U.S. patent number RE44,681 [Application Number 13/863,646] was granted by the patent office on 2013-12-31 for compositions and methods for inhibiting growth of smad4-deficient cancers.
This patent grant is currently assigned to Biogen Idec MA Inc.. The grantee listed for this patent is Biogen Idec MA Inc.. Invention is credited to Louise A. Koopman, Shelia M. Violette.
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United States Patent |
RE44,681 |
Violette , et al. |
December 31, 2013 |
**Please see images for:
( Certificate of Correction ) ** |
Compositions and methods for inhibiting growth of SMAD4-deficient
cancers
Abstract
The present invention is in the fields of cell biology,
immunology and oncology. The invention relates to the discovery
that there is a relationship between the expression levels of the
tumor suppressor gene smad4 (also known as dpc4) and integrin
.alpha..sub..nu..beta..sub.6, and the responsiveness of patient
populations to .alpha..sub..nu..beta..sub.6-active compounds and
compositions (e.g., antibodies and other ligands that bind
.alpha..sub..nu..beta..sub.6), particularly in cancer cells from
such patient populations, more particularly on carcinomas such as
pancreatic carcinomas. The invention thus provides methods for
determining the responsiveness of tumor cells (particularly those
from pancreatic tumors) to such .alpha..sub..nu..beta..sub.6-active
compounds and compositions by examining the expression of
.sub..nu..beta..sub.6 and smad4 by the tumor cells, as well as
methods of diagnosis and treatment/prevention of tumor progression
using ligands, including antibodies and molecule drugs, that bind
to integrin .alpha..sub..nu..beta..sub.6 on the surfaces of tumor
cells and/or that block one or more components of the TGF-.beta.
pathway, particularly in smad4-deficient tumor cells.
Inventors: |
Violette; Shelia M. (Lexington,
MA), Koopman; Louise A. (Chestnut Hill, MA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Biogen Idec MA Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
Biogen Idec MA Inc. (Cambridge,
MA)
|
Family
ID: |
38923823 |
Appl.
No.: |
13/863,646 |
Filed: |
April 16, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
60819359 |
Jul 10, 2006 |
|
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Reissue of: |
11822859 |
Jul 10, 2007 |
7927590 |
Apr 19, 2011 |
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Current U.S.
Class: |
424/130.1;
530/387.1 |
Current CPC
Class: |
G01N
33/5091 (20130101); C07K 16/2839 (20130101); A61K
39/39558 (20130101); G01N 33/5011 (20130101); A61K
38/1793 (20130101); A61P 43/00 (20180101); G01N
33/57407 (20130101); G01N 33/574 (20130101); A61P
35/00 (20180101); A61K 39/39558 (20130101); A61K
2300/00 (20130101); C07K 2317/73 (20130101); A61K
2039/505 (20130101) |
Current International
Class: |
A61K
39/395 (20060101); C07K 16/00 (20060101) |
References Cited
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|
Primary Examiner: Aeder; Sean
Attorney, Agent or Firm: Fish & Richardson P.C.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application
No. 60/819,359, filed Jul. 10, 2006, which is incorporated herein
by reference in its entirety.
Claims
What is claimed is:
1. A method of inhibiting growth of .[.a cell from.]. a
.Iadd.smad4-deficient .Iaddend.tumor .[.that is smad4 deficient.].,
comprising: (a) determining the level of expression of smad4 in a
cell from said .[.cancer.]. .Iadd.smad4-deficient tumor.Iaddend.;
and (b) treating .[.a cancer cell that is deficient in smad4
expression.]. .Iadd.the smad4-deficient tumor .Iaddend.with .[.one
or more.]. .Iadd.an .Iaddend..alpha.v.beta.6 antagonist antibody
that selectively binds to the integrin .alpha.v.beta.6 .[.in said
cancer cell.]., wherein said .[.one or more.]. antibody is an
antibody .[.from.]. .Iadd.produced by, or a humanized version of an
antibody produced by, .Iaddend.a hybridoma selected from the group
consisting of .[.2A1 (deposited under ATCC Accession No. ATCC
PTA-3896), 2E5 (deposited under ATCC Accession No. ATCC
PTA-3897),.]. 1A8 (deposited under ATCC Accession No. ATCC
PTA-3647), 2B10 .Iadd.(.Iaddend.deposited under ATCC Accession No.
ATCC PTA-3648), 2B1 .Iadd.(.Iaddend.deposited under ATCC Accession
No. ATCC PTA-3646), 1G10 (deposited under ATCC Accession No. ATCC
PTA-3898), 7G5 (deposited under ATCC Accession No. ATCC PTA-3899),
8G6 (deposited under ATCC Accession No. ATCC PTA-3645), .Iadd.and
.Iaddend.3G9 (deposited under ATCC Accession No. ATCC PTA-3649),
.[.or a humanized version thereof.]. wherein said treatment results
in the growth inhibition of said .[.cancer cell.].
.Iadd.smad4-deficient tumor.Iaddend..
2. The method of claim 1, wherein said tumor is a carcinoma.
3. The method of claim 2, wherein said carcinoma is an
adenocarcinoma.
4. The method of claim 2, wherein said carcinoma is selected from
the group consisting of a pancreatic carcinoma, a colorectal
carcinoma, a cervical carcinoma, .Iadd.a .Iaddend.squamous cell
carcinoma, a head and neck carcinoma, a liver carcinoma, an ovarian
carcinoma and a lung carcinoma.
5. The method of claim 2, wherein said carcinoma is a pancreatic
carcinoma.
6. The method of claim .[.4.]. .Iadd.2.Iaddend., wherein said
.[.squamous cell.]. carcinoma is an esophageal carcinoma.
7. The method of claim 2, wherein said carcinoma is a colorectal
carcinoma.
8. The method of claim 2, wherein said carcinoma is a cervical
carcinoma.
9. The method of claim 2, wherein said carcinoma is a head and neck
carcinoma.
10. The method of claim 1, wherein said .[.monoclonal.]. antibody
is .Iadd.an antibody produced by the hybridoma .Iaddend.3G9
.Iadd.(deposited under ATCC Accession No. ATCC PTA-3649) or a
humanized version thereof.Iaddend..
11. The method of claim 1, wherein said .[.monoclonal.]. antibody
is .Iadd.an antibody produced by the hybridoma .Iaddend.8G6
.Iadd.(deposited under ATCC Accession No. ATCC PTA-3645) or a
humanized version thereof.Iaddend..
12. The method of claim 1, wherein said .[.monoclonal.]. antibody
is a humanized monoclonal antibody.
13. The method of claim 12, wherein said humanized monoclonal
antibody is hu3G9 (BG00011).
14. The method of claim 12, wherein said humanized monoclonal
antibody is hu8G6.
15. The method of claim 1, wherein said antibody is conjugated with
at least one detectable label.
16. The method of claim 15, wherein said detectable label is
selected from the group consisting of a chromogenic label, an
enzyme label, a radioisotopic label, a non-radioactive isotopic
label, a fluorescent label, a toxic label, a chemiluminescent
label, an X-radiographic label, a spin label and a nuclear magnetic
resonance contrast agent label.
17. The method of claim 16, wherein said .Iadd.detectable label is
a .Iaddend.chromogenic label .[.is.]. selected from the group
consisting of diaminobenzidine and 4
hydroxyazo-benzene-2-carboxylic acid.
18. The method of claim 16, wherein said .Iadd.detectable label is
an .Iaddend.enzyme label .[.is.]. selected from the group
consisting of malate dehydrogenase, staphylococcal nuclease, delta
5 steroid isomerase, yeast alcohol dehydrogenase, alpha glycerol
phosphate dehydrogenase, triose phosphate isomerase, peroxidase,
alkaline phosphatase, asparaginase, glucose oxidase, .beta.
galactosidase, ribonuclease, urease, catalase, glucose 6 phosphate
dehydrogenase, glucoamylase and acetylcholine esterase.
19. The method of claim 16, wherein said .Iadd.detectable label is
a .Iaddend.radioisotopic label .[.is.]. selected from the group
consisting of .[.3H, 111 In, 125I, 131I, 32P, 35S, 14C, 51Cr, 57To,
58Co, 59Fe, 75Se, 152Eu, 90Y, 67Cu, 217Ci, 211At, 212Pb, 47Sc .].
.Iadd..sup.3H, .sup.111In, .sup.125I, .sup.131I, .sup.32P,
.sup.35S, .sup.14C, .sup.51Cr, .sup.57To, .sup.58Co, .sup.59Fe,
.sup.75Se, .sup.152Eu, .sup.90Y, .sup.67Cu, .sup.217Ci, .sup.211At,
.sup.212Pb, .sup.47Sc, .Iaddend.and .[.109Pd .].
.Iadd..sup.109Pd.Iaddend..
20. The method of claim 16, wherein said .Iadd.detectable label is
a .Iaddend.non-radioactive isotopic label .[.is.]. selected from
the group consisting of .[.157Gd, 55Mn, 162Dy, 52Tr, 56Fe, 99mTc.].
.Iadd..sup.157Gd, .sup.55Mn, .sup.162Dy, .sup.52Tr, .sup.56Fe,
.sup.99mTc, .Iaddend.and .[.112In.]. .Iadd..sup.112In.Iaddend..
21. The method of claim 16, wherein said .Iadd.detectable label is
a .Iaddend.fluorescent label .[.is.]. selected from the group
consisting of a .[.152Eu.]. .Iadd..sup.152Eu .Iaddend.label, a
fluorescein label, an isothiocyanate label, a rhodamine label, a
phycoerythrin label, a phycocyanin label, an allophycocyanin label,
a Green Fluorescent Protein (GFP) label, an ophthaldehyde label and
a fluorescamine label.
22. The method of claim 16, wherein said .Iadd.detectable label is
a .Iaddend.toxic label .[.is.]. selected from the group consisting
of a diphtheria toxin label, a ricin label and a cholera toxin
label.
23. The method of claim 16, wherein said .Iadd.detectable label is
a .Iaddend.chemiluminescent label .[.is.]. selected from the group
consisting of a luminol label, an isoluminol label, an aromatic
acridinium ester label, an imidazole label, an acridinium salt
label, an oxalate ester label, a luciferin label, a luciferase
label and an aequorin label.
24. The method of claim 16, wherein said .Iadd.detectable label is
an .Iaddend.X-radiographic label .Iadd.that .Iaddend.is barium or
cesium.
25. The method of claim 16, wherein said .Iadd.detectable label is
a .Iaddend.spin label .Iadd.that .Iaddend.is deuterium.
26. The method of claim 16, wherein said .Iadd.detectable label is
a .Iaddend.nuclear magnetic resonance contrast agent label .[.is.].
selected from the group consisting of Gd, Mn and .[.iron.].
.Iadd.Fe.Iaddend..
27. A method of increasing the responsiveness of a smad4-deficient
cancer .[.cell.]. to treatment with a growth-inhibiting
chemotherapeutic .[.compounds.]. .Iadd.compound.Iaddend.,
comprising: (a) determining the level of expression of smad4 in a
cell from said .Iadd.smad4-deficient .Iaddend.cancer; and (b)
treating .[.a.]. .Iadd.the smad4-deficient .Iaddend.cancer .[.cell
that is deficient in smad4 expression.]. with .[.one or more.].
.Iadd.an .Iaddend..alpha.v.beta.6-antagonist .[.antibodies.].
.Iadd.antibody .Iaddend.that selectively .[.bind.]. .Iadd.binds
.Iaddend.to the integrin .alpha.v.beta.6 .[.in said cancer cell.].,
wherein said .[.one or more.]. antibody is an antibody .[.from.].
.Iadd.produced by, or a humanized verison of an antibody produced
by, .Iaddend.a hybridoma selected from the group consisting of
.[.2A1 (deposited under ATCC Accession No. ATCC PTA-3896), 2E5
(deposited under ATCC Accession No. ATCC PTA-3897),.]. 1A8
(deposited under ATCC Accession No. ATCC PTA-3647), 2B10
.Iadd.(.Iaddend.deposited under ATCC Accession No. ATCC PTA-3648),
2B1 .Iadd.(.Iaddend.deposited under ATCC Accession No. ATCC
PTA-3646), 1G10 (deposited under ATCC Accession No. ATCC PTA-3898),
7G5 (deposited under ATCC Accession No. ATCC PTA-3899), 8G6
(deposited under ATCC Accession No. ATCC PTA-3645), .Iadd.and
.Iaddend.3G9 (deposited under ATCC Accession No. ATCC PTA-3649),
.[.or a humanized version thereof.]. wherein said treatment with
said .[.blocking antibodies.]. .Iadd.antibody .Iaddend.results in
increased responsiveness of said .Iadd.smad4-deficient
.Iaddend.cancer .[.cell.]. to .[.one or more.]. .Iadd.the
.Iaddend.growth-inhibiting chemotherapeutic .[.compounds.].
.Iadd.compound .Iaddend.as compared to the growth inhibition of
said .Iadd.smad4-deficient .Iaddend.cancer .[.cell produced by.].
.Iadd.treated with .Iaddend.said chemotherapeutic .[.agent.].
.Iadd.compound .Iaddend.alone.
28. The method of claim 27, wherein said growth-inhibiting
chemotherapeutic compound is selected from the group consisting of
cisplatin, carboplatin, oxaliplatin, paclitaxel, gemcitabine,
adriamycin, melphalan, methotrexate, 5-fluorouracil, etoposide,
mechlorethamine, cyclophosphamide, bleomycin, a calicheamicin, a
maytansine, a trichothene, CC1065, diphtheria A chain, Pseudomonas
aeruginosa exotoxin A chain, ricin A chain, abrin A chain, modeccin
A chain, alpha-sarcin, an Aleuritesfordii protein, a dianthin
protein, a Phytolaca americana protein, .[.momordica.].
.Iadd.Momordica .Iaddend.charantia inhibitor, curcin, crotin,
.[.sapaonaria.]. .Iadd.Sapaonaria .Iaddend.officinalis inhibitor,
gelonin, mitogellin, restrictocin, phenomycin, enomycin, a
tricothecene, a ribonuclease, and a deoxyribonuclease.
29. The method of claim 28, wherein said growth-inhibiting
chemotherapeutic compound is gemcitabine, adriamycin or
paclitaxel.
.Iadd.30. The method of claim 1, wherein said antibody is an
antibody produced by the hybridoma 1A8 (deposited under ATCC
Accession No. ATCC PTA-3647) or a humanized version
thereof..Iaddend.
.Iadd.31. The method of claim 27, wherein said antibody is hu3G9
(BG00011)..Iaddend.
.Iadd.32. The method of claim 27, wherein said antibody is
hu8G6..Iaddend.
.Iadd.33. The method of claim 27, wherein said antibody is an
antibody produced by the hybridoma 3G9 (deposited under ATCC
Accession No. ATCC PTA-3649) or a humanized version
thereof..Iaddend.
.Iadd.34. The method of claim 27, wherein said antibody is an
antibody produced by the hybridoma 8G6 (deposited under ATCC
Accession No. ATCC PTA-3645) or a humanized version
thereof..Iaddend.
.Iadd.35. The method of claim 27, wherein said antibody is an
antibody produced by the hybridoma 1A8 (deposited under ATCC
Accession No. ATCC PTA-3647) or a humanized version
thereof..Iaddend.
.Iadd.36. The method of claim 1, wherein the antibody comprises a
heavy chain and a light chain, wherein the heavy and light chains
comprise complementarity determining regions (CDRs) 1, 2, and 3 as
follows: heavy chain CDR 1 comprising an amino acid sequence set
forth in SEQ ID NO.: 3; heavy chain CDR 2 comprising an amino acid
sequence set forth in SEQ ID NO.: 9; heavy chain CDR 3 comprising
an amino acid sequence set forth in SEQ ID NO.: 15; light chain CDR
1 comprising an amino acid sequence set forth in SEQ ID NO.: 20;
light chain CDR 2 comprising an amino acid sequence set forth in
SEQ ID NO.: 25; and light chain CDR 3 comprising an amino acid
sequence set forth in SEQ ID NO.: 31..Iaddend.
.Iadd.37. The method of claim 1, wherein the antibody comprises a
heavy chain and a light chain, wherein the heavy and light chains
comprise CDRs 1, 2, and 3 as follows: heavy chain CDR 1 comprising
an amino acid sequence set forth in SEQ ID NO.: 1; heavy chain CDR
2 comprising an amino acid sequence set forth in SEQ ID NO.: 6;
heavy chain CDR 3 comprising an amino acid sequence set forth in
SEQ ID NO.: 12; light chain CDR 1 comprising an amino acid sequence
set forth in SEQ ID NO.: 18; light chain CDR 2 comprising an amino
acid sequence set forth in SEQ ID NO.: 24; and light chain CDR 3
comprising an amino acid sequence set forth in SEQ ID NO.:
28..Iaddend.
.Iadd.38. The method of claim 1, wherein the antibody comprises a
heavy chain and a light chain, wherein the heavy and light chains
comprise CDRs 1, 2, and 3 as follows: heavy chain CDR 1 comprising
an amino acid sequence set forth in SEQ ID NO.: 2; heavy chain CDR
2 comprising an amino acid sequence set forth in SEQ ID NO.: 7;
heavy chain CDR 3 comprising an amino acid sequence set forth in
SEQ ID NO.: 13; light chain CDR 1 comprising an amino acid sequence
set forth in SEQ ID NO.: 19; light chain CDR 2 comprising an amino
acid sequence set forth in SEQ ID NO.: 24; and light chain CDR 3
comprising an amino acid sequence set forth in SEQ ID NO.:
29..Iaddend.
.Iadd.39. The method of claim 27, wherein the antibody comprises a
heavy chain and a light chain, wherein the heavy and light chains
comprise CDRs 1, 2, and 3 as follows: heavy chain CDR 1 comprising
an amino acid sequence set forth in SEQ ID NO.: 3; heavy chain CDR
2 comprising an amino acid sequence set forth in SEQ ID NO.: 9;
heavy chain CDR 3 comprising an amino acid sequence set forth in
SEQ ID NO.: 15; light chain CDR 1 comprising an amino acid sequence
set forth in SEQ ID NO.: 20; light chain CDR 2 comprising an amino
acid sequence set forth in SEQ ID NO.: 25; and light chain CDR 3
comprising an amino acid sequence set forth in SEQ ID NO.:
31..Iaddend.
.Iadd.40. The method of claim 27, wherein the antibody comprises a
heavy chain and a light chain, wherein the heavy and light chains
comprise CDRs 1, 2, and 3 as follows: heavy chain CDR 1 comprising
an amino acid sequence set forth in SEQ ID NO.: 1; heavy chain CDR
2 comprising an amino acid sequence set forth in SEQ ID NO.: 6;
heavy chain CDR 3 comprising an amino acid sequence set forth in
SEQ ID NO.: 12; light chain CDR 1 comprising an amino acid sequence
set forth in SEQ ID NO.: 18; light chain CDR 2 comprising an amino
acid sequence set forth in SEQ ID NO.: 24; and light chain CDR 3
comprising an amino acid sequence set forth in SEQ ID NO.:
28..Iaddend.
.Iadd.41. The method of claim 27, wherein the antibody comprises a
heavy chain and a light chain, wherein the heavy and light chains
comprise CDRs 1, 2, and 3 as follows: heavy chain CDR 1 comprising
an amino acid sequence set forth in SEQ ID NO.: 2; heavy chain CDR
2 comprising an amino acid sequence set forth in SEQ ID NO.: 7;
heavy chain CDR 3 comprising an amino acid sequence set forth in
SEQ ID NO.: 13; light chain CDR 1 comprising an amino acid sequence
set forth in SEQ ID NO.: 19; light chain CDR 2 comprising an amino
acid sequence set forth in SEQ ID NO.: 24; and light chain CDR 3
comprising an amino acid sequence set forth in SEQ ID NO.:
29..Iaddend.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is in the fields of cell biology, immunology
and oncology. The invention relates to the discovery that there is
a relationship between the expression levels of the tumor
suppressor gene smad4 (also known as dpc4) and integrin
.alpha..sub..nu..beta..sub.6, and the responsiveness of patient
populations to .alpha..sub..nu..beta..sub.6-active compounds and
compositions (e.g., antibodies and other ligands that bind
.alpha..sub..nu..beta.6), particularly in cancer cells from such
patient populations, more particularly on carcinomas such as
pancreatic carcinomas. The invention thus provides methods for
determining the responsiveness of tumor cells (particularly those
from pancreatic tumors) to such .alpha..sub..nu..beta..sub.6-active
compounds and compositions by examining the expression of
.alpha..sub..nu..beta..sub.6 and smad4 by the tumor cells, as well
as methods of diagnosis and treatment/prevention of tumor
progression using ligands, including antibodies and small molecule
drugs, that bind to integrin .alpha..sub..nu..beta..sub.6 on the
surfaces of tumor cells and/or that block one or more components of
the TGF-.beta. pathway, particularly in smad4-deficient tumor
cells.
2. Related Art
Integrins are cell surface glycoprotein receptors which bind
extracellular matrix proteins and mediate cell-cell and
cell-extracellular matrix interactions (generally referred to as
cell adhesion events) (Ruoslahti, E., J Clin. Invest. 87:1-5
(1991); Hynes, R. O., Cell 69:11-25 (1992)). These receptors are
composed of noncovalently associated alpha (.alpha.) and beta
(.beta.) chains which combine to give a variety of heterodimeric
proteins with distinct cellular and adhesive specificities (Albeda,
S. M., Lab. Invest. 68:4-14 (1993)). Recent studies have implicated
certain integrins in the regulation of a variety of cellular
processes including cellular adhesion, migration, invasion,
differentiation, proliferation, apoptosis and gene expression
(Albedo, S. M., Lab. Invest. 68:4-14 (1993); Juliano, R., Cancer
Met. Rev. 13:25-30 (1994); Ruoslahti, E. and Reed, J. C., Cell
77:477-478 (1994); and Ruoslahti, E. and Giancotti, F. G., Cancer
Cells 1:119-126 (1989); Plow, Haas et al. 2000; van der Flier and
Sonnenberg 2001).
The .alpha..sub..nu..beta..sub.6 receptor is one member of a family
of integrins that are expressed as cell surface heterodimeric
proteins (Busk, M. et al., J. Biol. Chem. 267(9):5790-5796 (1992)).
While the .alpha..sub..nu. subunit can form a heterodimer with a
variety of .beta. subunits (.beta..sub.1, .beta..sub.3,
.beta..sub.5, .beta..sub.6 and .beta..sub.8), the .beta..sub.6
subunit can only be expressed as a heterodimer with the
.alpha..sub..nu. subunit. The .alpha..sub..nu..beta..sub.6 integrin
is known to be a fibronectin-, latency associated peptide (LAP)-
and tenascin C-binding cell surface receptor, interacting with the
extracellular matrix through the RGD tripeptide binding sites
thereon (Busk, M. et al., J. Biol. Chem. 267:5790-5796 (1992);
Weinacker, A. et al., J. Biol. Chem. 269:6940-6948 (1994); Prieto,
A. L. et al., Proc. Natl. Acad. Sci. USA 90:10154-10158 (1993)).
Although the .alpha..sub..nu..beta..sub.6 integrin was first
identified and sequenced more than 10 years ago, the biological
significance of .alpha..sub..nu..beta..sub.6, especially in
disease, is still under investigation. The expression of
.alpha..sub..nu..beta..sub.6 is restricted to epithelial cells
where it is expressed at relatively low levels in healthy tissue
and significantly upregulated during development, injury, and wound
healing (Breuss, J. M. et al., J. Histochem. Cytochem. 41:1521-1527
(1993); Breuss, J. M. et al., J Cell Sci. 108:2241-2251 (1995);
Koivisto, L. et al., Cell Adhes. Communic. 7:245-257 (1999);
Zambruno, G. et al., J. Cell Biol. 129(3):853-865 (1995); Hakkinen,
L. et al., J. Histochem. Cytochem. 48(6):985-998 (2000)). An
increasing number of recent reports demonstrate that
.alpha..sub..nu..beta..sub.6 is upregulated on cancers of
epithelial origin, including colon carcinoma (Niu, J. et al, Int.
