 SDM, sc 133, sc 133 TMT, sc
133 WDS, sc 133 TMT/WDS, sc 188, sc 254, sc 264 or sc 298.
[0084] Another 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 selected from antibodies c 264 RAD, sc 264
RAD/ADY, sc 188 SDM, sc 133, sc 133 TMT, sc 133 WDS, sc 133
TMT/WDS, sc 188, sc 254, sc 264 or sc 298.
[0085] Another 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.
[0086] 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.
[0087] In addition, one embodiment of the invention is a method of
producing an antibody by culturing host cells under conditions
wherein a nucleic acid molecule is expressed to produce the
antibody, followed by recovery of the antibody.
[0088] 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.
[0089] According to another aspect, the invention includes a method
of antagonising the biological activity of .alpha.V.beta.6
comprising administering an antagonist as described herein. The
method may include selecting an animal in need of treatment for an
.alpha.V.beta.6 related disease or disorder, and administering to
the animal a therapeutically effective dose of an antagonist of the
biological activity of .alpha.V.beta.6.
[0090] Another aspect of the invention includes a method of
antagonising the biological activity of .alpha.V.beta.6 comprising
administering an antibody as described hereinabove. The method may
include selecting an animal in need of treatment for an
.alpha.V.beta.6 related disease or disorder, and administering to
said animal a therapeutically effective dose of an antibody which
antagonises the biological activity of .alpha.V.beta.6.
[0091] According to another aspect there is provided a method of
treating an .alpha.V.beta.6 related disease or disorder in a mammal
comprising administering a therapeutically effective amount of an
antagonist of the biological activity of .alpha.V.beta.6. The
method may include selecting an animal in need of treatment for an
.alpha.V.beta.6 related disease or disorder, and administering to
said animal a therapeutically effective dose of an antagonist of
the biological activity of .alpha.V.beta.6.
[0092] According to another aspect there is provided a method of
treating an .alpha.V.beta.6 disease or disorder in a mammal
comprising administering a therapeutically effective amount of an
antibody which antagonizes the biological activity of
.alpha.V.beta.6. The method may include selecting an animal in need
of treatment for an .alpha.V.beta.6 related disease or disorder,
and administering to said animal a therapeutically effective dose
of an antibody which antagonises the biological activity of
.alpha.V.beta.6. The antibody can be administered alone, or can be
administered in combination with additional antibodies or
chemotherapeutic drug or radiation therapy.
[0093] According to another aspect there is provided a method of
treating cancer in a mammal comprising administering a
therapeutically effective amount of an antagonist of the biological
activity of .alpha.V.beta.6. The method may include selecting an
animal in need of treatment for cancer, and administering to said
animal a therapeutically effective dose of an antagonist which
antagonises the biological activity of .alpha.V.beta.6. The
antagonist can be administered alone, or can be administered in
combination with additional antibodies or chemotherapeutic drug or
radiation therapy.
[0094] According to another aspect there is provided a method of
treating cancer in a mammal comprising administering a
therapeutically effective amount of an antibody which antagonizes
the biological activity of .alpha.V.beta.6. The method may include
selecting an animal in need of treatment for cancer, and
administering to said animal a therapeutically effective dose of an
antibody which antagonises the biological activity of
.alpha.V.beta.6. The antibody can be administered alone, or can be
administered in combination with additional antibodies or
chemotherapeutic drug or radiation therapy.
[0095] According to another aspect of the invention there is
provided the use of an antagonist of the biological activity of
.alpha.V.beta.6 for the manufacture of a medicament for the
treatment of an .alpha.V.beta.6 related disease or disorder.
[0096] According to another aspect of the invention there is
provided the use of an antibody which antagonizes the biological
activity of .alpha.V.beta.6 for the manufacture of a medicament for
the treatment of an .alpha.V.beta.6 related disease or
disorder.
[0097] In a preferred embodiment the present invention is
particularly suitable for use in antagonizing .alpha.V.beta.6, in
patients with a tumor which is dependent alone, or in part, on
.alpha.V.beta.6 integrin.
[0098] Another embodiment of the invention includes an assay kit
for detecting .alpha.V.beta.6 in mammalian tissues, cells, or body
fluids to screen for an .alpha.V.beta.6 related disease or
disorder. The kit includes an antibody that binds to
.alpha.V.beta.6 and a means for indicating the reaction of the
antibody with .alpha.V.beta.6, if present. The antibody may be a
monoclonal antibody. In one embodiment, the antibody that binds
.alpha.V.beta.6 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.
[0099] Further embodiments, features, and the like regarding
anti-.alpha.V.beta.6 antibodies are provided in additional detail
below.
Sequence Listing
[0100] Embodiments of the invention include the specific
anti-.alpha.V.beta.6 antibodies listed below in Table 1. This table
reports the identification number of each anti-.alpha.V.beta.6
antibody, along with the SEQ ID number of the variable domain of
the corresponding heavy chain and light chain genes. Each antibody
has been given an identification number that includes the letters
"sc" followed by a number. TABLE-US-00001 TABLE 1 mAb SEQ ID ID
No.: Sequence NO: sc 49 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 sc 58 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 sc 97 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 sc 133 Nucleotide sequence
encoding the variable region of the heavy chain 13 Amino acid
sequence encoding the variable region of the heavy chain 14
Nucleotide sequence encoding the variable region of the light chain
15 Amino acid sequence encoding the variable region of the light
chain 16 sc 161 Nucleotide sequence encoding the variable region of
the heavy chain 17 Amino acid sequence encoding the variable region
of the heavy chain 18 Nucleotide sequence encoding the variable
region of the light chain 19 Amino acid sequence encoding the
variable region of the light chain 20 sc 188 Nucleotide sequence
encoding the variable region of the heavy chain 21 Amino acid
sequence encoding the variable region of the heavy chain 22
Nucleotide sequence encoding the variable region of the light chain
23 Amino acid sequence encoding the variable region of the light
chain 24 sc 254 Nucleotide sequence encoding the variable region of
the heavy chain 25 Amino acid sequence encoding the variable region
of the heavy chain 26 Nucleotide sequence encoding the variable
region of the light chain 27 Amino acid sequence encoding the
variable region of the light chain 28 sc 264 Nucleotide sequence
encoding the variable region of the heavy chain 29 Amino acid
sequence encoding the variable region of the heavy chain 30
Nucleotide sequence encoding the variable region of the light chain
31 Amino acid sequence encoding the variable region of the light
chain 32 sc 277 Nucleotide sequence encoding the variable region of
the heavy chain 33 Amino acid sequence encoding the variable region
of the heavy chain 34 Nucleotide sequence encoding the variable
region of the light chain 35 Amino acid sequence encoding the
variable region of the light chain 36 sc 298 Nucleotide sequence
encoding the variable region of the heavy chain 37 Amino acid
sequence encoding the variable region of the heavy chain 38
Nucleotide sequence encoding the variable region of the light chain
39 Amino acid sequence encoding the variable region of the light
chain 40 sc 320 Nucleotide sequence encoding the variable region of
the heavy chain 41 Amino acid sequence encoding the variable region
of the heavy chain 42 Nucleotide sequence encoding the variable
region of the light chain 43 Amino acid sequence encoding the
variable region of the light chain 44 sc 374 Nucleotide sequence
encoding the variable region of the heavy chain 45 Amino acid
sequence encoding the variable region of the heavy chain 46
Nucleotide sequence encoding the variable region of the light chain
47 Amino acid sequence encoding the variable region of the light
chain 48 sc 188 Nucleotide sequence encoding the variable region of
the heavy chain 70 SDM Amino acid sequence encoding the variable
region of the heavy chain 71 Nucleotide sequence encoding the
variable region of the light chain 72 Amino acid sequence encoding
the variable region of the light chain 73 sc 264 Nucleotide
sequence encoding the variable region of the heavy chain 74 RAD
Amino acid sequence encoding the variable region of the heavy chain
75 Nucleotide sequence encoding the variable region of the light
chain 76 Amino acid sequence encoding the variable region of the
light chain 77 sc 133 Nucleotide sequence encoding the variable
region of the heavy chain 78 TMT Amino acid sequence encoding the
variable region of the heavy chain 79 Nucleotide sequence encoding
the variable region of the light chain 80 Amino acid sequence
encoding the variable region of the light chain 81 sc 133
Nucleotide sequence encoding the variable region of the heavy chain
82 WDS Amino acid sequence encoding the variable region of the
heavy chain 83 Nucleotide sequence encoding the variable region of
the light chain 84 Amino acid sequence encoding the variable region
of the light chain 85 sc 133 Nucleotide sequence encoding the
variable region of the heavy chain 86 TMT/ Amino acid sequence
encoding the variable region of the heavy chain 87 WDS Nucleotide
sequence encoding the variable region of the light chain 88 Amino
acid sequence encoding the variable region of the light chain 89 sc
264 Nucleotide sequence encoding the variable region of the heavy
chain 90 ADY Amino acid sequence encoding the variable region of
the heavy chain 91 Nucleotide sequence encoding the variable region
of the light chain 92 Amino acid sequence encoding the variable
region of the light chain 93 sc 264 Nucleotide sequence encoding
the variable region of the heavy chain 94 RAD/ADY Amino acid
sequence encoding the variable region of the heavy chain 95
Nucleotide sequence encoding the variable region of the light chain
96 Amino acid sequence encoding the variable region of the light
chain 97
DEFINITIONS
[0101] 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.
[0102] 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.
[0103] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0104] 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.
[0105] An antagonist 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 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.)
[0106] Disease-related aberrant activation or expression of
".alpha.V.beta.6" may be any abnormal, undesirable or pathological
cell adhesion, for example tumor-related cell adhesion. Cell
adhesion-related diseases include, but are not limited to,
non-solid tumors such as leukemia, multiple myeloma or lymphoma,
and also solid tumors 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, oesophageal, bladder, lung, uterus, vulva,
endometrium, kidney, colorectum, pancreas, pleural/peritoneal
membranes, salivary gland, and epidermous.
[0107] A compound refers to any small molecular weight compound
with a molecular weight of less than about 2000 Daltons.
[0108] The term ".alpha.V.beta.6" refers to the heterodimer
integrin molecule consisting of an .alpha.V chain and a .beta.6
chain.
[0109] The term "neutralizing" when referring to a targeted binding
agent, such as an antibody, relates to the ability of said targeted
binding agent to eliminate, or significantly reduce, the activity
of a target antigen. Accordingly, a "neutralizing"
anti-.alpha.V.beta.6 antibody is capable of eliminating or
significantly reducing the activity of .alpha.V.beta.6. A
neutralizing .alpha.V.beta.6 antibody may, for example, act by
blocking the binding of TGF.beta.LAP to the integrin
.alpha.V.beta.6. By blocking this binding, .alpha.V.beta.6 mediated
cell adhesion is significantly, or completely, eliminated. Ideally,
a neutralizing antibody against .alpha.V.beta.6 inhibits cell
adhesion.
[0110] The term "isolated polynucleotide" as used herein shall mean
a polynucleotide that has been isolated from its naturally
occurring environment. Such polynucleotides may be genomic, cDNA,
or synthetic. Isolated polynucleotides preferably are not
associated with all or a portion of the polynucleotides they
associate with in nature. The isolated polynucleotides may be
operably linked to another polynucleotide that it is not linked to
in nature. In addition, isolated polynucleotides preferably do not
occur in nature as part of a larger sequence.
[0111] The term "isolated protein" referred to herein means a
protein that has been isolated from its naturally occurring
environment. Such proteins may be derived from genomic DNA, cDNA,
recombinant DNA, recombinant RNA, or synthetic origin or some
combination thereof, which by virtue of its origin, or source of
derivation, the "isolated protein" (1) is not associated with
proteins found in nature, (2) is free of other proteins from the
same source, e.g. free of murine proteins, (3) is expressed by a
cell from a different species, or (4) does not occur in nature.
[0112] 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.
[0113] The term "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.
[0114] 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] The term "selectively hybridize" referred to herein means to
detectably and specifically bind. Polynucleotides, oligonucleotides
and fragments thereof selectively hybridize to nucleic acid strands
under hybridization and wash conditions that minimize appreciable
amounts of detectable binding to nonspecific nucleic acids. High
stringency conditions can be used to achieve selective
hybridization 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%.
[0119] The term "CDR region" or "CDR" is intended to indicate the
hypervariable regions of the heavy and light chains of the
immunoglobulin as defined by Kabat et al., 1991 (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 recognizes.
[0120] Among the six short CDR sequences, 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).
[0121] 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 (HCDR refers to a variable heavy chain CDR), and a set of
LCDRs refers to LCDR1, LCDR2 and LCDR3 (LCDR refers to a variable
light chain CDR). Unless otherwise stated, a "set of CDRs" includes
HCDRs and LCDRs.
[0122] 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 at 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.
[0123] 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.
[0124] 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."
[0125] 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.
[0126] 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.
[0127] 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".
[0128] 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.
[0129] 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 about 75%, more preferably at least 80%, 90%, 95%, and
most preferably about 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.
[0130] 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., (1991) Science
253:164. 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.
[0131] A further aspect of the invention is a targeting binding
agent or an antibody molecule comprising a VH domain that has at
least about 60, 70, 80, 85, 90, 95, 98 or about 99% amino acid
sequence identity with a VH domain of any of antibodies shown in
Table 1, the appended sequence listing, an antibody described
herein, or with an HCDR (e.g., HCDR1, HCDR2, or HCDR3) shown in
Table 8 or Table 29. The targeting binding agent or antibody
molecule may optionally also comprise a VL domain that has at least
about 60, 70, 80, 85, 90, 95, 98 or about 99% amino acid sequence
identity with a VL domain any of antibodies shown in Table 1, the
appended sequence listing, an antibody described herein, or with an
LCDR (e.g., LCDR1, LCDR2, or LCDR3) shown in Table 9 or Table 30.
Algorithms that can be used to calculate % identity of two amino
acid sequences comprise e.g. BLAST (Altschul et al., (1990) J. Mol.
Biol. 215: 405-410), FASTA (Pearson and Lipman (1988) PNAS USA 85:
2444-2448), or the Smith-Waterman algorithm (Smith and Waterman
(1981) J. Mol. Biol. 147: 195-197), e.g. employing default
parameters. In some embodiments, the targeting binding agent or
antibody that shares amino acid sequence identity as describes
above, exhibits substantially the same activity as the antibodies
referenced. For instance, substantially the same activity comprises
at least one activity that differed from the activity of the
references antibodies by no more that about 50%, 40%, 30%, 20%,
10%, 5%, 2%, 1% or less.
[0132] An antigen binding site is generally formed by the variable
heavy (VH) and variable light (VL) immunoglobulin domains, with the
antigen-binding interface formed by six surface polypeptide loops,
termed complimentarily determining regions (CDRs). There are three
CDRs in each VH (HCDR1, HCDR2, HCDR3) and in each VL (LCDR1, LCDR2,
LCDR3), together with framework regions (FRs).
[0133] 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 (e.g. from Table 1) may
be paired with the VL domain (e.g. from Table 1), so that an
antibody antigen-binding site is formed comprising both the VH and
VL domains. Analogous embodiments are provided for the other VH and
VL domains disclosed herein. In other embodiments, VH chains in
Table 8 or Table 29 are paired with a heterologous VL domain.
Light-chain promiscuity is well established in the art. Again,
analogous embodiments are provided by the invention for the other
VH and VL domains disclosed herein. Thus, the VH of the parent or
of any of antibodies chain on Table 9 or Table 30 may be paired
with the VL of the parent or of any of antibodies on Table 1 or
other antibody.
[0134] An antigen binding site may comprise a set of H and/or L
CDRs of the parent antibody or any of antibodies in Table 1 with 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 set of H and/or L CDRs.
Alternatively, an antigen binding site may comprise a set of H
and/or L CDRs of the parent antibody or any of antibodies Table 1
with as many as twenty, sixteen, ten, nine or fewer, e.g. one, two,
three, four or five, amino acid substitutions within the disclosed
set of H and/or L CDRs. Such modifications may potentially be made
at any residue within the set of CDRs.
[0135] 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 muteins 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.
[0136] A further aspect of the invention is an antibody molecule
comprising a VH domain that has at least about 60, 70, 80, 85, 90,
95, 98 or about 99% amino acid sequence identity with a VH domain
of any of antibodies listed in Table 1, the appended sequence
listing or described herein, or with an HCDR (e.g., HCDR1, HCDR2,
or HCDR3) shown in Table 8 or Table 29. The antibody molecule may
optionally also comprise a VL domain that has at least 60, 70, 80,
85, 90, 95, 98 or 99% amino acid sequence identity with a VL domain
of any of the antibodies shown in Table 1, the appended sequence
listing or described herein, or with an LCDR (e.g., LCDR1, LCDR2,
or LCDR3) shown in Table 9 or Table 30. Algorithms that can be used
to calculate % identity of two amino acid sequences comprise e.g.
BLAST (Altschul et al., (1990) J. Mol. Biol. 215: 405-410), FASTA
(Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the
Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol. Biol.
147: 195-197), e.g. employing default parameters.
[0137] 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.V.beta.6 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 neutralization 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 complex; ability to bind to a
specified epitope; linear epitope, e.g. peptide sequence identified
using peptide-binding scan as described herein, 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.V.beta.6, or downstream molecule.
Such methods are also provided herein.
[0138] 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 P T
R, (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). 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.
[0139] 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.
[0140] 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
optimization 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.
[0141] The techniques required to make substitutions within amino
acid sequences of CDRs, antibody VH or VL domains and/or binding
agents generally are available in the art. Variant sequences may be
made, with substitutions that may or may not be predicted to have a
minimal or beneficial effect on activity, and tested for ability to
bind and/or neutralize and/or for any other desired property.
[0142] Variable domain amino acid sequence variants of any of the
VH and VL domains whose sequences are specifically disclosed herein
may be employed in accordance with the present invention, as
discussed.
[0143] 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 about 5, 6, 8 or 10 amino acids long,
preferably at least about 14 amino acids long, more preferably at
least about 20 amino acids long, usually at least about 50 amino
acids long, and even more preferably at least about 70 amino acids
long. The term "analog" as used herein refers to polypeptides which
are comprised of a segment of at least about 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.V.beta.6, under suitable binding
conditions, (2) ability to block appropriate ligand/.alpha.V.beta.6
binding, or (3) ability to inhibit .alpha.V.beta.6 activity.
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.
[0144] 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.
[0145] As used herein, the term "antibody" 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. An antibody typically has a tetrameric
form, comprising two identical pairs of polypeptide chains, each
pair having one "light" and one "heavy" chain. The variable regions
of each light/heavy chain pair form an antibody binding site.
[0146] As used herein, a "targeted binding agent" is an agent, e.g.
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. As described below,
a targeted binding agent may comprise at least one antigen binding
domain of an antibody, wherein said domain is fused or contained
within a heterologous protein.
[0147] "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, 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).
[0148] An antibody may be oligoclonal, a polyclonal antibody, a
monoclonal 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. The term antibody also includes binding
fragments of the antibodies of the invention; exemplary fragments
include Fv, Fab, Fab', single stranded antibody (svFC), dimeric
variable region (Diabody) and disulphide stabilized variable region
(dsFv).
[0149] 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 stabilized 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 which the cysteine residue(s) of the constant
domains bear a free thiol group.
[0150] 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.
[0151] 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.
[0152] "Active" or "activity" in regard to a .alpha.V.beta.6
heterodimeric polypeptide refers to a portion of an .alpha.V.beta.6
heterodimeric polypeptide that has a biological or an immunological
activity of a native .alpha.V.beta.6 polypeptide. "Biological" when
used herein refers to a biological function that results from the
activity of the native .alpha.V.beta.6 polypeptide. A preferred
.alpha.V.beta.6 biological activity includes, for example,
.alpha.V.beta.6 induced cell adhesion.
[0153] "Mammal" when used herein refers to any animal that is
considered a mammal. Preferably, the mammal is human.
[0154] 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.
[0155] "Fv" when used herein refers to the minimum fragment of an
antibody that retains both antigen-recognition and antigen-binding
sites.
[0156] "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.
[0157] The term "mAb" refers to monoclonal antibody.
[0158] "Liposome" when used herein refers to a small vesicle that
may be useful for delivery of drugs that may include the
.alpha.V.beta.6 polypeptide of the invention or antibodies to such
an .alpha.V.beta.6 polypeptide to a mammal.
[0159] "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.
[0160] 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; sensitizers;
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.
[0161] 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).
[0162] 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.
[0163] The term "patient" includes human and veterinary
subjects.
Human Antibodies and Humanization of Antibodies
[0164] 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.
[0165] 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.).
[0166] 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.
[0167] In an alternative approach, others, including GenPharm
International, Inc., have utilized 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. No.
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.
[0168] 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).
[0169] Human antibodies can also be derived by in vitro methods.
Suitable examples include but are not limited to phage display
(CAT, Morphosys, Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion
(formerly Proliferon), Affimed) ribosome display (CAT), yeast
display, and the like.
Preparation of Antibodies
[0170] Antibodies, as described herein, were prepared through the
utilization of the XenoMouse.RTM. technology, as described below.
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
utilized 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.
[0171] 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 immunized with an
antigen of interest (e.g. .alpha.V.beta.6), lymphatic cells (such
as B-cells) are recovered from the hyper-immunized 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.V.beta.6. Further,
provided herein are characterization of the antibodies produced by
such cell lines, including nucleotide and amino acid sequence
analyses of the heavy and light chains of such antibodies.
[0172] 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.V.beta.6 immunogen. The supernatants might also be screened
for immunoreactivity against fragments of .alpha.V.beta.6 to
further map the different antibodies for binding to domains of
functional interest on .alpha.V.beta.6. The antibodies may also be
screened against other related human integrins and against the rat,
the mouse, and non-human primate, such as Cynomolgus monkey,
orthologues of .alpha.V.beta.6, the last to determine species
cross-reactivity. B cells from wells containing antibodies of
interest may be immortalized by various methods including fusion to
make hybridomas either from individual or from pooled wells, or by
infection with EBV or transfection by known immortalizing genes and
then plating in suitable medium. Alternatively, single plasma cells
secreting antibodies with the desired specificities are then
isolated using a .alpha.V.beta.6-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.V.beta.6
antigen.
[0173] 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.V.beta.6-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.
[0174] In general, antibodies produced by the fused hybridomas were
human IgG2 heavy chains with fully human kappa or lambda light
chains. Antibodies described herein possess human IgG4 heavy chains
as well as IgG2 heavy chains. Antibodies can also be of other human
isotypes, including IgG1. The antibodies possessed high affinities,
typically possessing a Kd of from about 10.sup.-6 through about
10.sup.-12 M or below, when measured by solid phase and solution
phase techniques. Antibodies possessing a Kd of at least 10.sup.-11
M are preferred to inhibit the activity of .alpha.V.beta.6.
[0175] As will be appreciated, antibodies 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.
[0176] 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. Cell
lines of particular preference are selected through determining
which cell lines have high expression levels and produce antibodies
with constitutive .alpha.V.beta.6 binding properties.
[0177] Based on the ability of mAbs to significantly neutralize
.alpha.V.beta.6 activity (as demonstrated in the Examples below),
these antibodies will have therapeutic effects in treating symptoms
and conditions resulting from .alpha.V.beta.6 expression. In
specific embodiments, the antibodies and methods herein relate to
the treatment of symptoms resulting from .alpha.V.beta.6 induced
cell adhesion or signaling induced as a result of .alpha.V.beta.6
interaction with it s ligands
[0178] According to another aspect of the invention there is
provided a pharmaceutical composition comprising an antagonist of
the biological activity of .alpha.V.beta.6, 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.V.beta.6, and a pharmaceutically
acceptable carrier. In one embodiment the antagonist comprises an
antibody.
[0179] Anti-.alpha.V.beta.6 antibodies are useful in the detection
of .alpha.V.beta.6 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.V.beta.6
activity (as demonstrated in the Examples below),
anti-.alpha.V.beta.6 antibodies have therapeutic effects in
treating symptoms and conditions resulting from .alpha.V.beta.6
expression. In specific embodiments, the antibodies and methods
herein relate to the treatment of symptoms resulting from
.alpha.V.beta.6 induced cell adhesion. Further embodiments involve
using the antibodies and methods described herein to treat an
.alpha.V.beta.6 related disease or disorder including neoplastic
diseases, such as, melanoma, small cell lung cancer, non-small cell
lung cancer, glioma, hepatocellular (liver) carcinoma, thyroid
tumor, gastric (stomach) cancer, prostate cancer, breast cancer,
ovarian cancer, bladder cancer, lung cancer, glioblastoma,
endometrial cancer, kidney cancer, colon cancer, and pancreatic
cancer.
