U.S. patent application number 11/832570 was filed with the patent office on 2009-05-07 for methods and compositions for inhibition of metastasis.
This patent application is currently assigned to Scripps Research Institute. Invention is credited to Brunhilde Felding-Habermann, Kim D. Janda, Alan Saven.
Application Number | 20090117096 11/832570 |
Document ID | / |
Family ID | 35056641 |
Filed Date | 2009-05-07 |
United States Patent
Application |
20090117096 |
Kind Code |
A1 |
Felding-Habermann; Brunhilde ;
et al. |
May 7, 2009 |
METHODS AND COMPOSITIONS FOR INHIBITION OF METASTASIS
Abstract
This invention generally relates to methods of producing an
antibody phage population having affinity for a tumor cell target
expressing a metastatic phenotype. The invention further relates to
antibody compositions that specifically bind to a cell surface
receptor on the metastatic cell.
Inventors: |
Felding-Habermann; Brunhilde;
(San Diego, CA) ; Janda; Kim D.; (La Jolla,
CA) ; Saven; Alan; (Del Mar, CA) |
Correspondence
Address: |
FENWICK & WEST LLP
SILICON VALLEY CENTER, 801 CALIFORNIA STREET
MOUNTAIN VIEW
CA
94041
US
|
Assignee: |
Scripps Research Institute
|
Family ID: |
35056641 |
Appl. No.: |
11/832570 |
Filed: |
August 1, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11056825 |
Feb 11, 2005 |
7271245 |
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11832570 |
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60626726 |
Nov 10, 2004 |
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60544807 |
Feb 13, 2004 |
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Current U.S.
Class: |
424/130.1 ;
435/320.1; 435/325; 530/387.1; 536/23.53 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 2317/73 20130101; A61P 35/02 20180101; C07K 2317/21 20130101;
C07K 2317/34 20130101; A61P 13/08 20180101; C07K 16/2848 20130101;
A61P 35/04 20180101; A61K 2039/505 20130101; C07K 2317/622
20130101 |
Class at
Publication: |
424/130.1 ;
530/387.1; 536/23.53; 435/320.1; 435/325 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/00 20060101 C07K016/00; C07H 21/04 20060101
C07H021/04; C12N 15/63 20060101 C12N015/63; C12N 5/00 20060101
C12N005/00 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with Government support by Grant
Nos. NCI-CA95458 and NIAID-AI147027, awarded by the National
Institutes of Health and Grant No. DAMD 17-99-1-9368, awarded by
the U.S. Army Breast Cancer Research Program. The Government has
certain rights in this invention.
Claims
1. An isolated antibody which specifically binds to an activated
.alpha.v.beta.3 integrin receptor which is differentially produced
on a cell in a metastatic state compared to a similar,
non-metastatic cell.
2. (canceled)
3. The antibody of claim 1 comprising SEQ ID NO: 4.
4. The antibody of claim 1 comprising an R-G-D sequence in a
complementary determining region (CDR).
5. The antibody of claim 4, wherein the complementary determining
region is CDR-H3.
6. The antibody of claim 1, wherein said metastatic cell targets to
a tissue selected from breast, brain, lung, liver, or bone.
7. A pharmaceutical composition comprising said antibody of claim
1.
8. An isolated antibody which specifically binds to an activated
.alpha.v.beta.3 integrin receptor and does not bind to a
non-activated .alpha.v.beta.3 integrin receptor.
9. The antibody of claim 8, wherein the activated .alpha.v.beta.3
integrin receptor is differentially produced on a cell in a
metastatic state compared to a similar, non-metastatic cell.
10. The antibody of claim 8, wherein the antibody is a ligand
mimetic.
11. A method for treating a disease state in a mammal comprising
administering to the mammal an isolated antibody which specifically
binds to an activated .alpha.v.beta.3 integrin receptor which is
differentially produced on a cell in a metastatic state as compared
to a similar, non-metastatic cell.