J. Cancer 92:40-48 (2001); Bates, R. C. et al., J. Clin. Invest.
115:339-347 (2005)), ovarian cancer (Ahmed, N. et al., J. Cell.
Biochem. 84:675-686 (2002); Ahmed, N. et al., J. Histochem.
Cytochem. 50:1371-1379 (2002); Ahmed, N. et al., Carcinogen.
23:237-244 (2002)), squamous cell carcinoma (Koivisto, L. et al.,
Exp. Cell Res. 255:10-17 (2000); Xue, H. et al., Biochem. Biophys.
Res. Comm. 288:610-618 (2001); Thomas, G. J. et al., J. Invest.
Derinatol. 117:67-73 (2001); Thomas, G. J. et al., Int. J. Cancer
92:641-650 (2001); Ramos, D. M. et al., Matrix Biol. 21:297-307
(2002); (Agrez, M. et al., Br. J. Cancer 81:90-97 (1999); Hamidi,
S. et al., Br. J. Cancer 82(8):1433-1440 (2000); Kawashima, A. et
al., Pathol. Res. Pract. 99(2):57-64 (2003)), and breast cancer
(Arihiro, K. et al., Breast Cancer 7:19-26 (2000)). It has also
been reported that the cc subunit may be involved in tumor
metastasis, and that blocking this subunit consequently may prevent
metastasis (for review, see Imhof, B. A. et al., in: "Attempts to
Understand Metastasis Formation I," U. Gunthert and W. Birchmeier,
eds., Berlin: Springer-Verlag, pp. 195-203 (1996)).
The .alpha..sub..nu..beta..sub.6 integrin may have multiple
regulatory functions in tumor cell biology. Recent studies have
demonstrated that the extracellular and cytoplasmic domains of the
.beta..sub.6 subunit mediate different cellular activities. The
extracellular and transmembrane domains have been shown to mediate
TGF-.beta. activation and adhesion (Sheppard, D., Cancer and
Metastasis Rev. 24:395-402 (2005); Munger, J. S. et al., Cell
96:319-328 (1999)). The cytoplasmic domain of the .beta..sub.6
subunit contains a unique 11-amino acid sequence that is important
in mediating .alpha..sub..nu..beta..sub.6 regulated cell
proliferation, MMP production, migration, and pro-survival (Li, X.
et al., J. Biol. Chem. 278(43):41646-41653 (2003); Thomas, G. J. et
al., J Invest. Derm. 117(1):67-73 (2001); Thomas, G. J. et al., Br.
J. Cancer 87(8):859-867 (2002); Janes, S. M. and Watt, F. M., J.
Cell Biol 166(3):419-431 (2004)). The .beta..sub.6 subunit has been
cloned, expressed and purified (Sheppard et al., U.S. Pat. No.
6,787,322 B2, the disclosure of which is incorporated herein by
reference in its entirety), and function-blocking antibodies that
selectively bind to the .alpha..sub..nu..beta..sub.6 integrin have
been reported (Weinreb et al., J. Biol. Chem. 279:17875-17877
(2004), the disclosure of which is incorporated herein by reference
in its entirety). Antagonists of (.alpha..sub..nu..beta..sub.6
(including certain monoclonal antibodies) have also been suggested
as possible treatments for certain forms of acute lung injury and
fibrosis (see U.S. Pat. No. 6,692,741 B2 and WO 99/07405, the
disclosures of which are incorporated herein by reference in their
entireties).
.alpha..sub..nu..beta..sub.6 can bind to several ligands including
fibronectin, tenascin, and the latency associated peptide-1 and -3
(LAP1 and LAP3), the N-terminal 278 amino acids of the latent
precursor form of TGF-.beta.1 through a direct interaction with an
arginine-glycine-aspartate ("RGD") motif (Busk. M. et al., J. Biol.
Chew. 267(9):5790-5796 (1992); Yokosaki, Y. et al., J. Biol. Chem.
271(39):24144-24150 (1996); Huang, X. Z. et al., J. Cell. Sci.
111:2189-2195 (1998); Munger, J. S. et al., Cell 96:319-328
(1999)). The TGF-.beta. cytokine is synthesized as a latent complex
which has the N-terminal LAP non-covalently associated with the
mature active C-terminal TGF-.beta. cytokine. The latent TGF-.beta.
complex cannot bind to its cognate receptor and thus is not
biologically active until converted to an active form
(Barcellos-Hoff, M. H., J. Mamm. Gland Biol. 1(4):353-363 (1996);
Gleizes, P. E. et al., Stem Cells 15(3):190-197 (1997); Munger, J.
S. et al., Kid. Int 51:1376-1382 (1997); Khalil, N., Microbes
Infect. 1(15): 1255-1263 (1999)). .alpha..sub..nu..beta..sub.6
binding to LAP1 or LAP3 leads to activation of the latent precursor
form of TGF-.beta.1 and TGF-.beta.3 (Munger, J. S. et al., Cell
96:319-328 (1999)), proposed as a result of a conformational change
in the latent complex allowing TGF-.beta. to bind to its receptor.
Thus, upregulated expression of .alpha..sub..nu..beta..sub.6 can
lead to local activation of TGF-.beta. which in turn can activate a
cascade of downstream events.
The TGF-.beta.1 cytokine is a pleiotropic growth factor that
regulates cell proliferation, differentiation, and immune responses
(Wahl, S. M., J. Exp. Med. 180:1587-1590 (1994); Massague, J.,
Annu. Rev. Biochem. 67:753-791 (1998); Chen, W. and Wahl, S. M.,
TGF-.beta.: Receptors, Signaling Pathways and Autoimmunity, Basel:
Karger, pp. 62-91 (2002); Thomas, D. A. and Massague, J., Cancer
Cell 8:369-380 (2005)). The role that TGF-.beta.1 plays in cancer
is two-sided. TGF-.beta. is recognized to tumor suppressor and
growth inhibitory activity yet, many tumors evolve a resistance to
growth suppressive activities of TGF-.beta.1 (Yingling, J. M. et
al., Nature Rev. Drug Discov. 3(12):1011-1022 (2004); Akhurst, R.
J. et al., Trends Cell Biol. 11(11):S44-S51 (2001); Balmain, A. and
Akhurst, R. J., Nature 428(6980):271-272 (2004)). In established
tumors, TGF-.beta.1 expression and activity has been implicated in
promoting tumor survival, progression, and metastases (Akhurst, R.
J. et al., Trends Cell Biol. 11(11): S44-S51 (2001); Muraoka, R. S.
et al., J. Clin. Invest. 109 (12):1551 (2002); Yang, Y. A. et al.,
J. Clin. Invest. 109(12): 1607-1615 (2002)). This is postulated to
be mediated by both autocrine and paracrine effects in the local
tumor-stromal environment including the effects of TGF-.beta. on
immune surveillance, angiogenesis, and increased tumor interstitial
pressure. Several studies have now shown the anti-tumor and
anti-metastatic effects of inhibiting TGF-.beta.1 (Akhurst, R. J.,
J. Clin. Invest. 109(12):1533-1536 (2002); Muraoka, R. S. et al.,
J. Clin. Invest. 109(12):1551 (2002); Yingling, J. M. et al., Nat.
Rev. Drug Discov. 3(12):1011-1022 (2004); Yang, Y. A. et al., J.
Clin. Invest. 109(12):1607-1615 (2002); Halder, S. K. et al.,
Neoplasia 7(5):509-521 (2005); Tyer, S. et al., Cancer Biol. Ther.
4(3):261-266 (2005)).
Increased expression of .alpha..sub..nu..beta..sub.6 on tumors,
particularly at the tumor-stromal interface, may reflect a unique
mechanism for local activation of TGF-.beta.1 and the ability to
promote tumor survival, invasion, and metastasis. The high level of
expression in human metastases infers a potential role for
.alpha..sub..nu..beta..sub.6 in establishing metastases which is
consistent with previous reports that .alpha..sub..nu..beta..sub.6
can mediate epithelial to mesenchymal transition, tumor cell
invasion in vitro, and expression correlated with metastases in a
mouse model (Bates, R. C. et al., J. Clin. Invest. 115(2):339-347
(2005); Thomas, G. J. et al., Br. J. Cancer 87(8):859-867 (2002);
Morgan, M. R. et al., J. Biol. Chem. 279(25):26533-26539 (2004)).
We have previously described the generation of potent and selective
anti-.alpha..sub..nu..beta..sub.6 monoclonal antibodies (mAbs) that
bind to both the human and murine forms of
.alpha..sub..nu..beta..sub.6 and block the binding of
.alpha..sub..nu..beta..sub.6 to its ligands and
.alpha..sub..nu..beta..sub.6 mediated activation of TGF-.beta.1
(Weinreb, P. H. et al., J. Biol. Chem. 279(17):17875-17887
(2004)).
The generation of potent and selective
anti-.alpha..sub..nu..beta..sub.6 monoclonal antibodies (mAbs) that
bind to both the human and murine forms of
.alpha..sub..nu..beta..sub.6 and block the binding of
.alpha..sub..nu..beta..sub.6 to its ligands and
.alpha..sub..nu..beta..sub.6 mediated activation of TGF-.beta.1 has
been previously described (Weinreb, P. H. et al., J. Biol. Chem.
279(17):17875-17887 (2004); see also U.S. patent application Ser.
No. 11/483,190 by Violette et al., entitled
"Anti-.alpha..sub..nu..beta..sub.6 Antibodies and Uses Thereof,"
filed on Jul. 10, 2006, which is incorporated herein by reference
in its entirety). As also described in PCT Publication WO
03/100033, herein incorporated in its entirety by reference, high
affinity antibodies against .alpha..sub..nu..beta..sub.6, including
the identification and analysis of key amino acid residues in the
complementary determining regions (CDRs) of such antibodies, were
discovered and characterized. In particular, these high affinity
antibodies (a) specifically bind to .alpha..sub..nu..beta..sub.6;
(b) inhibit the binding of .alpha..sub..nu..beta..sub.6 to its
ligand such as LAP, fibronectin, vitronectin, and tenascin with an
IC.sub.50 value lower than that of 10D5 (International Patent
Application Publication WO 99/07405); (c) block activation of
TGF-.beta.; (d) contain certain amino acid sequences in the CDRs
that provide binding specificity to .alpha..sub..nu..beta..sub.6;
(e) specifically bind to the .beta..sub.6 subunit; and/or (f)
recognize .alpha..sub..nu..beta..sub.6 in immunostaining
procedures, such as immunostaining of paraffin-embedded
tissues.
WO 03/100033 also describes the discovery that antibodies that bind
to .alpha..sub..nu..beta..sub.6 can be grouped into biophysically
distinct classes and subclasses. One class of antibodies exhibits
the ability to block binding of a ligand (e.g., LAP) to
.alpha..sub..nu..beta..sub.6 (blockers). This class of antibodies
can be further divided into subclasses of cation-dependent blockers
and cation-independent blockers. Some of the cation-dependent
blockers contain an arginine-glycine-aspartate (RGD) peptide
sequence, whereas the cation-independent blockers do not contain an
RGD sequence. Another class of antibodies exhibits the ability to
bind to .alpha..sub..nu..beta..sub.6 and yet does not block binding
of .alpha..sub..nu..beta..sub.6 to a ligand (nonblockers).
Furthermore, WO 03/100033 discloses antibodies comprising heavy
chains and light chains whose complementarity determining regions
(CDR) 1, 2 and 3 consist of certain amino acid sequences that
provide binding specificity to .alpha..sub..nu..beta..sub.6. WO
03/100033 also provides for antibodies that specifically bind to
.alpha..sub..nu..beta..sub.6 but do not inhibit the binding of
.alpha..sub..nu..beta..sub.6 to latency associated peptide (LAP) as
well as antibodies that bind to the same epitope.
WO 03/100033 further discloses cells of hybridomas 6.1A8, 6.2B10,
6.3G9, 6.8G6, 6.2B1, 6.2A1, 6.2E5, 7.1G10, 7.7G5, and 7.1C5,
isolated nucleic acids comprising a coding sequences and isolated
polypeptides comprising amino acid sequences of the
anti-.alpha..sub..nu..beta..sub.6 antibodies. In particular, WO
03/100033 discloses anti-.alpha..sub..nu..beta..sub.6 antibodies
comprising heavy and light chain polypeptide sequences as
antibodies produced by hybridomas 6.1A8, 6.3G9, 6.8G6, 6.2B1,
6.2B10, 6.2A1, 6.2E5, 7.1G10, 7.7G5, or 7.1C5. Several of the
hybridomas were deposited at the American Type Culture Collection
("ATCC"; P.O. Box 1549, Manassas, Va. 20108, USA) under the
Budapest Treaty. In particular, hybridoma clones 6.3G9 and 6.8G6
were deposited on Aug. 16, 2001, and have accession numbers MCC
PTA-3649 and PTA-3645, respectively. The murine antibodies produced
by hybridomas 6.3G9 and 6.8G6 are being further explored in the
present application for their potential development as humanized
antibodies.
The murine monoclonal antibody 3G9 is a murine IgG1, kappa antibody
isolated from the .beta..sub.6 integrin -/- mouse (Huang et al., J.
Cell Biol. 133:921-928 (1996)) immunized with human soluble
.alpha..sub..nu..beta..sub.6. The 3G9 antibody specifically
recognizes the .alpha..sub..nu..beta..sub.6 integrin epitope which
is expressed at upregulated levels during injury, fibrosis and
cancer (see, e.g., Thomas et al., J. Invest. Dermatology 117:67-73
(2001); Brunton et al., Neoplasia 3: 215-226 (2001); Agrez et al.,
Int. J. Cancer 81:90-97 (1999); Breuss, J. Cell Science
108:2241-2251 (1995)). It does not bind to other .alpha..sub..nu.
integrins and is cross-reactive to both human and murine molecules.
The murine monoclonal antibody 3G9 has been described to block the
binding of .alpha..sub..nu..beta..sub.6 to LAP as determined by
blocking of ligand binding either to purified human soluble
.alpha..sub..nu..beta..sub.6 or to .beta..sub.6-expressing cells,
thereby inhibiting the pro-fibrotic activity of TGF-.beta. receptor
activation (see WO 03/100033). It has also been shown to inhibit
.alpha..sub..nu..beta..sub.6-mediated activation of TGF-.beta. with
an IC.sub.50 value lower than one of the known
.alpha..sub..nu..beta..sub.6 antibodies, 10D5 (Huang et al., J.
Cell Sci. 111:2189-2195 (1998)).
The murine monoclonal antibody 8G6 is a murine IgG1, kappa antibody
which also recognizes the .alpha..sub..nu..beta..sub.6 integrin
epitope, as described in WO 03/100033. The murine monoclonal
antibody 8G6 is a cation-dependent, high affinity blocker of
.alpha..sub..nu..beta..sub.6 displaying the ability to inhibit
.alpha..sub..nu..beta..sub.6--mediated activation of TGF-.beta.
with an IC.sub.50 value lower than 10D5 (see WO 03/100033).
Both the 3G9 and 8G6 murine antibodies were effective in preventing
fibrosis of the kidney and lung, as described in WO 03/100033.
Furthermore, the murine antibody 3G9 was able to effectively
inhibit tumor growth in a human tumor xenograft model, suggesting
the potential role of .alpha..sub..nu..beta..sub.6 in cancer
pathology and the effectiveness of such blockade using antibodies
directed at .alpha..sub..nu..beta..sub.6.
Smad4 is a component of the Smad pathway that is involved in signal
transduction in the TGF-.beta. pathway (Levy, L. and Hill, C. S.,
Molec. Cell. Biol. 25:8108-8125 (2005); Fukuchi, M. et al., Cancer
95:737-743 (2002)). This gene, also known as dpc4 (for "decreased
in pancreatic carcinoma"), appears to be a tumor suppressor gene,
and a decrease in smad4 expression has been observed in a variety
of primary carcinomas, including pancreatic carcinomas (Luttges, J.
et al., Am. J. Pathol. 158:1677-1683 (2001); Subramanian, G. et
al., Cancer Res. 64:5200-5211 (2004)), esophageal carcinomas
(Fukuchi, M. et al., Cancer 95:737-743 (2002), cervical carcinomas
(Maliekal, T. T. et al., Oncogene 22:4889-4897 (2003), and other
primary human cancers (Iacobuzio-Donahue, C. A. et al., Clin. Canc.
Res. 10:1597-1604 (2004), as well as in cell line cancer models
including of pancreatic cancers (Lohr, M. et al., Cancer Res.
61:550-555 (2001); Yasutome, M. et al., Clin. Exp. Metastasis
22:461-473 (2005)), and of colon cancers (Levy, L., and Hill, C.
S., Molec. Cell. Biol. 25:8108-8125 (2005)). A reduced expression
of smad4 in tumors has been associated with poor prognosis for
patient survival, particularly in patients with smad4-deficient
pancreatic adenocarcinomas (Liu, F., Clin. Cancer Res. 7:3853-3856
(2001); Tascilar, M. et al., Clin. Cancer Res. 7:4115-4121 (2001);
Toga, T. et al., Anticancer Res. 24:1173-1178 (2004)). The
mechanism of the tumor suppressive activity of the smad4 gene
product is poorly understood, but it is thought that it may act as
a "switch" regulating the growth-suppressive and growth-activating
activities of certain components of the TGF-.beta. signaling
pathway (for reviews, see Akhurst, A. J., J. Clin. Invest.
109:1533-1536 (2002); Bachman, K. E., and Park, B. H., Curr. Opin.
Oncol. 17:49-54 (2004); Bierie, B., and Moses, H. L., Nature Rev.
Cancer 6:506-520 (2006)).
BRIEF SUMMARY OF THE INVENTION
The present invention relates to methods of cancer diagnosis,
treatment and prevention using .alpha..sub..nu..beta..sub.6-binding
ligands, such as .alpha..sub..nu..beta..sub.6-binding antibodies.
In particular, the invention relates to the discovery of a
correlation between reduced expression of smad4 and increased
expression of integrin .alpha..sub..nu..beta..sub.6 in tumor cells,
and the propensity of tumor cells with such expression patterns to
be more likely to respond to .alpha..sub..nu..beta..sub.6-active
compounds and to TGF-.beta.-inhibitory ligands.
Thus, in one embodiment, the invention provides methods of
characterizing a tumor, e.g., identifying a tumor that is more
likely to progress to a metastatic or invasive tumor, or
identifying a tumor for treatment with a preselected agent, e.g.,
an anti-.alpha..sub..nu..beta..sub.6 agent described herein,
comprising: (a) obtaining from a patient a cancerous tissue sample
comprising a tumor or a portion thereof, and optionally a
noncancerous tissue sample; (b) determining the level of expression
of smad4 in cells from said cancerous tissue sample and optionally
from said noncancerous tissue sample; (c) contacting cells from
said tissue sample or samples with one or more ligands that bind to
one or more subunits of integrin .alpha..sub..nu..beta..sub.6; and
(d) determining the level of expression of integrin
.alpha..sub..nu..beta..sub.6 in said cells from said cancerous
tissue sample, and optionally from said cells from said
noncancerous tissue sample, and comparing the level of expression
in said cancerous tissue sample with a reference sample, e.g., the
level of expression in said noncancerous tissue sample, thereby
characterizing the tumor. A level of expression in the cancerous
sample which meets or exceeds a reference value indicates the
presence of a tumor more likely to progress to a metastatic or
invasive tumor. For example, a decrease in smad4 expression and a
concomitant increase in the level of expression of integrin
.alpha..sub..nu..beta..sub.6 in cells from said cancerous tissue
sample, relative to the levels of expression of smad4 and integrin
.alpha..sub..nu..beta..sub.6 in said non-cancerous tissue sample,
indicates the presence in said patient of a tumor that is more
likely to progress to a metastatic or invasive tumor. In certain
such embodiments of the invention, the tumor cell is a carcinoma,
such as an adenocarcinoma. In more particular embodiments, the
carcinoma is a pancreatic carcinoma, a colorectal carcinoma, a
cervical carcinoma, a squamous cell carcinoma (such as an
esophageal carcinoma), a head and neck carcinoma, a liver
carcinoma, an ovarian carcinoma and a lung carcinoma. More
particularly, the carcinoma is a pancreatic carcinoma, a colorectal
carcinoma, a cervical carcinoma, or a head and neck carcinoma.
Suitable embodiments according to this aspect of the invention use
.alpha..sub..nu..beta..sub.6 integrin-binding ligands which are
.alpha..sub..nu..beta..sub.6-binding antibodies or
.alpha..sub..nu..beta..sub.6 epitope-binding fragments thereof.
According to certain such embodiments, the antibodies are
monoclonal antibodies (which may be chimeric, primatized, human or
humanized), including those disclosed in U.S. patent application
publication no. U.S. 2005/0255102 A1, the disclosure of which is
incorporated herein in its entirety. Suitable such antibodies
include, but are not limited to, the
.alpha..sub..nu..beta..sub.6-binding monoclonal antibodies
designated 1A8, 3G9, 8G6, 2B1, 2B10, 2A1, 2E5, 1G10, 7G5, 1C5, 10D5
(ATCC deposit no. HB12382) and CS.beta.6, as well as fragments,
chimeras and hybrids thereof. Particularly suitable for use in such
embodiments of the invention are monoclonal antibodies 3G9 and 8G6.
Also particularly suitable for use in such embodiments of the
invention are humanized monoclonal antibodies, such as the
humanized 3G9 antibody designated hu3G9 (BG00011) and the humanized
8G6 antibody designated hu8G6. In certain such aspects of the
invention, the ligand is conjugated with at least one detectable
label, such as a chromogenic label (e.g., diaminobenzidine or
4-hydroxyazo-benzene-2-carboxylic acid), an enzyme label (e.g.,
malate dehydrogenase, staphylococcal nuclease, delta-5-steroid
isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate
dehydrogenase, those phosphate isomerase, peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, .beta.-galactosidase,
ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,
glucoamylase and acetylcholine esterase), a radioisotopic label
(e.g., .sup.3H, .sup.111In, .sup.125I, .sup.131I, .sup.32P,
.sup.35S, .sup.14C, .sup.51Cr, .sup.57To, .sup.58Co, .sup.59Fe,
.sup.75Se, .sup.152Eu, .sup.90Y, .sup.67Cu, .sup.217Ci, .sup.211At,
.sup.212Pb, .sup.47Sc and .sup.109Pd), a non-radioactive isotopic
label (e.g., .sup.157Gd, .sup.55Mn, .sup.162Dy, .sup.52Tr,
.sup.56Fe, .sup.99mTc and .sup.112In), a fluorescent label (e.g., a
.sup.152Eu label, a fluorescein label, an isothiocyanate label, a
rhodamine label, a phycoerythrin label, a phycocyanin label, an
allophycocyanin label, a Green Fluorescent Protein (GFP) label, an
o-phthaldehyde label and a fluorescamine label), a toxic label
(e.g., a diphtheria toxin label, a ricin label and a cholera toxin
label), a chemiluminescent label (e.g., a luminol label, an
isoluminol label, an aromatic acridinium ester label, an imidazole
label, an acridinium salt label, an oxalate ester label, a
luciferin label, a luciferase label and an aequorin label), an
X-radiographic label (e.g., barium or cesium), a spin label (e.g.,
deuterium) or a nuclear magnetic resonance contrast agent label
(e.g., Gd, Mn and iron).