Therapeutic Administration and Formulations
[0180] Embodiments of the invention include sterile pharmaceutical
formulations of anti-.alpha.V.beta.6 antibodies that are useful as
treatments for diseases. Such formulations would inhibit the
binding of ligands to the .alpha.V.beta.6 integrin, thereby
effectively treating pathological conditions where, for example,
tissue .alpha.V.beta.6 is abnormally elevated. Anti-.alpha.V.beta.6
antibodies preferably possess adequate affinity to potently inhibit
.alpha.V.beta.6 activity, 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.
[0181] 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.
[0182] 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 tumor site, or by sustained release systems
as noted below. The antibody is preferably administered
continuously by infusion or by bolus injection.
[0183] 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.
[0184] 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.
[0185] Sterile compositions for injection can be formulated
according to conventional pharmaceutical practice as described in
Remington: The Science and Practice of Pharmacy (20th 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.
[0186] 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).
[0187] 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 a.mu.gregate 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 a.mu.gregation 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.
[0188] 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.
In one embodiment, said antibody anybody half life is greater than
15 days, greater than 20 days, greater than 25 days, greater than
30 days, greater than 35 days, greater than 40 days, greater than
45 days, greater than 2 months, greater than 3 months, greater than
4 months, or greater than 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 derivatization 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.
[0189] 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.
[0190] 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.
[0191] 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 0.001 mg/kg
to up to 100 mg/kg or more, 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.
[0192] 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. 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
[0193] In accordance with the present invention and based on the
activity of the antibodies that are produced and characterized
herein with respect to .alpha.V.beta.6, 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, generation of
peptide therapeutics, .alpha.V.beta.6 binding domains in novel
scaffolds, gene therapies, particularly intrabodies, antisense
therapeutics, and small molecules.
[0194] 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.
[0195] Bispecific antibodies can be generated that comprise (i) two
antibodies one with a specificity to .alpha.V.beta.6 and another to
a second molecule that are conjugated together, (ii) a single
antibody that has one chain specific to .alpha.V.beta.6 and a
second chain specific to a second molecule, or (iii) a single chain
antibody that has specificity to .alpha.V.beta.6 and the other
molecule. Such bispecific antibodies can be generated using
techniques that are well known; 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
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)).
[0196] In connection with immunotoxins, antibodies can 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.
[0197] 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 & Ma.mu.gos (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
agent 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).
[0198] In addition to antibody sequences and/or an antigen-binding
site, a 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.
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 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.
Combinations
[0199] The anti-tumor treatment 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 tumor agents:
[0200] (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);
[0201] (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;
[0202] (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 (AZDO530; 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 antibodies to
Heparanase);
[0203] (iv) 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-EGFR inhibitor Bevacizumab (Avastin.TM.), 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), 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)
and cyclin dependent kinase inhibitors such as CDK2 and/or CDK4
inhibitors;
[0204] (v) 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 and WO 98/13354
and compounds that work by other mechanisms (for example linomide,
inhibitors of integrin .alpha.V.beta.3 function and
angiostatin);
[0205] (vi) 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;
[0206] (vii) antisense therapies, for example those which are
directed to the targets listed above, such as ISIS 2503, an
anti-ras antisense;
[0207] (viii) 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
[0208] (ix) immunotherapy approaches, including for example ex-vivo
and in-vivo approaches to increase the immunogenicity of patient
tumour cells, such as transfection with cytokines such as
interleukin 2, interleukin 4 or granulocyte-macrophage colony
stimulating factor, approaches to decrease T-cell anergy,
approaches using transfected immune cells such as
cytokine-transfected dendritic cells, approaches using
cytokine-transfected tumor cell lines and approaches using
anti-idiotypic antibodies.
[0209] 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.
EXAMPLES
[0210] 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
[0211] Immunizations were conducted using soluble .alpha.V.beta.6
and cell-bound .alpha.V.beta..sub.36 (CHO transfectants expressing
human .alpha.V.beta.6 at the cell surface), respectively. For the
generation of CHO transfectants, human full length
.alpha.V.beta..sub.36 cDNA was inserted into the pcDNA 3 expression
vector. CHO cells were transiently transfected via electroporation.
Expression of human .alpha.V.beta.6 on the cell surface at the
level suitable for immunogen purpose was confirmed by
Fluorescene-Activated Cell Sorter (FACS) analysis. Ten .mu.g/mouse
of soluble protein for Campaign 1, and 3.times.10.sup.6 cells/mouse
of transfected CHO cells for Campaign 2, were used for initial
immunization in XenoMouse.TM. 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, thirteen subsequent boost immunizations of
five .mu.g/mouse were administered for groups one and two (soluble
antigen), and nine subsequent boost immunizations of
1.5.times.10.sup.6 cells/mouse were administered for groups three
and four (cell-bound antigen). The immunization programs are
summarized in Table 2. TABLE-US-00002 TABLE 2 Summary of
Immunization Programs No of Immunization Campaign Group Immunogen
Strain mice routes 1 1 Soluble .alpha.V.beta.6 XMG2/k 10 IP, Tail,
BIP, twice/wk, x 6 wks 1 2 Soluble .alpha.V.beta.6 XMG1/k1 10 IP,
Tail, BIP, twice/wk, x 6 wks 2 3 Cell-bound XMG2/k 10 IP, Tail,
BIP, .alpha.V.beta.6 (CHO twice/wk, x transfectants) 6 wks 2 4
Cell-bound XMG1/k1 10 IP, Tail, BIP, .alpha.V.beta.6 (CHO twice/wk,
x transfectants) 6 wks
Selection of Animals for Harvest by Titer
[0212] Titers of the antibody against human .alpha.V.beta.6 were
tested by ELISA assay for mice immunized with soluble antigen.
Titers of the antibody for mice immunized with native (cell-bound)
antigen were tested by FACS. The ELISA and FACS analyses showed
that there were some mice which appeared to be specific for
.alpha.V.beta.6. Therefore, at the end of the immunization program,
twenty mice were selected for harvest, and lymphocytes were
isolated from the spleens and lymph nodes of the immunized mice, as
described in Example 2.
Example 2
Recovery of Lymphocytes and B-Cell Isolations
[0213] Immunized mice were sacrificed by cervical dislocation, and
the draining lymph nodes harvested and pooled from each cohort. The
lymphoid cells were dissociated by grinding in DMEM to release the
cells from the tissues and the cells were suspended in DMEM. B
cells were enriched by negative selection in IgM and positive
selection on IgG. The cells were cultured to allow B cell expansion
and differentiation into antibody-secreting plasma cells.
[0214] Antibody-secreting plasma cells were grown as routine in the
selective medium. Exhaustive supernatants collected from the cells
that potentially produce anti-human .alpha.V.beta.6 antibodies were
subjected to subsequent screening assays as detailed in the
examples below.
Example 3
Binding to Cell-Bound .alpha.V.beta.6
[0215] The binding of secreted antibodies to .alpha.V.beta.6 was
assessed. Binding to cell-bound .alpha.V.beta.6 was assessed using
an FMAT macroconfocal scanner, and binding to soluble
.alpha.V.beta.6 was analyzed by ELISA, as described below.
[0216] Supernatants collected from harvested cells were tested to
assess the binding of secreted antibodies to HEK 293 cells stably
overexpressing .alpha.V.beta.6. A parental 293F cell line was used
as a negative control. Cells in Freestyle media (Invitrogen) were
seeded into 384-well FMAT plates in a volume of 50 .mu.L/well at a
density of 2500 cells/well for the stable transfectants, and at a
density of 22,500 cells/well for the parental cells, and cells were
incubated overnight at 37.degree. C. Then, 10 .mu.L/well of
supernatant was added, and plates were incubated for approximately
one hour at 4.degree. C., after which 10 .mu.L/well of anti-human
IgG-Cy5 secondary antibody was added at a concentration of 2.8
.mu.g/ml (400 ng/ml final concentration). Plates were then
incubated for one hour at 4.degree. C., and fluorescence was read
using an FMAT macroconfocal scanner (Applied Biosystems). FMAT
results for 11 antibodies are summarized in Table 3.
[0217] Additionally, antibody binding to soluble .alpha.V.beta.6
was analyzed by ELISA. Costar medium binding 96-well plates (Costar
catalog #3368) were coated by incubating overnight at 4.degree. C.
with .alpha.V.beta.6 at a concentration of 5 .mu.g/ml in TBS/1 mM
MgCl.sub.2 buffer for a total volume of 50 .mu.L/well. Plates were
then washed with TBS/1 mM MgCl.sub.2 buffer, and blocked with 250
.mu.L of 1.times.PBS/1% milk for thirty minutes at room
temperature. Ten .mu.L of supernatant was then added to 40 .mu.L
TBS/1 mM MgCl.sub.2/1% milk and incubated for one hour at room
temperature. Plates were washed and then incubated with
goat-anti-human IgG Fc-peroxidase at 0.400 ng/ml in TBS/1 mM
MgCl.sub.2/1% milk, and incubated for one hour at room temperature.
Plates were washed and then developed with 1-Step TMB substrate.
The ELISA results for one of the antibodies are shown in Table 3.
TABLE-US-00003 TABLE 3 Binding of Supernatants to Cell-Bound and
Soluble .alpha.V.beta.6 ELISA FMAT Data data mAb Count FL1 FL1
.times. Count OD sc 049 185 4377.73 809880 ND sc 058 ND ND ND 1.79
sc 188 127 628.04 79761 ND sc 097 98 1237.18 121243 ND sc 277 28
382.31 10704 ND sc 133 82 709.82 58205 ND sc 161 23 725.21 16679 ND
sc 254 174 9179.65 1597259 ND sc 264 63 734.29 46260 ND sc 298 102
2137.94 218069 ND sc 374 174 4549.65 791639 ND sc 320 141 3014.63
425062 ND Negative Control (Blank): 0 0 0 0.21 Positive Control 67
659.49 44185 6.00 (2077z - 1 ug/mL):
Example 4
Inhibition of Cell Adhesion
[0218] In order to determine the relative potency of the different
antibody-containing supernatants, the antibodies were assessed for
their ability to inhibit TGF.beta.LAP-mediated adhesion of
.alpha.V.beta.6-positive HT29 cells. Plates were coated overnight
with 10 .mu.g/ml TGF.beta.LAP, and pre-blocked with 3% BSA/PBS for
1 hour prior to the assay. Cells were then pelleted and washed
twice in HBBS, after which the cells were then resuspended in HBSS
at appropriate concentrations. The cells were incubated in the
presence of appropriate antibodies at 4.degree. C. for 30 minutes
in a V-bottom plate. The antigen coating solution was removed and
the plates were blocked with 100 .mu.L of 3% BSA for one hour at
room temperature. Plates were 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 30 minutes. 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 results for twelve
antibodies are summarized in Table 4. Those antibodies shown ranged
in potency from 62% inhibition to over 100% inhibition, relative to
coated and uncoated control wells on the plate which were used to
represent the maximum and minimum adhesion values that could be
obtained in the assay. TABLE-US-00004 TABLE 4 Adhesion Assay
Antibody Assay 1% Assay 2% Average % ID Inhibition Inhibition
Inhibition sc 049 80% 98% 89% sc 058 77% 46% 62% sc 097 96% 106%
101% sc 133 99% 106% 103% sc 161 98% 106% 102% sc 188 99% 103% 101%
sc 254 98% 106% 102% sc 264 98% 100% 99% sc 277 98% 101% 100% sc
298 98% 102% 100% sc 320 97% 97% 97% sc 374 118% 89% 104%
Example 5
Cross-Reactivity to Macaque .alpha.V.beta.6 and Human .alpha.V
[0219] Cross-reactivity of the antibody-containing supernatants to
macaque .alpha.V.beta.6 was tested on the supernatants using FACS
analysis on HEK-293 cells transiently transfected with cynomolgus
.alpha.V and cynomolgus .beta.6.
[0220] Cross-reactivity to human .alpha.V was also tested. For this
assay, cross-reactivity was tested on the supernatants using FACS
analysis on parental A375M cells, which express .alpha.V.beta.3 and
.alpha.V.beta.5, but not .alpha.V.beta.6. This screen was designed
to show that the antibodies were specifically recognizing either
the .beta.6 chain or the .beta.6 chain in combination with
.alpha.V. The human .alpha.V assay was run at the same time as the
macaque .alpha.V.beta.6 cross-reactivity screen.
[0221] The assays were performed as follows. A375M cells that were
approximately 75% confluent were labeled with CFSE intracellular
dye by dissociating and then pelleting cells (equivalent to 250,000
to 300,000 cells per well) in a falcon tube, then resuspending in
0.125 .mu.M CFSE in FACS buffer to a final volume of 100 .mu.L for
every 250,000 cells, and then by incubating at 37.degree. C. for
five minutes. The cells were then pelleted, the supernatant
discarded, and resuspended in FACS buffer and incubated for 30
minutes at 37.degree. C. Cells were then washed twice with FACS
buffer and resuspended in a final volume of 100 .mu.L FACS buffer
per well.
[0222] HEK-293 cells were transiently transfected with cynomolgus
.alpha.V and cynomolgus .beta.6. After 48 hours, the cells were
collected and resuspended in FACS buffer to reach a final
concentration of approximately 50,000 cells in 100 .mu.L.
[0223] Approximately 100,000 cells total, comprising a 50:50 mix of
CFSE-labeled A375M cells and transfected 293 cells, were used in
each reaction as follows. 100 .mu.L of CFSE-labeled A375M cells and
100 .mu.L of 293 cells were dispensed into a V-bottom plate. The
cells in the plate were pelleted at 1500 rpm for 3 minutes, and
then resuspended in 100 .mu.L FACS buffer. The pelleting step was
repeated, and the FACS buffer supernatant was removed. The
harvested antibody-containing supernatants, or control primary
antibodies were added in a volume of 50 .mu.L and the cells were
resuspended. Primary antibody controls were murine .alpha.V.beta.6
(Cat#MAB2077z, Chemicon) and an anti-.alpha.V recombinant. The
plate was incubated on ice for 45 minutes, after which 100 .mu.L
FACS buffer was added to dilute the primary antibody. The cells
were then pelleted by centrifuging at 1500 rpm for 3 minutes, and
the pellet was resuspended in 100 .mu.L FACS buffer. The pelleting
step was repeated, and the FACS buffer supernatant was removed.
Cells were then resuspended in the appropriate secondary antibody
(5 .mu.g/ml) with 7AAD dye (10 .mu.g/ml), and stained on ice for 7
minutes. Then 150 .mu.L of FACS buffer was added and the cells were
pelLeted at 1500 rpm for 3 minutes, after which the cells were
washed in 100 .mu.L FACS buffer, pelleted, and then resuspended in
250 .mu.L buffer and added to FACS tubes. Samples were analyzed on
a high throughput FACS machine and analyzed using Cell Quest Pro
software.
[0224] The results for twelve antibodies are summarized in Table 5,
and demonstrate that the antibodies shown were able to specifically
bind to macaque .alpha.V.beta.6 but were not able to
non-specifically bind human .alpha.V on the parental A375M cells.
TABLE-US-00005 TABLE 5 Cross-Reactivity to Macaque .alpha.V.beta.6
and Human .alpha.V Mac AVB6 A375M A375M Antibody ID % Cells Shifted
Mac AVB6 GeoMean % Cells Shifted GeoMean sc 049 23% 30.19 20% 1.74
sc 058 25% 22.77 18% 1.78 sc 097 35% 37.04 24% 1.84 sc 133 32%
35.22 24% 1.79 sc 161 14% 32.98 11% 16.68 sc 188 18% 23.9 13% 1.65
sc 254 59% 78.49 55% 2.31 sc 264 55% 66.38 46% 2.35 sc 277 35%
33.35 23% 1.86 sc 298 53% 63.08 45% 2.14 sc 320 19% 33.45 15% 23.18
sc 374 51% 61.79 39% 2.14 Human IgG Isotype 1% (day 1) 9.54 (day 1)
5% (day 1) 1.66 (day 1) Control 0% (day 2) 7.39 (day 2) 1% (day 2)
7.23 (day 2) Mouse IgG2 with Murine Secondary 1% (day 1) 8.85 (day
1) 4% (day 1) 1.67 (day 1) Antibody 0% (day 2) 11.21 (day 2) 3%
(day 2) 11.16 (day 2) Positive Control 2077z 42% (day 1) 55.52 (day
1) 30% (day 1) 2.03 (day 1) (1 ug/ml) 11% (day 2) 28.11 (day 2) 5%
(day 2) 15.36 (day 2)
Example 6
.alpha.V.beta.6-Specific Hemolytic Plaque Assay
[0225] Antibody-secreting plasma cells were selected from each
harvest for the production of recombinant antibodies. Here, a
fluorescent plaque assay was used to identify single plasma cells
expressing antibodies against .alpha.V.beta.6. Then, the single
cells were subjected to reverse transcription and polymerase chain
reaction to rescue and amplify the variable heavy and variable
light chains that encoded the initial antibody specificity, as
described in Example 7. The preparation of a number of specialized
reagents and materials needed to conduct the
.alpha.V.beta.6-specific hemolytic plaque assay are described
below.
[0226] Biotinylation of Sheep red blood cells (SRBC). SRBC were
stored in RPMI media as a 25% stock. A 250 .mu.l SRBC packed-cell
pellet was obtained by aliquoting 1.0 mL of the stock into a 15-mL
falcon tube, spinning down the cells and removing the supernatant.
The cell pellet was then re-suspended in 4.75 mL PBS at pH 8.6 in a
50 mL tube. In a separate 50 mL tube, 2.5 mg of Sulfo-NHS biotin
was added to 45 mL of PBS at pH 8.6. Once the biotin had completely
dissolved, 5 mL of SRBCs was added and the tube was rotated at room
temperature for 1 hour. The SRBCs were centrifuged at 3000 g for 5
minutes. The supernatant was drawn off and 25 mL PBS at pH 7.4 was
added as a wash. The wash cycle was repeated 3 times, then 4.75 mL
immune cell media (RPMI 1640 with 10% FCS) was added to the 250
.mu.l biotinylated-SRBC (B-SRBC) pellet to gently re-suspend the
B-SRBC (5% B-SRBC stock). The stock was stored at 4.degree. C.
until needed.
[0227] Streptavidin (SA) coating of B-SRBC. One mL of the 5% B-SRBC
stock was transferred into to a fresh eppendorf tube. The B-SRBC
cells were pelleted with a pulse spin at 8000 rpm (6800 rcf) in a
microfuge. The supernatant was then drawn off, the pellet was
re-suspended in 1.0 mL PBS at pH 7.4, and the centrifugation was
repeated. The wash cycle was repeated 2 times, then the B-SRBC
pellet was resuspended in 1.0 mL of PBS at pH 7.4 to give a final
concentration of 5% (v/v). 10 .mu.l of a 10 mg/mL Streptavidin
(CalBiochem, San Diego, Calif.) stock solution was added. The tube
was mixed and rotated at RT for 20 minutes. The washing steps were
repeated and the SA-SRBC were re-suspended in 1 mL PBS pH 7.4 (5%
(v/v)).
[0228] Human .alpha.V.beta.6 coating of SA-SRBC. Soluble antigen
(lacking the transmembrane domain) was used for coating the SRBC.
Both chains were used because .alpha.V.beta.6 is only presented on
the cell surface as a dimer. The SA-SRBC were coated with the
biotinylated-.alpha.V.beta.6 at 50 .mu.g/mL, mixed and rotated at
room temperature for 20 minutes. The SRBC were washed twice with
1.0 mL of PBS at pH 7.4 as above. The Ag-coated SRBC were
re-suspended in RPMI (+10% FCS) to a final concentration of 5%
(v/v).
[0229] Determination of the quality of .alpha.V.beta.6-SRBC by
immunofluorescence (IF). 10 .mu.l of 5% SA-SRBC and 10 .mu.l of 5%
Ag-coated SRBC were each added to separate fresh 1.5 mL eppendorf
tube containing 40 .mu.l of PBS. Human anti-.alpha.V.beta.6
antibodies were added to each sample of SRBCs at 50 .mu.g/mL. The
tubes were rotated at room temperature for 25 min, and the cells
were then washed three times with 100 .mu.l of PBS. The cells were
re-suspended in 50 .mu.l of PBS and incubated with 2 .mu.g/mL
Gt-anti Human IgG Fc antibody conjugated to the photostable
fluorescent dye Alexa488 (Molecular Probes, Eugene, Oreg.). The
tubes were rotated at room temperature for 25 min, followed by
washing with 100 .mu.l PBS and re-suspension in 10 .mu.l PBS. 10
.mu.l of the stained cells were spotted onto a clean glass
microscope slide, covered with a glass coverslip, observed under
fluorescent light, and scored on an arbitrary scale of 0-4 to
assess the quality of the isolated cells.
[0230] Preparation of Plasma Cells. The Contents of a Single B Cell
Culture Well previously identified as neutralizing for
.alpha.V.beta.6 activity (therefore containing a B cell clone
secreting the immunoglobulin of interest), was harvested. The B
cell culture present in the well was recovered by addition of
RPMI+10% FCS at 37.degree. C. The cells were re-suspended by
pipetting and then transferred to a fresh 1.5 mL eppendorf tube
(final volume approximately 500-700 .mu.l). The cells were
centrifuged in a microfuge at 1500 rpm (240 rcf) for 2 minutes at
room temperature, then the tube was rotated 180 degrees and
centrifuged again for 2 minutes at 1500 rpm. The freeze media was
drawn off and the immune cells were resuspended in 100 .mu.l RPMI
(10% FCS), then centrifuged. This washing with RPMI (10% FCS) was
repeated and the cells re-suspended in 60 .mu.l RPMI (FCS) and
stored on ice until ready to use.
[0231] Performance of the Hemolytic Plaque Assay. To the 60 .mu.l
sample of immune cells was added 60 .mu.l each of
.alpha.V.beta.6-coated SRBC (5% v/v stock), 4.times. guinea pig
complement (Sigma, Oakville, ON) stock prepared in RPMI (FCS), and
4.times. enhancing sera stock (1:900 in RPMI (FCS)). The mixture
(3-5 .mu.l) was spotted onto plastic lids from 100 mm Falcon tissue
culture plates and the spots were covered with undiluted paraffin
oil. The slides were incubated at 37.degree. C. for a minimum of 45
minutes.
[0232] Analysis of Plaque assay results. The coating of the sheep
red blood cells with the catalytic domain of human .alpha.V.beta.6
was successful. These Ag-coated red blood cells were then used to
identify antigen-specific plasma cells from the wells shown below
in Table 6. These cells were then isolated by micromanipulation.
After micromanipulation to rescue the antigen-specific plasma
cells, the genes encoding the variable region genes were rescued by
RT-PCR on a single plasma cell, as described further in Example 7.
TABLE-US-00006 TABLE 6 Plaque Assay Results Parent Plate ID Plaque
Assay Plate Row Column Assay Single Cells 68 B 10 Fluorescent 45-57
296 D 10 Fluorescent 58-59 318 F 1 Hemolytic 60-62 612 G 1
Fluorescent 187-189 752 D 12 Fluorescent 95-100 762 D 8 Fluorescent
277-286 766 B 5 Fluorescent 132-143, 147-150 827 E 12 Fluorescent
159-170 659 F 11 Fluorescent 252-263 761 H 3 Fluorescent 264-276
765 A 8 Fluorescent 287-298 652 D 2 Fluorescent 374-379, 392-397
806 A 6 Fluorescent 312-321
Example 7
Recombinant Protein Isolation
[0233] After isolation of the desired single plasma cells from
Example 4, mRNA was extracted and reverse transcriptase PCR was
conducted to generate cDNA encoding the variable heavy and light
chains of the antibody secreted by each cell. The human variable
heavy chain cDNA was digested with restriction enzymes that were
added during the PCR and the product of this reaction were cloned
into an IgG2 expression vector with compatible overhangs for
cloning. This vector was generated by cloning the constant domain
of human IgG2 into the multiple cloning site of pcDNA3.1+/Hygro
(Invitrogen, Burlington, Ontario, Canada). The human variable light
chain cDNA was digested with restriction enzymes that were added
during the PCR reaction and the products of this reaction were
cloned into an IgKappa or IgLamda expression vector with compatible
overhangs for cloning. This vector was generated by cloning the
constant domain of human IgK or IgL into the multiple cloning site
of pcDNA3.1+/Neo (Invitrogen).