12. The method of claim 11, wherein the disease state is neoplastic
disease, solid tumor, hematological malignancy, leukemia,
colorectal cancer, benign or malignant breast cancer, uterine
cancer, uterine leiomyomas, ovarian cancer, endometrial cancer,
polycystic ovary syndrome, endometrial polyps, prostate cancer,
prostatic hypertrophy, pituitary cancer, adenomyosis,
adenocarcinomas, meningioma, melanoma, bone cancer, multiple
myeloma, CNS cancer, glioma, or astroblastoma.
13. The method of claim 12, wherein the neoplastic disease is tumor
cell metastasis in said mammal.
14. The method of claim 13, wherein the disease state is breast
cancer metastasis in said mammal.
15. The method of claim 11, wherein the antibody comprises SEQ ID
NO: 4.
16. An isolated Bc-12 polynucleotide comprising a nucleotide
sequence that has at least 90% identity to SEQ ID NO: 1.
17. An isolated polypeptide comprising an amino acid sequence that
has at least 90% sequence identity to SEQ ID NO: 2 and shares a
biological function with Bc-12 scFv polypeptide encoded by Bc-12
polynucleotide deposited as ATCC No. PTA-6303.
18. A vector comprising the polynucleotide of claim 16.
19. An expression vector comprising the polynucleotide of claim 16
in which the nucleotide sequence of the polynucleotide is
operatively linked with a regulatory sequence that controls
expression of the polynucleotide in a host cell.
20. A host cell comprising the polynucleotide of claim 16, or
progeny of the cell.
21. An isolated Bc-15 polynucleotide comprising a nucleotide
sequence that has at least 90% identity to SEQ ID NO: 3.
22. An isolated polypeptide comprising an amino acid sequence that
has at least 90% sequence identity to SEQ ID NO: 4 and shares a
biological function with Bc-15 scFv polypeptide encoded by Bc-15
polynucleotide deposited as ATCC No. PTA-6304.
23. A vector comprising the polynucleotide of claim 21.
24. An expression vector comprising the polynucleotide of claim 21
in which the nucleotide sequence of the polynucleotide is
operatively linked with a regulatory sequence that controls
expression of the polynucleotide in a host cell.
25. A host cell comprising the polynucleotide of claim 21, or
progeny of the cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Application No.
60/544,807, filed Feb. 13, 2004, and U.S. Application No.
60/626,726, filed Nov. 10, 2004, the entire disclosures of which
are incorporated herein by reference.
FIELD
[0003] This invention generally relates to methods of producing an
antibody phage population having affinity for a tumor cell target
expressing a metastatic phenotype. The invention further relates to
antibody compositions that specifically bind to a cell surface
receptor on the metastatic cell.
BACKGROUND
[0004] Breast cancer metastasis to lungs, liver, bone and brain is
the primary cause of death in breast cancer patients. It involves
cancer cell dissemination via the blood stream and depends on
adhesive and invasive tumor cell functions and their ability to
survive and proliferate at the target site. Bogenrieder et al.,
Oncogene 22: 6524-6536, 2003. These events are supported by
integrins, a family of transmembrane adhesion receptors composed of
.alpha. and .beta. subunits. Felding-Habermann, Clin. Exp.
Metastasis 20: 203-213, 2003; Hood et al., Nat. Rev. Cancer 2:
91-100, 2002. Integrins exist in distinct states of activation,
which determine the affinity for ligand and regulate whether
soluble ligands are bound, which matrix proteins are recognized,
and the degree to which cells can adhere, migrate and arrest under
dynamic flow conditions as found in the circulation. Tadokoro et
al., Science 302: 103-106, 2003; Shattil et al., J. Clin. Invest.
100:1-5, 1997; Felding-Habermann, et al., Proc. Natl. Acad. Sci. U.
S. A 98: 1853-1858, 2001.
[0005] An integrin found on breast cancer cells, but not on normal
breast epithelium is .alpha.v.beta.3. Expressio[n of this receptor
correlates with invasion and cancer progression. Liapis et al.,
Diagn. Mol. Pathol. 5: 127-135, 1996; Pignatelli et al., Hum.