In related embodiments, the invention provides therapeutic methods
for eliminating tumors from patients, e.g, wherein the tumors have
an increased potential to become metastatic or invasive. Such
methods comprise, e.g., characterizing a tissue sample as described
herein, for example: (a) identifying, in a tissue sample from a
patient, a tumor that is more likely to progress to a metastatic or
invasive tumor, according to the methods for identifying such
tumors that are described above; and (b) administering to a
patient, in which such a tumor has been identified, a
therapeutically effective amount of one or more ligands that binds
to one or more subunits of integrin .alpha..sub..nu..beta..sub.6 on
one or more .alpha..sub..nu..beta..sub.6-positive metastatic tumor
cells, wherein the binding of the ligand to the integrin results in
the death, chemosensitization or decreased invasiveness of said
metastatic tumor cell. In a preferred embodiment, the same agent,
e.g., the same antibody is used to characterize the tumor and treat
the patient. In other embodiments, different agents or antibodies
are used. For example, characterizing can be performed with an
antibody having a first label, and treating can be performed with
an antibody having a second label, e.g., a therapeutic agent.
In related embodiments, the invention provides methods of reducing
or preventing the progression of a pre-metastatic tumor to a
metastatic tumor in a patient, comprising (a) characterizing a
tissue sample as described herein, e.g., identifying, in a tissue
sample from a patient, a tumor that is more likely to progress to a
metastatic or invasive tumor, according to the methods for
identifying such tumors that are described above; and (b)
administering to the patient a therapeutically effective amount of
one or more ligands that binds to one or more subunits of integrin
.alpha..sub..nu..beta..sub.6 on one or more cells in the
pre-metastatic or pre-invasive tumor, wherein the binding of the
ligand to the integrin results in the reduction or prevention of
invasion of cells of the pre-metastatic cancer into tissue areas
surrounding the primary tumor. In therapeutic embodiments according
to this aspect of the invention, the
.alpha..sub..nu..beta..sub.6-binding ligands (e.g.,
.alpha..sub..nu..beta..sub.6-binding antibodies) can be conjugated
with or bound to one or more cytotoxic compounds or agents which
lead to or cause the death of the cell or tissue upon binding of
the .alpha..sub..nu..beta..sub.6-binding ligand-toxic compound
conjugate to one or more .alpha..sub..nu..beta..sub.6 integrins on
the cell or tissue. In some embodiments, the antibody used to
characterize the tumor will lack the cytoxic compound or agent. In
additional therapeutic embodiments of the invention, the
.alpha..sub..nu..beta..sub.6-binding ligands (e.g.,
.alpha..sub..nu..beta..sub.6-binding antibodies) are administered
to a patient in conjunction with one or more such cytotoxic
compounds or agents. Cytotoxic compounds or agents which can be
suitably used according to these aspects of the invention include,
but are not limited to, cytotoxic agents (e.g., cisplatin,
carboplatin, oxaliplatin, paclitaxel, melphalan, doxorubicin,
methotrexate, 5-fluorouracil, etoposide, mechlorethamine,
cyclophosphamide, bleomycin, a calicheamicin, a maytansine, a
trichothene, CC1065, diphtheria A chain, Pseudomonas aeruginosa
exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain,
alpha-sarcin, Aleurites fordii proteins, dianthin proteins,
Phytolaca americana proteins, momordica charantia inhibitors,
curcin, crotin, sapaonaria officinalis inhibitors, gelonin,
mitogellin, restrictocin, adriamycin (also known as, and used
interchangeably herein with, doxorubicin), gemcitabine, phenomycin,
enomycin, tricothecenes, ribonucleases and deoxyribonucleases),
radioisotopes (such as .sup.211At, .sup.131I, .sup.125I, .sup.90Y,
.sup.186Re, .sup.188Re, .sup.153Sm, .sup.212Bi, .sup.32P, and
radioactive isotopes of Lu) and prodrug-activating enzymes (such as
alkaline phosphatase, arylsulfatase, cytosine deaminase, proteases,
D-alanylcarboxy-peptidases, carbohydrate-cleaving enzymes,
P-lactamase and penicillin amidase. Cytotoxic agents can also be
agents which recruit particular cells or generally increase the
immune response to a tumor. In certain embodiments, the one or more
.alpha..sub..nu..beta..sub.6 integrin-binding ligands are
administered to the patient in the form of a pharmaceutical
composition comprising an effective amount of one or more of the
.alpha..sub..nu..beta..sub.6 integrin-binding ligands and one or
more pharmaceutically acceptable carriers or excipients. The one or
more .alpha..sub..nu..beta..sub.6 integrin-binding ligands and/or
one or more pharmaceutical compositions comprising the one or more
.alpha..sub..nu..beta..sub.6 integrin-binding ligands can be
administered to the patient by any suitable mode of administering
pharmaceutical compositions, including but not limited to oral
administration, parenteral administration (including, for example,
injection via an intramuscular, intravenous, intraarterial,
intraperitoneal, or subcutaneous route), intracranial
administration, transdermal administration, intrapulmonary
administration and intranasal administration.
In additional embodiments, the present invention provides methods
of diagnosing or identifying a tumor, such as a carcinoma (e.g., an
adenocarcinoma), that is more likely to progress to an invasive
carcinoma, and/or that is more likely to respond to treatment with
a ligand that binds to one or more subunits of integrin
.alpha..sub..nu..beta..sub.6. Suitable such methods may comprise,
for example, (a) obtaining from a patient a cancerous epithelial
tissue sample comprising a tumor or a portion thereof, and a
noncancerous epithelial tissue sample; (b) determining the level of
expression of smad4 in cells from said tissue samples; (c)
contacting the tissue samples with one or more ligands that binds
to one or more subunits of integrin .alpha..sub..nu..beta..sub.6;
and (d) determining the level of expression of integrin
.alpha..sub..nu..beta..sub.6 in the tissue samples, wherein a
decrease in the level of expression of smad4 and a concomitant
increase in the level expression of integrin
.alpha..sub..nu..beta..sub.6 in the cancerous tissue sample,
relative to the levels of expression of smad4 and integrin
.alpha..sub..nu..beta..sub.6 in the noncancerous tissue sample,
indicates the presence in the patient of a tumor that: (a) has an
increased likelihood of progressing from an in situ or noninvasive
form, to an invasive, metastatic form; and/or (b) is more likely to
respond to treatment with one or more of the above-referenced
treatment methods that relies upon the binding of an
.alpha..sub..nu..beta..sub.6-binding ligand, particularly an
.alpha..sub..nu..beta..sub.6-binding ligand that is conjugated to
or that is administered in conjunction with one or more cytotoxic
compounds or agents such as those described above. Such methods are
suitable for diagnosing or identifying a variety of tumors,
including but not limited to those involving the epithelial tissues
noted above. In certain such embodiments, the ligand that binds to
one or more subunits of integrin .alpha..sub..nu..beta..sub.6 is an
.alpha..sub..nu..beta..sub.6 integrin-binding antibody (which may
be a monoclonal antibody such as those described above) or an
.alpha..sub..nu..beta..sub.6 epitope-binding fragment thereof.
Particularly suitable for use in such diagnostic methods of the
invention are .alpha..sub..nu..beta..sub.6-binding ligands (e.g.,
antibodies) that are detectably labeled, i.e., that comprise, are
conjugated to, or are bound with at least one detectable label such
as a chromogenic label (e.g., diaminobenzidine or
4-hydroxyazobenzene-2-carboxylic acid), an enzyme label (e.g.,
malate dehydrogenase, staphylococcal nuclease, delta-5-steroid
isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate
dehydrogenase, triose phosphate isomerase, peroxidase, alkaline
phosphatase, asparaginase, glucose oxidase, .beta.-galactosidase,
ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,
glucoamylase or acetylcholine esterase), a radioisotopic label g,
.sup.3H, .sup.11In, .sup.125I, .sup.131I, .sup.32P, .sup.35S,
.sup.14C, .sup.51Cr, .sup.57To, .sup.58Co, .sup.59Fe, .sup.75Se,
.sup.152Eu, .sup.90Y, .sup.67Cu, .sup.217Ci, .sup.211At,
.sup.212Pb, .sup.47Sc or .sup.109Pd), a non-radioactive isotopic
label (e.g., .sup.157Gd, .sup.55Mn, .sup.162Dy, .sup.52Tr,
.sup.56Fe, .sup.99mTc or .sup.112In), a fluorescent label (e.g., a
.sup.152Eu label, a fluorescein label, an isothiocyanate label, a
rhodamine label, a phycoerythrin label, a phycocyanin label, an
allophycocyanin label, a Green Fluorescent Protein (GFP) label, an
o-phthaldehyde label or a fluorescamine label), a toxic label
(e.g., a diphtheria toxin label, a ricin label or a cholera toxin
label), a chemiluminescent label (e.g., a luminol label, an
isoluminol label, an aromatic acridinium ester label, an imidazole
label, an acridinium salt label, an oxalate ester label, a
luciferin label, a luciferase label or an aequorin label), an
X-radiographic label (e.g., barium or cesium), a spin label (e.g.,
deuterium) and a nuclear magnetic resonance contrast agent label
(e.g., Gd, Mn and iron).
In one aspect, the invention features a method of inhibiting growth
of a cell from a tumor that is smad4 deficient. The method
includes, for example, determining the level of expression of smad4
in a cell from the tumor, and treating a tumor cell that is
deficient in smad4 expression with one or more agents that cause
growth inhibition or death of the tumor cell. The one or more
agents can be, for example, a ligand that binds to one or more
subunits of integrin .alpha..sub..nu..beta..sub.6, such as an
anti-.alpha..sub..nu..beta..sub.6 antibody, or an
.alpha..sub..nu..beta..sub.6 epitope-binding fragment thereof. In
another embodiment, the one or more agents inhibits the TGF-.beta.
signaling pathway in the cell. Such agents include, e.g., protein
kinase molecules, small molecule therapeutic compounds, or soluble
TGF-.beta. receptor polypeptides.
In another aspect, the invention features a method of
chemosensitizing a smad-4-deficient tumor cell to treatment with a
growth-inhibiting chemotherapeutic compound. The method includes,
for example, determining the level of expression of smad4 in a cell
from the tumor, and treating a tumor cell deficient for smad4
expression with one or more agents, such that the treatment results
in increased responsiveness to one or more growth-inhibiting
chemotherapeutic compounds. The one or more agents can be, for
example, ligands that bind to one or more subunits of integrin
.alpha..sub..nu..beta..sub.6, or agents that inhibit the TGF-.beta.
signaling pathway.
Other preferred embodiments of the present invention will be
apparent to one of ordinary skill in light of the following
drawings and description of the invention, and of the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a composite of photomicrographs depicting the levels of
.alpha..sub..nu..beta..sub.6 expression (dark areas) in certain
human carcinomas that have metastasized to either lymph node (FIGS.
1A-1D) or lung (FIGS. 1E-1F), from the indicated primary tumor
sites.
FIG. 2 is a composite of photomicrographs depicting the levels of
.alpha..sub..nu..beta..sub.6 expression (dark areas) in certain
human carcinomas that have metastasized from the indicated primary
tumor site to the indicated metastatic tumor site.
FIG. 3 is a composite of two photomicrographs depicting the levels
of .alpha.v.beta..sub.6 expression (dark areas) observed in tissue
sections of ductal carcinoma in situ (DCIS; BrCa 19) (FIG. 3A) and
invasive breast carcinoma (BrCa 23) (FIG. 3B).
FIG. 4 is a composite of photomicrographs depicting the levels of
.alpha.v.beta.6 expression (dark areas) observed in matched samples
of primary and metastic pancreatic ductal adenocarcinoma tumors
from three different patients. FIGS. 4A-4C: expression in primary
tumor samples from three different patients.
FIGS. 4D-4F: expression in matched lymph node metastases from these
same three patients. FIGS. 4G-4H:: expression in normal pancreatic
tissue obtained from two of the three patients.
FIG. 5 is a composite of photomicrographs depicting the levels of
.alpha.v.beta.6 expression (dark areas) observed in matched samples
of primary and metastatic pancreatic adenocarcinoma tumors from
five different patients. FIGS. 5A-5E: expression in primary tumor
samples from five different patients, three with tumors
characterized as adenosquamous (FIGS. 5A-5C), and two with tumors
characterized as poorly differentiated (FIGS. 5D-5E). FIGS. 5F-5J:
expression in matched lymph node metastases from these same five
patients. FIGS. 5K-5L: expression in normal pancreatic tissue
obtained from two of the five patients.
FIG. 6 demonstrates the ability of an anti-.alpha..nu..beta.6
monoclonal antibody (3G9) to inhibit tumor growth in the BxPC-3
mouse xenograft model of human pancreatic cancer. FIG. 6A:
photomicrograph of a section of xenograft tumor stained via
immunohistochemistry with an anti-.alpha.v.beta.6 monoclonal
antibody (3G9). FIG. 6B: BxPC-3 xenograft tumor growth curves
during treatment with .alpha.v.beta.6 mAb 3G9 (.tangle-solidup.),
soluble TGFbRII-Fc-Ig fusion protein (), or vehicle PBS
(.box-solid.). FIG. 6C: scatter plot of individual tumor sizes at
the end of the study (day 66).
FIG. 7 is a series of photomicrographs of pancreatic tissue/tumor
sections from three different patient samples, probed for
expression of integrin .alpha.v.beta.6 (top three panels) or of
smad4 (bottom three panels).
FIG. 8 is a series of photomicrographs of pancreatic tissue/tumor
sections from three different patient samples (and different from
those in FIG. 7), probed for expression of integrin .alpha.v.beta.6
(top three panels) or of smad4 (bottom three panels).
FIG. 9 is a series of photomicrographs of pancreatic tissue/tumor
sections from three different patient samples (and different from
those in FIGS. 7 and 8), probed for expression of integrin
.alpha.v.beta.6 (top three panels) or of smad4 (bottom three
panels).
FIG. 10 is a series of photomicrographs of pancreatic tissue/tumor
sections from two different patient samples (and different from
those in FIGS. 7-9), probed for expression of integrin
.alpha.v.beta.6 (top two panels) or of smad4 (bottom two
panels).
FIG. 11 is a series of photomicrographs of pancreatic tissue/tumor
sections from two different patient samples (and different from
those in FIGS. 7-10), which are heterogeneous ("+/-") with respect
to expression of integrin .alpha.v.beta.6, probed for expression of
integrin .alpha.v.beta.6 (top two panels) or of smad4 (bottom two
panels).
FIG. 12 is a series of photomicrographs of pancreatic tissue/tumor
sections from a single patient sample (different from those in FIG.
11), which are heterogeneous ("+/-") with respect to expression of
integrin .alpha.v.beta.6, probed for expression of integrin
.alpha.v.beta.6 or of smad4.
FIG. 13 is a Table summarizing the results depicted in FIGS.
7-12.
FIG. 14 is a summary of .alpha.v.beta.6 and smad4 expression levels
in a panel of pancreatic cancer cell lines.
FIG. 15 is a composite of photomicrographs depicting the levels of
.alpha.v.beta.6 and smad4 expression in pancreatic cancers as
assayed by Folio array.
FIG. 16 is a schematic diagram summarizing .alpha.v.beta.6 and
smad4 expression levels in 23 different pancreatic tumor tissue
samples.
FIG. 17 is a schematic diagram summarizing .alpha.v.beta.6 and
smad4 expression levels in 70 different pancreatic ductal
adenocarcinoma (PDAC) samples assayed on Biomax tissue
microarrays.
FIG. 18 is a schematic diagram summarizing .alpha.v.beta.6 and
smad4 expression levels in 50 different PDAC samples assayed on
Leiden arrays.
FIG. 19 is a schematic diagram summarizing .alpha.v.beta.6 and
smad4 expression levels in 9 different PDAC metastases samples
assayed on Leiden arrays.
FIGS. 20A and B are bar graphs summarizing the results of
.alpha.v.beta.6 and smad4 expression levels in PDACs as measured by
Biomax array and Leiden array, respectively.
FIG. 21 is a line graph demonstrating the response of BxPC 3 human
pancreatic adenocarcinoma cells, subcutaneously implanted into
athymic nude mice, to mAb 3G9 and gemcitabine, either as single
agents or in combination.
FIG. 22 is a line graph demonstrating the response of BxPC 3 human
pancreatic adenocarcinoma cells, subcutaneously implanted into
athymic nude mice, to mAb 3G9 and gemcitabine, either as single
agents or in combination, expressing the results of test animals as
a percentage of control (vehicle) results.
FIG. 23 is a scatter plot demonstrating the response of BxPC 3
human pancreatic adenocarcinoma cells, subcutaneously implanted
into athymic nude mice, to mAb 3G9 and gemcitabine, either as
single agents or in combination, at day 45 of the treatment
regimen.
FIG. 24 is a line graph demonstrating change in host animal body
weight in response to treatment with mAb 3G9 or gemcitabine.
FIG. 25 is a line graph demonstrating the response of BxPC 3 human
pancreatic adenocarcinoma cells, subcutaneously implanted into
athymic nude mice, to mAb 3G9 and doxorubicin, either as single
agents or in combination.
FIG. 26 is a line graph demonstrating the response of BxPC 3 human
pancreatic adenocarcinoma cells, subcutaneously implanted into
athymic nude mice, to mAb 3G9 and doxorubicin, either as single
agents or in combination, expressing the results of test animals as
a percentage of control (vehicle) results.
FIG. 27 is a scatter plot demonstrating the response of BxPC 3
human pancreatic adenocarcinoma cells, subcutaneously implanted
into athymic nude mice, to mAb 3G9 and doxorubicin, either as
single agents or in combination, at day 45 of the treatment
regimen.
FIG. 28 is a line graph demonstrating change in host animal body
weight in response to treatment with mAb 3G9 or doxorubicin.
FIG. 29A is a line graph demonstrating the response of Su86.86
human pancreatic adenocarcinoma cells, subcutaneously implanted
into athymic nude mice, to mAb 3G9 and gemcitabine, either as
single agents or in combination, and to soluble TGF-.beta. receptor
II/Fc fusion protein (TGF-.beta. RII-Fc). FIG. 29B is a line graph
demonstrating the response of Su86.86 human pancreatic
adenocarcinoma cells, subcutaneously implanted into athymic nude
mice, to mAb 3G9 and gemcitabine, either as single agents or in
combination, and to TGF-.beta. RII-Fc expressing the results of
test animals as a percentage of control (vehicle) results.
FIG. 30A is a line graph demonstrating the response of Panc04 human
pancreatic adenocarcinoma cells, subcutaneously implanted into
athymic nude mice, to mAb 3G9 and TGF-.beta. RII-Fc, and to either
agent in combination with gemcitabine. FIG. 30B is a line graph
demonstrating the response of Panc04 human pancreatic
adenocarcinoma cells, subcutaneously implanted into athymic nude
mice, to mAb 3G9 and TGF-.beta. RII-Fc, and to either agent in
combination with gemcitabine. FIG. 30B is a line graph
demonstrating the response of Panc04 human pancreatic
adenocarcinoma cells, subcutaneously implanted into athymic nude
mice, to mAb 3G9 and TGF-.beta. RII-Fc, and to either agent in
combination with gemcitabine, expressing the results of test
animals as a percentage of control (vehicle) results.
FIG. 31A is a line graph demonstrating the response of Capan-2
human pancreatic adenocarcinoma cells, subcutaneously implanted
into athymic nude mice, to mAb 3G9, TGF-.beta. RII-Fc, and
gemcitabine, and to each of mAb 3G9 and TGF-.beta. RII-Fc in
combination with gemcitabine. FIG. 31B is a line graph
demonstrating the response of Capan-2 human pancreatic
adenocarcinoma cells, subcutaneously implanted into athymic nude
mice, to mAb 3G9, TGF-.beta. RII-Fc, and gemcitabine, and to each
of mAb 3G9 and TGF-.beta. RII-Fc in combination with gemcitabine,
expressing the results of test animals as a percentage of control
(vehicle) results.
FIGS. 32A-32C are line graphs demonstrating the response of SW1990
human pancreatic adenocarcinoma cells, subcutaneously implanted
into athymic nude mice, to mAb 3G9 and gemcitabine, each alone and
in combination, expressing the results of test animals as a
percentage of control (vehicle) results.
FIG. 33A is a line graph demonstrating the response of Capan-1
human pancreatic adenocarcinoma cells, subcutaneously implanted
into athymic nude mice, to mAb 3G9, TGF-.beta. RII-Fc, and
gemcitabine, and to each of mAb 3G9 and TGF-.beta. RII-Fc in
combination with gemcitabine. FIG. 33B is a line graph
demonstrating the response of Capan-1 human pancreatic
adenocarcinoma cells, subcutaneously implanted into athymic nude
mice, to mAb 3G9, TGF-.beta. RIT-Fc, and gemcitabine, and to each
of mAb 3G9 and TGF-.beta. RII-Fc in combination with gemcitabine,
expressing the results of test animals as a percentage of control
(vehicle) results.
FIG. 34 is a bar graph demonstrating the response of ASPC-1 human
pancreatic adenocarcinoma cells orthotopically implanted into
athymic nude mice to mAb 3G9, TGF-.beta. RII-Fc, and gemcitabine,
and to each of mAb 3G9 and TGF-.beta. RII -Fc in combination with
gemcitabine.
FIG. 35 is a Table summarizing the results depicted in FIGS.
29-34.
FIGS. 36A-36D are Kaplan-Meier curves representing the cumulative
survival of 26 patients from Leiden University Medical Center with
pancreatic ductal adenocarcinoma in function of retention of avb6
or deficiencies of avb6 expression, or retention of smad4 or
deficiencies of smad4 expression, and combinations of these
phenotypes. Only data from primary tumors was analyzed.
FIG. 37 is a Kaplan-Meier curve representing the cumulative
survival of patients with pancreatic ductal adenocarcinoma in
function of retention of smad4 or deficiencies of smad4 expression.
Only data from primary tumors was analyzed. Log Rank
(Mantel-Cox).
FIG. 38 is a line graph demonstrating the response of SW1990 human
pancreatic adenocarcinoma cells, subcutaneously implanted into
athymic nude mice (metastatic site: spleen), to mAb 3G9 and soluble
TGF-.beta. receptor II/Fc fusion protein (TGF-.beta.RII-Fc).
FIG. 39 is a line graph demonstrating the response of SW1990 human
pancreatic adenocarcinoma cells, subcutaneously implanted into
athymic nude mice (metastatic site: spleen), to mAb 3G9 and soluble
TGF-.beta.RII-Fc, expressing the results of test animals as a
percentage of control (vehicle) results.
FIG. 40 is a scatter plot demonstrating the response of SW1990
human pancreatic adenocarcinoma cells, subcutaneously implanted
into athymic nude mice (metastatic site: spleen), to mAb 3G9 and
soluble TGF-.beta.RII-Fc, at day 42 of the treatment regimen.
FIG. 41 is a composite figure, showing: A: an autoradiograph of a
Western blot showing smad4 expression in SW1990 cell subpopulations
sorted based on .alpha.v.beta.6 expression; B: immunohistochemistry
of .alpha.v.beta.6 expression in implanted SW1990 tumors; and C-E:
FACS-mediated cell sorting of SW1990 cell populations by smad4 and
.alpha.v.beta.6 expression.