[0234] The heavy chain and the light chain expression vectors were
then co-transfected using lipofectamine into a 60 mm dish of 70%
confluent human embryonal kidney (HEK) 293 cells. The transfected
cells secreted a recombinant antibody with the identical
specificity as the original plasma cell for 24 to 72 hours. The
supernatant (3 mL) was harvested from the HEK 293 cells and the
secretion of an intact antibody was demonstrated with a sandwich
ELISA to specifically detect human IgG. Specificity was confirmed
through binding of the recombinant antibody to .alpha.V.beta.6
using ELISA. The rescued clones secreting antibody that could bind
to the target antigen are summarized in Table 7. TABLE-US-00007
TABLE 7 Secretion and Binding Data for the Recombinant Antibodies
Parent Plate ID Antibody Plate Row Column ID 68 B 10 49 296 D 10 58
612 G 1 188 752 D 12 97 762 D 8 277 766 B 5 133 827 E 12 161 659 F
11 254 761 H 3 264 765 A 8 298 652 D 2 374 806 A 6 320
Example 8
Purification of Recombinant Antibodies
[0235] For larger scale production of the anti-.alpha.V.beta.6
antibodies, heavy and light chain expression vectors 2.5 .mu.g of
each chain/dish) were lipofected into ten 100 mm dishes that were
70% confluent with HEK 293 cells. The transfected cells were
incubated at 37.degree. C. for 4 days, the supernatant (6 mL) was
harvested and replaced with 6 mL of fresh media. At day 7, the
supernatant was removed and pooled with the initial harvest (120 mL
total from 10 plates). The antibodies were purified from the
supernatant using Protein-A Sepharose (Amersham Biosciences,
Piscataway, N.J.) affinity chromatography (1 mL). The antibodies
were eluted from the Protein-A column with 500 .mu.L of 0.1 M
Glycine pH 2.5. The eluate was dialyzed in PBS pH 7.4 and filter
sterilized. The antibodies were analyzed by non-reducing SDS-PAGE
to assess purity and yield. Protein concentration was determined by
determining the optical density at 280 nm.
Example 9
Structural Analysis OF .alpha.V.beta.6 Antibodies
[0236] 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.V.beta.6
antibodies is provided in the sequence listing with nucleotide and
amino acid sequences for each gamma and kappa/lambda chain
combination. The variable heavy sequences were analyzed to
determine the VH family, the D-region sequence and the J-region
sequence. The sequences were then translated to determine the
primary amino acid sequence and compared to the germline VH, D and
J-region sequences to assess somatic hypermutations.
[0237] Table 8 is a table comparing the antibody heavy chain
regions to their cognate germ line heavy chain region. Table 9 is a
table comparing the antibody kappa or lambda light chain regions to
their cognate germ line light chain region. 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.V.beta.6 was studied in detail. These assays revealed
several points specific to anti-.alpha.V.beta.6 antibodies.
[0238] According the sequencing data, the primary structure of the
heavy chains of sc 298 and sc 374 are similar, but not identical.
sc 254 is structurally different from the other two. It should also
be appreciated that where a particular antibody differs from its
respective germline sequence at the amino acid level, the antibody
sequence can be mutated back to the germline sequence. 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 9 shows that the light chain sequence of sc 298 (SEQ
ID NO.: 40) differs from the corresponding germline sequence (SEQ
ID NO.:68) by a Val to Ala mutation (mutation 1) in the FR1 region,
via a Leu to Ala mutation (mutation 2) in the CDR1 region and an
Asn to Ser in the FR3 region. Thus, the amino acid or nucleotide
sequence encoding the light chain of sc 298 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 sc 298 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 sc 298 can be modified to change mutation 3 to yield
the germline sequence at the site of mutation 3. Still further
again, the amino acid or nucleotide sequence encoding the light
chain of sc 298 can be modified to change mutation 1, mutation 2
and mutation 3 to yield the germline sequence at the sites of
mutations 1, 2 and 3. Still further again, the amino acid or
nucleotide sequence encoding the light chain of sc 298 can be
modified to change any combination of mutation 1, mutation 2 and
mutation 3. In another example, heavy chain of sc 264 (SEQ ID NO:
30) differs from it's germline (SEQ ID NO: 55) at position 61. Thus
the amino acid or nucleotide sequence encoding the heavy chain of
sc 264 can be modified from a N to Y to yield the germline
sequence. Tables 10-13 below illustrate the position of such
variations from the germline for sc 133, sc 188 and sc 264. Each
row represents a unique combination of germline and non-germline
residues at the position indicated by bold type. Particular
examples of an antibody sequence that can be mutated back to the
germline sequence include: sc 133 where the L at amino acid 70 of
the heavy chain is mutated back to the germline amino acid of M
(referred to herein as sc 133 TMT); sc 133 where the N at amino
acid 93 of the light chain is mutated back to the germline amino
acid of D (referred to herein as sc 133 WDS); and sc 264 where the
A at amino acid 84 of the light chain is mutated back to the
germline amino acid of D (referred to herein as sc 264 ADY).
[0239] In one embodiment, the invention features modifying one or
more of the amino acids in the CDR regions, i.e., CDR1, CDR2 and/or
CDR3. In one example, the CDR3 of the heavy chain of an antibody
described herein is modified. Typically, the amino acid is
substituted with an amino acid having a similar side chain (a
conservative amino acid substitution) or can be substituted with
any appropriate amino acid such as an alanine or a leucine. In one
embodiment, the sc 264 CDR3, VATGRGDYHFYAMDV (amino acid residues
100-114 of SEQ ID NO: 30), can be modified at one or more amino
acids. Applicants have already demonstrated that the CDR3 region
can be modified without adversely affecting activity, i.e., see sc
264 RAD where the second G in the CDR3 region is substituted for an
A. Other modifications within the CDR3 region are also
envisaged.
[0240] In another embodiment, the sc 133 CDR3 region, RLDV, can be
modified at one or more amino acids including substituting the L
for an A and/or the V for an A. Means of substituting amino acids
are well known in the art and include site-directed
mutagenesis.
[0241] In another embodiment, the invention includes replacing any
structural liabilities in the sequence that might affect the
heterogeneity or specificity of binding of the antibodies of the
invention. In one example, the antibody sc 264 has an RGD sequence
in the CDR3 region that might cause cross-reactive binding.
Therefore the glycine residue in the RGD can be replaced with an
alanine (sc 264 RAD). TABLE-US-00008 TABLE 8 Heavy chain analysis
SEQ Chain ID Name NO: V D J FR1 CDR1 FR2 CDR2 49 Germline
QVQLVQSGAEVKK GYTFTG WVRQAPG WINPNSGGT PGASVKVSCKAS YYMH QGLEWMG
NYAQKFQG sc 133 14 VH1-2 5-12 JH6B QVQLVQSGAEVKK GYTFTG WVRQAPG
WINPKSGDT PGASVKVSCKAS YYMH QGLEWMG NYAQKFQG 50 Germline
EVQLVESGGGLVK GFTFSS WVRQAPG SISSSSSYI PGGSLRLSCAAS YSMN KGLEWVS
YYADSVKG sc 320 42 VH3-21 D1-1 JH6B EVQLVESGGGLVK GYTFTN WVRQAPG
SISISSSYI PGGSLRLSCAAS YIMH KGLEWVS YYADSVKG 51 Germline
EVQLLESGGGLVQ GFTFSS WVRQAPG AISGSGGST PGGSLRLSCAAS YAMS KGLEWVS
YYADSVKG sc 58 6 VH3-23 D5-5 JH6B EVQLLESGGGLVQ GFTFSS WVRQAPG
AISGSGGST PGGSLRLSCAAS YVMS KGLEWVS YYADSVKG 52 Germline
QVQLVESGGGVVQ GFTFSS WVRQAPG VIWYDGSNK PGRSLRLSCAAS YGMH KGLEWVA
YYADSVKG sc 298 38 VH3-33 D6-6 JH6B QVQLVESGGGVVQ GFTFSS WVRQAPG
VIWYGGSNK PGRSLRLSCAAS YGMH KGLEWVA YYADSVKG sc 374 46 VH3-33 D6-6
JH6B QVQLVESGGGVVQ GFTFSS WVRQAPG VIWYDGSNK PGRSLRLSCAAS YGMH
KGLEWVA YYADSVKG 53 Germline QVQLQESGPGLVK GGSISS WIRQHPG YIYYSGSTY
PSQTLSLTCTVS GGYYWS KGLEWIG YNPSLKS sc 254 26 VH4-31 D6-13 JH6B
QVQLQESGPGLVK GGSISS WIRQHPG YIYYSGSTY PSQTLSLTCTVS GGYYWS KGLEWIG
YNPSLKS 54 Germline QVQLQESGPGLVK GGSISS WIRQHPG YIYYSGSTY
PSQTLSLTCTVS GGYYWS KGLEWIG YNPSLKS sc 49 2 VH4-31 D3-3 JH4B
QVQLQESGPGLVK GGSIRS WIRQHPG NIYYSGSTY PSQTLSLTCTVA GDYYWS KGLEWIG
YNPSLKS 55 Germline QVQLQESGPGLVK GGSISS WIRQHPG YIYYSGSTY
PSQTLSLTCTVS GGYYWS KGLEWIG YNPSLKS Sc 264 30 VH4-31 D4-17 JH6B
QVQLQESGPGLVK GGSISS WIRQHPG YIYYSGRTY PSQTLSLTCTVS GGYYWS KGLEWIG
NNPSLKS 56 Germline QVQLQESGPGLVK GGSISS WIRQHPG YIYYSGSTY
PSQTLSLTCTVS GGYYWS KGLEWIG YNPSLKS Sc 188 22 VH4-31 D4-23 JH6B
QVQLQESGPGLVK GGSISS WIRQHPG YIYYSGSTS PSQTLSLTCTVS GVYYWT NGLEWIG
YNPSLKS 57 Germline EVQLVQSGAEVKK GYSFTS WVRQMPG IIYPGDSDT
PGESLKISCKGS YWIG KGLEWMG RYSPSFQG Sc 97 10 VH5-51 D3-22 JH3B
EVQLVQSGAEVKK GYSFTS WVRQMPG IIYPGDSDT PGESLKISCKGS YWIG KGLEWMG
RYSPSFQG 58 Germline EVQLVQSGAEVKK GYSFTS WVRQMPG IIYPGDSDT
PGESLKISCKGS YWIG KGLEWMG RYSPSFQG Sc 277 34 VH5-51 D3-10 JH6B
EVQLVQSGAEVKK GYSFPS WVRQMPG IIYPGDSDT PGESLKISCKGS YWIG KGLEWMG
RYSPSFQG 59 Germline EVQLVQSGAEVKK GYSFTS WVRQMPG IIYPGDSDT
PGESLKISCKGS YWIG KGLEWMG RYSPSFQG Sc 161 18 VH5-51 D6-13 JH6C
EVQLVQSGAEVKK GYSFTS WVRQMPG IIYPGDSDT PGESLKISCKGS YWIG KGLEWMG
RYSPSFQG SEQ Chain ID Name NO: FR3 CDR3 FR4 49 RVTMTRDTSISTAYME
RL-- WGQGTT LSRLRSDDTAVYYCAR VTVSS sc 133 14 RVTLTRDTSTSTAYME RLDV
WGQGTT LSRLRSDDTAVYYCAR VTVSS 50 RFTISRDNAKNSLYLQ -- WGQGTT
MNSLRAEDTAVYYCAR VQLERYYYY VTVSS YGMDV sc 320 42 RFTISRDNAKNSLYLQ
DPVPLERRD WGQGTT MNSLRAEDTAVYYCAR YYYGMDV VTVSS 51 RFTISRDNSKNTLYLQ
- WGQGTT MNSLRAEDTAVYYCAK VDTAMVYYG VTVSS MDV sc 58 6
RFTISRDNSKNTLYLQ GVDTAMVTY WGQGTT MNSLRAEDTAVYYCAK GMDV VTVSS 52
RFTISRDNSKNTLYLQ -IAAR-- WGQGTT MNSLRAEDTAVYYCAR YYYYYGMDV VTVSS sc
298 38 RFTISRDNSKNTLYLQ DLAARRGDY WGQGTT MNSLRAEDTAVYYCAR YYYGMDV
VTVSS sc 374 46 RFTISRDNSKNTLYLQ TEGTAARLY WGQGTT MNSLRAEDTAVYYCAR
YYYGMDV VTVSS 53 RVTISVDTSKNQFSLK --GIAAAG-- WGQGTT
LSSVTAADTAVYYCAR YYYYYGMDV VTVSS sc 254 26 RVTISVDTSKNQFSLK
YRGPAAGRG WGQGTT LSSVTAADTAMYYCAR DFYYFGMDV VTVSS 54
RVTISVDTSKNQFSLK --- WGQGTL LSSVTAADTAVYYCAR ITIFGVFDY VTVSS sc 49
2 RITISVATSRNQFSLK GGAITIFGV WGQGTL LTSVTAADTAVYYCAR FDY VTVSS 55
RVTISVDTSKNQFSLK VAT--- WGQGTT LSSVTAADTAVYYCAR YYYYYGMDV VTVSS Sc
264 30 RVTISVDTSKNQFSLK VATGRGDYH WGQGTT LSSVTAADTAVYYCAR FYAMDV
VTVSS 56 RVTISVDTSKNQFSLK --- WGQGTT LSSVTAADTAVYYCAR LRYYYYYGM
VTVSS DV Sc 188 22 RVTISVDTSKKQFSLN EGPLRGDYY WGQGTT
LTSVTAADTAVYYCAR YGLDV VTVSS 57 QVTISADKSISTAYLQ --- WGQGTM
WSSLKASDTAMYYCAR SSGYYYAFDI VTVSSA Sc 97 10 QVILSADKSISTAYLQ
HDESSGYYY WGQGTM WSSLKASDTAMYYCAR VFDI VTVSSA 58 QVTISADKSISTAYLQ
-----GMDV WGQGTT WSSLKASDTAMYYCAR VTVSS Sc 277 34 QVTISADKSISTAYLQ
HPMEDGMDV WGQGTT WSSLKASDTAMYYCAR VTVSS 59 QVTISADKSISTAYLQ
-GIAAAG- WGKGTT WSSLKASDTAMYYCAR YYYGMDV VTVSSA Sc 161 18
QVTISADKSISTAYLQ HGIAAAGFY WGQGTT WSSLKASDTAMYYCAR YYYMDV
VTVSSA
[0242] TABLE-US-00009 TABLE 9 Light chain analysis SEQ Chain ID
Name NO: J FR1 CDR1 FR2 CDR2 FR3 CDR3 J V Kappa 60 Germline
DIVMTQTPLSLS KSSQSLLH WYLQKPGQ EVSN GVPDRFSGSGSGTDFT MQSTQL FGQGTK
VTPGQPASISC SDGKTYLY PPQLLIY RFS LKISRVEAEDVGVYYC PWT VEIK Sc 254
28 A2 JK1 DIVMTQTPLSLS KSSQSLLN WYLQKPGQ EVSN GVPDRFSGSGSGTDFT
MQGIQL FGQGTK VTPGQPASIFC SDGKTYLC PPQLLIY RFS LKISRVEAEDVGVYYC
PWAF VEIK 61 Germline EIVLTQSPGTLS RASQSVSS WYQQKPGQ GASS
GIPDRFSGSGSGTDFT QQYGSS FGQGTK LSPGERATLSC SYLA APRLLIY RAT
LTISRLEPEDFAVYYC PWT VEIK sc 188 24 A27 JK1 EIVLTQSPGTLS RAGQTISS
WYQQKPGQ GASS GIPDRFSGSGSGTDFT QQYGSS FGQGTK LSPGERATLSC RYLA
APRPLIY RAT LTISRLEPEDFAVYYC PRT VEIK sc 374 48 A27 JK1
EIVLTQSPGTLS RASQSVSS WYQQKPGQ GASS DIPDRFSGSGSGTDFT QQYGSS FGQGTK
LSPGERATLSC SYLA APRLLIY RAT LTISRLEPEDFAVYYC PWT VEIK 62 Germline
EIVLTQSPGTLS RASQSVSS WYQQKPGQ GASS GIPDRFSGSGSGTDFT QQYGSS FGQGTK
LSPGERATLSC SYLA APRLLIY RAT LTISRLEPEDFAVYYC PYT LEIK Sc 49 4 A27
JK2 EIVLTQSPGTLS RASQSVSS WYQQKPGQ GASS GIPDRFSGSGSGTDFT QQYGSS
FGQGTK LSPGERATLSC SYLA APRLLIY RAT LTISRLEPEDFAVYYC PCS LEIK 63
Germline EIVLTQSPGTLS RASQSVSS WYQQKPGQ GASS GIPDRFSGSGSGTDFT
QQYGSS FGPGTK LSPGERATLSC SYLA APRLLIY RAT LTISRLEPEDFAVYYC PFT
VDIKR sc 161 20 A27 JK3 EIVLTQSPDTLS RASQNVNR WYQQKPGQ GTSN
GIPDRFSGSGSGTDFT QQCGSL FGPGTK LSPGERASLSC NYLV APRLLIY RAT
LTISRLEPEDFAVYYC PFT VDIKR V Lambda 64 Germline QSVLTQPPSVSA
SGSSSNIG WYQQLPGT DNNK GIPDRFSGSKSGTSAT GTWDSS FGTGTK APGQKVTISC
NNYVS APKLLIY RPS LGITGLQTGDEADYYC LSA-YV VTV sc 133 16 V1-19 JL1
QSVLTQPPSVSA SGSSSNIG WYQQLPGT DNNK GIPDRFSGSKSGTSAT GTWNSS FGTGTK
APGQKVTISC NNYVS APKLLIY RPS LGITGLQTGDEADYYC LSAGYV VTV 65
Germline QSVLTQPPSVSA SGSSSNIG WYQQLPGT DNNK GIPDRFSGSKSGTSAT
GTWDSS FGGGTK APGQKVTISC NNYVS APKLLIY RPS LGITGLQTGDEADYYC LSAVV
LTVL sc 320 44 V1-19 JL2 QSVLTQPPSMSA SGSSSNIG WYQQLPGT DNNK
GIPDRFSGSKSGTSAT GTWDSS FGGGTK APGQKVTISC NNYVS APKLLIY RPS
LGITGLQTGDEADYYC LSAGV LTVL 66 Germline SYELTQPPSVSV SGDALPKK
WYQQKSGQ EDSK GIPERFSGSSSGTMAT YSTDSS FGGGTK SPGQTARITC YAY APVLVIY
RPS LTISGAQVEDEADYYC GNHVV LTVL sc 277 36 V2-7 JL2 SYELTQPPSVSV
SGDALPKK WYQQKSGQ DDNK GIPERFSGSSSGTMAT YSTDSS FGGGTK SPGQTARITC
YAF APVLVIY RPS LTITGAQVEDEADYYC GHHV LTVL sc 97 12 V2-7 JL2
SYELTQPPSVSV SGDALPKK WYQQKSGQ EDIK GIPERFSGSSSGTMAT YSTDSS FGGGTK
SPGQTARITC YAY APVLVIY RPS LTISGAQVEDEADYYC GNHWVF LTVL 67 Germline
SYELTQPPSVSV SGDALPKK WYQQKSGQ EDSK GIPERFSGSSSGTMAT YSTDSS FGGGTK
SPGQTARITC YAY APVLVIY RPS LTISGAQVEDEADYYC GNHVV LTVL sc 58 8 V2-7
JL3 SYELTQPPSVSV SGDALPKK WYQQKSGQ DDSK GIPERFSGSSSGTMAT YSTDSS
FGGGTK SPGQTRITC YAY APVLVIY RPS LTISGAQVEDEADYYC GNHRV LTVL 68
Germline SSELTQDPAVSV QGDSLRSY WYQQKPGQ GKNN GIPDRFSGSSSGNTAS
NSRDSS FGGGTK ALGQTVRITC YAS APVLVIY RPS LTITGAQAEDEADYYC GNHVV
LTVL sc 298 40 V2-13 JL2 SSELTQDPVVSV QGDSLRSY WYQQKPGQ GKNN
GIPDRFSGSNSGNTAS NSRDSS FGGGTK ALGQTVRITC YLS APVLVIY RPS
LTITGAQAEDEADYYC GNHL LTVL 69 Germline SYELTQPSSVSV SGDVLAKK
WFQQKPGQ KDSE GIPERFSGSSSGTTVT YSAADN FGGGTK SPGQTARITC YAR APVLVIY
RPS LTISGAQVEDEADYYC NVV LTVL sc 264 32 V2-19 JL2 SYELTQPSSVSV
SGDVLAKK WFHQKPGQ KDSE GIPERFSGSSSGTTVT YSAADN FGGGTK SPGQTARITC
SAR APVLVIY RPS LTISGAQVEDEAAYYC NLV LTVL
[0243] TABLE-US-00010 TABLE 10 Exemplary Mutations of sc 133 Heavy
Chain (SEQ ID NO: 14) to Germline (SEQ ID NO: 49) at the indicated
Residue Number 54 57 70 76 N G M T N G L I N G L T N D M I N D L I
N D M T N D L T K G M I K G M T K G L I K G L T K D M I K D L I K D
M T
[0244] TABLE-US-00011 TABLE 11 Exemplary Mutations of sc 188 Light
Chain (SEQ ID NO: 24) to Germline (SEQ ID NO: 61) at the indicated
Residue Number 26 28 29 32 47 G S V S L G S V S P G S V R P G S V R
L G S V R L G S V S P G S I R P G S I R L G T V R L G T V S P G T V
S L G T I R P G T I R L G T I S L S S V S P S S V R P S S V R L S S
V R L S S V S P S S I R P S S I R L S T V R L S T V S P S T V S L S
T I R P S T I R L S T I S L
[0245] TABLE-US-00012 TABLE 12 Exemplary Mutations of sc 188 Heavy
Chain (SEQ ID NO: 22) to Germline (SEQ ID NO: 56) at the indicated
Residue Number 33 37 45 60 78 83 85 G S K Y N K S G S K Y N K T G S
K Y N N S G S K Y N N T G S K Y K N S G S K Y K N T G S K Y K K S G
S K Y K K T G S K S N K S G S K S N K T G S K S N N S G S K S N N T
G S K S K N S G S K S K N T G S K S K K S G S K S K K T G S N Y N K
S G S N Y N K T G S N Y N N S G S N Y N N T G S N Y K N S G S N Y K
N T G S N Y K K S G S N Y K K T G S N S N K S G S N S N K T G S N S
N N S G S N S N N T G S N S K N S G S N S K N T G S N S K K S G S N
S K K T V S K Y N K S V S K Y N K T V S K Y N N S V S K Y N N T V S
K Y K N S V S K Y K N T V S K Y K K S V S K Y K K T V S K S N K S V
S K S N K T V S K S N N S V S K S N N T V S K S K N S V S K S K N T
V S K S K K S V S K S K K T V S N Y N K S V S N Y N K T V S N Y N N
S V S N Y N N T V S N Y K N S V S N Y K N T V S N Y K K S V S N Y K
K T V S N S N K S V S N S N K T V S N S N N S V S N S N N T V S N S
K N S V S N S K N T V S N S K K S V S N S K K T G I K Y N K S G I K
Y N K T G I K Y N N S G I K Y N N T G I K Y K N S G I K Y K N T G I
K Y K K S G I K Y K K T G I K S N K S G I K S N K T G I K S N N S G
I K S N N T G I K S K N S G I K S K N T G I K S K K S G I K S K K T
G I N Y N K S G I N Y N K T G I N Y N N S G I N Y N N T G I N Y K N
S G I N Y K N T G I N Y K K S G I N Y K K T G I N S N K S G I N S N
K T G I N S N N S G I N S N N T G I N S K N S G I N S K N T G I N S
K K S G I N S K K T V I K Y N K S V I K Y N K T V I K Y N N S V I K
Y N N T V I K Y K N S V I K Y K N T V I K Y K K S V I K Y K K T V I
K S N K S V I K S N K T V I K S N N S V I K S N N T V I K S K N S V
I K S K N T V I K S K K S V I K S K K T V I N Y N K S V I N Y N K T
V I N Y N N S V I N Y N N T V I N Y K N S V I N Y K N T V I N Y K K
S V I N Y K K T V I N S N K S V I N S N K T V I N S N N S V I N S N
N T V I N S K N S V I N S K N T V I N S K K S V I N S K K T
[0246] TABLE-US-00013 TABLE 13 Exemplary Mutations of sc 264 Light
Chain (SEQ ID NO: 32) to Germline (SEQ ID NO: 69) at the indicated
Residue Number 31 36 84 Y H A Y H D Y Q A S H D S Q D S Q A
Example 10
Potency Determination of .alpha.V.beta.6 Antibodies
[0247] To discriminate antibodies based on their ability to prevent
the adhesion of HT29 cells to TGF.beta.LAP, the following adhesion
assay was performed.