Pathol. 23: 1159-1166, 1992. Human breast cancer cells can express
.alpha.v.beta.3 in an activated or a non-activated functional
state. Activated .alpha.v.beta.3 supports breast cancer cell
attachment during blood flow, and strongly promotes invasive tumor
cell migration. In particular, only the activated state supports
target organ colonization by circulating breast cancer cells in a
mouse model, and metastatic cells isolated from breast cancer
patient blood express .alpha.v.beta.3 in a constitutively activated
form. Felding-Habermann et al.; Proc. Natl. Acad. Sci. U.S.A 98:
1853-1858, 2001; Rolli et al., Proc. Natl. Acad. Sci. U.S. A 100:
9482-9487, 2003. Integrin transition between distinct states of
activation is associated with conformational changes within the
heterodimer. Calzada et al., J. Biol. Chem. 277: 39899-39908, 2002;
Beglova et al., Nat. Struct. Biol. 9: 282-287, 2002; Xiong et al.,
Science 294: 339-345, 2001; Xiong et al., Science 296: 151-155,
2002; Pampori et al., J. Biol. Chem. 274: 21609-21616, 1999.
[0006] Like other integrins, .alpha.v.beta.3 can exist in distinct
states of activation and functional affinity. The activated or high
affinity state of .alpha.v.beta.3 has a unique molecular
conformation that is distinct from the non-activated state. Xiong
et al., Science, 294: 339-345, 2001; Xiong et al., Science, 296:
151-155, 2002; Xiong et al., Blood, 102: 1155-1159, 2003.
[0007] In contrast to the non-activated receptor, activated
.alpha.v.beta.3 is functionally characterized primarily through its
ability to bind soluble ligand proteins, to support tumor cell
interaction with platelets during blood flow and thereby mediated
tumor cell arrest under conditions as found in the vasculature, and
to promote invasive tumor cell and endothelial cell migration very
strongly. The latter is probably very important for angiogenesis.
The ability of the activated .alpha.v.beta.3 integrin to bind
soluble ligands is a key property that enables the ligand-mimetic
scFv antibodies to bind specifically and exclusively to the
activated conformation of .alpha.v.beta.3.
[0008] No therapy is known today that prevents cancer, for example,
breast cancer, from becoming systemic, and there is little
understanding of even how to design and test such drugs; yet
metastases ultimately are responsible for much of the suffering and
mortality from breast cancer. A need exists to identify and target
molecular and functional markers that identify metastatic breast
cancer cells and to generate reagents for their specific
inhibition.
SUMMARY
[0009] The invention is generally related to methods of producing
an antibody phage population having affinity for a tumor cell
target which is a tumor cell expressing a metastatic phenotype. The
tumor cell expressing the metastatic phenotype can be a cell line
expressing an activated cell surface receptor, for example, an
activated integrin receptor or an .alpha.v.beta.3 integrin
receptor. The invention further relates to an antibody composition
that specifically binds to a cell surface receptor on a metastatic
cell. The antibody composition specifically binds to an activated
cell surface receptor on a metastatic cell, for example, an
activated integrin receptor or an .alpha.v.beta.3 integrin
receptor. The invention further relates to methods for alleviating
a disease state in a mammal by treatment with a cancer therapeutic
comprising the step of administering to the mammal a therapeutic
amount of the pharmaceutical composition of the antibody
composition. The invention further relates to methods of detecting
an activated cell surface receptor on a metastatic tumor cell
surface in a mammalian tissue sample and to methods of identifying
cells liable to undergo metastasis associated with a disease state
comprising contacting a patient suspected of being at risk for
metastasis with the antibody composition, the antibody having
associated therewith an imaging moiety.