FIG. 42 is a line graph demonstrating the response of Detroit 562
human pharyngeal carcinoma cells, subcutaneously implanted into
athymic nude mice, to murine mAb 3G9 and soluble TGF-.beta.
receptor II/Fc fusion protein (TGF-.beta.RII-Fc).
FIG. 43 is a scatter plot demonstrating the response of Detroit 562
human pharyngeal carcinoma cells, subcutaneously implanted into
athymic nude mice, to murine mAb 3G9, mAb 4B4, and soluble
TGF-.beta. receptor II/Fc fusion protein (TGF-.beta.RII-Fc), at day
39 of the treatment regimen.
FIG. 44 is a line graph demonstrating the response of Detroit 562
human pharyngeal carcinoma cells, subcutaneously implanted into
athymic nude mice, to murine mAb 3G9, mAb 4B4, and soluble
TGF-.beta. receptor II/Fc fusion protein (TGF-.beta.RII-Fc).
FIG. 45 is a line graph demonstrating the response of Detroit 562
human pharyngeal carcinoma cells, subcutaneously implanted into
athymic nude mice, to murine mAb 3G9, mAb 4B4, mAb 8G6, and soluble
TGF-.beta. receptor II/Fc fusion protein (TGF-.beta.RII-Fc).
FIG. 46 is a scatter plot demonstrating the response of Detroit 562
human pharyngeal carcinoma cells, subcutaneously implanted into
athymic nude mice, to murine mAb 3G9, mAb 4B4, mAb 8G6, and soluble
TGF-.beta. receptor II/Fc fusion protein (TGF-.beta.RII-Fc), at day
34 of the treatment regimen.
FIG. 47 is a pair of line graphs demonstrating the response of
Detroit 562 human pharyngeal carcinoma cells, subcutaneously
implanted into athymic nude mice, to murine mAb 3G9 (top) and to
humanized mAb 3G9 (BG00011) (bottom).
FIG. 48 is a scatter plot demonstrating the response of Detroit 562
human pharyngeal carcinoma cells, subcutaneously implanted into
athymic nude mice, to murine mAb 3G9 (top) and to humanized mAb 3G9
(BG00011) (bottom).
FIG. 49 is a line graph demonstrating the response of Detroit 562
human pharyngeal carcinoma cells, subcutaneously implanted into
athymic nude mice, to murine mAb 3G9 and to humanized mAb 3G9
(BG00011), expressing the results of test animals as a percentage
of control (vehicle as negative; TGF-.beta. RII-Fc as positive)
results.
FIG. 50 is a line graph demonstrating the response of Detroit 562
human pharyngeal carcinoma cells, subcutaneously implanted into
athymic nude mice, to wildtype murine mAb 3G9.
FIG. 51 is a line graph demonstrating the response of Detroit 562
human pharyngeal carcinoma cells, subcutaneously implanted into
athymic nude mice, to aglycosyl murine mAb 3G9.
FIG. 52 is a scatter plot demonstrating the response of Detroit 562
human pharyngeal carcinoma cells, subcutaneously implanted into
athymic nude mice, to wildtype murine mAb 3G9 and to algycosyl
murine mAb 3G9.
FIG. 53 is a line graph demonstrating the response of Detroit 562
human pharyngeal carcinoma cells, subcutaneously implanted into
athymic nude mice, to wildtype murine mAb 3G9 and to algycosyl
murine mAb 3G9, expressing the results of test animals as a
percentage of control (vehicle) results.
FIG. 54 is a Table summarizing the results of FIGS. 43-52.
DETAILED DESCRIPTION OF THE INVENTION
Unless defined otherwise, all technical and scientific terms used
herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described
hereinafter.
Definitions
About: As used herein when referring to any numerical value, the
term "about" means a value of .+-.10% of the stated value (e.g.,
"about 50.degree. C." encompasses a range of temperatures from
45.degree. C. to 55.degree. C., inclusive; similarly, "about 100
mM" encompasses a range of concentrations from 90 mM to 110 mM,
inclusive).
Antagonist: As used herein, the term "antagonist" refers to a
compound, molecule, moiety or complex that reduces, substantially
reduces or completely inhibits the biological and/or physiological
effects of the .alpha..sub..nu..beta..sub.6 integrin in a cell,
tissue or organism. Antagonists, which may be ligands for
.alpha..sub..nu..beta..sub.6, may carry out such effects in a
variety of ways, including but not limited to competing with
another ligand for binding to .alpha..sub..nu..beta..sub.6 on the
cell surface; interacting with .alpha..sub..nu..beta..sub.6 in such
a way as to reduce, substantially reduce or inhibit the ability of
the integrin to bind other ligands; binding to and inducing a
conformational change in cell surface .alpha..sub..nu..beta..sub.6
such that the integrin assumes a structure to which other ligands
can no longer bind (or can bind only with reduced or substantially
reduced affinity and/or efficiency); inducing a physiological
change (e.g., increase in intracellular signaling complexes;
increase in transcriptional inhibitors; reduction in cell surface
.alpha..sub..nu..beta..sub.6 expression; etc.) in cells, tissues or
organisms such that the binding of other ligands, or the
physiological signal induced by such ligands upon binding to the
.alpha..sub..nu..beta..sub.6 on the cell, is reduced, substantially
reduced or completely inhibited; and other mechanisms by which
antagonists may carry out their activities, that will be familiar
to the ordinarily skilled artisan. As the ordinarily skilled
artisan will understand, an antagonist may have a similar structure
to another .alpha..sub..nu..beta..sub.6-binding moiety (e.g., an
.alpha..sub..nu..beta..sub.6-binding ligand) that it antagonizes
(e.g., the antagonist may be a mutein, variant, fragment or
derivative of the agonist), or may have a wholly unrelated
structure.
Bound: As used herein, the term "bound" refers to binding or
attachment that may be covalent, e.g., by chemically coupling, or
non-covalent, e.g., ionic interactions, hydrophobic interactions,
hydrogen bonds, etc. Covalent bonds can be, for example, ester,
ether, phosphoester, thioester, thioether, urethane, amide, amine,
peptide, imide, hydrazone, hydrazide, carbon-sulfur bonds,
carbon-phosphorus bonds, and the like. The term "bound" is broader
than and includes terms such as "coupled," "conjugated" and
"attached."
Conjugate/conjugation: As used herein, "conjugate" refers to the
product of covalent attachment of a moiety, e.g., a chemical or
radioisotope, to a ligand that binds to
.alpha..sub..nu..beta..sub.6, e.g., an
.alpha..sub..nu..beta..sub.6-binding antibody or fragment thereof.
"Conjugation" refers to the formation of a conjugate as defined in
the previous sentence. Any method normally used by those skilled in
the art of conjugation of chemicals or radioisotopes to
biologically active materials, such as proteins or polypeptides
(including antibodies) can be used in the present invention.
Disease, disorder, condition: As used herein, the terms "disease"
or "disorder" refer to any adverse condition of a human or animal
including tumors, cancer, allergies, addiction, autoimmunity,
infection, poisoning or impairment of optimal mental or bodily
function. "Conditions" as used herein includes diseases and
disorders but also refers to physiologic states. For example,
fertility is a physiologic state but not a disease or disorder.
Compositions of the invention suitable for preventing pregnancy by
decreasing fertility would therefore be described as a treatment of
a condition (fertility), but not a treatment of a disorder or
disease. Other conditions are understood by those of ordinary skill
in the art.
Effective Amount: As used herein, the term "effective amount"
refers to an amount of a given compound, conjugate or composition
that is necessary or sufficient to realize a desired biologic
effect. An effective amount of a given compound, conjugate or
composition in accordance with the methods of the present invention
would be the amount that achieves this selected result, and such an
amount can be determined as a matter of routine by a person skilled
in the art, using assays that are known in the art and/or that are
described herein, without the need for undue experimentation. For
example, an effective amount for treating or preventing cancer
metastasis could be that amount necessary to prevent migration and
invasion of a tumor cell across the basement membrane or across an
endothelial layer in vivo. The term is also synonymous with
"sufficient amount." The effective amount for any particular
application can vary depending on such factors as the disease,
disorder or condition being treated, the particular composition
being administered, the route of administration, the size of the
subject, and/or the severity of the disease or condition. One of
ordinary skill in the art can determine empirically the effective
amount of a particular compound, conjugate or composition of the
present invention, in accordance with the guidance provided herein,
without necessitating undue experimentation.
One, a, or an: When the terms "one," "a," or "an" are used in this
disclosure, they mean "at least one" or "one or more," unless
otherwise indicated. An such, the terms "a" (or "an"), "one or
more," and "at least one" can be used interchangeably herein.
Peptide, polypeptide, protein: As used herein, the term
"polypeptide" is intended to encompass a singular "polypeptide" as
well as plural "polypeptides," and refers to a molecule composed of
monomers (amino acids) linearly linked by amide bonds (also known
as peptide bonds). The term "polypeptide" refers to any chain or
chains of two or more amino acids, and does not refer to a specific
length of the product. Thus, peptides, dipeptides, tripeptides,
oligopeptides, "protein," "amino acid chain," or any other term
used to refer to a chain or chains of two or more amino acids, are
included within the definition of "polypeptide," and the term
"polypeptide" may be used instead of, or interchangeably with any
of these terms. The term "polypeptide" is also intended to refer to
the products of post-expression modifications of the polypeptide,
including without limitation glycosylation, acetylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, or modification
by non-naturally occurring amino acids. A polypeptide may be
derived from a natural biological source or produced by recombinant
technology, but is not necessarily translated from a designated
nucleic acid sequence. It may be generated in any manner, including
by chemical synthesis. In accordance with this definition,
polypeptides used in the present invention may be of a size of
about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50
or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000
or more, or 2,000 or more amino acids. Polypeptides may have a
defined three-dimensional structure, although they do not
necessarily have such structure. Polypeptides with a defined
three-dimensional structure are referred to as folded, and
polypeptides which do not possess a defined three-dimensional
structure, but rather can adopt a large number of different
conformations, and are referred to at unfolded. As used herein, the
term glycoprotein refers to a protein coupled to at least one
carbohydrate moiety that is attached to the protein via an
oxygen-containing or a nitrogen-containing side chain of an amino
acid residue, e.g., a serine residue or an asparagine residue.
Preferred polypeptides used in accordance with the invention
include polypeptides that are ligands or that bind to an
.alpha..sub..nu..beta..sub.6 integrin on the surface of a cell,
including but not limited to antibodies (especially monoclonal
antibodies) that recognize and bind to one or more epitopes on
.alpha..sub..nu..beta..sub.6.
By an "isolated" polypeptide or a fragment, variant, or derivative
thereof is intended a polypeptide that is not in its natural
milieu. No particular level of purification is required. For
example, an isolated polypeptide can be removed from its native or
natural environment. Recombinantly produced polypeptides and
proteins expressed in host cells are considered isolated for
purposed of the invention, as are native or recombinant
polypeptides which have been separated, fractionated, or partially
or substantially purified by any suitable technique.
Also included as polypeptides of the present invention are
fragments, derivatives, analogs, or variants of the foregoing
polypeptides, and any combination thereof. The terms "fragment,"
"variant," "derivative" and "analog" when referring to
anti-.alpha..sub..nu..beta..sub.6 antibodies or antibody
polypeptides include any polypeptides which retain at least some of
the antigen-binding properties of the corresponding native antibody
or polypeptide, i.e., those polypeptides that retain the ability to
bind to one or more epitopes on an .alpha..sub..nu..beta..sub.6
integrin. Fragments of polypeptides of the present invention
include proteolytic fragments, as well as deletion fragments, in
addition to specific antibody fragments discussed elsewhere herein.
Variants of anti-.alpha..sub..nu..beta..sub.6 antibodies and
antibody polypeptides useful in accordance with the present
invention include fragments as described above, and also
polypeptides with altered amino acid sequences due to amino acid
substitutions, deletions, or insertions. Variants may occur
naturally or be non-naturally occurring Non-naturally occurring
variants may be produced using art-known mutagenesis techniques.
Variant polypeptides may comprise conservative or non-conservative
amino acid substitutions, deletions or additions. Derivatives of
anti-.alpha..sub..nu..beta..sub.6 antibodies and antibody
polypeptides useful in accordance with the present invention are
polypeptides which have been altered so as to exhibit additional
features not found on the native polypeptide. Examples include
fusion proteins. Variant polypeptides may also be referred to
herein as "polypeptide analogs." As used herein a "derivative" of
an anti-.alpha..sub..nu..beta..sub.6 antibody or antibody
polypeptide refers to a subject polypeptide having one or more
residues chemically derivatized by reaction of a functional side
group. Also included as "derivatives" are those peptides which
contain one or more naturally occurring amino acid derivatives of
the twenty standard amino acids. For example, 4-hydroxyproline may
be substituted for proline; 5-hydroxylysine may be substituted for
lysine; 3-methylhistidine may be substituted for histidine;
homoserine may be substituted for serine; and ornithine may be
substituted for lysine.
smad4: As used herein, the tumor suppressor gene smad4 is
synonymous with other designations for the same tumor suppressor
gene that are known to those of skill in the art, including but not
limited to madh4 and dpc4. As is conventional, the products of the
expression of this gene is designated herein as SMAD4, which is
synonymous with the corresponding other designations for the
expression product of this gene that are known to those of skill in
the art, including but not limited to MADH4 and DPC4.
Substantially, substantial: As used herein, conjugation of a
protein is said not to interfere "substantially" with the ability
of the protein to bind to its receptor(s) if the rate and/or amount
of binding of a conjugated protein to a receptor is not less than
about 40%, about 50%, about 60%, about 65%, about 70%, about 75%,
about 80%, about 85%, about 90%, about 91%, about 92%, about 93%,
about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or
about 100% or more, of the binding rate and/or amount of the
corresponding cytokine, chemokine, growth factor or polypeptide
hormone that has not been conjugated.
Treatment: As used herein, the terms "treatment," "treat,"
"treated" or "treating" refer to prophylaxis and/or therapy,
particularly wherein the object is to prevent or slow down (lessen)
an undesired physiological change or disorder, such as the
progression of multiple sclerosis. Beneficial or desired clinical
results include, but are not limited to, alleviation of symptoms,
diminishment of extent of disease, stabilized (i.e., not worsening)
state of disease, delay or slowing of disease progression,
amelioration or palliation of the disease state, and remission
(whether partial or total), whether detectable or undetectable.
"Treatment" can also mean prolonging survival as compared to
expected survival if not receiving treatment. Those in need of
treatment include those already with the condition or disorder as
well as those prone to have the condition or disorder or those in
which the condition or disorder is to be prevented. By "subject" or
"individual" or "animal" or "patient" or "mammal," is meant any
subject, particularly a mammalian subject, for whom diagnosis,
prognosis, or therapy is desired. Mammalian subjects include humans
and other primates, domestic animals, farm animals, and zoo,
sports, or pet animals such as dogs, cats, guinea pigs, rabbits,
rats, mice, horses, cattle, cows, and the like.
Overview
The present invention is based at least in part upon the findings
that there is an inverse relationship between the level of
expression of the tumor suppressor gene smad4 and the integrin
.alpha..sub..nu..beta..sub.6 in certain tumor cells, and that the
levels of expression of these two markers can be used to determine
or predict the susceptibility to treatment with anti-integrin
ligands, and with agents that antagonize the TGF-.beta. signalling
pathway, of such tumor cells. Specifically, it has been found that
tumor cells that display decreased levels of expression of smad4
concomitantly express increased amounts of the integrin
.alpha..sub..nu..beta..sub.6; therefore, smad-4-deficient tumor
cells have a higher propensity to be responsive to ligands that
bind to integrin .alpha..sub..nu..beta..sub.6, compared to tumor
cells that display higher expression levels of smad4 (and,
concomitantly, lower levels of surface integrin
.alpha..sub..nu..beta..sub.6). In related embodiments, it has also
been found by the present inventors that smad-4-deficient tumor
cells are also more likely to be responsive to ligands that
antagonize one or more components of the TGF-.beta. signalling
pathway, compared to tumor cells that display higher expression
levels of smad4. As used herein, a tumor cell that is "responsive"
to a ligand that binds to integrin .alpha..sub..nu..beta..sub.6, or
to a ligand that antagonizes one or more components of the
TGF-.beta. pathway, refers to a tumor cell in which the growth,
division, and/or other metabolic pathways are adversely affected
such that the cell is inhibited from growing or dividing and/or
undergoes apoptosis or other forms of cell death.
In other embodiments, the invention also provides methods using
identification of this differential expression in determining the
invasive and/or metastatic potential of tumor cells and in
identifying those carcinomas, such as certain adeno-carcinomas
(including pancreatic carcinomas), that may be more likely to
rapidly progress and which should be aggressively treated in a
patient. The invention also provides methods of identifying those
tumors in which the cells making up the tumor may be more likely to
respond to treatment with one or more ligands that bind to integrin
.alpha..sub..nu..beta..sub.6. The invention also provides methods
of diagnosis and treatment/prevention of tumor metastasis.
Determination of Expression of smad4 and Integrin
.alpha..sub..nu..beta..sub.6
In one embodiment, the present invention is directed to methods for
identifying tumors and tumor cells, particularly carcinoma cells
such as pancreatic carcinoma cells, that are more likely to be
responsive to ligands that bind to integrin .alpha.v.beta.6, and/or
to ligands that antagonize one or more components of the TGF-.beta.
signaling pathway. Such methods include, for example, determining
the levels of expression of the tumor suppressor gene smad4 by the
tumor cells, wherein a reduced expression of smad4 indicates that
the tumor cell is more likely to be responsive to ligands that bind
to integrin .alpha.v.beta.6, and/or to ligands that antagonize one
or more components of the TGF-.beta. signalling pathway. In certain
such embodiments, the level of expression of smad4 in cells of a
tumor, e.g., a carcinoma, is determined in tissue sections obtained
from a patient suffering from such a tumor, wherein a decrease in
the expression of smad4 in the tumor cells relative to that in
non-tumor tissue samples (ideally, from the same organ in the same
patient) indicates that the tumor is more likely to be responsive
to ligands that bind to integrin .alpha.v.beta.6, and/or to ligands
that antagonize one or more components of the TGF-.beta. signalling
pathway. In this way, appropriate and aggressive protocols for
treating such tumors in a patient can rapidly be identified and
implemented, thereby providing an increased likelihood of positive
treatment outcomes for cancer patients.
In each such embodiment, the invention relies upon identification
or exploitation of the decreased expression of smad4, and the
concomitant increased expression of .alpha..sub..nu..beta..sub.6,
in certain tumor cells. The level of expression of smad4 can be
readily determined using methods that are well-known in the art for
measuring gene expression, including, for example, hybridization or
PCR/RT-PCR using known genetic probes or primers specific for the
smad4 gene (see, e.g., Maliekal, T. T. et al., Oncogene
22:4889-4897 (2003); Iacobuzio-Donahue, C. A. et al., Clin. Cancer
Res. 10:1597-1604 (2004)), northern blotting (see, e.g., Yasutome,
M. et al., Clin. Exper. Metast. 22:461-473 (2005)), and
immunohistochemical analysis with anti-SMAD4 antibodies (see, e.g.,
Luttges, J. et al., Am. J. Pathol. 158:1677-1683 (2001); Fukuchi,
M. et al., Cancer 95:737-743 (2002); Subramanian, G. et al., Cancer
Res. 64:5200-5211 (2004); Levy, L. and Hill, C. S., Mol. Cell.
Biol. 25:8108-8125 (2005); Toga, T. et al., Anticancer Res.
24:1173-1178 (2004)). Other methods suitable for detecting the
level of smad4 gene expression in a given cell, tissue, organ or
biological sample will be familiar to those of ordinary skill in
the art. Similarly, the level of expression of
.alpha..sub..nu..beta..sub.6 can be readily determined using
methods that are well-known in the art for measuring integrin
expression. In certain such embodiments, such determinations are
accomplished by contacting the tissue, tumor or tumor cells with
one or more ligands that binds to integrin
.alpha..sub..nu..beta..sub.6 in the tissue, tumor or tumor
cells.
In certain such embodiments, the tissue, tumor or tumor cells are
carcinoma tissues, tumors or tumor cells, including those from
carcinomas such as adenocarcinomas. In more particular embodiments,
the carcinoma is pancreatic carcinoma, a colorectal carcinoma, a
cervical carcinoma, a squamous cell carcinoma (such as an
esophageal carcinoma), a head and neck carcinoma, a liver
carcinoma, an ovarian carcinoma and a lung carcinoma. More
particularly, the carcinoma is a pancreatic carcinoma, a colorectal
carcinoma, a cervical carcinoma, or a head and neck carcinoma.
In certain embodiments of the invention, the ligands that bind to
.alpha..sub..nu..beta..sub.6 are antagonists of
.alpha..sub..nu..beta..sub.6. Such antagonists include but are not
limited to antibodies which specifically bind to
.alpha..sub..nu..beta..sub.6; antibodies which specifically bind to
.beta..sub.6; antibodies that bind to .alpha..sub..nu.; antibodies
that bind to ligands for .alpha..sub..nu..beta..sub.6; ligands for
.alpha..sub..nu..beta..sub.6; antisense nucleic acids; and peptide,
non-peptide, and peptidomimetic analogs of such ligands.
In certain such embodiments of the present invention, the ligand
that binds to integrin .alpha..sub..nu..beta..sub.6 is an antibody
that binds to integrin .alpha..sub..nu..beta..sub.6, or integrin
.alpha..sub..nu..beta..sub.6-binding fragments, variants, or
derivatives thereof. Such antibodies may bind to one subunit of the
integrin (e.g., antibodies that bind to an epitope located on the
.alpha..sub..nu. subunit or to an epitope that is located on the
.beta..sub.6 subunit), or to both subunits (e.g., antibodies that
bind to an epitope that is located in a region of the integrin
heterodimer that bridges both the .alpha..sub..nu. and .beta..sub.6
subunits). Unless specifically referring to full-sized antibodies
such as naturally occurring antibodies, the term
".alpha..sub..nu..beta..sub.6 antibodies" encompasses full-sized
antibodies as well as .alpha..sub..nu..beta..sub.6-binding
fragments, variants, analogs, or derivatives of such antibodies,
e.g., naturally occurring antibody or immunoglobulin molecules or
engineered antibody molecules or fragments that bind antigen in a
manner similar to antibody molecules. Antibodies can be synthetic,
monoclonal, or polyclonal and can be made by techniques well known
in the art. For therapeutic applications, "human" monoclonal
antibodies having human constant and variable regions are often
preferred so as to minimize the immune response of a patient
against the antibody. Such antibodies can be generated by
immunizing transgenic animals which contain human immunoglobulin
genes (see, e.g., Jakobovits et al., Ann. N.Y. Acad. Sci.
764:525-535 (1995)). In connection with synthetic and
semi-synthetic antibodies, such terms are intended to cover but are
not limited to antibody fragments, isotype switched antibodies,
humanized antibodies (e.g., mouse-human, human-mouse, and the
like), hybrids, antibodies having plural specificities, fully
synthetic antibody-like molecules, and the like.