[0248] Nunc MaxiSorp (Nunc) plates were coated overnight with 50
.mu.L of 10 g/ml TGF Beta1 LAP (TGF.beta.LAP), and pre-blocked with
3% BSA/PBS for 1 hour prior to the assay. HT29 cells grown to the
optimal density were then pelleted and washed twice in HBBS (with
1% BSA and without Mn.sup.2+), after which the cells were then
resuspended in HBSS at 30,000 cell per well. The coating liquid was
removed from the plates, which were then blocked with 100 .mu.L 3%
BSA at room temperature for 1 hour and thereafter washed twice with
PBS.
[0249] Antibody titrations were prepared in 1:3 serial dilutions in
a final volume of 30 .mu.L and at two times the final
concentration. Care was taken to ensure that the PBS concentration
in the control wells matched the PBS concentration in the most
dilute antibody well. 30 .mu.L of cells were added to each well,
and the cells were incubated in the presence of the antibodies at
4.degree. C. for 40 minutes in a V-bottom plate. The cell-antibody
mixtures were transferred to the coated plate and the plate was
incubated at 37.degree. C. for 40 minutes. 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 15 minutes. The cells were
allowed to thaw at room temperature, 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. An estimated IC.sub.50 value for each mAb was calculated
based on the maximal and minimal amount of cell adhesion possible
in the assay, as determined by positive and negative control wells.
The results for twelve antibodies are summarized in Table 14.
TABLE-US-00014 TABLE 14 Adhesion Assay Results (Estimated IC.sub.50
Values) n = 1 (ng/mL) n = 2 (ng/mL) n = 3 (ng/mL) sc 049 >5000
>5000 >5000 sc 058 4065 2028 3259 sc 097 1006 281 536 sc 133
25 16 30 sc 161 2408 137 ND sc 188 41 26 ND sc 254 63 37 37 sc 264
26 14 18 sc 277 1455 540 720 sc 298 29 25 33 sc 320 648 381 415 sc
374 277 300 549 Positive 226 185 286 Control 2077Z
Example 11
Competition Assay
[0250] To establish that the antibodies were specifically capable
of blocking .alpha.V.beta.6 integrin binding to soluble
TGF.beta.LAP, a competition assay was run with the purified
antibodies to measure their ability to bind to .alpha.V.beta.6 and
block its binding to a GST-LAP peptide.
[0251] Medium binding 96-well plates (Costar, catalog #3368) were
coated with 50 .mu.L/well of 10 .mu.g/ml GST-LAP in PBS and 0.05%
sodium azide, and incubated overnight at 4.degree. C. The plates
were then washed three times using 300 .mu.L/well of assay diluent
(1% milk in TBS (50 mM Tris, 50 mM NaCl, 1 mM MgCl.sub.2 and 1 mM
CaCl.sub.2, pH 6.9), after which the plates were blocked using 300
.mu.L/well 5% milk in TBS and incubated for 30 minutes at room
temperature. The mAbs (in 1:3 serial dilutions ranging from 10
.mu.g/ml to 0.01 .mu.g/ml) were incubated overnight with
.alpha.V.beta.6 (250 ng/ml in assay diluent containing 0.05% sodium
azide). The following day, 50 .mu.L/well of the pre-incubated
primary antibody was transferred to the GST-LAP peptide-coated
plate and incubated for one hour at room temperature. The wells
were then washed three times using 300 .mu.L/well of assay diluent.
Then, to detect the amount of .alpha.V.beta.6 bound to the plates,
mAb 2075 (Chemicon) was added at a concentration of 1 .mu.g/ml in
assay diluent (50 .mu.L/well) and incubated for one hour at room
temperature. The wells were then washed three times using 300
.mu.L/well of assay diluent, and incubated with goat anti-mouse IgG
Fc-peroxidase at 400 ng/ml in assay diluent (50 .mu.L/well) for one
hour at room temperature. The wells were then washed three times
using 300 .mu.L/well of assay diluent, and developed using 1-step
TMB (Neogen) at a total volume of 50 .mu.L/well. After 15 minutes,
the developing reaction was quenched with 50 .mu.L/well of 1N
Hydrochloric acid. The plates were read at 450 nm, and the results
for five of the antibodies are summarized in FIG. 1, which shows
that the antibodies were able to inhibit .alpha.V.beta.6 binding to
GST-LAP.
Example 12
Cross-Reactivity to .alpha.V.beta.3 or .alpha.V.beta.5
Integrins
[0252] To establish that the antibodies were functional only
against .alpha.V.beta.6 integrin and not .alpha.V.beta.3 or
.alpha.V.beta.5 integrins, the following assay was performed to
test the ability of the antibodies to inhibit the adhesion of A375M
cells to an osteopontin peptide.
[0253] Assay plates were coated with osteopontin peptide. Two
fragments of osteopontin were used: OPN 17-168 and OPN 17-314.
Assay plates were pre-blocked with 3% BSA/PBS for one hour prior to
the assay. The A375M cells were removed from a culture flask,
pelleted and washed twice with HBSS containing 1% BSA and 1 mM
Ca.sup.2+ and 1 mM Mg.sup.2+. The cells were then resuspended in
HBSS at a concentration of 30,000 cells per well. The coating
liquid containing the osteopontin fragments was removed, and the
plates were blocked with 100 .mu.L of 3% BSA for one hour at room
temperature. The coated plates were washed twice with HBSS
containing 1% BSA. Antibody titrations were prepared in 1:4 serial
dilutions in a final volume of 30 .mu.L and at twice the final
concentration. The resuspended cells were added to the wells
containing the titrated antibody in a V-bottom plate, and the cells
and antibodies were co-incubated at 4.degree. C. for 40 minutes.
The cell-antibody mixture was then transferred to the coated plate,
which was thereafter incubated at 37.degree. C. for 40 minutes. The
cells on the coated plates were next washed four times in warm
HBSS, and the cells in the plates were then frozen at -80.degree.
C. for 15 minutes. The cells were allowed to thaw at room
temperature, 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.
[0254] The results for five of the antibodies are summarized in
Table 15. A commercially available .alpha.V integrin specific
antibody was used as a positive control in this assay and exhibited
about 90% inhibition of adhesion. A commercially available
.alpha.V.beta.6 antibody served as a negative control in this assay
for adhesion to .alpha.V.beta.3 or .alpha.V.beta.5 integrins. All
antibodies were tested at a concentration of 5 .mu.g/ml and none of
the test antibodies could block adhesion to .alpha.V.beta.3 or
.alpha.V.beta.5 integrins. TABLE-US-00015 TABLE 15 Cross-Reactivity
to .alpha.V.beta.3 or .alpha.V.beta.5 Integrins Percent Antibody ID
Inhibition sc 133 3 sc 188 -2 sc 254 -5 sc 264 3 sc 298 9 .alpha.V
Control 89 .alpha.V.beta.6 Control 11 Human IgG Control 3 Mouse IgG
Control 5
Example 13
Cross-Reactivity to .alpha.4.beta.1 Integrin
[0255] To establish that the antibodies were functional only
against the .alpha.V.beta.6 integrin and not the .alpha.4.beta.1
integrin, an assay was performed to test the ability of the
antibodies to inhibit the adhesion of J6.77 cells to the CS-1
peptide of fibronectin. The assay was performed as described in
Example 12 above, with the exception that J6.77 cells were used for
binding and the CS-1 peptide of fibronectin was used to coat the
assay plates.
[0256] The results for 11 of the antibodies are summarized in Table
16. A commercially available .beta.1 integrin specific antibody was
used as a positive control in this assay and exhibited 97%
inhibition of adhesion. A commercially available .alpha.V.beta.6
specific antibody served as a negative control in this assay for
adhesion to .alpha.4.beta.1. All antibodies were used at 5 .mu.g/ml
and none of the test antibodies could block adhesion to
.alpha.4.beta.1. TABLE-US-00016 TABLE 16 Cross-Reactivity to
.alpha.4.beta.1 Integrin Percent Antibody at 5 ug/ml Inhibition sc
58 -14 sc 97 -7 sc 133 -15 sc 161 12 sc 188 -10 sc 254 0 sc 264 -8
sc 277 -17 sc 298 -7 sc 320 -8 sc 374 -8 Human IgG1 -6 Human IgG2
-9 Anti-beta1 integrin antibody 97 Anti-.alpha.V.beta.6 integrin
antibody -15 No CS-1 or antibody on plates 12 CS-1 fragment coated
plates without 10 antibody
Example 14
Cross-Reactivity to .alpha.5.beta.1 Integrin
[0257] To establish that the antibodies were functional only
against the .alpha.V.beta.6 integrin and not the .alpha.5.beta.1
integrin, an adhesion assay was performed to test the ability of
the antibodies to inhibit the adhesion of K562 cells to
fibronectin.
[0258] Assay plates were coated with the FN9-10 peptide of
fibronectin at a concentration of 12.5 .mu.g/mL. Assay plates were
pre-blocked with 3% BSA/PBS for one hour prior to the assay. The
K562 cells were removed from a culture flask, pelleted and washed
twice with HBSS containing 1% BSA and 1 mM Mn.sup.2+. The cells
were then resuspended in HBSS at a concentration of 30,000 cells
per well. The coating liquid containing the osteopontin fragments
was removed, and the plates were blocked with 100 .mu.L of 3% BSA
for one hour at room temperature. The coated plates were washed
twice with HBSS containing 1% BSA. Antibody titrations were
prepared in 1:4 serial dilutions in a final volume of 30 .mu.L and
at twice the final concentration. The resuspended cells were added
to the wells containing the titrated antibody in a V-bottom plate,
and the cells and antibodies were co-incubated at 4.degree. C. for
60 minutes. The cell-antibody mixture was then transferred to the
coated plate, which was thereafter incubated at 37.degree. C. for
40 minutes. The cells on the coated plates were next washed four
times in warm HBSS, and the cells in the plates were then frozen at
-80.degree. C. for 15 minutes. The cells were allowed to thaw at
room temperature, 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.
[0259] The results for five of the antibodies are summarized in
Table 17. Test antibodies were compared to a commercially available
.beta.5.beta.1 antibody as a positive control and an
.alpha.V.beta.6 specific antibody as a negative control. None of
the test antibodies were able to block adhesion in the assay at the
5 .mu.g/ml concentration used in this assay. TABLE-US-00017 TABLE
17 Cross-Reactivity to .alpha.5.beta.1 Integrin Percent Antibody ID
Inhibition sc 133 -12 sc 188 5 sc 254 -9 sc 264 -4 sc 298 2
.alpha.V.beta.6 Control 7 .alpha.5.beta.1 Control 78 Human IgG 11
Control
Example 15
Cross-Reactivity to Murine and Cynomolgus .alpha.V.beta.6
Integrin
[0260] In order to determine whether the antibodies exhibited
cross-reactivity to mouse .alpha.V.beta.6 or Cynomolgus
.alpha.V.beta.6, the following assay was performed.
[0261] Cross-reactivity of the mAbs to macaque and mouse
.alpha.V.beta.6 was tested on the purified mAbs using FACS analysis
on HEK-293 cells transiently transfected with cynomolgus or mouse
.alpha.V, .beta.6, or .alpha.V.beta.6. Approximately 48 hours after
transfection, the cells were collected and resuspended in FACS
buffer to reach a final concentration of approximately 50,000 cells
in 100 .mu.L.
[0262] Approximately 100,000 cells total, were used in each
reaction as follows. 200 .mu.L of 293 cells were dispensed into a
V-bottom plate. The cells in the plate were pelleted at 1500 rpm
for 3 minutes, and then resuspended in 100 .mu.L FACS buffer. The
pelleting step was repeated, and the FACS buffer supernatant was
removed. The purified mAbs, or control primary antibodies were
added in a volume of 50 .mu.L and the cells were resuspended.
Primary antibody controls were murine .alpha.V.beta.6
(Cat#MAB2077z, Chemicon) and anti-.alpha.V and anti-.beta.6
recombinants. The plate was incubated on ice for 45 minutes, after
which 100 .mu.L FACS buffer was added to dilute the primary
antibody. The cells were then pelleted by centrifuging at 1500 rpm
for 3 minutes, and the pellet was resuspended in 100 .mu.L FACS
buffer. The pelleting step was repeated, and the FACS buffer
supernatant was removed. Cells were then resuspended in the
appropriate secondary antibody (5 .mu.g/ml) with 7AAD dye (10
.mu.g/ml), and stained on ice for 7 minutes. Then 150 .mu.L of FACS
buffer was added and the cells were pelleted at 1500 rpm for 3
minutes, after which the cells were washed in 100 .mu.L FACS
buffer, pelleted, and then resuspended in 250 .mu.L buffer and
added to FACS tubes. Samples were analyzed on a high throughput
FACS machine and analyzed using Cell Quest Pro software.
[0263] The results are summarized in Table 18, and demonstrate that
mAb sc 133 and mAb sc 188 were clearly cross-reactive with mouse
and Cynomolgus .alpha.V.beta.6 and 6. mAb sc 254 appeared to
cross-react with .beta.6, .alpha.V, and .alpha.V.beta.6. mAbs sc
264 and 298 had high levels of binding to parental cells making
species cross-reactivity difficult to discern. TABLE-US-00018 TABLE
18 Cross-Reactivity with Mouse and Cynomolgus .alpha.V.beta.6 Mouse
Mouse Mouse Cynomolgus Cynomolgus Cynomolgus Antibodies Parental
alphaV beta6 alphaVbeta6 alphaV beta6 alphaVbeta6 Cells alone 0 0 0
0 1 0 0 Gt anti 0 0 0 0 0 0 0 Mouse anti 0 1 11 45 0 5 20
alphaVbeta6 anti alphaV 68 68 63 59 68 69 67 anti beta6 0 0 0 0 0 0
0 Gt anti 0 0 0 0 0 0 0 Human Human 0 1 0 1 1 1 0 IgG1 sc.133 2 4
19 49 5 10 28 sc.188 1 3 29 51 2 17 27 sc.254 8 13 21 50 16 19 26
sc.264 74 71 68 63 70 75 54 sc.298 49 45 52 53 48 52 38 Data
represent percent of cells shifted
Example 16
Internalization Assay
[0264] The internalization of the antibodies was tested using a
K562 cell line that stably expressed human .alpha.V.beta.6.
Internalization of the purified antibodies was compared to a
commercially available .alpha.V.beta.6 antibody that was not
internalized in this assay.
[0265] The results are summarized in Table 19. TABLE-US-00019 TABLE
19 Summary of the Internalization Assay Concentration Percent
Antibody (ug/mL) Internalization sc 133 10 28% sc 133 1 30% sc 188
10 38% sc 188 1 34% sc 254 10 49% sc 254 1 39% sc 264 10 76% sc 264
1 77% sc 298 10 28% sc 298 1 26%
Example 17
High Resolution Biacore Analysis
[0266] High resolution Biacore analysis using a soluble
.alpha.V.beta.6 protein to bind antibodies immobilized on CM5 chips
was performed for 5 of the .alpha.V.beta.6 antibodies to estimate
their affinity for soluble antigen.
[0267] The Biacore analysis was performed as follows. A
high-density goat .alpha. human IgG antibody surface over two CM5
Biacore chips was prepared using routine amine coupling. Each mAb
was diluted in degassed HBS-P running buffer containing 100
.mu.g/ml BSA, 1 mM MgCl.sub.2, and 1 mM CaCl.sub.2 to a
concentration of approximately 1 .mu.g/mL. More precisely, mAb sc
133 was diluted to 0.98 .mu.g/mL, mAb sc 188 was diluted to 0.96
.mu.g/mL, mAb sc 264 was diluted to 0.94 .mu.g/mL, mAb sc 254.2 was
diluted to 0.87 .mu.g/mL, and mAb sc 298 was diluted to 1.6
.mu.g/mL. Then, a capture level protocol was developed for each mAb
by capturing each mAb over a separate flow cell at a 10 .mu.L/min
flow rate at the concentrations listed above. mAbs sc 133, sc 298,
and sc 254.2 were captured for 30 seconds while mAbs sc 188 and sc
264 were captured for 1 minute. A 4-minute wash step at 50
.mu.L/min followed to stabilize the mAb baseline.
[0268] Soluble .alpha.V.beta.6 was injected for 4 minutes at a
concentration range of 116-3.6 nM for mAbs sc 133, sc 188, sc 264,
and sc 298, and 233-3.6 nM for mAb sc 254.2, with a 2.times. serial
dilution for each concentration range. A 10-minute dissociation
followed each antigen injection. The antigen samples were prepared
in the HBS-P running described above. All samples were randomly
injected in triplicate with several mAb capture/buffer inject
cycles interspersed for double referencing. The high-density goat
.alpha. mouse antibody surfaces were regenerated with one 18-second
pulse of 146 mM phosphoric acid (pH 1.5) after each cycle at a flow
rate of 100 .mu.L/min. A flow rate of 50 .mu.L/min was used for all
antigen injection cycles.
[0269] The data were then fit to a 1:1 interaction model with the
inclusion of a term for mass transport using CLAMP. The resulting
binding constants are listed in Table 20. The mAbs are listed from
highest to lowest affinity. TABLE-US-00020 TABLE 20 Affinity
Determination Results for Cloned and Purified mAbs Derived from
High Resolution Biacore .TM.. Antibody R.sub.max k.sub.a
(M.sup.-1s.sup.-1) k.sub.d (s.sup.-1) K.sub.D (nM) sc 264 96 5.85
.times. 10.sup.4 3.63 .times. 10.sup.-4 6.2 sc 298 77 5.65 .times.
10.sup.4 1.18 .times. 10.sup.-3 21.0 sc 188 76 4.52 .times.
10.sup.4 9.56 .times. 10.sup.-4 21.2 sc 133 96 5.73 .times.
10.sup.4 1.89 .times. 10.sup.-3 33.0 sc 254.2 53, 45 5.73 .times.
10.sup.4 5.64 .times. 10.sup.-4 34.9
Example 18
Binding Affinity Analysis Using FACS
[0270] As an alternative to Biacore, FACS analysis was also used to
estimate the binding affinity of one of the antibodies to K562
cells that stably express the human .alpha.V.beta.6 antigen. The
amount of antigen was titrated to generate a binding curve and
estimate the binding affinity to the antigen.
[0271] K562 cells expressing .alpha.V.beta.6 were resuspended in
filtered HBS buffer containing 1 mM of MgCl.sub.2 and 1 mM of
CaCl.sub.2 at a concentration of approximately 6 million cells/mL.
The cells were kept on ice. Purified mAb sc 188 was serially
diluted by a factor of 1:2 in HBS across 11 wells in a 96-well
plate. The 12.sup.th well in each row contained buffer only.
Titrations were done in triplicate. Additional HBS and cells were
added to each well so that the final volume was 300 .mu.L/well and
each well contained approximately 120,000 cells. The final
molecular concentration range for mAb sc 188 was 4.9-0.019 nM. The
plates were placed into a plate shaker for 5 hours at 4.degree. C.,
after which the plates were spun and washed three times with HBS,
following which, 200 .mu.L of 131 nM Cy5 goat .alpha.-human
polyclonal antibody (Jackson Laboratories, #109-175-008) were added
to each well. The plates were then shaken for 40 minutes at
4.degree. C., and thereafter were spun and washed once again three
times with HBS. The Geometric Mean Fluorescence (GMF) of 20,000
"events" for each mAb concentration was recorded using a
FACSCalibur instrument, and the corresponding triplicate titration
points were averaged to give one GMF point for each mAb
concentration. A plot of the averaged GMF as a function of
molecular mAb concentration with Scientist software was fit
nonlinearly using the equation: F = P ' .times. ( K D + L T + n M )
- ( K D + L T + n M ) 2 - 4 .times. n M L T 2 + B ##EQU1##
[0272] In the above equation, F=geometric 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 mAb sc 188 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.
[0273] The resulting plot with its nonlinear fits (red line) is
shown in FIG. 2. Table 21 lists the resulting K.sub.D for mAb sc
188 along with the 95% confidence interval of the fit. These
results for mAb sc 188 indicate binding to one type of
receptor.
[0274] Binding affinity for sc 188 as determined by FACS was
significantly tighter than as determined by Biacore (Example 17).
There are at least 2 possible explanations for the difference in
K.sub.D values for sc 188. The first reason is that the two assays
used different forms of the antigen for the measurement--Biacore
used soluble antigen and the FACs analysis used a cell-bound form
of the antigen. The second reason is that the antibodies that were
tested were raised against the cell-bound form of the antigen and
may not bind with as high an affinity to the soluble antigen as
they do to the cell-bound antigen. TABLE-US-00021 TABLE 21 Binding
Affinity Analysis Using FACS Antibody K.sub.D (pM) 95% CI (pM) sc
188 51.9 .+-.22.7
Example 19
CDC Assay
[0275] The purified antibodies described in the examples above are
of the IgG1 isotype and can have effector function. In order to
determine the ability of these antibodies to mediate
complement-dependent cytotoxicity (CDC), the following assay was
performed using 293 cells stably expressing .alpha.V.beta.6
(293-10A11) and parental 293 cells (293F).
[0276] For calcein staining of cells, aliquots of approximately
25.times.10e6 each of HT29, 293-10A11, and 293F cells were
individually resuspended in 3 ml serum-free RPMI media. 45 .mu.L of
1 mM calcein was then added to each 3 ml sample of cells, and the
samples were incubated at 37.degree. C. for 45 minutes. The cells
were centrifuged at 1200.times.RPM for 3 minutes, the supernatant
was discarded and the cells were resuspended in each respective
cell line's culture media. The centrifugation step was repeated and
the cells were resuspended to give a final concentration of about
100,000 cells in 50 .mu.L media.
[0277] Serial 1:2 dilutions of each antibody were prepared in a
v-bottom 96-well plate, with concentrations ranging from 20
.mu.g/ml to 0.625 .mu.g/ml in a volume of 50 .mu.L. Then, 100,000
of the cells prepared as described above were added in a volume of
50 .mu.L to the antibody-containing plates, and the resulting
mixture was incubated on ice for two hours. Following the
incubation, the cells were pelleted, and the supernatant was
discarded. The cells were resuspended in 100 .mu.L of their
respective media containing 10% human sera (ABI donor #27), and
incubated at 37.degree. C. for 30 minutes. The cells were then
centrifuged, and 80 L of the supernatant was transferred to a FMAT
plate. The plate was read on a Tecan reader using an excitation
wavelength of 485 nm and an emission wavelength of 530 nm.
[0278] The results are summarized in FIGS. 3A-3E, and demonstrate
that each purified antibody tested is capable of mediating CDC in
293 cells stably expressing .alpha.V.beta.6 integrin.
Example 20
Site-Directed Mutagenesis
[0279] One of the antibodies (sc 264) prepared from the
immunizations (Example 1) showed strong functional blocking
activity in vitro in the TGF.beta.LAP binding inhibition assay (see
Example 4), but exhibited cross-reactive binding to
non-.alpha.V.beta.6 expressing cell lines (see Example 15). This
antibody, sc 264, has an RGD sequence in the CDR3 region, which is
presumed to be responsible for the cross-reactive binding.
Therefore, site-directed mutagenesis was used to replace the
glycine residue in the RGD with an alanine (sc 264 RAD).
[0280] A second antibody (sc 188) has a glycosylation site within
the FR3 region. This site was eliminated through site-directed
mutagenesis with a substitution from NLT to KLT (sc 188 SDM). The
mutated versions of these two antibodies were then expressed and
purified as described in Examples 7 and 8, and the purified
antibodies were analyzed as described in the following
examples.
Example 21
Binding Assay to Test Cross-Reactive Binding of Mutant
Antibodies
[0281] A binding assay was performed to test whether the
cross-reactive binding observed in Example 15 was reduced because
of site-directed mutagenesis of sc 264. Binding of the antibodies
was analyzed on 293T and 293F cell lines to test whether removing
the RGD site from sc 264 would result in decreased binding compared
with the original antibody.