[0010] Cells or tumor cells with a non-activated .alpha.v.beta.3
integrin receptor refers to a conformation of .alpha.v.beta.3 that
is unable to bind soluble ligands and does not support tumor cell
arrest under dynamic flow conditions as during blood flow under
conditions found in the vasculature. This non-activated
conformation of .alpha.v.beta.3 can be associated with
non-metastatic tumor cells and it is not recognized by an antibody,
for example, scFv antibodies Bc-12 or Bc-15.
[0011] In one embodiment, an antibody which specifically binds to
an activated .alpha.v.beta.3 integrin receptor which is
differentially produced on a cell in a metastatic state compared to
a similar, non-metastatic cell. In a detailed embodiment, the
antibody comprises scFv antibody Bc-12. In a further detailed
embodiment, the antibody comprises SEQ ID NO: 2. In a detailed
embodiment, the antibody comprises scFv antibody Bc-15. In a
further detailed embodiment, the antibody comprises SEQ ID NO: 4.
In a further embodiment, the antibody comprises an R-G-D sequence
in a complementary determining region (CDR). In a detailed aspect,
the CDR can be CDR-H3. In a further detailed aspect, the metastatic
cell targets to a tissue selected from breast, brain, lung, liver,
or bone. In a further detailed aspect, a pharmaceutical composition
comprises the antibody.
[0012] The present invention further provides a cDNA encoding scFv
Bc-12 in a phage display vector for expression and production of
scFv antibody Bc-12. In a detailed embodiment, the cDNA encoding
scFv Bc-12 comprises SEQ ID NO: 1. The cDNA encoding scFv Bc-12 has
an ATCC accession number PTA-6303, date of deposit: Nov. 12, 2004.
The present invention further provides a cDNA encoding scFv Bc-15
in a phage display vector for expression and production of scFv
antibody Bc-15. In a detailed embodiment, the cDNA encoding scFv
Bc-15 comprises SEQ ID NO: 2. The cDNA encoding scFv Bc-15 has an
ATCC accession number PTA-6304, date of deposit: Nov. 12, 2004.
[0013] In another embodiment, an antibody specifically binds to an
activated .alpha.v.beta.3 integrin receptor and does not bind to a
non-activated .alpha.v.beta.3 integrin receptor. In a further
aspect, the activated .alpha.v.beta.3 integrin receptor is
differentially produced on a cell in a metastatic state compared to
a similar, non-metastatic cell.
[0014] In another embodiment, an antibody comprises a ligand
mimetic which specifically binds to an activated .alpha.v.beta.3
integrin receptor which is differentially produced on a cell in a
metastatic state compared to a similar, non-metastatic cell.
[0015] In another embodiment, a method for treating a disease state
in a mammal comprises administering to the mammal an antibody which
specifically binds to an activated .alpha.v.beta.3 integrin
receptor which is differentially produced on a cell in a metastatic
state as compared to a similar, non-metastatic cell. In a detailed
aspect, the disease state is neoplastic disease, solid tumor,
hematological malignancy, leukemia, colorectal cancer, benign or
malignant breast cancer, uterine cancer, uterine leiomyomas,
ovarian cancer, endometrial cancer, polycystic ovary syndrome,
endometrial polyps, prostate cancer, prostatic hypertrophy,
pituitary cancer, adenomyosis, adenocarcinomas, meningioma,
melanoma, bone cancer, multiple myeloma, CNS cancer, glioma, or
astroblastoma. In a further detailed aspect, the neoplastic disease
is tumor cell metastasis in the mammal or the neoplastic disease
state is breast cancer metastasis in the mammal.
[0016] In a detailed embodiment, a method for treating a disease
state in a mammal comprises administering to the mammal an antibody
comprising SEQ ID NO: 2 or SEQ ID NO: 4. In a detailed aspect, the
disease state is neoplastic disease, solid tumor, hematological
malignancy, leukemia, colorectal cancer, breast cancer, uterine
cancer, uterine leiomyomas, ovarian cancer, endometrial cancer,
polycystic ovary syndrome, endometrial polyps, prostate cancer,
prostatic hypertrophy, pituitary cancer, adenomyosis,
adenocarcinomas, meningioma, melanoma, bone cancer, multiple
myeloma, CNS cancer, glioma, or astroblastoma. In a further
detailed aspect, the neoplastic disease is tumor cell metastasis in
the mammal or the neoplastic disease state is breast cancer
metastasis in the mammal.