The terms "antibody" and "immunoglobulin" are used interchangeably
herein. An antibody or immunoglobulin comprises at least the
variable domain of a heavy chain, and normally comprises at least
the variable domains of a heavy chain and a light chain. Basic
immunoglobulin structures in vertebrate systems are relatively well
understood. See, e.g., Harlow et al., Antibodies: A Laboratory
Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988). As
will be understood by those of ordinary skill, the terms "antibody"
and "immunoglobulin" comprise various broad classes of polypeptides
that can be distinguished biochemically. Those skilled in the art
will appreciate that heavy chains are classified as gamma, mu,
alpha, delta, or epsilon, (.gamma., .mu., .alpha., .delta.,
.epsilon.) with some subclasses among them (e.g.,
.gamma.1-.gamma.4). It is the nature of this chain that determines
the "class" of the antibody as IgG, IgM, IgA IgG, or IgE,
respectively. The immunoglobulin subclasses (isotypes) e.g.,
IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, etc. are
well characterized and are known to confer functional
specialization. Modified versions of each of these classes and
isotypes are readily discernable to the skilled artisan in view of
the instant disclosure and, accordingly, are within the scope of
the instant invention.
Antibodies that bind to .alpha..sub..nu..beta..sub.6 , or
.alpha..sub..nu..beta..sub.6-binding fragments, variants, or
derivatives thereof, that are suitable for use in the present
invention include but are not limited to polyclonal, monoclonal,
multispecific, human, humanized, primatized, or chimeric
antibodies, single chain antibodies, epitope-binding fragments,
e.g., Fab, Fab' and F(ab').sub.2, Fd, Fvs, single-chain Fvs (scFv),
single-chain antibodies, disulfide-linked Fvs (sdFv), fragments
comprising either a V.sub.L or V.sub.H domain, fragments produced
by a Fab expression library, and anti-idiotypic (anti-Id)
antibodies (including, e.g., anti-Id antibodies to
anti-.alpha..sub..nu..beta..sub.6 antibodies disclosed herein).
ScFv molecules are known in the art and are described, e.g., in
U.S. Pat. No. 5,892,019. Immunoglobulin or antibody molecules of
the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA,
and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or
subclass of immunoglobulin molecule.
Antibody fragments, including single-chain antibodies, may comprise
the variable region(s) alone or in combination with the entirety or
a portion of the following: hinge region, C.sub.H1, C.sub.H2, and
C.sub.H3 domains. Also included in the invention are
antigen-binding fragments also comprising any combination of
variable region(s) with a hinge region, C.sub.H1, C.sub.H2, and
C.sub.H3 domains. Antibodies or immunospecific fragments thereof
for use in the diagnostic and therapeutic methods disclosed herein
may be from any animal origin including birds and mammals.
Preferably, the antibodies are human, murine, rat, donkey, rabbit,
goat, guinea pig, camel, llama, horse, bovine or chicken
antibodies. Most preferably, the antibodies are human, humanized or
primatized antibodies, or chimeric antibodies, particularly
monoclonal antibodies. As used herein, "human" antibodies include
antibodies having the amino acid sequence of a human immunoglobulin
and include antibodies isolated from human immunoglobulin libraries
or from animals transgenic for one or more human immunoglobulins
and that do not express endogenous immunoglobulins, as described
infra and, for example in, U.S. Pat. No. 5,939,598 by Kucherlapati
et al. As used herein, the term "chimeric antibody" will be held to
mean any antibody wherein the immunoreactive region or site is
obtained or derived from a first species and the constant region
(which may be intact, partial or modified in accordance with the
instant invention) is obtained from a second species. In preferred
embodiments the target binding region or site will be from a
non-human source (e.g. mouse or primate) and the constant region is
human.
Particularly preferred antibodies for use in accordance with the
present invention are anti-.alpha..sub..nu..beta..sub.6 monoclonal
antibodies such as those disclosed in Weinreb et al., J. Biol.
Chem. 279(17):17875-17877 (2004) (the disclosure of which is
incorporated herein by reference in its entirety), including
monoclonal antibodies 6.8G6 ("8G6") and 6.3G9 ("3G9") disclosed
therein. Additional antibodies that bind to
.alpha..sub..nu..beta..sub.6 and that therefore are suitable for
use in accordance with the present invention include antibodies (or
fragments, variants or derivatives thereof) that bind to the
.beta..sub.6 subunit of integrin .alpha..sub..nu..beta..sub.6 (and
that are therefore considered "anti-.beta..sub.6 antibodies"), such
as those disclosed in Weinacker et al., J. Cell Biol. 269:1-9
(1994), which is incorporated herein by reference in its entirety;
and in U.S. Pat. No. 6,692,741 B2, which is incorporated herein by
reference in its entirety, particularly at columns 2-3 and 7-8
thereof, including the monoclonal antibody designated 10D5 (ATCC
deposit no. HB12382, deposited Aug. 6, 1997, American Type Culture
Collection, P.O. Box 1549, Manassas, Va. 20108) (see U.S. Pat. No.
6,692,741 at col. 3, lines 7-13, and at cols. 7-8) and CS.beta.6
(see U.S. Pat. No. 6,692,741 at cols. 7-8). Suitable embodiments
according to this aspect of the invention use
.alpha..sub..nu..beta..sub.6 integrin-binding ligands which are
.alpha..sub..nu..beta..sub.6-binding antibodies or
.alpha..sub..nu..beta..sub.6 epitope-binding fragments thereof.
Additional antibodies suitable for use in accordance with this
aspect of the invention include, but are not limited to, the
.alpha..sub..nu..beta..sub.6-binding monoclonal antibodies
disclosed in U.S. patent application publication no. US
2005/0255102 A1, the disclosure of which is incorporated herein in
its entirety, including those designated therein as 3G9, 8G6, 1A8,
2B1, 2B10, 2A1, 2E5, 1G10, 7G5, 1C5, as well as fragments, chimeras
and hybrids thereof. Particularly suitable antibodies for use in
accordance with the present invention are monoclonal antibodies
2B1, 3G9 and 8G6.
In some embodiments, the antibodies comprise the same heavy and
light chain polypeptide sequences as an antibody produced by
hybridoma 6.1A8, 6.3G9, 6.8G6, 6.2B1, 6.2B10, 6.2A1, 6.2E5, 7.1G10,
7.7G5, or 7.1C5. Particularly suitable antibodies for use in
accordance with the present invention are monoclonal antibodies
that comprise the same heavy and light chain polypeptide sequences
as 2B1 antibodies produced by hybridoma 6.2B1 (ATCC deposit no.
PTA-3646, deposited Aug. 16, 2001, American Type Culture
Collection, P.O. Box 1549, Manassas, Va. 20108), 8G6 antibodies
produced by hybridoma 6.8G6 (ATCC deposit no. PTA-3645, deposited
Aug. 16, 2001, American Type Culture Collection, P.O. Box 1549,
Manassas, Va. 20108) and 3G9 antibodies produced by hybridoma 6.3G9
(ATCC deposit no. PTA-3649, deposited Aug. 16, 2001, American Type
Culture Collection, P.O. Box 1549, Manassas, Va. 20108) (see
published U.S. Appl. No. U.S. 2005/0255102 A1, the disclosure of
which is incorporated herein by reference in its entirety,
particularly at page 1, paragraph 0008; at page 2, paragraphs 0032
and 0036; and in the Examples at pages 6-14), and the antibody
designated as 10D5 (the hybridoma secreting which antibody was
deposited on Aug. 6, 1997, as ATCC deposit no. HB12382, American
Type Culture Collection, P.O. Box 1549, Manassas, Va. 20108) (see
U.S. Pat. No. 6,692,741, the disclosure of which is incorporated
herein by reference in its entirety, particularly at col. 3, lines
7-13, and at cols. 7-8).
In some embodiments, the antibodies comprise a heavy chain whose
complementarity determining regions (CDR) 1, 2 and 3 consist
essentially (i.e., with the exception of some conservative
variations) of the sequences shown in Table 1 below. In certain
such embodiments, the antibodies comprise a heavy chain whose CDR1
consists essentially of any one of SEQ ID NOs:1-5; whose CDR2
consists essentially of any one of SEQ ID NOs: 6-11; and whose CDR3
consists essentially of any one of SEQ ID NOs:12-17; and/or a light
chain whose CDRs 1, 2 and 3 consist essentially of any one of the
sequences of SEQ ID NOs:18-23, 24-27, and 28-33, respectively.
TABLE-US-00001 TABLE 1 Antibody Amino Acid Sequence SEQ ID NO:
Heavy Chain CDR1 Sequences 8G6 SYTFTDYAMH 1 1A8 SYTFTDYTMH 2 2B1
GFTFSRYVMS 3 3G9 GFTFSRYVMS 3 2A1 GYDFNNDLIE 4 2G2 GYAFTNYLIE 5
Heavy Chain CDR2 Sequences 8G6 VISTYYGNTNYNQKFKG 6 1A8
VIDTYYGKTNYNQKFEG 7 2B1 SISSG-GSTYYPDSVKG 8 3G9 SISSG-GRMYYPDTVKG 9
2A1 VINPGSGRTNYNEKFKG 10 2G2 VISPGSGIINYNEKFKG 11 Heavy Chain CDR3
Sequences 8G6 GGLRRGDRPSLRYAMDY 12 1A8 GGFRRGDRPSLRYAMDS 13 2B1
GAIYDG-----YYVFAY 14 3G9 GSIYDG-----YYVFPY 15 2A1 IYYGPH-----SYAMDY
16 2G2 ID-YSG-----PYAVDD 17 Light Chain CDR1 Sequences 8G6
RASQSVSTSS-YSYMY 18 1A8 RASQSVSIST-YSYIH 19 2B1 SASSSVSSS----YLY 20
3G9 SANSSVSSS----YLY 21 2A1 KASLDVRTAVA 22 2G2 KASQAVNTAVA 23 Light
Chain CDR2 Sequences 8G6 YASNLES 24 1A8 YASNLES 24 2B1 STSNLAS 25
3G9 STSNLAS 25 2A1 SASYRYT 26 2G2 SASYQYT 27 Light Chain CDR3
Sequences 8G6 QHNWEIPFT 28 1A8 QHSWEIPYT 29 2B1 HQWSSYPPT 30 3G9
HQWSTYPPT 31 2A1 QQHYGTPWT 32 2G2 QHHYGVPWT 33
In other related embodiments, the monoclonal antibodies used in
accordance with the present invention are chimeric antibodies,
i.e., those in which a cognate antibody from one species (e.g.,
murine, rat or rabbit) is altered by recombinant DNA technology
such that part or all of the hinge and/or constant regions of the
heavy and/or light chains are replaced with the corresponding
components of an antibody from another species (e.g., human).
Generally, the variable domains of the engineered antibody remain
identical or substantially so to the variable domains of the
cognate antibody. Such an engineered antibody is called a chimeric
antibody and is less antigenic than the cognate antibody when
administered to an individual of the species from which the hinge
and/or constant region is derived (e.g., a human). Methods of
making chimeric antibodies are well known in the art.
In other related embodiments, the monoclonal antibodies used in
accordance with the present invention are fully human antibodies.
Methods for producing such fully human monoclonal antibodies are
well known in the art (see, e.g., U.S. 2005/0255102 A1 at page 4,
paragraphs 0069-0070, which are incorporated herein by
reference).
In other related embodiments, the monoclonal antibodies used in
accordance with the present invention are humanized versions of
cognate anti-.alpha..sub.84 .beta..sub.6 antibodies derived from
other species. A humanized antibody is an antibody produced by
recombinant DNA technology, in which some or all of the amino acids
of a human immunoglobulin light or heavy chain that are not
required for antigen binding (e.g., the constant regions and the
framework regions of the variable domains) are used to substitute
for the corresponding amino acids from the light or heavy chain of
the cognate, nonhuman antibody. By way of example, a humanized
version of a murine antibody to a given antigen has, on both of its
heavy and light chain: (a) constant regions of a human antibody,
(b) framework regions from the variable domains of a human
antibody; and (c) CDRs from the murine antibody. When necessary,
one or more residues in the human framework regions can be changed
to residues at the corresponding positions in the murine antibody
so as to preserve the binding affinity of the humanized antibody to
the antigen. This change is sometimes called "back mutation."
Humanized antibodies generally are less likely to elicit an immune
response in humans as compared to chimeric human antibodies because
the former contain considerably fewer non-human components. Methods
for producing such humanized monoclonal antibodies are well known
in the art (see, e.g., U.S. 2005/0255102 A1 at pages 4-5,
paragraphs 0072-0077, which are incorporated herein by
reference).
In additional such embodiments, the humanized antibodies comprise
one or more CDRs in the heavy and/or light chain that are derived
from the corresponding CDRs in the heavy and/or light chain of a
different antibody. One suitable non-limiting example of such an
antibody is a humanized 3G9 antibody comprising a light chain CDR1
that has the sequence of the light chain CDR1 derived from the 2B1
antibody (SEQ ID NO:20) instead of the sequence of the light chain
CDR1 for the deposited 3G9 antibody (SEQ ID NO:21). Such a
humanized 3G9 antibody having a light chain CDR1 sequence set forth
in SEQ ID NO:20 is designated herein as hu3G9 (or BG00011). Another
suitable non-limiting example of such an antibody is a humanized
8G6 antibody comprising a light chain CDR1 that has the sequence of
the light chain CDR1 derived from the 2B1 antibody (SEQ ID NO:20)
instead of the sequence of the light chain CDR1 for the deposited
8G6 antibody (SEQ ID NO: 18) Such a humanized 8G6 antibody having a
light chain CDR1 sequence set forth in SEQ ID NO:20 is designated
herein as hu8G9. Additional examples of such derivative antibodies,
in which one or more heavy chain and/or light chain CDRs has been
replaced with one or more corresponding heavy chain and/or light
chain CDRs from another antibody, and which are suitable for use in
accordance with the present invention, will be readily apparent to
those of ordinary skill in view of the sequences depicted in Table
1 and the guidance provided herein. Suitable methods for preparing
such humanized antibodies, including such derivative humanized
antibodies, are familiar to those of ordinary skill and are set
forth, for example, in U.S. published application no. 2005/0255102
A1, the disclosure of which is incorporated herein by reference in
its entirety.
In certain embodiments, the ligands, e.g., the antibodies, that
bind to .alpha..sub..nu..beta..sub.6 can be used in unconjugated
form. In such embodiments, the tumor cells are contacted with the
ligand binding to .alpha..sub..nu..beta..sub.6, under conditions
favoring binding of the ligand to .alpha..sub..nu..beta..sub.6 on
the cell surface. Suitable such conditions are well-known in the
art, and are described in detail in the Examples hereinbelow. In
certain such embodiments, the binding of the ligand sensitizes the
tumor cell to which the ligand has bound to the action of one or
more toxic compounds, i.e., the binding of the ligand to
.alpha.v.beta.6 on the tumor cell surface induces the tumor cell to
become more sensitive to the growth-inhibiting and/or
death-inducing actions of one or more toxins Thus, in additional
embodiments, the invention provides methods of inhibiting the
growth of a tumor cell, or of killing a tumor cell, comprising (a)
contacting the tumor cell with one or more ligands (e.g., one or
more antibodies or fragments thereof that bind to
.alpha..sub..nu..beta..sub.6 on the cell surface, under conditions
favoring the binding of the ligand .alpha..sub..nu..beta..sub.6 on
the cell surface; and then (b) contacting the tumor cell with one
or more toxic compounds that inhibits the growth of and/or kills
the tumor cell. Suitable toxic compounds for use in accordance with
this aspect of the invention, and appropriate dosages and
administration regimens therefor, are known in the art and are
described in detail elsewhere herein.
In related embodiments, the invention provides methods of
sensitizing tumor cells to one or more toxic compounds by treating
the tumor cells with one or more agents that modulates (e.g.,
inhibits or activates) one or more components of the TGF-.beta.
signaling pathway. Suitable such agents include, for example,
TGF-.beta. or a fragment thereof, a soluble TGF-.beta. receptor or
a fragment thereof, or a fusion protein comprising a soluble
TGF-.beta. receptor or a fragment thereof (e.g., a TGF-.beta. type
II receptor fragment fused to the Fc region of an antibody, to
produce a soluble "TGF-.beta. RII-Fc" polypeptide). In use, the
binding or internalization of the TGF-.beta.-modulating agent
sensitizes the tumor cell by which the agent has been bound or
internalized to the action of one or more toxic compounds, i.e.,
induces the tumor cell to become more sensitive to the
growth-inhibiting and/or death-inducing actions of one or more
toxins. Thus, in additional embodiments, the invention provides
methods of inhibiting the growth of a tumor cell, or of killing a
tumor cell, comprising (a) contacting the tumor cell with one or
more agents that modulates (e.g., inhibits or activates) one or
more components of the TGF-.beta. signaling pathway, under
conditions favoring the binding or internalization of the agent by
the tumor cell; and then (b) contacting the tumor cell with one or
more toxic compounds that inhibits the growth of and/or kills the
tumor cell. Suitable toxic compounds for use in accordance with
this aspect of the invention, and appropriate dosages and
administration regimens therefor, are known in the art and are
described in detail elsewhere herein.
Conjugates an Other Modifications of
.alpha..sub..nu..beta..sub.6-Binding Ligands
As noted above, in certain embodiments, the ligands, e.g., the
antibodies, that bind to .alpha..sub..nu..beta..sub.6 can be used
in unconjugated form. In other embodiments, the ligands, e.g., the
antibodies, that bind to .alpha..sub..nu..beta..sub.6 can be
conjugated, e.g., to a detectable label, a drug, a prodrug or an
isotope.
In certain methods of the invention described in more detail below,
such as methods of detecting .alpha..sub..nu..beta..sub.6
expression in cells or tissues in conjunction with detection of
smad4 expression as a measure of the potential of tumor cells to be
responsive to .alpha..sub..nu..beta..sub.6-binding ligands and/or
TGF-.beta.-blocking agents, the
.alpha..sub..nu..beta..sub.6-binding ligands (e.g., antibodies) are
conjugated to one or more detectable labels. For such uses, the
.alpha..sub..nu..beta..sub.6-binding ligands, e.g.,
.alpha..sub..nu..beta..sub.6-binding antibodies, may be detectably
labeled by covalent or non-covalent attachment of a chromogenic,
enzymatic, radioisotopic, isotopic, fluorescent, toxic,
chemiluminescent, nuclear magnetic resonance contrast agent or
other label.
Examples of suitable chromogenic labels include diaminobenzidine
and 4-hydroxyazo-benzene-2-carboxylic acid.
Examples of suitable enzyme labels include malate dehydrogenase,
staphylococcal nuclease, .DELTA.-5-steroid isomerase, yeast-alcohol
dehydrogenase, .alpha.-glycerol phosphate dehydrogenase, triose
phosphate isomerase, peroxidase, alkaline phosphatase,
asparaginase, glucose oxidase, .beta.-galactosidasce, ribonuclease,
urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase,
and acetylcholine esterase.
Examples of suitable radioisotopic labels include .sup.3H,
.sup.111In, .sup.125I, .sup.131I, .sup.32P, .sup.35S, .sup.14C,
.sup.51Cr, .sup.57To, .sup.58Co, .sup.59Fe, .sup.75Se, .sup.152Eu,
.sup.90Y, .sup.67Cu, .sup.217Ci, .sup.211At, .sup.212Pb, .sup.47Sc,
.sup.109Pd, etc. .sup.111In is a preferred isotope where in vivo
imaging is used since its avoids the problem of dehalogenation of
the .sup.125I or .sup.131I-labeled
.alpha..sub..nu..beta..sub.6-binding ligands by the liver. In
addition, this radionucleotide has a more favorable gamma emission
energy for imaging (Perkins et al., Eur. J. Nucl. Med. 10:296-301
(1985); Carasquillo et al., J. Nucl. Med. 28:281-287 (1987)). For
example, .sup.111In coupled to monoclonal antibodies with
1-(P-isothiocyanatobenzyl)-DPTA has shown little uptake in
non-tumorous tissues, particularly the liver, and therefore
enhances specificity of tumor localization (Esteban et al., J.
Nucl. Med. 28:861-870 (1987)).
Examples of suitable non-radioactive isotopic labels include
.sup.157Gd, .sup.55Mn, .sup.162Dy, .sup.52Tr, .sup.56Fe.
Examples of suitable fluorescent labels include an .sup.152Eu
label, a fluorescein label, an isothiocyanate label, a rhodamine
label, a phycoerythrin label, a phycocyanin label, an
allophycocyanin label, a Green Fluorescent Protein (GFP) label, an
o-phthaldehyde label, and a fluorescamine label. Examples of
suitable toxin labels include diphtheria toxin, ricin, and cholera
toxin.
Examples of chemiluminescent labels include a luminol label, an
isoluminol label, an aromatic acridinium ester label, an imidazole
label, an acridinium salt label, an oxalate ester label, a
luciferin label, a luciferase label, and an aequorin label.
Examples of nuclear magnetic resonance contrasting agents include
heavy metal nuclei such as Gd, Mn, and iron.
Typical techniques for binding the above-described labels to
.alpha..sub..nu..beta..sub.6-binding ligands, e.g.,
.alpha..sub..nu..beta..sub.6-binding antibodies, are provided by
Kennedy et al., Clin. Chim. Acta 70:1-31 (1976), and Schurs et al.,
Clin. Chim. Acta 81:1-40 (1977). Coupling techniques mentioned in
the latter are the glutaraldehyde method, the periodate method, the
dimaleimide method, the m-maleimidobenzyl-N-hydroxy-succinimide
ester method, all of which methods are incorporated by reference
herein.
For use in certain therapeutic approaches of the invention such as
ablation of tumor cells, or prevention of tumor cell growth,
invasion or metastasis, the .alpha..sub..nu..beta..sub.6-binding
ligands can be conjugated to one or more drugs, prodrugs or
isotopes. Preferred such conjugates comprise one or more ligands,
e.g., one or more antibodies or fragments, derivatives or variants
thereof, that bind to .alpha..sub..nu..beta..sub.6, conjugated to
one or more cytotoxic agents; such conjugates are useful in the
methods of treatment and prevention of tumor metastasis provided by
the invention. According to certain such embodiments of the
invention, the .alpha..sub..nu..beta..sub.6-binding ligand, e.g.,
antibody, is conjugated to a cytotoxic agent. Cytotoxic, e.g.,
chemotherapeutic, agents useful in the generation of
.alpha..sub..nu..beta..sub.6-binding ligand-cytotoxic agent
conjugates are well known in the art, and include but are not
limited to cisplatin, carboplatin, oxaliplatin, paclitaxel,
gemcitabine, adriamycin (doxorubicin), melphalan, methotrexate,
5-fluorouracil, etoposide, mechlorethamine, cyclophosphamide,
bleomycin, a calicheamicin, a maytansine, a trichothene.
Particularly suitable for use in accordance with this aspect of the
invention are paclitaxel, gemcitabine and adriamycin (doxorubicin).
Other chemotherapeutic agents suitable for use in accordance with
this aspect of the invention are well-known and will be familiar to
the ordinarily skilled artisan.
Thus, the use of conjugates of one or more
.alpha..sub..nu..beta..sub.6-binding ligand, e.g., one or more
.alpha..sub..nu..beta..sub.6-binding antibodies, and one or more
small molecule toxins, such as paclitaxel, adriamycin
(doxorubicin), gemcitabine, a calicheamicin, a maytansine (U.S.
Pat. No. 5,208,020), a trichothene, and CC1065, is also
contemplated herein. In one embodiment of the invention, the
.alpha..sub..nu..beta..sub.6-binding ligand is conjugated to one or
more maytansine molecules (e.g. about 1 to about 10 maytansine
molecules per .alpha..sub..nu..beta..sub.6-binding ligand).