[0282] 293T and 293F cells were spun down after collection and
resuspended in HBSS with 1% BSA and 1 mM CaCl.sub.2 and 1 mM
MgCl.sub.2 (wash buffer), so that at least 150,000 cells were used
in each reaction. Cells were divided between reactions in a
V-bottom 96-well plate (Sarstedt), and the cells in the plate were
pelleted at 1500 rpm for 3 minutes, after which the HBSS
supernatant was removed. The primary antibody was added at the
concentration indicated in Table 19 in a volume of 50 .mu.L, and
the cells were resuspended and thereafter incubated on ice for 60
minutes. After incubation, the cells were pelleted by
centrifugation at 1500 rpm for 3 minutes, resuspended in 100 .mu.L
wash buffer, and then pelleted again. Cells were then resuspended
in the appropriate secondary antibody at 2 .mu.g/ml with 10
.mu.g/ml 7AAD, and stained on ice for 7 minutes, after which 150
.mu.L of wash buffer was added, and cells were pelleted at 1500 rpm
for 3 minutes and then resuspended in 100 .mu.L of HBSS with 1%
BSA. Samples were read on a FACS machine with a HTS attachment and
the data was analyzed using Cell Quest Pro software. The results
are summarized in Table 22, and data appears as Geometric Mean
Shift values in arbitrary units. These data demonstrate that at all
concentrations tested, sc 264 RAD had significantly less binding to
parental 293T cells compared to the original mAb sc 264.
TABLE-US-00022 TABLE 22 Cross-reactivity of mutated antibodies to
parental cells. Concentration Antibody (ug/ml) 293T Cells
293T-.alpha.V.beta.6 Cells None n/a 3 2 Mouse IgG2a 20 27 8 Human
IgG1 20 4 4 Anti-aVb6 20 4 5 sc 264 20 433 6673 sc 264 RAD 20 44
7241 sc 188 20 27 6167 sc 188 SDM 20 25 6758 sc 264 5 88 6418 sc
264 RAD 5 13 6840 sc 188 5 9 5822 sc 188 SDM 5 9 6822 sc 264 1.25
24 6230 sc 264 RAD 1.25 7 4890 sc 188 1.25 6 6395 sc 188 SDM 1.25 5
4532
Example 22
Potency Analysis of Mutant Antibodies
[0283] In order to determine the concentration (IC.sub.50) of
mutant .alpha.V.beta.6 antibodies required to inhibit
TGF.beta.LAP-mediated adhesion of HT-29 cells, the following assay
was performed.
[0284] Nunc MaxiSorp (Nunc) plates were coated overnight with 50
.mu.L of 10 .mu.g/ml TGF Beta1 LAP (TGF.beta.LAP), and pre-blocked
with 3% BSA/PBS for 1 hour prior to the assay. HT29 cells grown to
the optimal density were then pelleted and washed twice in HBBS
(with 1% BSA and with 1 mM Ca and 1 mM Mg.sup.2+), after which the
cells were then resuspended in HBSS at 30,000 cell per well. The
coating liquid was removed from the plates, which were then blocked
with 100 .mu.L 3% BSA at room temperature for 1 hour and thereafter
washed twice with PBS.
[0285] Antibody titrations were prepared in 1:4 serial dilutions in
a final volume of 30 .mu.L and at two times the final
concentration. Care was taken to ensure that the PBS concentration
in the control wells matched the PBS concentration in the most
dilute antibody well. 30 .mu.L of cells were added to each well,
and the cells were incubated in the presence of the antibodies at
4.degree. C. for 40 minutes in a V-bottom plate. The cell-antibody
mixtures were transferred to the coated plate and the plate was
incubated at 37.degree. C. for 40 minutes. 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 15 minutes. The cells were
allowed to thaw at room temperature, 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 results for twelve antibodies are summarized in Table
23, and demonstrate that the IC.sub.50 of the mutant antibodies is
consistently less than that of each original antibody.
TABLE-US-00023 TABLE 23 Concentration (IC.sub.50) of mutant
antibodies required to inhibit TGF.beta.LAP-mediated adhesion of
HT29 cells. n = 1 (ng/ml) n = 2 (ng/ml) n = 3 (ng/ml) sc.264 113 96
55 sc.264 RAD 13 13 39 sc.264 57 89 46 sc.188 125 157 64 sc.188 SDM
22 24 67
Example 23
Cross-Reactivity of Mutant Antibodies to .alpha.4.beta.1
Integrin
[0286] To establish that the mutant antibodies were functional only
against the .alpha.V.beta.6 integrin and not the .beta.4.beta.1
integrin, an assay was performed to test the ability of the
antibodies to inhibit the adhesion of J6.77 cells to the CS-1
peptide of fibronectin. The assay was performed as described as
described below.
[0287] Assay plates were coated with the CS-1 peptide of
fibronectin. Assay plates were pre-blocked with 3% BSA/PBS for one
hour prior to the assay. The J6.77 cells were grown to confluency,
then removed from a culture flask, pelleted and washed three times
with HBSS. The cells were then resuspended in HBSS at a
concentration of 30,000 cells per well. The coating liquid
containing the fibronectin fragments was removed, and the plates
were blocked with 100 .mu.L of 3% BSA for one hour at room
temperature. The coated plates were washed three times with HBSS.
Antibody titrations were prepared in 1:4 serial dilutions in a
final volume of 30 .mu.L and at twice the final concentration. The
resuspended cells were added to the wells containing the titrated
antibody in a V-bottom plate, and the cells and antibodies were
co-incubated at 4.degree. C. for 40 minutes. The cell-antibody
mixture was then transferred to the coated plate, which was
thereafter incubated at 37.degree. C. for 40 minutes. The cells on
the coated plates were next washed four times in warm HBSS, and the
cells in the plates were then frozen at -80.degree. C. for 15
minutes. The cells were allowed to thaw at room temperature, 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.
[0288] The results for the two mutant antibodies and their
non-mutated counterparts are summarized in Table 24. A commercially
available .beta.1 integrin specific antibody was used as a positive
control in this assay and exhibited 95% inhibition of adhesion. A
commercially available .alpha.V.beta.6 specific antibody served as
a negative control in this assay for adhesion to .alpha.4.beta.1.
All antibodies were used at 5 .mu.g/ml and none of the test
antibodies could block adhesion to .alpha.4.beta.1. TABLE-US-00024
TABLE 24 Cross-Reactivity to .alpha.4.beta.1 Integrin Antibody at 5
ug/ml Percent Inhibition sc.188 2 sc.188 SDM -6 sc.264 -30 sc.264
RAD -2 Human IgG1 26 Human IgG2 13 Human IgG4 15 Anti-beta 1
Integrin 95
Example 24
Cross-Reactivity of Mutant Antibodies to .alpha.5.beta.1
Integrin
[0289] To establish that the mutant antibodies were functional only
against the .alpha.V.beta.6 integrin and not the .alpha.5.beta.1
integrin, an assay was performed to test the ability of the
antibodies to inhibit the adhesion of K562 cells to fibronectin.
The assay was performed as described as described in Example 14.
The results are summarized in Table 25, and demonstrate that none
of the tested antibodies could block adhesion to .alpha.5.beta.1.
TABLE-US-00025 TABLE 25 Cross-Reactivity to .alpha.5.beta.1
Integrin. Antibody ID % Inhibition sc 188 -5 sc 188 SDM -8 sc 264 3
sc 264 RAD 6 .alpha.V.beta.6 Control -16 .alpha.5.beta.1 Control 78
Human IgG -12 Control
Example 25
Cross-Reactivity of Mutant Antibodies to Mouse and Cynomolgus
.alpha.V.beta.6 Integrin
[0290] In order to determine if the mutant .alpha.V.beta.6-specific
antibodies exhibit cross-reactivity to mouse .alpha.V.beta.6 or
Cynomolgus .alpha.V.beta.6, the following assay was performed.
[0291] K562 parental cells, or K562 cells expressing Cynomolgus or
mouse .alpha.V.beta.6 were spun down after collection and
resuspended in HBSS with 1% BSA and 1 mM CaCl.sub.2 and 1 mM
MgCl.sub.2 (wash buffer), so that at least 150,000 cells were used
in each reaction. Cells were divided between reactions in a
V-bottom 96-well plate (Sarstedt), and the cells in the plate were
pelleted at 1500 rpm for 3 minutes, after which the HBSS
supernatant was removed. The primary antibody was added in a volume
of 50 .mu.L, and the cells were resuspended and thereafter
incubated on ice for 60 minutes. After incubation, the cells were
pelleted by centrifugation at 1500 rpm for 3 minutes, resuspended
in 100 .mu.L wash buffer, and then pelleted again. Cells were then
resuspended in the appropriate secondary antibody at 2 .mu.g/ml
with 10 .mu.g/ml 7AAD, and stained on ice for 7 minutes, after
which 150 .mu.L of wash buffer was added, and cells were pelleted
at 1500 rpm for 3 minutes and then resuspended in 100 .mu.L of HBSS
with 1% BSA. Samples were read on a FACS machine with a HTS
attachment and the data was analyzed using Cell Quest Pro software.
The results are summarized in Table 26, and data appears as
Geometric Mean Shift values in arbitrary units. These data
demonstrate that at the concentrations tested, sc 264 RAD and sc
188 SDM exhibit cross-reactivity to mouse and cynomolgus
.alpha.V.beta.6. TABLE-US-00026 TABLE 26 Cross-Reactivity with
Mouse and Cynomolgus .alpha.V.beta.6 Mouse Cynomolgus Antibodies
Parental alphaVbeta6 alphaVbeta6 Cells Alone 3 3 3 Gt anti Mouse 5
6 7 anti 15 122 84 alphaVbeta6 anti alphaV 109 144 163 anti beta6
26 43 37 Mouse IgG2a 23 36 25 Mouse IgG1 12 20 13 Gt anti Human 7
12 7 Human IgG1 46 108 54 sc 133 57 246 154 sc 188 55 227 139 sc
188 SDM 47 219 142 sc 254 98 260 190 sc 264 33 160 121 sc 264 RAD
48 196 139 sc 298 33 150 97
Example 26
Internalization Assay
[0292] The internalization of the mutant antibodies was tested
using a K562 cell line that stably expressed human .alpha.V.beta.6.
The assay was performed as described in Example 15. Internalization
of the purified antibodies was compared to a commercially available
.alpha.V.beta.6 antibody that was not internalized in this
assay.
[0293] The results are summarized in Table 27, and demonstrate that
the sc 264 RAD mutant antibody is internalized significantly less
than the non-mutated sc 264. TABLE-US-00027 TABLE 27 Summary of the
Internalization Assay Concentration Antibody (ug/ml) Percent
Internalization sc 264 10 75% sc 264 1 47% sc 264 RAD 10 42% sc 264
RAD 1 31% sc 188 10 18% sc 188 1 27% sc 188 SDM 10 22% sc 188 SDM 1
17%
Example 27
Binding Affinity Analysis of SC 264 Rad Using Facs
[0294] The binding affinity to .alpha.V.beta.6 of the sc 264 RAD
antibody was measured as described in Example 18. The results of
this assay are summarized in Table 28, and demonstrate that the sc
264 RAD antibody has an affinity <50 pM. TABLE-US-00028 TABLE 28
Binding Affinity Analysis Using FACS mAb Sample K.sub.D (pM) 95% CI
(pM) sc 264 RAD 46.3 .+-.15.9
Example 28
Comparison of the Activity of SC 264 RAD with SC 264 RAD/ADY
[0295] The activity of sc 264 RAD antibody and the germlined (GL)
version of 264RAD (containing the mutation A84D in the light
chain), 264 RAD/ADY were compared in a Detroit-562 adhesion
assay.
[0296] Plates were coated with 0.5 .mu.g/ml GST-TGF-b LAP fusion
protein at 4.degree. C. overnight and the following morning,
washed, and then blocked with 3% BSA/PBS for 1 hour. Detroit-562
cells (25000 cells per well) were then allowed to adhere to the
plates for 45 minutes at 37.degree. C. in HBSS containing 2 mM
MgCl.sub.2. After 45 minutes the plates were washed three times in
PBS and then fixed in ethanol. Cells were visualized by staining
with Hoescht and quantitated by counting the number of cells bound
per well on a Cellomics Arrayscan II.
The data shown in FIG. 5 indicates that both sc 264 RAD and sc 264
RAD/ADY have similar activity and that the ability to block
.alpha.V.beta.6 function is maintained in the modified
antibody.
Example 29
Growth Study
[0297] To establish that the antibodies 264RAD, 133 and 188 SDM
block .alpha.V.beta.6 function in vivo each were tested for the
ability to inhibit growth of .alpha.V.beta.6 positive tumour
xenograft. One such model is the Detroit-562 nasophayngeal cell
line, which expresses .alpha.V.beta.6 and also grows as a
sub-cutaeneous tumour xenograft.
[0298] Detroit 562 cells were cultured in EMEM with Earle's BSS and
2 mM L-Glu+1.0 mM sodium pyruvate, 0.1 mM NEAA+1.5 g/L sodium
bicarbonate+10% FBS. Cells were harvested and resuspended in 50%
PBS+50% matrigel. The suspension was then implanted at
5.times.10.sup.-6 per mouse in a volume of 0.1 ml within the right
flank. Animals were 6-8 week old NCR female nude mice. Dosing was
initiated when tumours reached 0.1 cm3 and dosed at 20 mg/kg once
weekly for the duration of the study.
[0299] All three antibodies inhibited tumour growth (see FIG. 4).
264RAD was the most effective, followed by 133, and 188. This data
clearly shows that the antibodies 264RAD, 133 and 188 are active in
vivo and are able reduce the growth of a tumour dependent on
.alpha.V.beta.6 signalling for growth.
INCORPORATION BY REFERENCE
[0300] 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 for all
purposes.
EQUIVALENTS
[0301] 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. TABLE-US-00029 TABLE 29 Exemplary Antibody
Heavy Chain Amino Acid Sequences SEQ Chain ID Name NO: FR1 CDR1 FR2
CDR2 FR3 CDR3 FR4 sc 264 75 QVQLQESGPGLVK GGSISSG WIRQHPGKGLEWIG
YIYYSGRTYNNP RVTISVDTSKNQFSLKLS VATGRADYH WGQGTTVT RAD PSQTLSLTCTVS
GYYWS SLKS SVTAADTAVYYCAR FYAMDV VSS Sc 264 95 QVQLQESGPGLVK
GGSISSG WIRQHPGKGLEWIG YIYYSGRTYNNP RVTISVDTSKNQFSLKLS VATGRADYH
WGQGTTVT RAD/ADY PSQTLSLTCTVS GYYWS SLKS SVTAADTAVYYCAR FYAMDV VSS
sc 188 71 QVQLQESGPGLVK GGSISSG WIRQHPGNGLEWIG YIYYSGSTSYNP
RVTISVDTSKKQFSLKLT EGPLRGDYY WGQGTTVT SDM PSQTLSLTCTVS VYYWT SLKS
SVTAADTAVYYCAR YGLDV VSS sc 133 79 QVQLVQSGAEVKK GYTFTGY
WVRQAPGQGLEWMG WINPKSGDTNYA RVTMTRDTSTSTAYMELS RLDV WGQGTTVT TMT
PGASVKVSCKAS YMH QKFQG RLRSDDTAVYYCAR VSS sc 133 83 QVQLVQSGAEVKK
GYTFTGY WVRQAPGQGLEWMG WINPKSGDTNYA RVTLTRDTSTSTAYMELS RLDV
WGQGTTVT WDS PGASVKVSCKAS YMH QKFQG RLRSDDTAVYYCAR VSS sc 133 87
QVQLVQSGAEVKK GYTFTGY WVRQAPGQGLEWMG WINPKSGDTNYA
RVTMTRDTSTSTAYMELS RLDV WGQGTTVT TMT/WDS PGASVKVSCKAS YMH QKFQG
RLRSDDTAVYYCAR VSS
[0302] TABLE-US-00030 TABLE 30 Exemplary Antibody Light Chain Amino
Acid Sequences SEQ Chain ID Name NO: FR1 CDR1 FR2 CDR2 FR3 CDR3 FR4
sc 264 77 SYELTQPSSVSVS SGDVLAK WFHQKPGQAPVLVIY KDSERPS
GIPERFSGSSSGTTVTLT YSAADNNLV FGGGTKLT RAD PGQTARITC KSAR
ISGAQVEDEAAYYC VL sc 264 97 SYELTQPSSVSVS SGDVLAK WFHQKPGQAPVLVIY
KDSERPS GIPERFSGSSSGTTVTLT YSAADNNLV FGGGTKLT RAD/ADY PGQTARITC
KSAR ISGAQVEDEADYYC VL sc 188 73 EIVLTQSPGTLSL RAGQTIS
WYQQKPGQAPRPLIY GASSRAT GIPDRFSGSGSGTDFTLT QQYGSSPRT FGQGTKVE SDM
SPGERATLSC SRYLA ISRLEPEDFAVYYC IK sc 133 81 QSVLTQPPSVSAA SGSSSNI
WYQQLPGTAPKLLIY DNNKRPS GIPDRFSGSKSGTSATLG GTWNSSLSA FGTGTKVT TMT
PGQKVTISC GNNYVS ITGLQTGDEADYYC GYV VL sc 133 85 QSVLTQPPSVSAA
SGSSSNI WYQQLPGTAPKLLIY DNNKRPS GIPDRFSGSKSGTSATLG GTWDSSLSA
FGTGTKVT WDS PGQKVTISC GNNYVS ITGLQTGDEADYYC GYV VL sc 133 89
QSVLTQPPSVSAA SGSSSNI WYQQLPGTAPKLLIY DNNKRPS GIPDRFSGSKSGTSATLG
GTWDSSLSA FGTGTKVT TMT/WDS PGQKVTISC GNNYVS ITGLQTGDEADYYC GYV
VL
[0303] TABLE-US-00031 <160> NUMBER OF SEQ ID NOS: 97
<210> SEQ ID NO 1 <211> LENGTH: 366 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 1
caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cc #tgtccctc
60 acctgcactg tcgctggtgg ctccatcaga agtggtgatt actactggag ct
#ggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggaacatct
attacagtgg ga #gcacctac 180 tacaacccgt ccctcaagag tcgaattacc
atttcagtag ccacgtctag ga #accagttc 240 tccctgaagc tgacctctgt
gactgccgcg gacacggccg tgtattactg tg #cgagaggg 300 ggagctatta
cgatttttgg agtgtttgac tactggggcc agggaaccct gg #tcaccgtc 360 tcctca
# # # 366 <210> SEQ ID NO 2 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 2 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Le
#u Val Lys Pro Ser Gln 1 5 # 10 # 15 Thr Leu Ser Leu Thr Cys Thr
Val Ala Gly Gl #y Ser Ile Arg Ser Gly 20 # 25 # 30 Asp Tyr Tyr Trp
Ser Trp Ile Arg Gln His Pr #o Gly Lys Gly Leu Glu 35 # 40 # 45 Trp
Ile Gly Asn Ile Tyr Tyr Ser Gly Ser Th #r Tyr Tyr Asn Pro Ser 50 #
55 # 60 Leu Lys Ser Arg Ile Thr Ile Ser Val Ala Th #r Ser Arg Asn
Gln Phe 65 #70 #75 #80 Ser Leu Lys Leu Thr Ser Val Thr Ala Ala As
#p Thr Ala Val Tyr Tyr 85 # 90 # 95 Cys Ala Arg Gly Gly Ala Ile Thr
Ile Phe Gl #y Val Phe Asp Tyr Trp 100 # 105 # 110 Gly Gln Gly Thr
Leu Val Thr Val Ser Ser 115 # 120 <210> SEQ ID NO 3
<211> LENGTH: 324 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 3 gaaattgtgt tgacgcagtc
tccaggcacc ctgtctttgt ctccagggga aa #gagccacc 60 ctctcctgca
gggccagtca gagtgttagc agcagctact tagcctggta cc #agcagaaa 120
cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac tg #gcatccca
180 gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag ca
#gactggag 240 cctgaagatt ttgcagtgta ttactgtcag cagtatggta
gctcaccgtg ca #gttttggc 300 caggggacca agctggagat caaa # # 324
<210> SEQ ID NO 4 <211> LENGTH: 108 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 4 Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Le #u Ser Leu Ser Pro Gly 1 5 #
10 # 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gl #n Ser Val Ser
Ser Ser 20 # 25 # 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gl #n
Ala Pro Arg Leu Leu 35 # 40 # 45 Ile Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Il #e Pro Asp Arg Phe Ser 50 # 55 # 60 Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Th #r Ile Ser Arg Leu Glu 65 #70 #75 #80 Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gl #n Tyr Gly Ser Ser Pro 85 #
90 # 95 Cys Ser Phe Gly Gln Gly Thr Lys Leu Glu Il #e Lys 100 # 105
<210> SEQ ID NO 5 <211> LENGTH: 366 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 5
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cc #tgagactc
60 tcctgtgcag cctctggatt cacctttagc agctatgtca tgagctgggt cc
#gccaggct 120 ccagggaagg ggctggagtg ggtctcagct attagtggta
gtggtggtag ca #catactac 180 gcagactccg tgaagggccg gttcaccatc
tccagagaca attccaagaa ca #cgctgtat 240 ctgcaaatga acagcctgag
agccgaggac acggccgtat attactgtgc ga #aaggtgtg 300 gatacagcta
tggttaccta cggtatggac gtctggggcc aagggaccac gg #tcaccgtc 360 tcctca
# # # 366 <210> SEQ ID NO 6 <211> LENGTH: 122
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 6 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Le
#u Val Gln Pro Gly Gly 1 5 # 10 # 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Ph #e Thr Phe Ser Ser Tyr 20 # 25 # 30 Val Met Ser Trp
Val Arg Gln Ala Pro Gly Ly #s Gly Leu Glu Trp Val 35 # 40 # 45 Ser
Ala Ile Ser Gly Ser Gly Gly Ser Thr Ty #r Tyr Ala Asp Ser Val 50 #
55 # 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Se #r Lys Asn Thr
Leu Tyr 65 #70 #75 #80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Th
#r Ala Val Tyr Tyr Cys 85 # 90 # 95 Ala Lys Gly Val Asp Thr Ala Met
Val Thr Ty #r Gly Met Asp Val Trp 100 # 105 # 110
Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 # 120 <210> SEQ
ID NO 7 <211> LENGTH: 324 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 7 tcctatgagc
tgacacagcc accctcggtg tcagtgtccc caggacaaac gg #ccaggatc 60
acctgctctg gagatgcatt gccaaaaaaa tatgcttatt ggtaccagca ga #agtcaggc
120 caggcccctg tgctggtcat ctatgacgac agcaaacgac cctccgggat cc
#ctgagaga 180 ttctctggct ccagctcagg gacaatggcc accttgacta
tcagtggggc cc #aggtggag 240 gatgaagctg actactactg ttactcaaca
gacagcagtg gtaatcatag gg #tgttcggc 300 ggagggacca agctgaccgt ccta #
# 324 <210> SEQ ID NO 8 <211> LENGTH: 108 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
8 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Se #r Val Ser Pro Gly Gln
1 5 # 10 # 15 Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Le #u Pro Lys
Lys Tyr Ala 20 # 25 # 30 Tyr Trp Tyr Gln Gln Lys Ser Gly Gln Ala Pr
#o Val Leu Val Ile Tyr 35 # 40 # 45 Asp Asp Ser Lys Arg Pro Ser Gly