[0017] In another embodiment, a cell line comprises a tumor cell
variant with a metastatic homing propensity to a target tissue. In
a further aspect, the tumor cell variant is derived from solid
tumor, hematological malignancy, leukemia, colorectal cancer,
breast cancer, uterine cancer, uterine leiomyomas, ovarian cancer,
endometrial cancer, polycystic ovary syndrome, endometrial polyps,
prostate cancer, prostatic hypertrophy, pituitary cancer,
adenomyosis, adenocarcinomas, meningioma, melanoma, bone cancer,
multiple myeloma, CNS cancer, glioma, or astroblastoma. In a
further aspect, the target tissue is selected from brain, liver,
lung, or bone.
[0018] In another embodiment a method of producing an antibody
phage population having affinity for a tumor cell target, comprises
providing a phage library derived from a blood lymphocyte cDNA
library from a cohort of cancer patients, subtracting the phage
library on a cell line expressing a non-metastatic phenotype,
selecting a first antibody phage population that do not bind to the
cell line expressing the non-metastatic phenotype, panning the
first antibody phage population on a cell line expressing a
metastatic phenotype, selecting a second antibody phage population
that binds to the cell line expressing the metastatic phenotype,
purifying an antibody phage clone that binds to the cell line
expressing the metastatic phenotype and binds to the tumor cell
target. In a further aspect, the cell line expressing the
metastatic phenotype is a cell line expressing an activated cell
surface receptor. In a further aspect, the activated cell surface
receptor is an activated integrin receptor. In a detailed aspect,
the activated cell surface receptor is an .alpha.v.beta.3 integrin
receptor. In a further aspect, the cell line expressing the
metastatic phenotype is a tumor cell variant with a metastatic
homing propensity to a target tissue. In a further aspect, the
target tissue is selected from brain, liver, lung, or bone. In a
further detailed aspect, the tumor cell target is a metastatic
cell. In a further detailed aspect, the metastatic cell is a
metastatic breast tumor cell. In a further detailed aspect, the
antibody phage population is a single chain (scFv) antibody phage
population.
[0019] In a further embodiment the method further comprises testing
the antibody phage clone for (a) binding to tumor cells expressing
activated integrin receptor on a cell surface; and (b) reduced
binding to tumor cells expressing non-activated integrin receptor
on a cell surface.
[0020] In a further embodiment the method further comprises
measuring binding efficiency of the antibody phage population for
the cell line expressing activated integrin receptor increased in
the presence of cations selected from Ca.sup.++, Mg.sup.++, or
Mn.sup.++.
[0021] In another embodiment, a method of detecting tumor cells in
a mammal by treatment with a cancer therapeutic comprises linking a
detectable marker to an antibody composition that specifically
binds to an activated integrin receptor, contacting the detectable
marker-antibody composition complex to the mammal or a mammalian
tissue, detecting binding of the detectable marker-antibody
composition complex to the tumor cell in the mammalian tissue. In a
detailed aspect, the tumor cells are metastatic tumor cells.
[0022] In another embodiment, a method for inducing or enhancing an
immune response to an antigen in a mammal comprises administering
to the mammal an antibody to a cell surface receptor on a
metastatic cell such that plasma concentration of the anti-cell
surface receptor antibody is maintained above detectable levels for
at least four months. In a further embodiment, the cell surface
receptor is an activated integrin receptor. In a further
embodiment, the activated integrin receptor is an .alpha.v.beta.3
integrin receptor. In a further aspect, the anti-cell surface
receptor antibody is administered multiple times such that plasma
concentration is maintained above detectable levels for at least
four months. In a further aspect, the anti-cell surface receptor
antibody is administered in an amount and at intervals such that
the plasma concentration of the anti-integrin receptor antibody in
the mammal is at least 2 .mu.g/ml for at least four months, or at
least 5 .mu.g/ml for at least four months or at least 10 .mu.g/ml
for at least four months. In a detailed aspect, the mammal is a
human. In a further detailed aspect, the antibody to an activated
integrin receptor is a human anti-activated integrin receptor
antibody. In a further detailed aspect, the antibody to an
activated integrin receptor is a humanized anti-activated integrin
receptor antibody. In a further detailed aspect, the antibody to an
activated integrin receptor is a human sequence anti-activated
integrin receptor antibody.