Maytansine may, for example, be converted to May-SS-Me which may be
reduced to May-SH3 and reacted with modified
.alpha..sub..nu..beta..sub.6-binding ligands (Chari et al. Cancer
Research 52: 127-131 (1992)) to generate a
maytansinoid-.alpha..sub..nu..beta..sub.6-binding ligand
conjugate.
Alternatively, the .alpha..sub..nu..beta..sub.6-binding ligand can
be conjugated to one or more calicheamicin molecules. The
calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations.
Structural analogues of calicheamicin which may be used include,
but are not limited to, .gamma..sub.1.sup.I, .alpha..sub.2.sup.I,
.alpha..sub.3.sup.I, N-acetyl-.gamma..sub.1.sup.I, PSAG and
.PHI..sub.1.sup.I (Hinman et al. Cancer Research 53: 3336-3342
(1993) and Lode et al. Cancer Research 58: 2925-2928 (1998)).
Enzymatically active toxins and fragments thereof which can be used
to produce conjugates with one or more
.alpha..sub..nu..beta..sub.6-binding ligands, e.g., one or more
.alpha..sub..nu..beta..sub.6-binding antibodies, include diphtheria
A chain, nonbinding active fragments of diphtheria toxin, exotoxin
A chain (from Pseudomonas aeruiginosa), ricin A chain, abrin A
chain, modeccin A chain, alpha-sarcin, Aleuiritesfordii proteins,
dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and
PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin and the tricothecenes. See, for example, WO
93/21232 published in the English language on Oct. 28, 1993, the
disclosure of which is incorporated herein by reference in its
entirety. Mytansinoids may also be conjugated to one or more
.alpha.v.beta.6-binding ligands, e.g., one or more
.alpha..sub..nu..beta..sub.6-binding antibodies. The present
invention further contemplates .alpha..sub..nu..beta..sub.6-binding
ligands conjugated with a compound with nucleolytic activity (e.g.,
a ribonuclease or a DNA endonuclease such as a deoxyribonuclease;
DNase).
A variety of radioactive isotopes are also available for the
production of radioconjugated .alpha..sub..nu..beta..sub.6-binding
ligands for use in therapeutic methods of the invention. Examples
include .sup.211At, .sup.131I, .sup.125I, .sup.90Y, .sup.186Re,
.sup.188Re, .sup.153Sm, .sup.212Bi, .sup.32P and radioactive
isotopes of Lu.
Conjugates of the .alpha..sub..nu..beta..sub.6-binding ligands and
cytotoxic agents may be made using a variety of bifunctional
protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)
propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)
cyclohexane-1-carboxylate, iminothiolane (IT), bifunctional
derivatives of imidoesters (such as dimethyl adipimidate HCl),
active esters (such as disuccinimidyl suberate), aldehydes (such as
glutaraldehyde), his-azido compounds (such as bis(p-azidobenzoyl)
hexanediamine), bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diusocyanates (such as
tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al., Science
238: 1098 (1987). .sup.14Carbon-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the .alpha..sub..nu..beta..sub.6-binding ligand.
See WO 94/11026. The linker may be a "cleavable linker"
facilitating release of the cytotoxic drug in the cell. For
example, an acid-labile linker, peptidase-sensitive linker,
dimethyl linker or disulfide-containing linker (Chari et al. Cancer
Research 52:127-131 (1992)) may be used.
Alternatively, a fusion protein comprising the
.alpha.v.beta.6-binding ligand and cytotoxic agent may be made,
e.g. by recombinant techniques or peptide synthesis.
In yet another embodiment, the .alpha..sub..nu..beta..sub.6-binding
ligand may be conjugated to a "receptor" (such streptavidin) for
utilization in "pretargeting" wherein the
.alpha..sub..nu..beta..sub.6-binding ligand-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g. avidin) which is conjugated to a
cytotoxic agent (e.g., a radionucleotide).
The .alpha..sub..nu..beta..sub.6-binding ligands of the present
invention may also be conjugated with a prodrug-activating enzyme
which converts a prodrug (e.g. a peptidyl chemotherapeutic agent,
see WO 81/01145) to an active drug. See, for example, WO 88/07378
and U.S. Pat. No. 4,975,278. The enzyme component of such
conjugates includes any enzyme capable of acting on a prodrug in
such a way so as to covert it into its more active, cytotoxic
form.
Enzymes that are useful in the method of this invention include,
but are not limited to, alkaline phosphatase useful for converting
phosphate-containing prodrugs into free drugs; arylsulfatase useful
for converting sulfate-containing prodrugs into free drugs;
cytosine deaminase useful for converting non-toxic 5-fluorocytosine
into the anti-cancer drug, 5-fluorouracil; proteases, such as
Serratia protease, thermolysin, subtilisin, carboxypeptidases and
cathepsins (such as cathepsins B and L), that are useful for
converting peptide-containing prodrugs into free drugs;
D-alanylcarboxypeptidases, useful for converting prodrugs that
contain D-amino acid substituents; carbohydrate-cleaving enzymes
such as O-galactosidase and neuraminidase useful for converting
glycosylated prodrugs into free drugs; P-lactamase useful for
converting drugs derivatized with P-lactams into free drugs; and
penicillin amidases, such as penicillin V amidase or penicillin G
amidase, useful for converting drugs derivatized at their amine
nitrogens with phenoxyacetyl or phenylacetyl groups, respectively,
into free drugs.
Enzymes can be covalently bound to the
.alpha..sub..nu..beta..sub.6-binding ligand by techniques well
known in the art such as the use of the heterobifunctional
crosslinking reagents. Alternatively, fusion proteins comprising at
least the antigen binding region of a
.alpha..sub..nu..beta..sub.6-binding ligand of the invention linked
to at least a functionally active portion of an enzyme can be
constructed using recombinant DNA techniques well known in the art
(see, e.g., Neuberger et al., Nature 312: 604-608 (1984)).
Disease Diagnosis and Prognosis
As noted above, it has now been found that cells from certain
tumors that display significantly decreased levels of smad4 gene
expression and significantly enhanced levels of integrin
.alpha..sub..nu..beta..sub.6 are more likely to be responsive to
ligands that bind to .alpha..sub..nu..beta..sub.6, and to agents
that block one or more components of the TGF-.beta. signalling
pathway, when compared to cells that are display higher levels of
smad4 gene expression. Thus, in one aspect, the invention provides
a method useful in determining the likelihood of a tumor cell to
respond to or to be chemosensitized by treatment with
anti-.alpha.v.beta.6 ligands (e.g., antibodies) or with
TGF-.beta.-blocking agents, including tumors from carcinomas such
as an adenocarcinoma. In more particular embodiments, the carcinoma
is a pancreatic carcinoma, a colorectal carcinoma, a cervical
carcinoma, a squamous cell carcinoma (such as an esophageal
carcinoma), a head and neck carcinoma, a liver carcinoma, an
ovarian carcinoma and a lung carcinoma. More particularly, the
carcinoma is a pancreatic carcinoma, a colorectal carcinoma, a
cervical carcinoma, or a head and neck carcinoma.
Methods according to this aspect of the invention involve assaying
the level of expression of smad4 in the tumor cells or in a tumor
tissue sample, and comparing these expression levels with a
standard smad4 expression level (e.g., in normal cells or tissues,
preferably obtained from the same animal, such as a human patient),
wherein a decrease in the expression of smad4 in a tumor or in the
cells thereof indicates that the tumor is more likely to be
responsive to the growth-inhibiting actions of anti-.alpha.v.beta.6
ligands (e.g., antibodies) or with TGF-.beta.-blocking agents, or
to be chemosensitized by such ligands and agents to subsequent (or
simultaneous) co-treatment with one or more chemotherapeutic
compounds or compositions. As noted above, decreased expression of
smad4 in tumors is often associated with a poor patient prognosis,
so the methods of the invention provide a way to rapidly diagnose
and aggressively treat such tumors with compounds and compositions
that are most likely to provide a specifically targeted therapeutic
approach, and thereby providing for a more favorable clinical
outcome for the patient.
By "assaying the levels of expression of smad4" is intended
qualitatively or quantitatively measuring or estimating the levels
of smad4 in a first biological sample (e.g., a tumor sample, a
tissue biopsy or aspirate, etc.) either directly (e.g., by
determining or estimating absolute level of expression of
.alpha..sub..nu..beta..sub.6 in the sample) or relatively (e.g., by
comparing the level of expression of smad4 in a first biological
sample to that in a second biological sample). Preferably, the
level of smad4 in the first biological sample is measured or
estimated and compared to that in a standard taken from a second
biological sample obtained from an individual not having a cancer
or pre-cancerous lesion. The levels of smad4 can be measured in a
given cell, tissue, organ or biological sample according to
art-known methods of measuring gene expression, including, for
example, hybridization or PCR/RT-PCR detection of the smad4 gene
using known genetic probes or primers (see, e.g., Maliekal, T. T.
et al., Oncogene 22:4889-4897 (2003); Iacobuzio-Donahue, C. A. et
al., Clin. Cancer Res. 10:1597-1604 (2004)), northern blotting
(see, e.g., Yasutome, M. et al., Clin. Exper. Metast. 22:461-473
(2005)), and immunohistochemical analysis with anti-SMAD4
antibodies (see, e.g., Luttges, J. et al., Am. J. Pathol.
158:1677-1683 (2001); Fukuchi, M. et al., Cancer 95:737-743 (2002);
Subramanian, G. et al., Cancer Res. 64:5200-5211 (2004); Levy, L.
and Hill, C. S., Mol. Cell. Biol. 25:8108-8125 (2005)). As will be
appreciated by one of ordinary skill in the art, once a standard
smad4 expression level is known for a given non-cancerous tissue,
it can be used repeatedly as a standard for comparison.
Similarly, by "assaying the levels of expression of
.alpha..sub..nu..beta..sub.6" is intended qualitatively or
quantitatively measuring or estimating the levels of
.alpha..sub..nu..beta..sub.6 in a first biological sample (e.g., a
tumor sample, a tissue biopsy or aspirate, etc.) either directly
(e.g., by determining or estimating absolute amount of
.alpha..sub..nu..beta..sub.6 in the sample) or relatively (e.g., by
comparing the level of expression of .alpha..sub..nu..beta..sub.6
in a first biological sample to that in a second biological
sample). Preferably, the level of .alpha..sub..nu..beta..sub.6 in
the first biological sample is measured or estimated and compared
to that in a standard taken from a second biological sample
obtained from an individual not having a cancer or pre-cancerous
lesion. As will be appreciated by one of ordinary skill in the art,
once a standard .alpha..sub..nu..beta..sub.6 expression level is
known for a given non-cancerous tissue, it can be used repeatedly
as a standard for comparison.
By "biological sample" is intended any biological sample obtained
from an individual (such as a patient), cell line, tissue culture,
or other source which may contain cells or cellular products such
as extracellular matrix. Such biological samples include mammalian
body tissues and cells, including leukocyte, ovary, prostate,
heart, placenta, pancreas, liver, spleen, lung, breast, head and
neck tissues (e.g., oral, pharyngeal, lingual and laryngeal
tissues), squamous cells (e.g., esophageal), endometrium, colon (or
colorectal), cervix, stomach and umbilical tissues which may
express .alpha..sub..nu..beta..sub.6. Methods for obtaining tissue
biopsies and body fluids from mammals are well known in the art.
Preferred mammals include monkeys, apes, cats, dogs, cows, pigs,
horses, rabbits and humans. Particularly preferred are humans.
Assaying .alpha..sub..nu..beta..sub.6 expression levels in a
biological sample can occur using any art-known method. Preferred
for assaying .alpha..sub..nu..beta..sub.6 expression levels in a
biological sample are immunological techniques. For example,
.alpha..sub..nu..beta..sub.6 expression in tissues can be studied
with classical immunohistological methods. In these, the specific
recognition is provided by a primary ligand, e.g., an antibody
(polyclonal or monoclonal), that binds to
.alpha..sub..nu..beta..sub.6. This primary ligand can be labeled,
e.g., with a fluorescent, chemiluminescent, phosphorescent,
enzymatic or radioisotopic label. Alternatively, these methods of
the invention can use a secondary detection system in which a
second ligand that recognizes and binds to the
.alpha..sub..nu..beta..sub.6-binding ligand, e.g., a so-called
"secondary" antibody which recognizes and binds to a first
.alpha..sub..nu..beta..sub.6-binding antibody, is detectably
labeled as described above. As a result, an immunohistological
staining of tissue section for pathological examination is
obtained. Alternatively, tissues and cell samples can also be
extracted, e.g., with urea and neutral detergent, for the
liberation of .alpha..sub..nu..beta..sub.6 protein for Western-blot
or dot/slot assay (Jalkanen, M., et al., J. Cell. Biol. 101:976-985
(1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096 (1987))
for direct quantitation, relative to a standard tissue or cell
sample known to have lower levels of expression of
.alpha..sub..nu..beta..sub.6.
As noted above, the methods of the present invention are useful for
detecting cancers in mammals, for determining the likelihood that a
given tumor cell will be responsive to treatment and/or
chemosensitization with anti-.alpha.v.beta.6 ligands and/or with
TGF-.beta.), and for treating carcinomas of a variety of types. In
particular the methods of the invention are useful in detecting
and/or treating cancers of epithelial tissues (i.e., carcinomas),
including of the pancreas, colon/rectum, cervix, esophagus (and
other squamous epithelial tissues), head and neck, liver, ovary and
lung. Particularly amenable to detection by the methods of the
present invention are invasive and/or metastatic adenocarcinomas,
including but not limited to pancreatic carcinomas, colorectal
carcinomas, cervical carcinomas, and head and neck carcinomas.
Early identification and treatment of such carcinomas is associated
with a better long-term prognosis for patients. For example, it has
been reported that if left untreated, a significant proportion of
noninvasive breast tumors become invasive and can lead to
metastatic cancers which have a much poorer prognosis (see
Sakorafas, G. H., and Tsiotou, A. G. H., Cancer Treatment Rev.
26:103-125 (2000)). In addition, as noted above, reduced expression
of smad4 in primary pancreatic adenocarcinomas has been associated
with poor prognosis for patients having such tumors (Liu, F., Clin.
Cancer Res. 7:3853-3856 (2001); Tascilar, M. et al., Clin. Cancer
Res. 7:4115-4121 (2001); Toga, T. et al., Anticancer Res.
24:1173-1178 (2004)). Thus, early diagnosis and initiation of
treatment, preferably according to the methods described herein, is
key to long-term survival of patients having smad4-deficient
cancers.
Accordingly, the present invention contemplates methods of treating
or preventing cancers by identifying smad4-deficient pre-cancerous
lesions or carcinomas in patients, and treating the patient to
eliminate the pre-cancerous lesion before it has the opportunity to
evolve into a more advance form of cancer. Such methods comprise,
for example, (a) obtaining a tissue sample that is suspected of
containing a cancer or a pre-cancerous lesion, and a tissue sample
that does not contain a cancer or pre-cancerous lesion (preferably
from the same tissue or organ as that suspected of containing a
cancer or pre-invasive lesion); and (b) determining the level of
smad4 expression in the tumor or tissue samples, or cells
therefrom, wherein a decrease in the level of smad4 expression in
the tissue sample containing the cancerous or pre-invasive lesion,
relative to the levels of smad4 expression in the non-cancerous
tissue sample (or cells thereof), is indicative of a cancer or a
precancerous lesion that is more likely to progressive to a more
invasive or aggressive form of cancer. In other related
embodiments, the invention contemplates methods of reducing or
preventing the progression of a pre-cancerous lesion to a tumor in
a patient, comprising identifying in a patient tumors (or cells
thereof) that have a reduced level of smad4 expression (preferably
using the methods described hereinabove), and administering to the
patient a therapeutically effective amount of one or more ligands
that binds to one or more subunits of integrin
.alpha..sub..nu..beta..sub.6 on one or more cells in the
pre-cancerous, wherein the binding of the ligand to the integrin
results in the death or inhibition of growth of the pre-cancerous
lesion, or in the chemosensitization of the pre-cancerous lesion to
the death-inducing or growth-inhibiting actions of one or more
chemotherapeutic compounds.
Suitable tissues and organs from which samples can be obtained for
use in accordance with these methods of the invention include, but
are not limited to, the epithelial tissues described elsewhere
herein. Cancers and tumors that may be advantageously treated or
prevented according to such methods of the invention include, but
are not necessarily limited to, carcinomas, particularly
adenocarcinomas, including the carcinomas and adenocarcinomas
described in detail elsewhere herein. Once such a carcinoma has
been detected according to the methods of the invention, it can
then be eliminated from the patient via surgical, chemotherapeutic,
radiological or other methods of cancer therapy that are well-known
in the art and that therefore will be familiar to those of ordinary
skill. Alternatively, such a carcinoma can be eliminated using the
methods of treatment of the present invention, by administering to
the patient, or to the organs or tissues of the patient, one or
more .alpha..sub..nu..beta..sub.6-binding ligands, such as one or
more .alpha..sub..nu..beta..sub.6-binding antibodies or fragments
thereof. In certain non-limiting examples of such embodiments, the
one or more .alpha..sub..nu..beta..sub.6-binding ligands have been
conjugated with one or more cytotoxic compounds or agents as
described in detail hereinabove. In additional non-limiting
examples of such embodiments, the one or more
.alpha..sub..nu..beta..sub.6-binding ligands, such as one or more
.alpha..sub..nu..beta..sub.6-binding antibodies or fragments
thereof, are administered to a subject, such as a patient, in
conjunction with one or more cytotoxic compounds or agents as
described in detail hereinabove. In other related embodiments, such
methods of the invention comprise administering one or more
TGF-.beta.-blocking agents, such as those described herein, which
inhibit the growth of the tumor cells and/or chemosensitize the
tumor cells to the actions of one or more toxic chermotherapeutic
compounds.
In related embodiments, the invention contemplates determining the
metastatic potential of a tumor or cancer cell by measuring the
levels of expression of smad4 and .alpha..sub..nu..beta..sub.6 by
the tumor or cancer cell. In such embodiments, tumor or cell
samples are obtained from a patient as described above and are
assayed according to the methods described herein for the levels of
expression of smad4 and .alpha..sub..nu..beta..sub.6 by the tumor
or cancer cell. According to these methods of the invention, there
is an inverse correlation between the level of expression of smad4
by the tumor or cancer cell and the level of expression of
.alpha..sub..nu..beta..sub.6 by the tumor or cancer cell that
provides a useful clinical picture that can be used to determine
the metastatic potential of a tumor or cancer cell. Specifically, a
decrease in the expression of smad4 by a tumor or cancer cell and a
concomitant increase in the expression of
.alpha..sub..nu..beta..sub.6 by the tumor or cancer cell direct
correlation between the level of expression of
.alpha..sub..nu..beta..sub.6 by the tumor or cancer cell indicates
that that tumor or cancer cell is more likely to metastasize to a
secondary locus from the primary tumor site. Hence, the levels of
expression of smad4 and of .alpha..sub..nu..beta..sub.6 by a tumor
or cancer cell can be used as a prognostic indicator of the
metastatic potential of a tumor or cancer cell, which can assist
cancer patients and their physicians in making appropriate
treatment decisions based on the present or predicted future
aggressiveness or invasiveness of the cancer.
In addition to assaying smad4 and .alpha..sub..nu..beta..sub.6
expression levels in a biological sample obtained from an
individual, such as a tissue or tumor cell sample, the level and
pattern of expression of .alpha..sub..nu..beta..sub.6 can also be
detected in vivo by imaging. In such methods of the invention, one
or more SMAD4-binding ligands and
.alpha..sub..nu..beta..sub.6-binding ligands, e.g., one or more
SMAD4-binding antibodies and one or more
.alpha..sub..nu..beta..sub.6-binding antibodies, are detectably
labeled with one or more labels suitable for in vivo imaging.
Suitable labels or markers for in vivo imaging include those
detectable by X-radiography, NMR or ESR. For X-radiography,
suitable labels include radioisotopes such as barium or cesium,
which emit detectable radiation but are not overtly harmful to the
subject. Suitable markers for NMR and ESR include those with a
detectable characteristic spin, such as deuterium.
A ligand binding to SMAD4 or .alpha..sub..nu..beta..sub.6, e.g., a
SMAD4- or an .alpha..sub..nu..beta..sub.6-binding antibody or
antibody fragment, which has been labeled with an appropriate
detectable imaging moiety, such as a radioisotope (for example,
.sup.131I, .sup.112In, .sup.99mTc), a radio-opaque substance, or a
material detectable by nuclear magnetic resonance, is introduced
(for example, parenterally, subcutaneously or intraperitoneally)
into the mammal to be examined for cancer or carcinoma in situ. It
will be understood in the art that the size of the subject and the
imaging system used will determine the quantity of imaging moiety
needed to produce diagnostic images. In the case of a radio-isotope
moiety, for a human subject, the quantity of radioactivity injected
will normally range from about 5 to 20 millicuries of .sup.99mTc.
The labeled ligand, e.g., SMAD4- or
.alpha..sub..nu..beta..sub.6-binding antibody or antibody fragment,
will then preferentially accumulate at the location of cells or
tissues which contain or express SMAD4 or
.alpha..sub..nu..beta..sub.6 integrin. In vivo tumor imaging is
then accomplished as described in S. W. Burchiel et al.,
"Immunopharmacokinetics of Radiolabelled Antibodies and Their
Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical
Detection of Cancer, S. W. Burchiel and B. A. Rhodes, eds., Masson
Publishing Inc. (1982)).
Therapeutic Methods
In additional embodiments of the invention, the methods of the
present invention can be used therapeutically regimens for treating
mammals afflicted with certain diseases, particularly with certain
carcinomas such as those described elsewhere herein. Such methods
of the invention are useful in treating cancer and associated
events, including tumor growth, metastasis and angiogenesis.
Particularly amenable to such an approach are those diseases or
cancers that are characterized by decreased levels of smad4
expression, and concomitantly increased levels of
.alpha..sub..nu..beta..sub.6 expression, in the tissues or cells of
a mammal suffering from the disease, and which are responsive to
treatments which target the tissues or cells expressing increased
levels of .alpha..sub..nu..beta..sub.6 and eliminate those tissues
or cells. Methods according to this aspect of the invention
comprise, for example, (a) identifying tumors or tumor cells with
decreased smad4 expression in a patient, and (b) treating the
patient with one or more .alpha..sub..nu..beta..sub.6-binding
ligands, such as one or more .alpha..sub..nu..beta..sub.6-binding
antibodies or fragments thereof, or with one or more
TGF-.beta.-blocking agents or fragments thereof. Alternative such
methods comprise, for example, (a) identifying tumors or tumor
cells with decreased smad4 expression in a patient; (b) treating
the patient with one or more .alpha..sub..nu..beta..sub.6-binding
ligands, such as one or more .alpha..sub..nu..beta..sub.6-binding
antibodies or fragments thereof, or with one or more
TGF-.beta.-blocking agents or fragments thereof, wherein the
binding of the .alpha..sub..nu..beta..sub.6-binding ligands or the
TGF-.beta.-blocking agents to the tumor cells chemosensitizes the
tumor cells to the growth-inhibiting and/or death-inducing actions
of one or more chemotherapeutic compounds; and (c) treating the
patient with one or more chemotherapeutic compounds that inhibits
the growth of, or kills, the tumor cells.
Diseases that are particularly treatable by these methods include
metastatic cancers of epithelial tissues (i.e., metastatic
carcinomas and/or adenocarcinomas), including of the pancreas,
colon/rectum, cervix, esophagus (and other squamous epithelial
tissues and organs), head and neck, liver, ovary and lung.