Ile Pro Gl #u Arg Phe Ser Gly Ser 50 # 55 # 60 Ser Ser Gly Thr Met
Ala Thr Leu Thr Ile Se #r Gly Ala Gln Val Glu 65 #70 #75 #80 Asp
Glu Ala Asp Tyr Tyr Cys Tyr Ser Thr As #p Ser Ser Gly Asn His 85 #
90 # 95 Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Va #l Leu 100 # 105
<210> SEQ ID NO 9 <211> LENGTH: 369 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: misc_feature <222> LOCATION:
(369)..(369) <223> OTHER INFORMATION: n is a, c, g, or #t
<400> SEQUENCE: 9 gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc
ccggggagtc tc #tgaagatc 60 tcctgtaagg gttctggata cagctttacc
agctactgga tcggctgggt gc #gccagatg 120 cccgggaaag gcctggagtg
gatggggatc atctatcctg gtgactctga ta #ccagatac 180 agcccgtcct
tccaaggcca ggtcatcctc tcagccgaca agtccatcag ca #ccgcctac 240
ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc ga #gacatgat
300 gaaagtagtg gttattacta tgtttttgat atctggggcc aagggacaat gg
#tcaccgtc 360 tcttcagcn # # # 369 <210> SEQ ID NO 10
<211> LENGTH: 123 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 10 Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Va #l Lys Lys Pro Gly Glu 1 5 # 10 # 15 Ser Leu Lys Ile
Ser Cys Lys Gly Ser Gly Ty #r Ser Phe Thr Ser Tyr 20 # 25 # 30 Trp
Ile Gly Trp Val Arg Gln Met Pro Gly Ly #s Gly Leu Glu Trp Met 35 #
40 # 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Ar #g Tyr Ser Pro
Ser Phe 50 # 55 # 60 Gln Gly Gln Val Ile Leu Ser Ala Asp Lys Se #r
Ile Ser Thr Ala Tyr 65 #70 #75 #80 Leu Gln Trp Ser Ser Leu Lys Ala
Ser Asp Th #r Ala Met Tyr Tyr Cys 85 # 90 # 95 Ala Arg His Asp Glu
Ser Ser Gly Tyr Tyr Ty #r Val Phe Asp Ile Trp 100 # 105 # 110 Gly
Gln Gly Thr Met Val Thr Val Ser Ser Al #a 115 # 120 <210> SEQ
ID NO 11 <211> LENGTH: 327 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (300)..(300) <223> OTHER
INFORMATION: n is c or t <400> SEQUENCE: 11 tcctatgagc
tgacacaacc accctcggtg tcagtgtccc caggacaaac gg #ccaggatc 60
acctgctctg gagatgcatt gccaaaaaaa tatgcttatt ggtaccagca ga #agtcaggc
120 caggcccctg ttctggtcat ctatgatgac atcaaacgac cctccgggat cc
#ctgagaga 180 ttctctggct ccagctcagg gacaatggcc accttgacta
tcagtggggc cc #aggtggag 240 gatgaagctg actactactg ttactcaaca
gacagcagtg gtaatcattg gg #ttttcttn 300 ggcggaggga ccaagctgac
cgtccta # # 327 <210> SEQ ID NO 12 <211> LENGTH: 109
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 12 Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Se
#r Val Ser Pro Gly Gln 1 5 # 10 # 15 Thr Ala Arg Ile Thr Cys Ser
Gly Asp Ala Le #u Pro Lys Lys Tyr Ala 20 # 25 # 30 Tyr Trp Tyr Gln
Gln Lys Ser Gly Gln Ala Pr #o Val Leu Val Ile Tyr 35 # 40 # 45 Glu
Asp Ile Lys Arg Pro Ser Gly Ile Pro Gl #u Arg Phe Ser Gly Ser 50 #
55 # 60 Ser Ser Gly Thr Met Ala Thr Leu Thr Ile Se #r Gly Ala Gln
Val Glu 65 #70 #75 #80 Asp Glu Ala Asp Tyr Tyr Cys Tyr Ser Thr As
#p Ser Ser Gly Asn His 85 # 90 # 95 Trp Val Phe Phe Gly Gly Gly Thr
Lys Leu Th
#r Val Leu 100 # 105 <210> SEQ ID NO 13 <211> LENGTH:
339 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 13 caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctggggcctc ag #tgaaggtc 60 tcctgcaagg cttctggata
caccttcacc ggctactata tgcactgggt gc #gacaggcc 120 cctggacaag
ggcttgagtg gatgggatgg atcaacccta aaagtggtga ca #caaactat 180
gcacagaagt ttcagggcag ggtcaccctg accagggaca cgtccaccag ca #cagcctac
240 atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc ga
#gaaggttg 300 gacgtctggg gccaagggac cacggtcacc gtctcctca # # 339
<210> SEQ ID NO 14 <211> LENGTH: 113 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 14 Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Va #l Lys Lys Pro Gly Ala 1 5 #
10 # 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Ty #r Thr Phe Thr
Gly Tyr 20 # 25 # 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gl #n
Gly Leu Glu Trp Met 35 # 40 # 45 Gly Trp Ile Asn Pro Lys Ser Gly
Asp Thr As #n Tyr Ala Gln Lys Phe 50 # 55 # 60 Gln Gly Arg Val Thr
Leu Thr Arg Asp Thr Se #r Thr Ser Thr Ala Tyr 65 #70 #75 #80 Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp Th #r Ala Val Tyr Tyr Cys 85 #
90 # 95 Ala Arg Arg Leu Asp Val Trp Gly Gln Gly Th #r Thr Val Thr
Val Ser 100 # 105 # 110 Ser <210> SEQ ID NO 15 <211>
LENGTH: 330 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 15 cagtctgtgt tgacgcagcc gccctcagtg
tctgcggccc caggacagaa gg #tcaccatc 60 tcctgctctg gaagcagctc
caacattggg aataattatg tatcctggta cc #agcagctc 120 ccaggaacag
cccccaaact cctcatttat gacaataata agcgaccctc ag #gaattcct 180
gaccgattct ctggctccaa gtctggcacg tcagccaccc tgggcatcac cg #gactccag
240 actggggacg aggccgatta ttactgcgga acatggaata gcagcctgag tg
#ctggttat 300 gtcttcggaa ctgggaccaa ggtcaccgtc # # 330 <210>
SEQ ID NO 16 <211> LENGTH: 110 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 16 Gln Ser
Val Leu Thr Gln Pro Pro Ser Val Se #r Ala Ala Pro Gly Gln 1 5 # 10
# 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Se #r Asn Ile Gly Asn
Asn 20 # 25 # 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Th #r Ala
Pro Lys Leu Leu 35 # 40 # 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser
Gly Il #e Pro Asp Arg Phe Ser 50 # 55 # 60 Gly Ser Lys Ser Gly Thr
Ser Ala Thr Leu Gl #y Ile Thr Gly Leu Gln 65 #70 #75 #80 Thr Gly
Asp Glu Ala Asp Tyr Tyr Cys Gly Th #r Trp Asn Ser Ser Leu 85 # 90 #
95 Ser Ala Gly Tyr Val Phe Gly Thr Gly Thr Ly #s Val Thr Val 100 #
105 # 110 <210> SEQ ID NO 17 <211> LENGTH: 375
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (375)..(375) <223> OTHER INFORMATION: n is a, c, g,
or #t <400> SEQUENCE: 17 gaggtgcagc tggtgcagtc tggagcagag
gtgaaaaagc ccggggagtc tc #tgaagatc 60 tcctgtaagg gttctggata
cagctttacc agctactgga tcggctgggt gc #gccagatg 120 cccgggaaag
gcctggagtg gatggggatc atctatcctg gtgactctga ta #ccagatat 180
agtccgtcct tccaaggcca ggtcaccatc tcagccgaca agtccatcag ca #ccgcctac
240 ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc ga
#gacatggt 300 atagcagcag ctggtttcta ctactactat atggacgtct
ggggccaagg ga #ccacggtc 360 accgtctcct cagcn # # # 375 <210>
SEQ ID NO 18 <211> LENGTH: 125 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 18 Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Va #l Lys Lys Pro Gly Glu 1 5 # 10
# 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Ty #r Ser Phe Thr Ser
Tyr 20 # 25 # 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Ly #s Gly
Leu Glu Trp Met 35 # 40 # 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp
Thr Ar #g Tyr Ser Pro Ser Phe 50 # 55 # 60 Gln Gly Gln Val Thr Ile
Ser Ala Asp Lys Se #r Ile Ser Thr Ala Tyr 65 #70 #75 #80 Leu Gln
Trp Ser Ser Leu Lys Ala Ser Asp Th #r Ala Met Tyr Tyr Cys 85 # 90 #
95 Ala Arg His Gly Ile Ala Ala Ala Gly Phe Ty #r Tyr Tyr Tyr Met
Asp 100 # 105 # 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Se #r
Ser Ala
115 # 120 # 125 <210> SEQ ID NO 19 <211> LENGTH: 327
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (325)..(325) <223> OTHER INFORMATION: n is a or c
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: (327)..(327) <223> OTHER INFORMATION: n is a, c, g,
or #t <400> SEQUENCE: 19 gaaattgtgt tgacgcagtc cccagacacc
ctgtctttgt ctccagggga aa #gagcctcc 60 ctctcctgca gggccagtca
gaatgttaac aggaactact tagtctggta cc #agcagaaa 120 cctggccagg
ctcccaggct cctcatctat ggtacatcca acagggccac tg #gcatccca 180
gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag ca #gactggag
240 cctgaagatt ttgcagttta ttactgtcag cagtgtggta gtttaccatt ca
#ctttcggc 300 cctgggacca aagtggatat caaangn # # 327 <210> SEQ
ID NO 20 <211> LENGTH: 109 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 20 Glu Ile Val Leu Thr
Gln Ser Pro Asp Thr Le #u Ser Leu Ser Pro Gly 1 5 # 10 # 15 Glu Arg
Ala Ser Leu Ser Cys Arg Ala Ser Gl #n Asn Val Asn Arg Asn 20 # 25 #
30 Tyr Leu Val Trp Tyr Gln Gln Lys Pro Gly Gl #n Ala Pro Arg Leu
Leu 35 # 40 # 45 Ile Tyr Gly Thr Ser Asn Arg Ala Thr Gly Il #e Pro
Asp Arg Phe Ser 50 # 55 # 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Th #r Ile Ser Arg Leu Glu 65 #70 #75 #80 Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gl #n Cys Gly Ser Leu Pro 85 # 90 # 95 Phe Thr
Phe Gly Pro Gly Thr Lys Val Asp Il #e Lys Arg 100 # 105 <210>
SEQ ID NO 21 <211> LENGTH: 372 <212> TYPE: DNA
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 21
caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cc #tgtccctc
60 acctgcactg tctctggtgg ctccatcagc agtggtgttt actactggac ct
#ggatccgc 120 cagcacccag ggaacggcct ggagtggatt ggctacatct
attacagtgg ga #gcacctcc 180 tacaacccgt ccctcaagag tcgagttacc
atatcagtag acacgtctaa ga #aacagttc 240 tccctgaacc tgacctctgt
gactgccgcg gacacggccg tgtattactg tg #cgagagaa 300 ggaccactac
ggggggacta ctactacggt ctggacgtct ggggccaagg ga #ccacggtc 360
accgtctcct ca # # # 372 <210> SEQ ID NO 22 <211>
LENGTH: 124 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 22 Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Le #u Val Lys Pro Ser Gln 1 5 # 10 # 15 Thr Leu Ser Leu Thr
Cys Thr Val Ser Gly Gl #y Ser Ile Ser Ser Gly 20 # 25 # 30 Val Tyr
Tyr Trp Thr Trp Ile Arg Gln His Pr #o Gly Asn Gly Leu Glu 35 # 40 #
45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Th #r Ser Tyr Asn Pro
Ser 50 # 55 # 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Th #r Ser
Lys Lys Gln Phe 65 #70 #75 #80 Ser Leu Asn Leu Thr Ser Val Thr Ala
Ala As #p Thr Ala Val Tyr Tyr 85 # 90 # 95 Cys Ala Arg Glu Gly Pro
Leu Arg Gly Asp Ty #r Tyr Tyr Gly Leu Asp 100 # 105 # 110 Val Trp
Gly Gln Gly Thr Thr Val Thr Val Se #r Ser 115 # 120 <210> SEQ
ID NO 23 <211> LENGTH: 324 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 23 gaaattgtgt
tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aa #gagccacc 60
ctctcctgca gggccggtca gactattagc agtcgctact tagcctggta cc #agcagaaa
120 cctggccagg ctcccaggcc cctcatctat ggtgcatcca gcagggccac tg
#gcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc
tcaccatcag ca #gactggag 240 cctgaagatt ttgcagtgta ttactgtcag
cagtatggta gctcacctcg ga #cgttcggc 300 caagggacca aggtggaaat caaa #
# 324 <210> SEQ ID NO 24 <211> LENGTH: 108 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
24 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Le #u Ser Leu Ser Pro
Gly 1 5 # 10 # 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Gly Gl #n Thr
Ile Ser Ser Arg 20 # 25 # 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gl #n Ala Pro Arg Pro Leu 35 # 40 # 45 Ile Tyr Gly Ala Ser Ser
Arg Ala Thr Gly Il #e Pro Asp Arg Phe Ser 50 # 55 # 60 Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Th #r Ile Ser Arg Leu Glu 65 #70 #75
#80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gl #n Tyr Gly Ser Ser
Pro 85 # 90 # 95 Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Il #e
Lys
100 # 105 <210> SEQ ID NO 25 <211> LENGTH: 384
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 25 caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcacagac cc #tgtccctc 60 acctgcactg tctctggtgg
ctccatcagc agtggtggtt actactggag ct #ggatccgc 120 cagcacccag
ggaagggcct ggagtggatt gggtacatct attacagtgg ga #gcacctac 180
tacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa ga #accagttc
240 tccctgaagc tgagctctgt gactgccgcg gacacggcca tgtattactg tg
#cgagatat 300 cgaggaccag cggctgggcg gggagacttc tactacttcg
gtatggacgt ct #ggggccaa 360 gggaccacgg tcaccgtctc ctca # # 384
<210> SEQ ID NO 26 <211> LENGTH: 128 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 26 Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Le #u Val Lys Pro Ser Gln 1 5 #
10 # 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gl #y Ser Ile Ser
Ser Gly 20 # 25 # 30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pr #o
Gly Lys Gly Leu Glu 35 # 40 # 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser
Gly Ser Th #r Tyr Tyr Asn Pro Ser 50 # 55 # 60 Leu Lys Ser Arg Val
Thr Ile Ser Val Asp Th #r Ser Lys Asn Gln Phe 65 #70 #75 #80 Ser
Leu Lys Leu Ser Ser Val Thr Ala Ala As #p Thr Ala Met Tyr Tyr 85 #
90 # 95 Cys Ala Arg Tyr Arg Gly Pro Ala Ala Gly Ar #g Gly Asp Phe
Tyr Tyr 100 # 105 # 110 Phe Gly Met Asp Val Trp Gly Gln Gly Thr Th
#r Val Thr Val Ser Ser 115 # 120 # 125 <210> SEQ ID NO 27
<211> LENGTH: 339 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (312)..(312) <223> OTHER
INFORMATION: n is c or t <400> SEQUENCE: 27 gatattgtga
tgacccagac tccactctct ctgtccgtca cccctggaca gc #cggcctcc 60
atcttctgca agtctagtca gagcctcctg aacagtgatg gaaagaccta tt #tgtgttgg
120 tacctgcaga agccaggcca gcctccacag ctcctgatct atgaagtttc ca
#accggttc 180 tctggagtgc cagataggtt cagtggcagc gggtcaggga
cagatttcac ac #tgaaaatc 240 agccgggtgg aggctgagga tgttggggtt
tattactgca tgcaaggtat ac #agcttccg 300 tgggcgttct tnggccaagg
gaccaaggtg gaaatcaaa # # 339 <210> SEQ ID NO 28 <211>
LENGTH: 113 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 28 Asp Ile Val Met Thr Gln Thr Pro
Leu Ser Le #u Ser Val Thr Pro Gly 1 5 # 10 # 15 Gln Pro Ala Ser Ile
Phe Cys Lys Ser Ser Gl #n Ser Leu Leu Asn Ser 20 # 25 # 30 Asp Gly
Lys Thr Tyr Leu Cys Trp Tyr Leu Gl #n Lys Pro Gly Gln Pro 35 # 40 #
45 Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn Ar #g Phe Ser Gly Val
Pro 50 # 55 # 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr As #p Phe
Thr Leu Lys Ile 65 #70 #75 #80 Ser Arg Val Glu Ala Glu Asp Val Gly
Val Ty #r Tyr Cys Met Gln Gly 85 # 90 # 95 Ile Gln Leu Pro Trp Ala
Phe Phe Gly Gln Gl #y Thr Lys Val Glu Ile 100 # 105 # 110 Lys
<210> SEQ ID NO 29 <211> LENGTH: 383 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 29
caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cc #tgtccctc
60 acctgcactg tctctggtgg ctccatcagc agtggtggtt actactggag ct
#ggatccgc 120 cagcacccag ggaagggcct ggagtggatt gggtacatct
attacagtgg ga #gaacctac 180 aacaacccgt ccctcaagag tcgagttacc
atatcagtag acacgtctaa ga #accagttc 240 tccctgaagt tgagttctgt
gactgccgcg gacacggccg tgtattactg tg #cgagagtg 300 gctacgggga
gaggggacta ccacttctac gctatggacg tctggggcca ag #ggaccacg 360
gtcaccgtct cctcagcctc cac # # 383 <210> SEQ ID NO 30
<211> LENGTH: 125 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 30 Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Le #u Val Lys Pro Ser Gln 1 5 # 10 # 15 Thr Leu Ser Leu
Thr Cys Thr Val Ser Gly Gl #y Ser Ile Ser Ser Gly 20 # 25 # 30 Gly
Tyr Tyr Trp Ser Trp Ile Arg Gln His Pr #o Gly Lys Gly Leu Glu 35 #
40 # 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Arg Th #r Tyr Asn Asn
Pro Ser 50 # 55 # 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Th #r
Ser Lys Asn Gln Phe 65 #70 #75 #80 Ser Leu Lys Leu Ser Ser Val Thr
Ala Ala As #p Thr Ala Val Tyr Tyr 85 # 90 # 95 Cys Ala Arg Val Ala
Thr Gly Arg Gly Asp Ty #r His Phe Tyr Ala Met
100 # 105 # 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Va #l Ser
Ser 115 # 120 # 125 <210> SEQ ID NO 31 <211> LENGTH:
318 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 31 tcctatgagc tgacacagcc atcctcagtg
tcagtgtctc cgggacagac ag #ccaggatc 60 acctgctcag gagatgtact
ggcaaaaaag tctgctcggt ggttccacca ga #agccaggc 120 caggcccctg
tactggtgat ttataaagac agtgagcggc cctcagggat cc #ctgagcgc 180
ttctccggct ccagctcagg gaccacagtc accttgacca tcagcggggc cc #aggttgag
240 gatgaggctg cctattactg ttactctgcg gctgacaaca atctggtatt cg
#gcggaggg 300 accaagctga ccgtccta # # # 318 <210> SEQ ID NO
32 <211> LENGTH: 106 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 32 Ser Tyr Glu Leu Thr
Gln Pro Ser Ser Val Se #r Val Ser Pro Gly Gln 1 5 # 10 # 15 Thr Ala
Arg Ile Thr Cys Ser Gly Asp Val Le #u Ala Lys Lys Ser Ala 20 # 25 #
30 Arg Trp Phe His Gln Lys Pro Gly Gln Ala Pr #o Val Leu Val Ile
Tyr 35 # 40 # 45 Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Gl #u Arg
Phe Ser Gly Ser 50 # 55 # 60 Ser Ser Gly Thr Thr Val Thr Leu Thr
Ile Se #r Gly Ala Gln Val Glu 65 #70 #75 #80 Asp Glu Ala Ala Tyr
Tyr Cys Tyr Ser Ala Al #a Asp Asn Asn Leu Val 85 # 90 # 95 Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 # 105 <210> SEQ ID NO 33
<211> LENGTH: 354 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 33 gaggtgcagc tggtgcagtc
tggagcagag gtgaaaaagc ccggggagtc tc #tgaagatc 60 tcctgtaagg
gttctggata cagctttccc agctactgga tcggctgggt gc #gccagatg 120
cccgggaagg gcctggagtg gatggggatc atctatcctg gtgactctga ta #ccagatac
180 agcccgtcct tccaaggcca ggtcaccatc tcagctgaca agtccatcag ca
#ccgcctac 240 ctgcagtgga gcagcctgaa ggcctcggac accgccatgt
attactgtgc ga #gacaccct 300 atggaggacg gtatggacgt ctggggccaa
gggaccacgg tcaccgtctc ct #ca 354 <210> SEQ ID NO 34
<211> LENGTH: 118 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 34 Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Va #l Lys Lys Pro Gly Glu 1 5 # 10 # 15 Ser Leu Lys Ile
Ser Cys Lys Gly Ser Gly Ty #r Ser Phe Pro Ser Tyr 20 # 25 # 30 Trp
Ile Gly Trp Val Arg Gln Met Pro Gly Ly #s Gly Leu Glu Trp Met 35 #
40 # 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Ar #g Tyr Ser Pro
Ser Phe 50 # 55 # 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Se #r
Ile Ser Thr Ala Tyr 65 #70 #75 #80 Leu Gln Trp Ser Ser Leu Lys Ala
Ser Asp Th #r Ala Met Tyr Tyr Cys 85 # 90 # 95 Ala Arg His Pro Met
Glu Asp Gly Met Asp Va #l Trp Gly Gln Gly Thr 100 # 105 # 110 Thr
Val Thr Val Ser Ser 115 <210> SEQ ID NO 35 <211>
LENGTH: 321 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 35 tcctatgagc tgacacagcc accctcggtg
tcagtgtccc caggacaaac gg #ccaggatc 60 acctgctctg gagatgcttt
gccaaaaaaa tatgcttttt ggtaccagca ga #agtcaggc 120 caggcccctg
tgctggtcat ctatgacgac aacaaacgac cctccgggat cc #ctgagaga 180
ttctctggct ccagctcagg gacaatggcc accttgacta tcactggggc cc #aggtggag
240 gatgaagctg actactactg ttactcaaca gacagcagtg gtcatcatgt at
#tcggcgga 300 gggaccaagc tgaccgtcct a # # 321 <210> SEQ ID NO
36 <211> LENGTH: 107 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 36 Ser Tyr Glu Leu Thr
Gln Pro Pro Ser Val Se #r Val Ser Pro Gly Gln 1 5 # 10 # 15 Thr Ala
Arg Ile Thr Cys Ser Gly Asp Ala Le #u Pro Lys Lys Tyr Ala 20 # 25 #
30 Phe Trp Tyr Gln Gln Lys Ser Gly Gln Ala Pr #o Val Leu Val Ile
Tyr 35 # 40 # 45 Asp Asp Asn Lys Arg Pro Ser Gly Ile Pro Gl #u Arg
Phe Ser Gly Ser 50 # 55 # 60 Ser Ser Gly Thr Met Ala Thr Leu Thr
Ile Th #r Gly Ala Gln Val Glu 65 #70 #75 #80 Asp Glu Ala Asp Tyr
Tyr Cys Tyr Ser Thr As #p Ser Ser Gly His His 85 # 90 # 95 Val Phe
Gly Gly Gly Thr Lys Leu Thr Val Le #u 100 # 105 <210> SEQ ID
NO 37 <211> LENGTH: 375 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 37
caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cc #tgagactc
60 tcctgtgcag cgtctggatt caccttcagt agctatggca tgcactgggt cc
#gccaggct 120 ccaggcaagg ggctggagtg ggtggcagtt atatggtatg
gtggaagtaa ta #aatactat 180 gcagactccg tgaagggccg attcaccatc
tccagagaca attccaagaa ca #cgctgtat 240 ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc ga #gagatctg 300 gcagctcgtc
ggggggacta ctactactac ggtatggacg tctggggcca ag #ggaccacg 360
gtcaccgtct cctca # # # 375 <210> SEQ ID NO 38 <211>
LENGTH: 125 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 38 Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Va #l Val Gln Pro Gly Arg 1 5 # 10 # 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Ph #e Thr Phe Ser Ser Tyr 20 # 25 # 30 Gly Met
His Trp Val Arg Gln Ala Pro Gly Ly #s Gly Leu Glu Trp Val 35 # 40 #
45 Ala Val Ile Trp Tyr Gly Gly Ser Asn Lys Ty #r Tyr Ala Asp Ser
Val 50 # 55 # 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Se #r Lys
Asn Thr Leu Tyr 65 #70 #75 #80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Th #r Ala Val Tyr Tyr Cys 85 # 90 # 95 Ala Arg Asp Leu Ala Ala
Arg Arg Gly Asp Ty #r Tyr Tyr Tyr Gly Met 100 # 105 # 110 Asp Val
Trp Gly Gln Gly Thr Thr Val Thr Va #l Ser Ser 115 # 120 # 125
<210> SEQ ID NO 39 <211> LENGTH: 321 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 39