[0023] In another embodiment, a method for treating a mammal for a
metastatic cancer disease, comprises administering to the mammal an
antibody to a cell surface receptor on a metastatic cell linked to
a cytotoxic agent such that the mammal is treated for the
metastatic cancer disease. In a further embodiment, the cell
surface receptor is an activated integrin receptor. In a further
aspect, the cytotoxic agent is a cytotoxic drug. In a further
aspect, the cytotoxic agent is a radioactive isotope.
[0024] In another embodiment, a method of detecting an activated
cell surface receptor on a metastatic tumor cell surface in a
mammalian tissue sample, comprises contacting the mammalian tissue
with a first human antibody immobilized to a solid phase, and a
second human antibody in solution, wherein the first and second
antibodies bind to different epitopes of the activated cell surface
receptor if present in the tissue sample; detecting binding of the
activated cell surface receptor to the first and second antibodies,
binding indicating presence of the activated cell surface receptor
in the tissue sample; wherein the first and second human antibodies
are produced by subcloning nucleic acids encoding the first and
second human antibodies provided by a phage library derived from a
blood lymphocyte cDNA library from a cohort of cancer patients,
subtracting the phage library on a cell line expressing a
non-metastatic phenotype; selecting a first antibody phage
population that do not bind to the cell line expressing the
non-metastatic phenotype; panning the first antibody phage
population on a cell line expressing a metastatic phenotype;
selecting a second antibody phage population that binds to the cell
line expressing the metastatic phenotype; purifying an antibody
phage clone that binds to the cell line expressing the metastatic
phenotype and binds to the tumor cell target.
[0025] In a detailed embodiment, the activated cell surface
receptor is an activated integrin receptor. In another detailed
embodiment, the activated integrin receptor is an .alpha.v.beta.3
integrin receptor. In another embodiment, the second antibody is
labelled and wherein the detecting step detects binding of the
second antibody to the activated cell surface receptor.
[0026] In another further embodiment, the tissue sample is
contacted with a first population of human antibodies immobilized
to the solid phase and a second population of human antibodies in
solution, wherein members from the first and second populations
bind to different epitopes on the activated cell surface receptor.
In another detailed aspect, the second population of human
antibodies is labeled. In another further aspect, the first and
second human antibodies each have an affinity of at least 10.sup.9
M.sup.-1 for their respective epitopes on the activated cell
surface receptor. In another detailed aspect, the first and second
human antibodies each have an affinity of at least 10.sup.10
M.sup.-1 for the activated cell surface receptor. In another
detailed aspect, the first and second human antibodies have an
affinity of a least 10.sup.11 M.sup.-1 for the activated cell
surface receptor. In a further embodiment, the binding of the first
and second human antibodies to the activated cell surface receptor
reaches equilibrium within an hour. In another detailed embodiment,
the first and second human antibodies were produced by expression
by expression of recombinant constructs in E. coli. In another
detailed aspect, at least 90% of molecules of the first and second
antibody are immunoreactive with the activated cell surface
receptor.