Particularly suitable for treatment by these methods of the present
invention are carcinomas of the pancreas, colon/rectum, cervix,
head and neck and esophagus. Preferred mammals for treatment
include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and
humans. Particularly preferred are humans.
In related embodiments, as described above, the invention provides
methods of reducing or preventing the progression of a
pre-metastatic tumor to a metastatic tumor in a patient, comprising
administering to the patient a therapeutically effective amount of
one or more ligands that binds to one or more subunits of integrin
.alpha..sub..nu..beta..sub.6 on one or more cells in the
pre-metastatic tumor, wherein the binding of the ligand to the
integrin results in the reduction or prevention of invasion of
cells of the pre-metastatic cancer into tissue areas surrounding
the primary tumor.
In carrying out these therapeutic methods of the invention,
.alpha..sub..nu..beta..sub.6-binding ligands, such as
.alpha..sub..nu..beta..sub.6-binding antibodies or fragments
thereof, or TGF-.beta.-blocking agents or fragments thereof, may be
administered to patients in the form of therapeutic formulations
(which are also referred to herein interchangeably and equivalently
as pharmaceutical compositions). Therapeutic formulations of the
.alpha..sub..nu..beta..sub.6-binding ligands or TGF-.beta.-blocking
agents or fragments thereof used in accordance with the present
invention are prepared for storage by mixing a
.alpha..sub..nu..beta..sub.6-binding ligand or TGF-.beta.-blocking
agent or fragment thereof having the desired degree of purity with
optional pharmaceutically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), for example in the form of lyophilized
formulations or aqueous solutions. In addition to the
pharmacologically active compounds such as the
.alpha..sub..nu..beta..sub.6-binding ligands or the
TGF-.beta.-blocking agents or fragments thereof, the compositions
used in the therapeutic methods of the invention can contain one or
more suitable pharmaceutically acceptable carriers comprising
excipients and auxiliaries that facilitate processing of the active
compounds into preparations that can be used pharmaceutically. The
pharmaceutical preparations of the present invention are
manufactured in a manner that is, itself, known, for example, by
means of conventional mixing, granulating, dragee-making,
dissolving, or lyophilizing processes. Thus, pharmaceutical
preparations for oral use can be obtained by combining the active
compounds with solid excipients, optionally grinding the resulting
mixture and processing the mixture of granules, after adding
suitable auxiliaries, if desired or necessary, to obtain tablets or
dragee cores.
Suitable excipients are, in particular, fillers such as
saccharides, for example, lactose or sucrose, mannitol or sorbitol,
cellulose preparations and/or calcium phosphates, for example,
tricalcium phosphate or calcium hydrogen phosphate, as well as
binders, such as starch paste, using, for example, maize starch,
wheat starch, rice starch, potato starch, gelatin, tragacanth,
methyl cellulose, hydroxypropylmethylcellulose, sodium
carboxy-methylcellulose, and/or polyvinyl pyrrolidone. If desired,
disintegrating agents can be added, such as the above-mentioned
starches and also carboxymethyl-starch, cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof, such as
sodium alginate. Auxiliaries are, above all, flow-regulating agents
and lubricants, for example silica, talc, stearic acid or salts
thereof, such as magnesium stearate or calcium stearate, and/or
polyethylene glycol. Dragee cores are provided with suitable
coatings, that, if desired, are resistant to gastric juices. For
this purpose, concentrated saccharide solutions can be used, which
may optionally contain gum arabic, talc, polyvinyl pyrrolidone,
polyethylene glycol, and/or titanium dioxide, lacquer solutions and
suitable organic solvents or solvent mixtures. In order to produce
coatings resistant to gastric juices, solutions of suitable
cellulose preparations, such as acetylcellulose phthalate or
hydroxypropylmethylcellulose phthalate, are used. Dye stuffs or
pigments can be added to the tablets or dragee coatings, for
example, for identification or in order to characterize
combinations of active compound doses.
Other pharmaceutical preparations that can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer such as glycerol or sorbitol. The
push-fit capsules can contain the active compounds in the form of
granules that may be mixed with fillers such as lactose, binders
such as starches, and/or lubricants such as talc or magnesium
stearate and, optionally, stabilizers. In soft capsules, the active
compounds are preferably dissolved or suspended in suitable liquids
such as fatty oils or liquid paraffin. In addition, stabilizers may
be added.
Suitable formulations for parenteral administration include aqueous
solutions of the active compounds in water-soluble form, for
example water-soluble salts and alkaline solutions. Alkaline salts
can include ammonium salts prepared, for example, with Tris,
choline hydroxide, bis-Tris propane, N-methylglucamine, or
arginine. In addition, suspensions of the active compounds as
appropriate oily injection suspensions can be administered.
Suitable lipophilic solvents or vehicles include fatty oils, for
example, sesame oil, or synthetic fatty acid esters, for example,
ethyl oleate or triglycerides or polyethylene glycol-400 (the
compounds are soluble in PEG-400). Aqueous injection suspensions
can contain substances that increase the viscosity of the
suspension, for example sodium carboxymethyl cellulose, sorbitol,
and/or dextran. Optionally, the suspension may also contain
stabilizers.
The compounds of the present invention may be administered to the
eye in animals and humans as a drop, or within ointments, gels,
liposomes, or biocompatible polymer discs, pellets or carried
within contact lenses. The intraocular composition may also contain
a physiologically compatible ophthalmic vehicle as those skilled in
the art can select using conventional criteria. The vehicles may be
selected from the known ophthalmic vehicles which include but are
not limited to water, polyethers such as polyethylene glycol 400,
polyvinyls such as polyvinyl alcohol, povidone, cellulose
derivatives such as carboxymethylcellulose, methylcellulose and
hydroxypropyl methylcellulose, petroleum derivatives such as
mineral oil and white petrolatum, animal fats such as lanolin,
vegetable fats such as peanut oil, polymers of acrylic acid such as
carboxylpolymethylene gel, polysaccharides such as dextrans and
glycosaminoglycans such as sodium chloride and potassium, chloride,
zinc chloride and buffer such as sodium bicarbonate or sodium
lactate. High molecular weight molecules can also be used.
Physiologically compatible preservatives which do not inactivate
the compounds of the present invention in the composition include
alcohols such as chlorobutanol, benzalkonium chloride and EDTA, or
any other appropriate preservative known to those skilled in the
art.
Lyophilized formulations of antibodies adapted for subcutaneous
administration are described in U.S. Pat. No. 6,267,958, the
disclosure of which is incorporated herein by reference in its
entirety. Such lyophilized formulations may be reconstituted with a
suitable diluent to a high protein concentration and the
reconstituted formulation may be administered subcutaneously to the
patient to be treated herein.
The .alpha..sub..nu..beta..sub.6-binding ligands may also be
entrapped in microcapsules prepared, for example, by coacervation
techniques or by interfacial polymerization, for example,
hydroxymethylcellulose or gelatin-microcapsules and
poly-(methylmethacylate) microcapsules, respectively, in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules) or
in macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).
Sustained-release preparations of
.alpha..sub..nu..beta..sub.6-binding ligands may be prepared.
Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
.alpha..sub..nu..beta..sub.6-binding ligand, which matrices are in
the form of shaped articles, e.g. films, or microcapsules. Examples
of sustained-release matrices include polyesters, hydrogels (for
example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma.-ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, 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.
The formulations to be used for in vivo administration must be
sterile. This is readily accomplished by filtration through sterile
filtration membranes.
The .alpha..sub..nu..beta..sub.6-binding ligand or
TGF-.beta.-blocking agents or fragments thereof may be administered
to the subject or patient by any suitable means, including
parenteral, intrapulmonary, intracranial, transdermal and
intranasal. Parenteral infusions include intramuscular,
intravenous, intraarterial, intraperitoneal, or subcutaneous
administration. In addition, the
.alpha..sub..nu..beta..sub.6-binding ligand or TGF-.beta.-blocking
agents or fragments thereof may suitably be administered by pulse
infusion, e.g., with declining doses of the
.alpha..sub..nu..beta..sub.6-binding ligand or TGF-.beta.-blocking
agent or fragments thereof. Preferably the dosing is given by
injections, most preferably intravenous or subcutaneous injections,
depending in part on whether the administration is brief or
chronic.
In certain exemplary embodiments of the invention, the
.alpha..sub..nu..beta..sub.6-binding ligands or TGF-.beta.-blocking
agents or fragments thereof are administered to the patient (e.g.,
intravenously) in a dosage of between about 1 mg/m.sup.2 and about
500 mg/m.sup.2. For instance, the
.alpha..sub..nu..beta..sub.6-binding ligand or TGF-.beta.-blocking
agent or fragments thereof may be administered in a dosage of about
1 mg/m.sup.2, 2 mg/m.sup.2, 3 mg/m.sup.2, 4 mg/m.sup.2, 5
mg/m.sup.2, 10 mg/m.sup.2, 15 mg/m.sup.2, 20 mg/m.sup.2, 25
mg/m.sup.2, 30 mg/m.sup.2, 35 mg/m.sup.2, 40 mg/m.sup.2, 45
mg/m.sup.2, 50 mg/m.sup.2, 55 mg/m.sup.2, 60 mg/m.sup.2, 65
mg/m.sup.2, 70 mg/m.sup.2, 75 mg/m.sup.2, 80 mg/m.sup.2, 85
mg/m.sup.2, 90 mg/m.sup.2, 95 mg/m.sup.2, 100 mg/m.sup.2, 105
mg/m.sup.2, 110 mg/m.sup.2, 115 mg/m.sup.2, 120 mg/m.sup.2, 125
mg/m.sup.2, 130 mg/m.sup.2, 135 mg/m.sup.2, 140 mg/m.sup.2, 145
mg/m.sup.2, 150 mg/m.sup.2, 155 mg/m.sup.2, 160 mg/m.sup.2, 165
mg/m.sup.2, 170 mg/m.sup.2, 175 mg/m.sup.2, 180 mg/m.sup.2, 185
mg/m.sup.2, 190 mg/m.sup.2, 195 mg/m.sup.2, 200 mg/m.sup.2, 205
mg/m.sup.2, 210 mg/m.sup.2, 215 mg/m.sup.2, 220 mg/m.sup.2, 225
mg/m.sup.2, 230 mg/m.sup.2, 235 mg/m.sup.2, 240 mg/m.sup.2, 245
mg/m.sup.2, 250 mg/m.sup.2, 255 mg/m.sup.2, 260 mg/m.sup.2, 265
mg/m.sup.2, 270 mg/m.sup.2, 275 mg/m.sup.2, 280 mg/m.sup.2, 285
mg/m.sup.2, 290 mg/m.sup.2, 295 mg/m.sup.2, 300 mg/m.sup.2, 305
mg/m.sup.2, 310 mg/m.sup.2, 315 mg/m.sup.2, 320 mg/m.sup.2, 325
mg/m.sup.2, 330 mg/m.sup.2, 335 mg/m.sup.2, 340 mg/m.sup.2, 345
mg/m.sup.2, 350 mg/m.sup.2, 355 mg/m.sup.2, 360 mg/m.sup.2, 365
mg/m.sup.2, 370 mg/m.sup.2, 375 mg/m.sup.2, 380 mg/m.sup.2, 385
mg/m.sup.2, 390 mg/m.sup.2, 395 mg/m.sup.2 or 400 mg/m.sup.2.
The .alpha..sub..nu..beta..sub.6-binding ligand or
TGF-.beta.-blocking agents or fragments thereof can be administered
according to a wide variety of dosing schedules. For example, the
.alpha..sub..nu..beta..sub.6-binding ligand or TGF-.beta.-blocking
agents or fragments thereof can be administered once daily for a
predetermined amount of time (e.g., four to eight weeks, or more),
or according to a weekly schedule (e.g., one day per week, two days
per week, three days per week, four days per week, five days per
week, six days per week or seven days per week) for a predetermined
amount of time (e.g., four to eight weeks, or more). A specific
example of a "once weekly" dosing schedule is administration of the
.alpha..sub..nu..beta..sub.6-binding ligand or TGF-.beta.-blocking
agents or fragments thereof on days 1, 8, 15 and 22 of the
treatment period. In alternative embodiments the
.alpha..sub..nu..beta..sub.6-binding ligand or TGF-.beta.-blocking
agents or fragments thereof may be administered intermittently over
a period of months. For example, the
.alpha..sub..nu..beta..sub.6-binding ligand or TGF-.beta.-blocking
agents or fragments thereof may be administered weekly for three
consecutive weeks biannually (i.e., repeat the weekly dosing
schedule every six months). It will be appreciated that such
administration regimens may be continued for extended periods (on
the order of years) to maintain beneficial therapeutic effects
provided by initial treatments. In yet other embodiments such
maintenance therapy may be effected following an acute dosing
regimen designed to reduce the immediate symptoms of the cancerous,
metastatic or in situ carcinoma condition.
The amount of .alpha..sub..nu..beta..sub.6-binding ligand or
TGF-.beta.-blocking agents or fragments thereof administered each
time throughout the treatment period can be the same;
alternatively, the amount administered each time during the
treatment period can vary (e.g., the amount administered at a given
time can be more or less than the amount administered previously).
For example, doses given during maintenance therapy may be lower
than those administered during the acute phase of treatment.
Appropriate dosing schedules depending on the specific
circumstances will be apparent to persons of ordinary skill in the
art.
In certain embodiments of the invention, multiple types or species
of .alpha..sub..nu..beta..sub.6-binding ligands are combined with
one another and administered to a patient to treat one or more
cancerous, metastatic or in situ carcinoma conditions. For example,
the invention contemplates the administration of two or more
different .alpha..sub..nu..beta..sub.6-binding antibodies and/or
TGF-.beta.-blocking agents or fragments thereof to a patient, such
as those disclosed herein. When multiple
.alpha..sub..nu..beta..sub.6-binding ligands and/or
TGF-.beta.-blocking agents or fragments thereof are administered to
a patient, the different .alpha..sub..nu..beta..sub.6-binding
ligands and/or TGF-.beta.-blocking agents or fragments thereof can
be administered together in a single pharmaceutical composition,
or, more preferably, can be administered sequentially in separate
dosages. The effective amount of such other agents depends on the
amount of .alpha..sub..nu..beta..sub.6-binding ligand and/or
TGF-.beta.-blocking agents or fragments thereof present in the
formulation, the type of disease or disorder or treatment, and
other factors.
The present invention also includes methods for treating cancerous
or metastatic conditions that comprise administering to a patient a
first agent in conjunction with a second agent, wherein the first
agent is a .alpha..sub..nu..beta..sub.6-binding ligand and the
second agent is an agent that is useful for treating one or more
cancerous, metastatic or in situ carcinoma conditions but that is
not necessarily a .alpha..sub..nu..beta..sub.6-binding ligand. In
other aspects, the invention provides methods for treating
cancerous or metastatic conditions that comprise administering to a
patient a first agent in conjunction with a second agent, wherein
the first agent is a TGF-.beta.-blocking agent or fragment thereof
and the second agent is an agent that is useful for treating one or
more cancerous, metastatic or in situ carcinoma conditions but that
is not necessarily a TGF-.beta.-blocking agent. By administering a
first agent "in conjunction with" a second agent is meant that the
first agent can be administered to the patient prior to,
simultaneously with, or after, administering the second agent to
the patient, such that both agents are administered to the patient
during the therapeutic regimen. For example, according to certain
such embodiments of the invention, a
.alpha..sub..nu..beta..sub.6-binding ligand is administered to a
patient in conjunction (i.e., before, simultaneously with, or
after) administration of an antagonist of one or more other
integrin receptors (e.g., .alpha..sub.1.beta..sub.1,
.alpha..sub.4.beta..sub.1, .alpha..sub..nu..beta..sub.8,
.alpha..sub..nu..beta..sub.5, .alpha..sub.5.beta..sub.1, etc.) to
the patient, including antibodies, polypeptide antagonists and/or
small molecule antagonists specific for one or more integrin
receptors (e.g., .alpha..sub.1.beta..sub.1,
.alpha..sub.4.beta..sub.1, .alpha..sub..nu..beta..sub.8,
.alpha..sub..nu..beta..sub.5, .alpha..sub.5.beta..sub.1, etc.)
which are known in the art.
In certain embodiments of this aspect of the invention, the second
agent that is administered in conjunction with an
.alpha..sub..nu..beta..sub.6-binding ligand or TGF-.beta.-blocking
agents or fragments thereof is, e.g., a steroid, a cytotoxic
compound (including those described elsewhere herein, and
particularly paclitaxel, gemicitabine or adriamycin (doxorubicin),
a radioisotope (including those described elsewhere herein), a
prodrug-activating enzyme (including those described elsewhere
herein), colchicine, oxygen, an antioxidant (e.g.,
N-acetylcysteine), a metal chelator (e.g., terathiomolybdate),
IFN-.beta., IFN-.gamma., alpha-antitrypsin and the like. Additional
second agents or compounds that can be administered to a patient in
conjunction with one or more first agents, such as one or more
.alpha..sub..nu..beta..sub.6-binding ligands, for therapeutic
purposes according to this aspect of the invention, will be
familiar to those of ordinary skill in the art; the use of such
additional second agents or compounds is therefore considered to be
encompassed by the present invention.
Diagnostic Kits
In additional embodiments, the present invention provides kits,
particularly kits useful in diagnosis and/or prognosis of diseases
or disorders such as cancers. Kits according to this aspect the
present invention may comprise at least one container containing
one or more of the above-described ligands, such as antibodies,
that bind to or recognize integrin .alpha.v.beta.6. These kits of
the invention may optionally further comprise at least one
additional container which may contain, for example, a reagent
(such as a buffered salt solution) for delivering the ligand (e.g.,
antibody) to a test sample such as an organ, tissue or cell sample
from a patient; at least one additional container containing, for
example, a reagent (including but not limited to the nucleic acid
reagents described above) suitable for determining the expression
of smad4 in a biological sample; TGF-.beta.; one or more
detergents, lysis agents, or other solutions suitable for
preparation of a biological sample for testing; and the like. Other
suitable additional components of such kits of the invention will
be familiar to those of ordinary skill in the art.
It will be readily apparent to one of ordinary skill in the
relevant arts that other suitable modifications and adaptations to
the methods and applications described herein are obvious and may
be made without departing from the scope of the invention or any
embodiment thereof. Having now described the present invention in
detail, the same will be more clearly understood by reference to
the following examples, which are included herewith for purposes of
illustration only and are not intended to be limiting of the
invention.
EXAMPLES
Example 1
.alpha..sub..nu..beta..sub.6 is Highly Expressed in Metastases
Relative to Primary Tumors
In the present experiments, we set out to study the expression of
.alpha..sub..nu..beta..sub.6 in a variety of cancers of epithelial
origin and on metastatic lesions and to determine if function
blocking .alpha..sub..nu..beta..sub.6 mAbs could inhibit the growth
of .alpha..sub..nu..beta..sub.6 expressing tumors in vivo. We
evaluated the in vitro and in vivo anti-tumor activity of our
anti-human .alpha..sub..nu..beta..sub.6 mAbs on a human pharyngeal
carcinoma, Detroit62, and compared this to the in vivo anti-tumor
activity of TGF.beta.RII:Fc. Our data support a role in human
cancer for .alpha..sub..nu..beta..sub.6 and potential for
therapeutic intervention with a function-blocking
.alpha..sub..nu..beta..sub.6 mAb.
A. Materials and Methods
For immunohistochemistry, tissue sections were deparaffinized in
xylene and ethanol, rehydrated in distilled water, and then
immersed in methanol containing 0.45% H.sub.2O. Tissues were
incubated with pepsin (00-3009, Zymed, San Francisco, Calif.) and
blocked with avidin and biotin (SP-2001; Vector Laboratories,
Burlingame, Calif.). Primary antibody was diluted in
phosphate-buffered saline (PBS) containing 0.1% bovine serum
albumin (BSA) and tissues were incubated overnight at 4.degree. C.
For immunostaining .beta.6 on mouse xenograft tissue, sections were
incubated with a human/mouse chimeric form of the
anti-.alpha..sub..nu..beta..sub.6 mAb, 2A1 (Weinreb, P. H. et al.,
J. Biol. Chem. 279(17):17875-17887 (2004)), and an anti-human
biotinylated secondary antibody (PK-6103, Vector Laboratories,
Burlingame, Calif.). For immunostaining .beta..sub.6 on human
tissue, sections were incubated with murine 2A1 and an
anti-mouse-biotinylated secondary antibody (PK-6102, Vector
Laboratories). Avidin-biotin complex-horseradish peroxidase (Vector
Kit, PK-6102) was applied to sections, incubated for 30 minutes at
room temperature, and 3,3'-diaminobenzidine (DAB) substrate was
prepared as directed (SK-4100, Vector Laboratories) and applied to
sections for five minutes at room temperature. Tissue sections were
stained with Mayer's Hematoxylin for 1 minute and rinsed in water
and PBS.
B. Results
1. .alpha..sub..nu..beta..sub.6 Expression in Metastases
.alpha..sub..nu..beta..sub.6 immunostaining was evaluated on a
variety of tumor metastases. 78% of the metastases (43/55) were
positively immunostained showing intense staining over a majority
of the metastases (FIGS. 1A-F; FIGS. 2A-I). This result was found
to be an increase in percent positive immunostaining in that only
head and neck, cervical and pancreatic tumors were found to have an
equivalent level of expression (Table 2):
TABLE-US-00002 TABLE 2 a.sub.v.beta..sub.6 Expression in Human
Tumors (immunohistochemistry) Tissue
.alpha..sub.v.beta..sub.6.sup.+/total %
.alpha..sub.v.beta..sub.6.sup.+ Oral 4/4 100% Cervical 79/98 81%
Pancreas 31/39 80% Skin 41/53 77% Larynx & pharynx 43/64 67%
Esophageal 35/56 63% Endometrium 17/32 53% Lung 28/70 40% Breast
41/101 41% Colorectal 181/488 37%
2. .alpha..sub..nu..beta..sub.6 Expression in Human Pancreatic
Tumor Samples, Patient-Matched Metastases and Mouse Xenograft Model
of Invasive Pancreatic Tumor
As noted above in Table 2, .alpha..sub..nu..beta..sub.6
immunostaining was positive on 80% of pancreatic tumors examined.
When samples from primary pancreatic tumors from eight different
patients were examined by immunohistochemistry, staining was
prominent in invasive regions of high grade tumors (FIGS. 4A-4C;
5A-5E). The primary tumor samples also had matched lymph node
metastases (FIGS. 4D-4F; 5F-5J), which also demonstrated strong
.alpha..sub..nu..beta..sub.6 staining, supporting the notion that
.alpha..sub..nu..beta..sub.6-positive cells have disseminated from
the primary tumor site. In normal pancreas (FIGS. 4G-4H; 5A-5E),
staining was confined to occasional cells on the surface layer.
To further examine the influence of .alpha..sub..nu..beta..sub.6
expression on tumor cell invasion, we used a BxPC-3 mouse tumor
xenograft as a model of invasive human pancreatic adenocarcinoma.