tcttctgagc tgactcagga ccctgttgtg tctgtggcct tgggacagac ag #tcaggatc
60 acttgccaag gcgacagcct cagaagctat tatttaagct ggtaccagca ga
#agccagga 120 caggcccctg tacttgtcat ctatggtaaa aacaaccggc
cctcagggat cc #cagaccga 180 ttctctggct ccaactcagg aaacacagct
tccttgacca tcactggggc tc #aggcggaa 240 gatgaggctg actattactg
taattcccgg gacagcagtg gtaaccatct gt #tcggcgga 300 gggaccaagc
tgaccgtcct a # # 321 <210> SEQ ID NO 40 <211> LENGTH:
107 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 40 Ser Ser Glu Leu Thr Gln Asp Pro Val Val Se
#r Val Ala Leu Gly Gln 1 5 # 10 # 15 Thr Val Arg Ile Thr Cys Gln
Gly Asp Ser Le #u Arg Ser Tyr Tyr Leu 20 # 25 # 30 Ser Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pr #o Val Leu Val Ile Tyr 35 # 40 # 45 Gly
Lys Asn Asn Arg Pro Ser Gly Ile Pro As #p Arg Phe Ser Gly Ser 50 #
55 # 60 Asn Ser Gly Asn Thr Ala Ser Leu Thr Ile Th #r Gly Ala Gln
Ala Glu 65 #70 #75 #80 Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg As
#p Ser Ser Gly Asn His 85 # 90 # 95 Leu Phe Gly Gly Gly Thr Lys Leu
Thr Val Le #u 100 # 105 <210> SEQ ID NO 41 <211>
LENGTH: 375 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (81)..(81) <223> OTHER INFORMATION: n
is c or t <220> FEATURE: <221> NAME/KEY: misc_feature
<222> LOCATION: (90)..(90) <223> OTHER INFORMATION: n
is a, c, g, or #t <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (105)..(105) <223> OTHER
INFORMATION: n is c or t <220> FEATURE: <221> NAME/KEY:
misc_feature <222> LOCATION: (300)..(300) <223> OTHER
INFORMATION: n is a, c, g, or #t <400> SEQUENCE: 41
gaggtgcagc tggtggagtc tgggggaggc ctggtcaagc ctggggggtc cc #tgagactc
60 tcctgtgcag cctctggata naccttcacn aactatatca tgcantgggt cc
#gccaggct 120 ccagggaagg ggctggagtg ggtctcatcc attagtatta
gtagtagtta ca #tatactac 180 gcagactcag tgaagggccg attcaccatc
tccagagaca acgccaagaa ct #cactgtat 240 ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc ga #gagatccn 300 gtaccactgg
aacgacgcga ctactactac ggtatggacg tctggggcca ag #ggaccacg 360
gtcaccgtct cctca # # # 375 <210> SEQ ID NO 42 <211>
LENGTH: 125 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 42 Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Le #u Val Lys Pro Gly Gly 1 5 # 10 # 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Ty #r Thr Phe Thr Asn Tyr 20 # 25 # 30 Ile Met
His Trp Val Arg Gln Ala Pro Gly Ly #s Gly Leu Glu Trp Val 35 # 40 #
45 Ser Ser Ile Ser Ile Ser Ser Ser Tyr Ile Ty #r Tyr Ala Asp Ser
Val 50 # 55 # 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Al #a Lys
Asn Ser Leu Tyr 65 #70 #75 #80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Th #r Ala Val Tyr Tyr Cys 85 # 90 # 95
Ala Arg Asp Pro Val Pro Leu Glu Arg Arg As #p Tyr Tyr Tyr Gly Met
100 # 105 # 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Va #l Ser
Ser 115 # 120 # 125 <210> SEQ ID NO 43 <211> LENGTH:
330 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 43 cagtctgtgt tgacgcagcc gccctcaatg
tctgcggccc caggacagaa gg #tcaccatc 60 tcctgctctg gaagcagctc
caacattggg aataattatg tatcctggta cc #agcagctc 120 ccaggaacag
cccccaaact cctcatttat gacaataata agcgaccctc ag #ggattcct 180
gaccgattct ctggctccaa gtctggcacg tcagccaccc tgggcatcac cg #gactccag
240 actggggacg aggccgatta ttactgcgga acatgggata gcagcctgag cg
#ctggggta 300 ttcggcggag ggaccaagct gaccgtccta # # 330 <210>
SEQ ID NO 44 <211> LENGTH: 110 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 44 Gln Ser
Val Leu Thr Gln Pro Pro Ser Met Se #r Ala Ala Pro Gly Gln 1 5 # 10
# 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Se #r Asn Ile Gly Asn
Asn 20 # 25 # 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Th #r Ala
Pro Lys Leu Leu 35 # 40 # 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser
Gly Il #e Pro Asp Arg Phe Ser 50 # 55 # 60 Gly Ser Lys Ser Gly Thr
Ser Ala Thr Leu Gl #y Ile Thr Gly Leu Gln 65 #70 #75 #80 Thr Gly
Asp Glu Ala Asp Tyr Tyr Cys Gly Th #r Trp Asp Ser Ser Leu 85 # 90 #
95 Ser Ala Gly Val Phe Gly Gly Gly Thr Lys Le #u Thr Val Leu 100 #
105 # 110 <210> SEQ ID NO 45 <211> LENGTH: 375
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 45 caggtgcagc tggtggagtc tgggggaggc
gtggtccagc ctgggaggtc cc #tgagactc 60 tcctgtgcag cgtctggatt
caccttcagt agctatggca tgcactgggt cc #gccaggct 120 ccaggcaagg
ggctggagtg ggtggcagtt atatggtatg atggaagtaa ta #aatactac 180
gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa ca #cgctgtat
240 ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc ga
#gaacggag 300 ggtatagcag ctcgtctcta ctactactac ggtatggacg
tctggggcca ag #ggaccacg 360 gtcaccgtct cctca # # # 375 <210>
SEQ ID NO 46 <211> LENGTH: 125 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 46 Gln Val
Gln Leu Val Glu Ser Gly Gly Gly Va #l Val Gln Pro Gly Arg 1 5 # 10
# 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ph #e Thr Phe Ser Ser
Tyr 20 # 25 # 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Ly #s Gly
Leu Glu Trp Val 35 # 40 # 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn
Lys Ty #r Tyr Ala Asp Ser Val 50 # 55 # 60 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Se #r Lys Asn Thr Leu Tyr 65 #70 #75 #80 Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp Th #r Ala Val Tyr Tyr Cys 85 # 90 #
95 Ala Arg Thr Glu Gly Ile Ala Ala Arg Leu Ty #r Tyr Tyr Tyr Gly
Met 100 # 105 # 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Va #l
Ser Ser 115 # 120 # 125 <210> SEQ ID NO 47 <211>
LENGTH: 324 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 47 gaaattgtgt tgacgcagtc tccaggcacc
ctgtctttgt ctccagggga aa #gagccacc 60 ctctcctgca gggccagtca
gagtgttagc agcagctact tagcctggta cc #agcagaaa 120 cctggccagg
ctcccaggct cctcatctat ggtgcatcca gcagggccac tg #acatccca 180
gacaggttca gtggcagtgg gtctgggaca gacttcactc tcaccatcag ca #gactggag
240 cctgaagatt ttgcagtgta ttactgtcag cagtatggta gctcaccgtg ga
#cgttcggc 300 caagggacca aggtggaaat caaa # # 324 <210> SEQ ID
NO 48 <211> LENGTH: 108 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 48 Glu Ile Val Leu Thr
Gln Ser Pro Gly Thr Le #u Ser Leu Ser Pro Gly 1 5 # 10 # 15 Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Gl #n Ser Val Ser Ser Ser 20 # 25 #
30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gl #n Ala Pro Arg Leu
Leu 35 # 40 # 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Asp Il #e Pro
Asp Arg Phe Ser 50 # 55 # 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Th #r Ile Ser Arg Leu Glu 65 #70 #75 #80 Pro Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gl #n Tyr Gly Ser Ser Pro 85 # 90 # 95
Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Il #e Lys 100 # 105
<210> SEQ ID NO 49 <211> LENGTH: 111 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 49 Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Va #l Lys Lys Pro Gly Ala 1 5 #
10 # 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Ty #r Thr Phe Thr
Gly Tyr 20 # 25 # 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gl #n
Gly Leu Glu Trp Met 35 # 40 # 45 Gly Trp Ile Asn Pro Asn Ser Gly
Gly Thr As #n Tyr Ala Gln Lys Phe 50 # 55 # 60 Gln Gly Arg Val Thr
Met Thr Arg Asp Thr Se #r Ile Ser Thr Ala Tyr 65 #70 #75 #80 Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp Th #r Ala Val Tyr Tyr Cys 85 #
90 # 95 Ala Arg Arg Leu Trp Gly Gln Gly Thr Thr Va #l Thr Val Ser
Ser 100 # 105 # 110 <210> SEQ ID NO 50 <211> LENGTH:
123 <212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 50 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Le
#u Val Lys Pro Gly Gly 1 5 # 10 # 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Ph #e Thr Phe Ser Ser Tyr 20 # 25 # 30 Ser Met Asn Trp
Val Arg Gln Ala Pro Gly Ly #s Gly Leu Glu Trp Val 35 # 40 # 45 Ser
Ser Ile Ser Ser Ser Ser Ser Tyr Ile Ty #r Tyr Ala Asp Ser Val 50 #
55 # 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Al #a Lys Asn Ser
Leu Tyr 65 #70 #75 #80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Th
#r Ala Val Tyr Tyr Cys 85 # 90 # 95 Ala Arg Val Gln Leu Glu Arg Tyr
Tyr Tyr Ty #r Tyr Gly Met Asp Val 100 # 105 # 110 Trp Gly Gln Gly
Thr Thr Val Thr Val Ser Se #r 115 # 120 <210> SEQ ID NO 51
<211> LENGTH: 121 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 51 Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Le #u Val Gln Pro Gly Gly 1 5 # 10 # 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Ph #e Thr Phe Ser Ser Tyr 20 # 25 # 30 Ala
Met Ser Trp Val Arg Gln Ala Pro Gly Ly #s Gly Leu Glu Trp Val 35 #
40 # 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Ty #r Tyr Ala Asp
Ser Val 50 # 55 # 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Se #r
Lys Asn Thr Leu Tyr 65 #70 #75 #80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Th #r Ala Val Tyr Tyr Cys 85 # 90 # 95 Ala Lys Val Asp Thr
Ala Met Val Tyr Tyr Gl #y Met Asp Val Trp Gly 100 # 105 # 110 Gln
Gly Thr Thr Val Thr Val Ser Ser 115 # 120 <210> SEQ ID NO 52
<211> LENGTH: 122 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 52 Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Va #l Val Gln Pro Gly Arg 1 5 # 10 # 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Ph #e Thr Phe Ser Ser Tyr 20 # 25 # 30 Gly
Met His Trp Val Arg Gln Ala Pro Gly Ly #s Gly Leu Glu Trp Val 35 #
40 # 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Ty #r Tyr Ala Asp
Ser Val 50 # 55 # 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Se #r
Lys Asn Thr Leu Tyr 65 #70 #75 #80 Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Th #r Ala Val Tyr Tyr Cys 85 # 90 # 95 Ala Arg Ile Ala Ala
Arg Tyr Tyr Tyr Tyr Ty #r Gly Met Asp Val Trp 100 # 105 # 110 Gly
Gln Gly Thr Thr Val Thr Val Ser Ser 115 # 120 <210> SEQ ID NO
53 <211> LENGTH: 125 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 53 Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Le #u Val Lys Pro Ser Gln 1 5 # 10 # 15 Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Gl #y Ser Ile Ser Ser Gly 20 # 25 #
30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pr #o Gly Lys Gly Leu
Glu 35 # 40 # 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Th #r Tyr
Tyr Asn Pro Ser 50
# 55 # 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Th #r Ser Lys Asn
Gln Phe 65 #70 #75 #80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala As
#p Thr Ala Val Tyr Tyr 85 # 90 # 95 Cys Ala Arg Gly Ile Ala Ala Ala
Gly Tyr Ty #r Tyr Tyr Tyr Gly Met 100 # 105 # 110 Asp Val Trp Gly
Gln Gly Thr Thr Val Thr Va #l Ser Ser 115 # 120 # 125 <210>
SEQ ID NO 54 <211> LENGTH: 119 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 54 Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Le #u Val Lys Pro Ser Gln 1 5 # 10
# 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gl #y Ser Ile Ser Ser
Gly 20 # 25 # 30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pr #o Gly
Lys Gly Leu Glu 35 # 40 # 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly
Ser Th #r Tyr Tyr Asn Pro Ser 50 # 55 # 60 Leu Lys Ser Arg Val Thr
Ile Ser Val Asp Th #r Ser Lys Asn Gln Phe 65 #70 #75 #80 Ser Leu
Lys Leu Ser Ser Val Thr Ala Ala As #p Thr Ala Val Tyr Tyr 85 # 90 #
95 Cys Ala Arg Ile Thr Ile Phe Gly Val Phe As #p Tyr Trp Gly Gln
Gly 100 # 105 # 110 Thr Leu Val Thr Val Ser Ser 115 <210> SEQ
ID NO 55 <211> LENGTH: 122 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 55 Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Le #u Val Lys Pro Ser Gln 1 5 # 10 # 15 Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Gl #y Ser Ile Ser Ser Gly 20 # 25 #
30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pr #o Gly Lys Gly Leu
Glu 35 # 40 # 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Th #r Tyr
Tyr Asn Pro Ser 50 # 55 # 60 Leu Lys Ser Arg Val Thr Ile Ser Val
Asp Th #r Ser Lys Asn Gln Phe 65 #70 #75 #80 Ser Leu Lys Leu Ser
Ser Val Thr Ala Ala As #p Thr Ala Val Tyr Tyr 85 # 90 # 95 Cys Ala
Arg Val Ala Thr Tyr Tyr Tyr Tyr Ty #r Gly Met Asp Val Trp 100 # 105
# 110 Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 # 120 <210>
SEQ ID NO 56 <211> LENGTH: 121 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 56 Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Le #u Val Lys Pro Ser Gln 1 5 # 10
# 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gl #y Ser Ile Ser Ser
Gly 20 # 25 # 30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pr #o Gly
Lys Gly Leu Glu 35 # 40 # 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly
Ser Th #r Tyr Tyr Asn Pro Ser 50 # 55 # 60 Leu Lys Ser Arg Val Thr
Ile Ser Val Asp Th #r Ser Lys Asn Gln Phe 65 #70 #75 #80 Ser Leu
Lys Leu Ser Ser Val Thr Ala Ala As #p Thr Ala Val Tyr Tyr 85 # 90 #
95 Cys Ala Arg Leu Arg Tyr Tyr Tyr Tyr Tyr Gl #y Met Asp Val Trp
Gly 100 # 105 # 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 # 120
<210> SEQ ID NO 57 <211> LENGTH: 120 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 57 Glu
Val Gln Leu Val Gln Ser Gly Ala Glu Va #l Lys Lys Pro Gly Glu 1 5 #
10 # 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Ty #r Ser Phe Thr
Ser Tyr 20 # 25 # 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Ly #s
Gly Leu Glu Trp Met 35 # 40 # 45 Gly Ile Ile Tyr Pro Gly Asp Ser
Asp Thr Ar #g Tyr Ser Pro Ser Phe 50 # 55 # 60 Gln Gly Gln Val Thr
Ile Ser Ala Asp Lys Se #r Ile Ser Thr Ala Tyr 65 #70 #75 #80 Leu
Gln Trp Ser Ser Leu Lys Ala Ser Asp Th #r Ala Met Tyr Tyr Cys 85 #
90 # 95 Ala Arg Ser Ser Gly Tyr Tyr Tyr Ala Phe As #p Ile Trp Gly
Gln Gly 100 # 105 # 110 Thr Met Val Thr Val Ser Ser Ala 115 # 120
<210> SEQ ID NO 58 <211> LENGTH: 113 <212> TYPE:
PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 58 Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Va #l Lys Lys Pro Gly Glu 1 5 # 10
# 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Ty #r Ser Phe Thr Ser
Tyr 20 # 25 # 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Ly #s Gly
Leu Glu Trp Met 35 # 40 # 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp
Thr Ar #g Tyr Ser Pro Ser Phe 50 # 55 # 60 Gln Gly Gln Val Thr Ile
Ser Ala Asp Lys Se #r Ile Ser Thr Ala Tyr 65 #70 #75 #80 Leu Gln
Trp Ser Ser Leu Lys Ala Ser Asp Th #r Ala Met Tyr Tyr Cys 85 # 90 #
95 Ala Arg Gly Met Asp Val Trp Gly Gln Gly Th #r Thr Val Thr Val
Ser 100 # 105 # 110 Ser <210> SEQ ID NO 59 <211>
LENGTH: 123 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 59 Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Va #l Lys Lys Pro Gly Glu 1 5 # 10 # 15 Ser Leu Lys Ile Ser
Cys Lys Gly Ser Gly Ty #r Ser Phe Thr Ser Tyr 20 # 25 # 30 Trp Ile
Gly Trp Val Arg Gln Met Pro Gly Ly #s Gly Leu Glu Trp Met 35 # 40 #
45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Ar #g Tyr Ser Pro Ser
Phe 50 # 55 # 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Se #r Ile
Ser Thr Ala Tyr 65 #70 #75 #80 Leu Gln Trp Ser Ser Leu Lys Ala Ser
Asp Th #r Ala Met Tyr Tyr Cys 85 # 90 # 95 Ala Arg Gly Ile Ala Ala
Ala Gly Tyr Tyr Ty #r Gly Met Asp Val Trp 100 # 105 # 110 Gly Lys
Gly Thr Thr Val Thr Val Ser Ser Al #a 115 # 120 <210> SEQ ID
NO 60 <211> LENGTH: 112 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 60 Asp Ile Val Met Thr
Gln Thr Pro Leu Ser Le #u Ser Val Thr Pro Gly 1 5 # 10 # 15 Gln Pro
Ala Ser Ile Ser Cys Lys Ser Ser Gl #n Ser Leu Leu His Ser 20 # 25 #
30 Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gl #n Lys Pro Gly Gln
Pro 35 # 40 # 45 Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn Ar #g Phe
Ser Gly Val Pro 50 # 55 # 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr As #p Phe Thr Leu Lys Ile 65 #70 #75 #80 Ser Arg Val Glu Ala
Glu Asp Val Gly Val Ty #r Tyr Cys Met Gln Ser 85 # 90 # 95 Ile Gln
Leu Pro Trp Thr Phe Gly Gln Gly Th #r Lys Val Glu Ile Lys 100 # 105
# 110 <210> SEQ ID NO 61 <211> LENGTH: 108 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
61 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Le #u Ser Leu Ser Pro
Gly 1 5 # 10 # 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gl #n Ser
Val Ser Ser Ser 20 # 25 # 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gl #n Ala Pro Arg Leu Leu 35 # 40 # 45 Ile Tyr Gly Ala Ser Ser
Arg Ala Thr Gly Il #e Pro Asp Arg Phe Ser 50 # 55 # 60 Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Th #r Ile Ser Arg Leu Glu 65 #70 #75
#80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gl #n Tyr Gly Ser Ser
Pro 85 # 90 # 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Il #e Lys
100 # 105 <210> SEQ ID NO 62 <211> LENGTH: 108
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 62 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Le
#u Ser Leu Ser Pro Gly 1 5 # 10 # 15 Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gl #n Ser Val Ser Ser Ser 20 # 25 # 30 Tyr Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Gl #n Ala Pro Arg Leu Leu 35 # 40 # 45 Ile
Tyr Gly Ala Ser Ser Arg Ala Thr Gly Il #e Pro Asp Arg Phe Ser 50 #
55 # 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Th #r Ile Ser Arg
Leu Glu 65 #70 #75 #80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gl
#n Tyr Gly Ser Ser Pro 85 # 90 # 95
Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Il #e Lys 100 # 105
<210> SEQ ID NO 63 <211> LENGTH: 109 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 63 Glu
Ile Val Leu Thr Gln Ser Pro Gly Thr Le #u Ser Leu Ser Pro Gly 1 5 #
10 # 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gl #n Ser Val Ser
Ser Ser 20 # 25 # 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gl #n
Ala Pro Arg Leu Leu 35 # 40 # 45 Ile Tyr Gly Ala Ser Ser Arg Ala
Thr Gly Il #e Pro Asp Arg Phe Ser 50 # 55 # 60 Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Th #r Ile Ser Arg Leu Glu 65 #70 #75 #80 Pro
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gl #n Tyr Gly Ser Ser Pro 85 #
90 # 95 Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Il #e Lys Arg 100 #
105 <210> SEQ ID NO 64 <211> LENGTH: 109 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
64 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Se #r Ala Ala Pro Gly
Gln 1 5 # 10 # 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Se #r Asn
Ile Gly Asn Asn 20 # 25 # 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro
Gly Th #r Ala Pro Lys Leu Leu 35 # 40 # 45 Ile Tyr Asp Asn Asn Lys
Arg Pro Ser Gly Il #e Pro Asp Arg Phe Ser 50 # 55 # 60 Gly Ser Lys
Ser Gly Thr Ser Ala Thr Leu Gl #y Ile Thr Gly Leu Gln 65 #70 #75
#80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Th #r Trp Asp Ser Ser
Leu 85 # 90 # 95 Ser Ala Tyr Val Phe Gly Thr Gly Thr Lys Va #l Thr
Val 100 # 105 <210> SEQ ID NO 65 <211> LENGTH: 110
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 65 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Se
#r Ala Ala Pro Gly Gln 1 5 # 10 # 15 Lys Val Thr Ile Ser Cys Ser
Gly Ser Ser Se #r Asn Ile Gly Asn Asn 20 # 25 # 30 Tyr Val Ser Trp
Tyr Gln Gln Leu Pro Gly Th #r Ala Pro Lys Leu Leu 35 # 40 # 45 Ile
Tyr Asp Asn Asn Lys Arg Pro Ser Gly Il #e Pro Asp Arg Phe Ser 50 #
55 # 60 Gly Ser Lys Ser Gly Thr Ser Ala Thr Leu Gl #y Ile Thr Gly
Leu Gln 65 #70 #75 #80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Th
#r Trp Asp Ser Ser Leu 85 # 90 # 95 Ser Ala Val Val Phe Gly Gly Gly
Thr Lys Le #u Thr Val Leu 100 # 105 # 110 <210> SEQ ID NO 66
<211> LENGTH: 108 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 66 Ser Tyr Glu Leu Thr Gln Pro
Pro Ser Val Se #r Val Ser Pro Gly Gln 1 5 # 10 # 15 Thr Ala Arg Ile
Thr Cys Ser Gly Asp Ala Le #u Pro Lys Lys Tyr Ala 20 # 25 # 30 Tyr
Trp Tyr Gln Gln Lys Ser Gly Gln Ala Pr #o Val Leu Val Ile Tyr 35 #
40 # 45 Glu Asp Ser Lys Arg Pro Ser Gly Ile Pro Gl #u Arg Phe Ser
Gly Ser 50 # 55 # 60 Ser Ser Gly Thr Met Ala Thr Leu Thr Ile Se #r
Gly Ala Gln Val Glu 65 #70 #75 #80 Asp Glu Ala Asp Tyr Tyr Cys Tyr
Ser Thr As #p Ser Ser Gly Asn His 85 # 90 # 95 Val Val Phe Gly Gly
Gly Thr Lys Leu Thr Va #l Leu 100 # 105 <210> SEQ ID NO 67
<211> LENGTH: 108 <212> TYPE: PRT <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 67 Ser Tyr Glu Leu Thr Gln Pro
Pro Ser Val Se #r Val Ser Pro Gly Gln 1 5 # 10 # 15 Thr Ala Arg Ile
Thr Cys Ser Gly Asp Ala Le #u Pro Lys Lys Tyr Ala 20 # 25 # 30 Tyr
Trp Tyr Gln Gln Lys Ser Gly Gln Ala Pr #o Val Leu Val Ile Tyr 35 #
40 # 45 Glu Asp Ser Lys Arg Pro Ser Gly Ile Pro Gl #u Arg Phe Ser
Gly Ser 50 # 55 # 60 Ser Ser Gly Thr Met Ala Thr Leu Thr Ile Se #r
Gly Ala Gln Val Glu 65 #70 #75 #80 Asp Glu Ala Asp Tyr Tyr Cys Tyr
Ser Thr As #p Ser Ser Gly Asn His 85 # 90 # 95
Val Val Phe Gly Gly Gly Thr Lys Leu Thr Va #l Leu 100 # 105
<210> SEQ ID NO 68 <211> LENGTH: 108 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 68 Ser
Ser Glu Leu Thr Gln Asp Pro Ala Val Se #r Val Ala Leu Gly Gln 1 5 #
10 # 15 Thr Val Arg Ile Thr Cys Gln Gly Asp Ser Le #u Arg Ser Tyr
Tyr Ala 20 # 25 # 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pr #o
Val Leu Val Ile Tyr 35 # 40 # 45 Gly Lys Asn Asn Arg Pro Ser Gly
Ile Pro As #p Arg Phe Ser Gly Ser 50 # 55 # 60 Ser Ser Gly Asn Thr
Ala Ser Leu Thr Ile Th #r Gly Ala Gln Ala Glu 65 #70 #75 #80 Asp
Glu Ala Asp Tyr Tyr Cys Asn Ser Arg As #p Ser Ser Gly Asn His 85 #
90 # 95 Val Val Phe Gly Gly Gly Thr Lys Leu Thr Va #l Leu 100 # 105
<210> SEQ ID NO 69 <211> LENGTH: 106 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 69 Ser
Tyr Glu Leu Thr Gln Pro Ser Ser Val Se #r Val Ser Pro Gly Gln 1 5 #
10 # 15 Thr Ala Arg Ile Thr Cys Ser Gly Asp Val Le #u Ala Lys Lys
Tyr Ala 20 # 25 # 30 Arg Trp Phe Gln Gln Lys Pro Gly Gln Ala Pr #o
Val Leu Val Ile Tyr 35 # 40 # 45 Lys Asp Ser Glu Arg Pro Ser Gly
Ile Pro Gl #u Arg Phe Ser Gly Ser 50 # 55 # 60 Ser Ser Gly Thr Thr
Val Thr Leu Thr Ile Se #r Gly Ala Gln Val Glu 65 #70 #75 #80 Asp
Glu Ala Asp Tyr Tyr Cys Tyr Ser Ala Al #a Asp Asn Asn Val Val 85 #
90 # 95 Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 # 105
<210> SEQ ID NO 70 <211> LENGTH: 372 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <400> SEQUENCE: 70
caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cc #tgtccctc
60 acctgcactg tctctggtgg ctccatcagc agtggtgttt actactggac ct
#ggatccgc 120 cagcacccag ggaacggcct ggagtggatt ggctacatct
attacagtgg ga #gcacctcc 180 tacaacccgt ccctcaagag tcgagttacc
atatcagtag acacgtctaa ga #aacagttc 240 tccctgaagc tgacctctgt
gactgccgcg gacacggccg tgtattactg tg #cgagagaa 300 ggaccactac
ggggggacta ctactacggt ctggacgtct ggggccaagg ga #ccacggtc 360
accgtctcct ca # # # 372 <210> SEQ ID NO 71 <211>
LENGTH: 124 <212> TYPE: PRT <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 71 Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Le #u Val Lys Pro Ser Gln 1 5 # 10 # 15 Thr Leu Ser Leu Thr
Cys Thr Val Ser Gly Gl #y Ser Ile Ser Ser Gly 20 # 25 # 30 Val Tyr
Tyr Trp Thr Trp Ile Arg Gln His Pr #o Gly Asn Gly Leu Glu 35 # 40 #
45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Th #r Ser Tyr Asn Pro
Ser 50 # 55 # 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Th #r Ser
Lys Lys Gln Phe 65 #70 #75 #80 Ser Leu Lys Leu Thr Ser Val Thr Ala
Ala As #p Thr Ala Val Tyr Tyr 85 # 90 # 95 Cys Ala Arg Glu Gly Pro
Leu Arg Gly Asp Ty #r Tyr Tyr Gly Leu Asp 100 # 105 # 110 Val Trp
Gly Gln Gly Thr Thr Val Thr Val Se #r Ser 115 # 120 <210> SEQ
ID NO 72 <211> LENGTH: 324 <212> TYPE: DNA <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 72 gaaattgtgt
tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aa #gagccacc 60
ctctcctgca gggccggtca gactattagc agtcgctact tagcctggta cc #agcagaaa
120 cctggccagg ctcccaggcc cctcatctat ggtgcatcca gcagggccac tg
#gcatccca 180 gacaggttca gtggcagtgg gtctgggaca gacttcactc
tcaccatcag ca #gactggag 240 cctgaagatt ttgcagtgta ttactgtcag
cagtatggta gctcacctcg ga #cgttcggc 300 caagggacca aggtggaaat caaa #
# 324 <210> SEQ ID NO 73 <211> LENGTH: 108 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
73 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Le #u Ser Leu Ser Pro
Gly 1 5 # 10 # 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Gly Gl #n Thr
Ile Ser Ser Arg 20 # 25 # 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gl #n Ala Pro Arg Pro Leu 35 # 40 # 45 Ile Tyr Gly Ala Ser Ser
Arg Ala Thr Gly Il #e Pro Asp Arg Phe Ser 50 # 55 # 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Th #r Ile Ser Arg Leu Glu
65 #70 #75 #80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gl #n Tyr
Gly Ser Ser Pro 85 # 90 # 95 Arg Thr Phe Gly Gln Gly Thr Lys Val
Glu Il #e Lys 100 # 105 <210> SEQ ID NO 74 <211>
LENGTH: 375 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 74 caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcacagac cc #tgtccctc 60 acctgcactg tctctggtgg
ctccatcagc agtggtggtt actactggag ct #ggatccgc 120 cagcacccag
ggaagggcct ggagtggatt gggtacatct attacagtgg ga #gaacctac 180
aacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa ga #accagttc
240 tccctgaagt tgagttctgt gactgccgcg gacacggccg tgtattactg tg
#cgagagtg 300 gctacgggga gagcggacta ccacttctac gctatggacg
tctggggcca ag #ggaccacg 360 gtcaccgtct cctca # # # 375 <210>
SEQ ID NO 75 <211> LENGTH: 125 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 75 Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Le #u Val Lys Pro Ser Gln 1 5 # 10
# 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gl #y Ser Ile Ser Ser
Gly 20 # 25 # 30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pr #o Gly
Lys Gly Leu Glu 35 # 40 # 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly
Arg Th #r Tyr Asn Asn Pro Ser 50 # 55 # 60 Leu Lys Ser Arg Val Thr
Ile Ser Val Asp Th #r Ser Lys Asn Gln Phe 65 #70 #75 #80 Ser Leu
Lys Leu Ser Ser Val Thr Ala Ala As #p Thr Ala Val Tyr Tyr 85 # 90 #
95 Cys Ala Arg Val Ala Thr Gly Arg Ala Asp Ty #r His Phe Tyr Ala
Met 100 # 105 # 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Va #l
Ser Ser 115 # 120 # 125 <210> SEQ ID NO 76 <211>
LENGTH: 318 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 76 tcctatgagc tgacacagcc atcctcagtg
tcagtgtctc cgggacagac ag #ccaggatc 60 acctgctcag gagatgtact
ggcaaaaaag tctgctcggt ggttccacca ga #agccaggc 120 caggcccctg
tactggtgat ttataaagac agtgagcggc cctcagggat cc #ctgagcgc 180
ttctccggct ccagctcagg gaccacagtc accttgacca tcagcggggc cc #aggttgag
240 gatgaggctg cctattactg ttactctgcg gctgacaaca atctggtatt cg
#gcggaggg 300 accaagctga ccgtccta # # # 318 <210> SEQ ID NO
77 <211> LENGTH: 106 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 77 Ser Tyr Glu Leu Thr
Gln Pro Ser Ser Val Se #r Val Ser Pro Gly Gln 1 5 # 10 # 15 Thr Ala
Arg Ile Thr Cys Ser Gly Asp Val Le #u Ala Lys Lys Ser Ala 20 # 25 #
30 Arg Trp Phe His Gln Lys Pro Gly Gln Ala Pr #o Val Leu Val Ile
Tyr 35 # 40 # 45 Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Gl #u Arg
Phe Ser Gly Ser 50 # 55 # 60 Ser Ser Gly Thr Thr Val Thr Leu Thr
Ile Se #r Gly Ala Gln Val Glu 65 #70 #75 #80 Asp Glu Ala Ala Tyr
Tyr Cys Tyr Ser Ala Al #a Asp Asn Asn Leu Val 85 # 90 # 95 Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 # 105 <210> SEQ ID NO 78
<211> LENGTH: 339 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 78 caggtgcagc tggtgcagtc
tggggctgag gtgaagaagc ctggggcctc ag #tgaaggtc 60 tcctgcaagg
cttctggata caccttcacc ggctactata tgcactgggt gc #gacaggcc 120
cctggacaag ggcttgagtg gatgggatgg atcaacccta aaagtggtga ca #caaactat
180 gcacagaagt ttcagggcag ggtcaccatg accagggaca cgtccaccag ca
#cagcctac 240 atggagctga gcaggctgag atctgacgac acggccgtgt
attactgtgc ga #gaaggttg 300 gacgtctggg gccaagggac cacggtcacc
gtctcctca # # 339 <210> SEQ ID NO 79 <211> LENGTH: 113
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 79 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Va
#l Lys Lys Pro Gly Ala 1 5 # 10 # 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Ty #r Thr Phe Thr Gly Tyr 20 # 25 # 30 Tyr Met His Trp
Val Arg Gln Ala Pro Gly Gl #n Gly Leu Glu Trp Met 35 # 40 # 45 Gly
Trp Ile Asn Pro Lys Ser Gly Asp Thr As #n Tyr Ala Gln Lys Phe 50 #
55 # 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Se #r Thr Ser Thr
Ala Tyr 65 #70 #75 #80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Th
#r Ala Val Tyr Tyr Cys
85 # 90 # 95 Ala Arg Arg Leu Asp Val Trp Gly Gln Gly Th #r Thr Val
Thr Val Ser 100 # 105 # 110 Ser <210> SEQ ID NO 80
<211> LENGTH: 333 <212> TYPE: DNA <213> ORGANISM:
Homo sapiens <400> SEQUENCE: 80 cagtctgtgt tgacgcagcc
gccctcagtg tctgcggccc caggacagaa gg #tcaccatc 60 tcctgctctg
gaagcagctc caacattggg aataattatg tatcctggta cc #agcagctc 120
ccaggaacag cccccaaact cctcatttat gacaataata agcgaccctc ag #gaattcct
180 gaccgattct ctggctccaa gtctggcacg tcagccaccc tgggcatcac cg
#gactccag 240 actggggacg aggccgatta ttactgcgga acatggaata
gcagcctgag tg #ctggttat 300 gtcttcggaa ctgggaccaa ggtcaccgtc cta #
# 333 <210> SEQ ID NO 81 <211> LENGTH: 111 <212>
TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE:
81 Gln Ser Val Leu Thr Gln Pro Pro Ser Val Se #r Ala Ala Pro Gly
Gln 1 5 # 10 # 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Se #r Asn
Ile Gly Asn Asn 20 # 25 # 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro
Gly Th #r Ala Pro Lys Leu Leu 35 # 40 # 45 Ile Tyr Asp Asn Asn Lys
Arg Pro Ser Gly Il #e Pro Asp Arg Phe Ser 50 # 55 # 60 Gly Ser Lys
Ser Gly Thr Ser Ala Thr Leu Gl #y Ile Thr Gly Leu Gln 65 #70 #75
#80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Th #r Trp Asn Ser Ser
Leu 85 # 90 # 95 Ser Ala Gly Tyr Val Phe Gly Thr Gly Thr Ly #s Val
Thr Val Leu 100 # 105 # 110 <210> SEQ ID NO 82 <211>
LENGTH: 339 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 82 caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctggggcctc ag #tgaaggtc 60 tcctgcaagg cttctggata
caccttcacc ggctactata tgcactgggt gc #gacaggcc 120 cctggacaag
ggcttgagtg gatgggatgg atcaacccta aaagtggtga ca #caaactat 180
gcacagaagt ttcagggcag ggtcaccctg accagggaca cgtccaccag ca #cagcctac
240 atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc ga
#gaaggttg 300 gacgtctggg gccaagggac cacggtcacc gtctcctca # # 339
<210> SEQ ID NO 83 <211> LENGTH: 113 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 83 Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Va #l Lys Lys Pro Gly Ala 1 5 #
10 # 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Ty #r Thr Phe Thr
Gly Tyr 20 # 25 # 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gl #n
Gly Leu Glu Trp Met 35 # 40 # 45 Gly Trp Ile Asn Pro Lys Ser Gly
Asp Thr As #n Tyr Ala Gln Lys Phe 50 # 55 # 60 Gln Gly Arg Val Thr
Leu Thr Arg Asp Thr Se #r Thr Ser Thr Ala Tyr 65 #70 #75 #80 Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp Th #r Ala Val Tyr Tyr Cys 85 #
90 # 95 Ala Arg Arg Leu Asp Val Trp Gly Gln Gly Th #r Thr Val Thr
Val Ser 100 # 105 # 110 Ser <210> SEQ ID NO 84 <211>
LENGTH: 333 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 84 cagtctgtgt tgacgcagcc gccctcagtg
tctgcggccc caggacagaa gg #tcaccatc 60 tcctgctctg gaagcagctc
caacattggg aataattatg tatcctggta cc #agcagctc 120 ccaggaacag
cccccaaact cctcatttat gacaataata agcgaccctc ag #gaattcct 180
gaccgattct ctggctccaa gtctggcacg tcagccaccc tgggcatcac cg #gactccag
240 actggggacg aggccgatta ttactgcgga acatgggata gcagcctgag tg
#ctggttat 300 gtcttcggaa ctgggaccaa ggtcaccgtc cta # # 333
<210> SEQ ID NO 85 <211> LENGTH: 111 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 85 Gln
Ser Val Leu Thr Gln Pro Pro Ser Val Se #r Ala Ala Pro Gly Gln 1 5 #
10 # 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Se #r Asn Ile Gly
Asn Asn 20 # 25 # 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Th #r
Ala Pro Lys Leu Leu 35 # 40 # 45 Ile Tyr Asp Asn Asn Lys Arg Pro
Ser Gly Il #e Pro Asp Arg Phe Ser 50 # 55 # 60 Gly Ser Lys Ser Gly
Thr Ser Ala Thr Leu Gl #y Ile Thr Gly Leu Gln 65 #70 #75 #80 Thr
Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Th #r Trp Asp Ser Ser Leu 85 #
90 # 95 Ser Ala Gly Tyr Val Phe Gly Thr Gly Thr Ly #s Val Thr Val
Leu 100 # 105 # 110 <210> SEQ ID NO 86 <211> LENGTH:
339
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 86 caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctggggcctc ag #tgaaggtc 60 tcctgcaagg cttctggata
caccttcacc ggctactata tgcactgggt gc #gacaggcc 120 cctggacaag
ggcttgagtg gatgggatgg atcaacccta aaagtggtga ca #caaactat 180
gcacagaagt ttcagggcag ggtcaccatg accagggaca cgtccaccag ca #cagcctac
240 atggagctga gcaggctgag atctgacgac acggccgtgt attactgtgc ga
#gaaggttg 300 gacgtctggg gccaagggac cacggtcacc gtctcctca # # 339
<210> SEQ ID NO 87 <211> LENGTH: 113 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 87 Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Va #l Lys Lys Pro Gly Ala 1 5 #
10 # 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Ty #r Thr Phe Thr
Gly Tyr 20 # 25 # 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gl #n
Gly Leu Glu Trp Met 35 # 40 # 45 Gly Trp Ile Asn Pro Lys Ser Gly
Asp Thr As #n Tyr Ala Gln Lys Phe 50 # 55 # 60 Gln Gly Arg Val Thr
Met Thr Arg Asp Thr Se #r Thr Ser Thr Ala Tyr 65 #70 #75 #80 Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp Th #r Ala Val Tyr Tyr Cys 85 #
90 # 95 Ala Arg Arg Leu Asp Val Trp Gly Gln Gly Th #r Thr Val Thr
Val Ser 100 # 105 # 110 Ser <210> SEQ ID NO 88 <211>
LENGTH: 333 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 88 cagtctgtgt tgacgcagcc gccctcagtg
tctgcggccc caggacagaa gg #tcaccatc 60 tcctgctctg gaagcagctc
caacattggg aataattatg tatcctggta cc #agcagctc 120 ccaggaacag
cccccaaact cctcatttat gacaataata agcgaccctc ag #gaattcct 180
gaccgattct ctggctccaa gtctggcacg tcagccaccc tgggcatcac cg #gactccag
240 actggggacg aggccgatta ttactgcgga acatgggata gcagcctgag tg
#ctggttat 300 gtcttcggaa ctgggaccaa ggtcaccgtc cta # # 333
<210> SEQ ID NO 89 <211> LENGTH: 111 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 89 Gln
Ser Val Leu Thr Gln Pro Pro Ser Val Se #r Ala Ala Pro Gly Gln 1 5 #
10 # 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Se #r Asn Ile Gly
Asn Asn 20 # 25 # 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Th #r
Ala Pro Lys Leu Leu 35 # 40 # 45 Ile Tyr Asp Asn Asn Lys Arg Pro
Ser Gly Il #e Pro Asp Arg Phe Ser 50 # 55 # 60 Gly Ser Lys Ser Gly
Thr Ser Ala Thr Leu Gl #y Ile Thr Gly Leu Gln 65 #70 #75 #80 Thr
Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Th #r Trp Asp Ser Ser Leu 85 #
90 # 95 Ser Ala Gly Tyr Val Phe Gly Thr Gly Thr Ly #s Val Thr Val
Leu 100 # 105 # 110 <210> SEQ ID NO 90 <211> LENGTH:
383 <212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 90 caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcacagac cc #tgtccctc 60 acctgcactg tctctggtgg
ctccatcagc agtggtggtt actactggag ct #ggatccgc 120 cagcacccag
ggaagggcct ggagtggatt gggtacatct attacagtgg ga #gaacctac 180
aacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa ga #accagttc
240 tccctgaagt tgagttctgt gactgccgcg gacacggccg tgtattactg tg
#cgagagtg 300 gctacgggga gaggggacta ccacttctac gctatggacg
tctggggcca ag #ggaccacg 360 gtcaccgtct cctcagcctc cac # # 383
<210> SEQ ID NO 91 <211> LENGTH: 125 <212> TYPE:
PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 91 Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Le #u Val Lys Pro Ser Gln 1 5 #
10 # 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gl #y Ser Ile Ser
Ser Gly 20 # 25 # 30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pr #o
Gly Lys Gly Leu Glu 35 # 40 # 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser
Gly Arg Th #r Tyr Asn Asn Pro Ser 50 # 55 # 60 Leu Lys Ser Arg Val
Thr Ile Ser Val Asp Th #r Ser Lys Asn Gln Phe 65 #70 #75 #80 Ser
Leu Lys Leu Ser Ser Val Thr Ala Ala As #p Thr Ala Val Tyr Tyr 85 #
90 # 95 Cys Ala Arg Val Ala Thr Gly Arg Gly Asp Ty #r His Phe Tyr
Ala Met 100 # 105 # 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Va
#l Ser Ser 115 # 120 # 125 <210> SEQ ID NO 92 <211>
LENGTH: 318 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 92 tcctatgagc tgacacagcc atcctcagtg
tcagtgtctc cgggacagac ag
#ccaggatc 60 acctgctcag gagatgtact ggcaaaaaag tctgctcggt ggttccacca
ga #agccaggc 120 caggcccctg tactggtgat ttataaagac agtgagcggc
cctcagggat cc #ctgagcgc 180 ttctccggct ccagctcagg gaccacagtc
accttgacca tcagcggggc cc #aggttgag 240 gatgaggctg actattactg
ttactctgcg gctgacaaca atctggtatt cg #gcggaggg 300 accaagctga
ccgtccta # # # 318 <210> SEQ ID NO 93 <211> LENGTH: 106
<212> TYPE: PRT <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 93 Ser Tyr Glu Leu Thr Gln Pro Ser Ser Val Se
#r Val Ser Pro Gly Gln 1 5 # 10 # 15 Thr Ala Arg Ile Thr Cys Ser
Gly Asp Val Le #u Ala Lys Lys Ser Ala 20 # 25 # 30 Arg Trp Phe His
Gln Lys Pro Gly Gln Ala Pr #o Val Leu Val Ile Tyr 35 # 40 # 45 Lys
Asp Ser Glu Arg Pro Ser Gly Ile Pro Gl #u Arg Phe Ser Gly Ser 50 #
55 # 60 Ser Ser Gly Thr Thr Val Thr Leu Thr Ile Se #r Gly Ala Gln
Val Glu 65 #70 #75 #80 Asp Glu Ala Asp Tyr Tyr Cys Tyr Ser Ala Al
#a Asp Asn Asn Leu Val 85 # 90 # 95 Phe Gly Gly Gly Thr Lys Leu Thr
Val Leu 100 # 105 <210> SEQ ID NO 94 <211> LENGTH: 375
<212> TYPE: DNA <213> ORGANISM: Homo sapiens
<400> SEQUENCE: 94 caggtgcagc tgcaggagtc gggcccagga
ctggtgaagc cttcacagac cc #tgtccctc 60 acctgcactg tctctggtgg
ctccatcagc agtggtggtt actactggag ct #ggatccgc 120 cagcacccag
ggaagggcct ggagtggatt gggtacatct attacagtgg ga #gaacctac 180
aacaacccgt ccctcaagag tcgagttacc atatcagtag acacgtctaa ga #accagttc
240 tccctgaagt tgagttctgt gactgccgcg gacacggccg tgtattactg tg
#cgagagtg 300 gctacgggga gagcggacta ccacttctac gctatggacg
tctggggcca ag #ggaccacg 360 gtcaccgtct cctca # # # 375 <210>
SEQ ID NO 95 <211> LENGTH: 125 <212> TYPE: PRT
<213> ORGANISM: Homo sapiens <400> SEQUENCE: 95 Gln Val
Gln Leu Gln Glu Ser Gly Pro Gly Le #u Val Lys Pro Ser Gln 1 5 # 10
# 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gl #y Ser Ile Ser Ser
Gly 20 # 25 # 30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pr #o Gly
Lys Gly Leu Glu 35 # 40 # 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly
Arg Th #r Tyr Asn Asn Pro Ser 50 # 55 # 60 Leu Lys Ser Arg Val Thr
Ile Ser Val Asp Th #r Ser Lys Asn Gln Phe 65 #70 #75 #80 Ser Leu
Lys Leu Ser Ser Val Thr Ala Ala As #p Thr Ala Val Tyr Tyr 85 # 90 #
95 Cys Ala Arg Val Ala Thr Gly Arg Ala Asp Ty #r His Phe Tyr Ala
Met 100 # 105 # 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr Va #l
Ser Ser 115 # 120 # 125 <210> SEQ ID NO 96 <211>
LENGTH: 318 <212> TYPE: DNA <213> ORGANISM: Homo
sapiens <400> SEQUENCE: 96 tcctatgagc tgacacagcc atcctcagtg
tcagtgtctc cgggacagac ag #ccaggatc 60 acctgctcag gagatgtact
ggcaaaaaag tctgctcggt ggttccacca ga #agccaggc 120 caggcccctg
tactggtgat ttataaagac agtgagcggc cctcagggat cc #ctgagcgc 180
ttctccggct ccagctcagg gaccacagtc accttgacca tcagcggggc cc #aggttgag
240 gatgaggctg actattactg ttactctgcg gctgacaaca atctggtatt cg
#gcggaggg 300 accaagctga ccgtccta # # # 318 <210> SEQ ID NO
97 <211> LENGTH: 106 <212> TYPE: PRT <213>
ORGANISM: Homo sapiens <400> SEQUENCE: 97 Ser Tyr Glu Leu Thr
Gln Pro Ser Ser Val Se #r Val Ser Pro Gly Gln 1 5 # 10 # 15 Thr Ala
Arg Ile Thr Cys Ser Gly Asp Val Le #u Ala Lys Lys Ser Ala 20 # 25 #
30 Arg Trp Phe His Gln Lys Pro Gly Gln Ala Pr #o Val Leu Val Ile
Tyr 35 # 40 # 45 Lys Asp Ser Glu Arg Pro Ser Gly Ile Pro Gl #u Arg
Phe Ser Gly Ser 50 # 55 # 60 Ser Ser Gly Thr Thr Val Thr Leu Thr
Ile Se #r Gly Ala Gln Val Glu 65 #70 #75 #80 Asp Glu Ala Asp Tyr
Tyr Cys Tyr Ser Ala Al #a Asp Asn Asn Leu Val 85 # 90 # 95 Phe Gly
Gly Gly Thr Lys Leu Thr Val Leu 100 # 105
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