Animals were implanted (day 0) subcutaneously on the flank with
5.times.10.sup.6 cells/mouse, suspended in sterile saline, using a
injection of 0.1 ml/mouse. At day 30, mice with established tumors
(.about.60-100 mm.sup.3) were pair-matched to each of three
treatment groups (PBS; mAb 3G9; soluble TGF-.beta. receptor II-Ig
fusion protein (solTGF.beta.RII -Fc)) for all studies. Test agents
were administered to mice intraperitoneally on a 3 times per week
treatment schedule. Mice were injected with 3G9 at 10 mg/kg,
solTGF.beta.RII-Fc at 2 mg/kg, or PBS (negative control). Tumor
growth was measured twice a week and tumor volume estimated
according to the formula: [(width).sup.2.times.length]/2. Tumors
from treatment groups were excised, fixed in 10% paraformaldehyde,
paraffin-embedded and sectioned for immunohistochemical analysis
using the non-blocking v6 chimeric mAb 6.2A1.
Treatment with anti-.alpha..sub..nu..beta..sub.6 mAb 3G9 had a
direct effect on tumor growth (FIGS. 6B, 6C), with significantly
reduced tumor growth observed after about 48 days of treatment with
the antibody. The level of growth inhibition observed with
solTGF.beta.RII-Fc was somewhat lower than that observed for 3G9.
These results indicate that the anti-.alpha..sub..nu..beta..sub.6
mAb 3G9 inhibits tumor growth in a xenograft model of human
pancreatic cancer, and suggest that such blocking antibodies could
be useful in inhibiting tumor growth, and by extension tumor
invasion, in primary human pancreatic adenocarcinomas.
Example 2
Inverse Relationship Between smad4 Gene Expression and Integrin
Expression in Primary Human Pancreatic Tumors
To examine the relationship between smad4 expression and
.alpha.v.beta.6 expression in primary tumor cells, sections of
human primary pancreatic tumors were obtained and stained via
immunohistochemistry for SMAD4 protein and for .alpha.v.beta.6.
Fourteen tumor samples from different patients were examined.
Results are shown in FIGS. 7-13.
Of the 14 tumor samples, 7 (50%) were found to have clearly
.alpha.v.beta.6+ tumor areas, while 3 of these showed heterogeneity
in expression (i.e., some sections of the tumor that were
.alpha.v.beta.6+, other sections in the same tumor that were
.alpha.v.beta.6-; FIGS. 8-9). Seven of the 14 tumors were found to
be .alpha.v.beta.6-negative.
In the 11 tumors (excluding the 3 that were heterogeneous in
.alpha.v.beta.6 expression), the following results were observed
FIGS. 7-10, 13:
(A) four were .alpha.v.beta.6+, and of these, three were smad4- and
one was smad4+; and
(B) seven were .alpha.v.beta.6-, and all seven of these were
smad4+.
Of the three tumors in which .alpha.v.beta.6 expression was
heterogeneous, the following results were observed (FIGS.
11-13):
(A) those areas of the tumors that were .alpha.v.beta.6+ were
smad4-; and
(B) those areas of the tumors that were .alpha.v.beta.6- were
smad4+.
These results support the conclusion that there is an inverse
relationship between the expression of smad4 and of integrin
.alpha.v.beta.6 by primary pancreatic tumor cells.
Example 3
Expression of .alpha.v.beta.6 and smad4 on Pancreatic Cancer Cell
Lines
Expression of .alpha..sub..nu..beta..sub.6 and smad4 were
determined for a panel of pancreatic cell lines. The results are
summarized in FIG. 14.
In a preliminary analyses of .alpha..sub..nu..beta..sub.6 and smad4
expression on primary pancreatic ductal adenocarcinomas (PDACs),
three sample sets were examined. In the first set, 10 PDAC samples
were tested by BioCat array, and all 10 (100%) were found to
express .alpha..sub..nu..beta..sub.6. In the second set, 14
pancreatic adenocarcinoma samples were tested by Cytomix assay, and
7 (50%) of these were .alpha..sub..nu..beta..sub.6 positive. In the
third set, 11 PDAC samples were tested by a Folio array (FIG. 15),
and of these 10 (91%) were .alpha.v.beta.6 positive.
For the second and third sample sets, smad4 expression was also
assayed. Fifteen lines were found to be .alpha.v.beta.6 positive,
and 11 of the lines were smad4 positive. Twelve lines were
.alpha.v.beta.6+/smad4-; 8 lines were .alpha.v.beta.6-/smad4+; and
3 lines were .alpha.v.beta.6+/smad4+. Two of the lines were
heterogeneous and had areas that stained as .alpha.v.beta.6+/smad4-
and .alpha.v.beta.6-/smad4+ (FIG. 16).
Seventy PDACs were examined by immunohistochemistry on Biomax
Tissue MicroArray (FIG. 17). 89% of these samples were determined
to be .alpha.v.beta.6 positive, 97% of which were smad4
downregulated. In total, 86% had a .alpha.v.beta.6+/smad4-
phenotype.
In a separate study of 50 PDACs examined by immunohistochemistry on
tissue arrays prepared at the Leiden University Medical Center
(LUMC) (hereafter, "Leiden arrays"), 88% were determined to be
.alpha.v.beta.6 positive, 59% of which were smad4 downregulated
(FIG. 18). In total, 52% had a .alpha.v.beta.6+/smad4-
phenotype.
In a study of 9 PDAC metastases examined using the Leiden arrays,
all were found to be .alpha.v.beta.6 positive (most were strongly
positive), and 78% had a .alpha.v.beta.6+/smad4- phenotype (FIG.
19).
The above results indicate that an .alpha.v.beta.6+/smad4-
phenotype is the predominant phenotype in primary human pancreatic
cancers.
A graphical representation of the results of .alpha.v.beta.6 and
smad4 expression analysis on PDAC as scored in the Leiden and
Biomax arrays is shown in FIG. 20. The differences in results
observed between the two different methods may be due to the lower
intensities of smad4 expression observed using the Biomax
arrays.
Example 4
Response of the Subcutaneously Implanted BxPC-3, Human Pancreatic
Adenocarcinoma, to .alpha..sub..nu..beta. mAb 3G9 and the
Chemotherapeutics, Adriamycin (Doxorubicin) and Gemcitabine, both
as Single Agents and in Combination
As a model for human tumor treatment, single or combination
chemotherapy was carried out in the BxPC-3 xenogeneic mouse model
of human pancreatic tumors. BxPC-3 is known to be smad4-deficient
due to an apparent homozygous deletion of the smad4 gene in these
cells (Subramanian, G. et al., Cancer Res. 64:5200-5211 (2004);
Yasutome, M. et al., Clin. Exp. Metastasis 22:461-473 (2005).
Materials and Methods:
1. Animals. 220 athymic nude female mice from Harlan Sprague Dawley
(Madison, Wis.), Rec. Mar. 13, 2006, DOB Jan. 30, 2006. Mice
started on study at 6-7 weeks of age. Animals are acclimated to the
lab for at least 5 days prior to implantation of tumor. Animals are
individually marked with BioMedic implantable ID chips. Data
collection performed using Onco Pharmacology Informatics (OPI).
2. Tumor. BXPC-3, cell line originally obtained from the University
of Arizona (Tucson, Ariz.). The cell line is a human pancreatic
adenocarcinoma. Donor line was passed for 5 generations in athymic
nude female mice prior to implantation into this study. Animals
implanted with a 3 mm.sup.3 fragment of tissue via trochar
subcutaneously into the right flank area. Treatments were initiated
on an established tumor with a minimum size of 100 milligrams and
three progressive growths.
TABLE-US-00003 Group # of # Treatment ** Mice 1. Vehicle Control,
Pyrogen-free PBS, 10 ml/kg/ini, i.p., 18 2x/wk plus 0.9% Sterile
Saline, 10 ml/kg/ini, i.v., Q4Dx3 2. 3G9, 10 mg/kg/inj., i.p.,
2x/wk 10 3. Gemcitabine, 100 mg/kg/inj, i.p., Q4Dx3 10 4.
Gemcitabine, 140 mg/kg/inj, i.p., Q4Dx3 10 5. Adriamycin
(doxorubicin), 3 mg/kg/inj, i.v., Q4Dx3 10 6. Adriamycin
(doxorubicin), 6 mg/kg/inj, i.v., Q4Dx3 10 7. 3G9, 10 mg/kg/inj.,
i.p., 2x/wk 10 plus Gemcitabine, 100 mg/kg/inj, i.p., Q4Dx3 8. 3G9,
10 mg/kg/inj., i.p., 2x/wk 10 plus Gemcitabine, 140 mg/kg/inj,
i.p., Q4Dx3 9. 3G9, 10 mg/kg/inj., i.p., 2x/wk 10 plus Adriamycin
(Dox), 3 mg/kg/inj, i.v., Q4Dx3 10. 3G9, 10 mg/kg/inj., i.p., 2x/wk
10 plus Adriamycin (Dox), 6 mg/kg/inj, i.v., Q4Dx3
3. Testing Schedule.
Day--1: Implant ID transponders subcutaneously in the left
flank.
Day 0: Implant tumor as described above. Run bacterial cultures on
the tumor implanted into mice. Record initial body weight of
animals.
Day2: Begin recording tumor size and body weight measurements and
continue every other day until staging day.
Staging Day (Treatment Day 1) (approximately Day 11-13 post
implantation): Select mice with tumors measuring a minimum size of
100 milligrams determined using vernier calipers. Randomize mice
into control and treatment groups and record body weights.
Administer i.p. doses on the schedule listed above. Doses are
blinded within each dosing regimen. Continue to record tumor size
and body weights on mice 2.times./week. Monitor the study daily and
make notations of any unusual observation on animals. Study will be
blinded until the termination of the study.
Midpoint Bleed: (Day 34/35 post implantation): Before first dose of
3G9 for the week collect serum from 5 animals/group from all
groups. 24 hours post dosing, collect serum from the remaining 5
animals/group from all groups. Use the 5 remaining animals/group
not used for the trough serum collection 24 hours earlier.
Final week of dosing (Day 44/45 post implantation): Before final
dose of 3G9 collect serum from 5 animals/group from all groups. 24
hours post dosing, collect serum from the remaining 5 animals/group
from all groups. Use the 5 remaining animals/group not used for the
trough serum collection 24 hours earlier.
Endpoints:
(a) Initial body weight
(b) Tumor size and body weight measurements twice weekly
(c) Serum 3G9 levels at midpoint and final week of dosing
At sacrifice:
7 tumors from each of the following groups are harvested; 1/2 of
each tumor preserved in 10% NBF, the other 1/2 frozen in OCT:
Group 1: Vehicle; Group 2: 3G9; Group 4: Gemcitabine (Gem) @140
mg/kg; Group 6: Doxorubicin (Dox) @ 6 mg/kg; Group 8: 3G9 plus Dox
@ 6 mg/kg; and Group 10: 3G9 plus Gem at 140 mg/kg.
Results
The results of these experiments are depicted in FIGS. 21-28. These
results demonstrate that 3G9 in combination with gemcitabine leads
to significantly increased growth inhibition, which seems to be
synergistic. The combined effect is even greater when 3G9 is
combined with doxorubicin (adriamycin), about 60% growth
inhibition. This combination chemotherapy was chosen because data
obtained earlier with this xenograft model by oncopharmacology (not
shown) showed that BxPC-3 was resistant to gemcitabine. Together,
these results demonstrate that the smad4-deficient cell line BxPC-3
is (a) responsive to treatment (measured as growth inhibition) with
anti-.alpha.v.beta.6 mAbs as single agents; and (b) is
chemosensitized by treatment with anti-.alpha.v.beta.6 mAbs to
subsequent (or simultaneous) treatment with small molecule agents
such as gemcitabine and doxorubicin.
Similar synergistic effects were observed in mice transplanted with
the pancreatic cancer cell lines Su86.6 (FIG. 29), Panc04 (FIG.
30), and Capan-2 (FIG. 31), when treated with a combination of 3G9
and gemcitabine (140 mg/kg). However, when mice transplanted with
the pancreatic cell line SW1990 were treated with the same
combination therapy, there was no significant difference in
therapeutic effect as compared with treatment with gemcitabine
alone (see FIG. 32C). When the gemcitabine concentration was
lowered to 100 mg/kg, treatment with gemcitabine alone was
significantly better than when with the combination therapy (FIG.
32C). When the gemcitabine concentration was decreased even further
to 80 mg/kg, treatment with the combination therapy was
significantly better than treatment with either gemcitabine or 3G9
alone (FIG. 32A), as observed with the other cell lines at the
higher gemcitabine concentration of 140 mg/kg. In a Capan-1
xenograft mode, a combination therapy of antibody 3G9 and
gemcitabine was only as effective as gemcitabine alone, and a
combination therapy of TGF-.beta. RII-Fc and gemcitabine (FIG.
33).
Gemcitabine and 3G9 significantly decreased tumor volume in an
orthotopic model of pancreatic cancer using cells from the cell
line ASPC-1. Treatment with gemcitabine alone or 3G9 alone did not
have a significant effect on tumor volume (FIG. 34).
Signaling studies were performed on treated xenograft tumors of
BxPC-3, Panc04 and Capan-2. Tumors were harvested at different
timepoints following treatment with 3G9 and gemcitabine for the
following analyses:
TABLE-US-00004 TABLE 3 Summary of signaling studies Read-out
Western IHC Tumor Interstitial Fluid Pressure Fibronectin X
Collagen I X SmaI X CD31 X Apoptosis Caspase X TUNEL X Survival
p-AKT X Proliferation p-ERK X Ki67 X Pathway p-smad-2 X FAK X
The results of the above studies are summarized in FIG. 35.
Example 5
Survival Phenotypes of a Cohort of 26 Patients of Leiden University
Medical Center
Survival data for patients diagnosed with pancreatic cancer is
shown in FIGS. 36 and 37. The four panels in FIGS. 36 and 37
express the data in terms of avb6 and smad4 expression.
Generally, patients with tumors that express smad4 survive longer
than patients with tumors that do not express smad4. Further,
patients with an .alpha.v.beta.6+/smad4- phenotype have a
significantly reduced survival.
Example 6
Reverse of Growth Inhibition of
smad4+/.alpha..sub..nu..beta..sub.6+ Tumor Cells by
anti-.alpha..sub..nu..beta..sub.6 Ligands
The human pancreatic cell line SW1990 was tested in the same
xenogeneic mouse system as described in Example 4. Results are
depicted in FIGS. 38-41.
The SW1990 cell line showed heterogeneity of .alpha.v.beta.6
expression. The cell line was sorted and enriched for
.alpha.v.beta.6 positive and .alpha.v.beta.6 negative cells (FIG.
41 C-E). Western blot analysis of the resulting variants showed
that the .alpha.v.beta.6 positive population was smad4+, while the
.alpha.v.beta.6 negative population was smad4- (FIG. 41A). Thus,
the .alpha.v.beta.6 positive SW1990 cell line does not fit the
profile of the BxPC-3 line in which we saw inhibition of tumor
growth by .alpha.v.beta.6 blocking mAb 3G9, especially in
combination with chemotreatment (Example 4). Since tissue sections
of the SW1990 xenograft showed .alpha.v.beta.6 expression (FIG.
41B), it is therefore expected that these cells in vivo are in fact
smad4+.
Mice implanted with SW1990 cells were then treated in vivo with
murine mAb 3G9 or with soluble TGF-.beta. RII-Fc as described in
Example 4, and were assessed for tumor growth over time. The
results are shown in FIGS. 39-41, which demonstrate that in
contrast to the pancreatic tumor line BxPC-3, SW1990 tumors are not
inhibited from growth by treatment with the anti-.alpha.v.beta.6
3G9 mAb, but instead demonstrate a slight increase in cell growth.
The inverse effect of treatment with 3G9 or sol TGF-.beta. RII on
growth in SW 1990 can be explained by reasoning that in the
presence of smad4, TGF-.beta. can induce a growth inhibitory
signal, which is abrogated when TGF-.beta. (activation) is blocked
by soluble TGF-.beta. RII and/or 3G9.
These results suggest that in order to be responsive to
.alpha.v.beta.6 ligands, tumor cells must be deficient in smad4
expression, since the smad4+ cell line SW 1990 was not
growth-inhibited by anti-.alpha.v.beta.6 mAbs, while the smad4-cell
line BxPC-3 was. These results thus are consistent with the
hypothesis and support the responsiveness in a selected set of
pancreatic cancer patients (smad4-/.alpha.v.beta.6+), indicating
that a useful clinical marker to predict the sensitivity or
responsiveness of tumor cells, particularly pancreatic tumor cells,
to .alpha.v.beta.6-active ligands or chemosenstitization therewith,
would be to determine the level of expression of smad4 in those
tumor cells.
Example 7
Growth Inhibition of Detroit 562 Human Pharyngeal Carcinoma Cells
by .alpha..sub..nu..beta..sub.6 mAbs
In a xenogeneic study, Detroit 562 human pharyngeal carcinoma cells
were implanted subcutaneously into mice according to the protocol
described in Example 4. Tumor growth was then assessed over time as
described in the earlier Examples. Results are shown in FIGS.
42-54. These results indicate that in the Detroit 562 model, murine
and humanized 3G9 anti-.alpha.v.beta.6 mAbs were able to
significantly inhibit the growth of tumors, while other
anti-.alpha.v.beta.6 mAbs (notably murine 4B4) were less
effective.
Example 8
Relevance of .alpha.v.beta.6 Blockade in Pancreatic Cancer
Smad4 is inactivated in about 55% of pancreatic adenocarcinomas
(Tascilar et al., Clin. Cancer Res. 4:4115-4121, 2001). Lohr et al.
(Cancer Res. 61:550-555, 2001) reported that TGF.beta. confers
desmoplastic potential in pancreatic cancer, which causes fibrosis.
This desmoplastic potential, however, correlated with smad4
expression. We observed that human pancreatic cell lines that were
smad4 null (AsPC-2, BxPC-3, Capan-1, and Capan-2), induced
desmoplasia when transplanted into mice, while cell lines that
carried wildtype smad4 (PaCa-2, PaCa-3, PaCa-44, and PANC-1) did
not induce desmoplasia (see Table 1 of Lohr et al.).
Summary of results: We have discovered that .alpha.v.beta.6 is
highly upregulated in pancreatic cancer. We also obtained efficacy
data with .alpha.v.beta.6 mAb (3G9) in vivo, determining single
agent efficacy in a pharyngeal cancer model and in two pancreatic
cancer models. We also observed that treatment with a combination
of 3G9 and gemcitabine demonstrated increased efficacy over
gemcitabine alone in 5 of 7 pancreatic xenograft models (FIG.
35).
Having now fully described the present invention in some detail by
way of illustration and example for purposes of clarity of
understanding, it will be obvious to one of ordinary skill in the
art that the same can be performed by modifying or changing the
invention within a wide and equivalent range of conditions,
formulations and other parameters without affecting the scope of
the invention or any specific embodiment thereof, and that such
modifications or changes are intended to be encompassed within the
scope of the appended claims.
All publications, patents and patent applications mentioned in this
specification are indicative of the level of skill of those skilled
in the art to which this invention pertains, and are herein
incorporated by reference to the same extent as if each individual
publication, patent or patent application was specifically and
individually indicated to be incorporated by reference.
SEQUENCE LISTINGS
1
33110PRTArtificialSynthetic 1Ser Tyr Thr Phe Thr Asp Tyr Ala Met
His 1 5 10 210PRTArtificialSynthetic 2Ser Tyr Thr Phe Thr Asp Tyr
Thr Met His 1 5 10 310PRTArtificialSynthetic 3Gly Phe Thr Phe Ser
Arg Tyr Val Met Ser 1 5 10 410PRTArtificialSynthetic 4Gly Tyr Asp
Phe Asn Asn Asp Leu Ile Glu 1 5 10 510PRTArtificialSynthetic 5Gly
Tyr Ala Phe Thr Asn Tyr Leu Ile Glu 1 5 10
617PRTArtificialSynthetic 6Val Ile Ser Thr Tyr Tyr Gly Asn Thr Asn
Tyr Asn Gln Lys Phe Lys 1 5 10 15 Gly 717PRTArtificialSynthetic
7Val Ile Asp Thr Tyr Tyr Gly Lys Thr Asn Tyr Asn Gln Lys Phe Glu 1
5 10 15 Gly 816PRTArtificialSynthetic 8Ser Ile Ser Ser Gly Gly Ser
Thr Tyr Tyr Pro Asp Ser Val Lys Gly 1 5 10 15
916PRTArtificialSynthetic 9Ser Ile Ser Ser Gly Gly Arg Met Tyr Tyr
Pro Asp Thr Val Lys Gly 1 5 10 15 1017PRTArtificialSynthetic 10Val
Ile Asn Pro Gly Ser Gly Arg Thr Asn Tyr Asn Glu Lys Phe Lys 1 5 10
15 Gly 1117PRTArtificialSynthetic 11Val Ile Ser Pro Gly Ser Gly Ile
Ile Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 Gly
1217PRTArtificialSynthetic 12Gly Gly Leu Arg Arg Gly Asp Arg Pro
Ser Leu Arg Tyr Ala Met Asp 1 5 10 15 Tyr
1317PRTArtificialSynthetic 13Gly Gly Phe Arg Arg Gly Asp Arg Pro
Ser Leu Arg Tyr Ala Met Asp 1 5 10 15 Ser
1412PRTArtificialSynthetic 14Gly Ala Ile Tyr Asp Gly Tyr Tyr Val
Phe Ala Tyr 1 5 10 1512PRTArtificialSynthetic 15Gly Ser Ile Tyr Asp
Gly Tyr Tyr Val Phe Pro Tyr 1 5 10 1612PRTArtificialSynthetic 16Ile
Tyr Tyr Gly Pro His Ser Tyr Ala Met Asp Tyr 1 5 10
1711PRTArtificialSynthetic 17Ile Asp Tyr Ser Gly Pro Tyr Ala Val
Asp Asp 1 5 10 1815PRTArtificialSynthetic 18Arg Ala Ser Gln Ser Val
Ser Thr Ser Ser Tyr Ser Tyr Met Tyr 1 5 10 15
1915PRTArtificialSynthetic 19Arg Ala Ser Gln Ser Val Ser Ile Ser
Thr Tyr Ser Tyr Ile His 1 5 10 15 2012PRTArtificialSynthetic 20Ser
Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu Tyr 1 5 10
2112PRTArtificialSynthetic 21Ser Ala Asn Ser Ser Val Ser Ser Ser
Tyr Leu Tyr 1 5 10 2211PRTArtificialSynthetic 22Lys Ala Ser Leu Asp
Val Arg Thr Ala Val Ala 1 5 10 2311PRTArtificialSynthetic 23Lys Ala
Ser Gln Ala Val Asn Thr Ala Val Ala 1 5 10
247PRTArtificialSynthetic 24Tyr Ala Ser Asn Leu Glu Ser 1 5
257PRTArtificialSynthetic 25Ser Thr Ser Asn Leu Ala Ser 1 5
267PRTArtificialSynthetic 26Ser Ala Ser Tyr Arg Tyr Thr 1 5
277PRTArtificialSynthetic 27Ser Ala Ser Tyr Gln Tyr Thr 1 5
289PRTArtificialSynthetic 28Gln His Asn Trp Glu Ile Pro Phe Thr 1 5
299PRTArtificialSynthetic 29Gln His Ser Trp Glu Ile Pro Tyr Thr 1 5
309PRTArtificialSynthetic 30His Gln Trp Ser Ser Tyr Pro Pro Thr 1 5
319PRTArtificialSynthetic 31His Gln Trp Ser Thr Tyr Pro Pro Thr 1 5
329PRTArtificialSynthetic 32Gln Gln His Tyr Gly Ile Pro Trp Thr 1 5
339PRTArtificialSynthetic 33Gln His His Tyr Gly Val Pro Trp Thr 1
5
* * * * *