[0027] In a further embodiment, a method of interfering with cells
liable to undergo metastasis associated with a disease state
comprises contacting a patient suspected of being at risk for
metastasis with an antibody which specifically binds to an
activated .alpha.v.beta.3 integrin receptor which is differentially
produced on a cell in a metastatic state compared to a similar,
non-metastatic cell, the antibody having associated therewith a
cytotoxic moiety. In a detailed embodiment, the antibody
composition comprises SEQ ID NO: 2. In a further detailed
embodiment, the antibody composition comprises SEQ ID NO: 4. In
another detailed embodiment, the cytotoxic moiety is a chemical
toxin. In further detailed embodiment, the cytotoxic moiety is a
biological toxin. In another detailed embodiment, the cytotoxic
moiety is a radioactive agent. In further detailed embodiment, the
association is a covalent bond. In another detailed embodiment, the
association is a ligand interaction. In a further detailed
embodiment, the association is a physical interaction. In a further
detailed embodiment, the association comprises containment within a
vessel. In another detailed embodiment, the vessel is a liposome or
other blood circulating vessel.
[0028] In a further embodiment, a method of identifying cells
liable to undergo metastasis associated with a disease state
comprises contacting a patient suspected of being at risk for
metastasis with an antibody which specifically binds to an
activated .alpha.v.beta.3 integrin receptor which is differentially
produced on a cell in a metastatic state compared to a similar,
non-metastatic cell, the antibody having associated therewith an
imaging moiety. In a further embodiment, the imaging moiety can be
imaged through magnetic resonance spectroscopy, X-ray spectroscopy,
or positron emission tomography (PET). In a detailed embodiment,
the association is a covalent bond. In a further detailed
embodiment, the association is a non-covalent bond.
[0029] A method for treating a mammal for a metastatic cancer
disease is provided which comprises administering to the mammal an
antibody to a cell surface receptor on a metastatic cell, and
inducing programmed cell death of the metastatic cell, such that
the mammal is treated for said metastatic cancer disease. In one
aspect, the cell surface receptor is an activated integrin
receptor. In a further aspect, the activated integrin receptor is
an .alpha.v.beta.3 integrin receptor.
[0030] The present invention provides an isolated Bc-12
polynucleotide comprising a nucleotide sequence that has at least
90% percent identity to SEQ ID NO: 1. The present invention further
provides an isolated polypeptide comprising a nucleotide sequence
that has at least 90% sequence identity to SEQ ID NO: 1 or shares a
biological function with Bc-12. In a detailed aspect, a vector
comprises the isolated Bc-12 polynucleotide with at least 90%
percent identity to SEQ ID NO: 1. In a detailed aspect, an
expression vector comprising the isolated Bc-12 polynucleotide with
at least 90% percent identity to SEQ ID NO: 1 in which the
nucleotide sequence of the polynucleotide is operatively linked
with a regulatory sequence that controls expression of the
polynucleotide in a host cell. In a detailed aspect, a host cell
comprising the isolated Bc-12 polynucleotide with at least 90%
percent identity to SEQ ID NO: 1, or progeny of the cell. In a
further aspect, an isolated Bc-12 polypeptide comprises the amino
acid sequence that has at least 90% identity to SEQ ID NO: 2.
[0031] The present invention provides an isolated Bc-15
polynucleotide comprising a nucleotide sequence that has at least
90% percent identity to SEQ ID NO: 3. The present invention further
provides an isolated polypeptide comprising a nucleotide sequence
that has at least 90% sequence identity to SEQ ID NO: 3 or shares a
biological function with Bc-15. In a detailed aspect, a vector
comprises the isolated Bc-15 polynucleotide with at least 90%
percent identity to SEQ ID NO: 3. In a detailed aspect, an
expression vector comprising the isolated Bc-15 polynucleotide with
at least 90% percent identity to SEQ ID NO: 3 in which the
nucleotide sequence of the polynucleotide is operatively linked
with a regulatory sequence that controls expression of the
polynucleotide in a host cell. In a detailed aspect, a host cell
comprising the isolated Bc-15 polynucleotide with at least 90%
percent identity to SEQ ID NO: 3, or progeny of the cell. In a
further aspect, an isolated Bc-15 polypeptide comprises the amino
acid sequence that has at least 90% identity to SEQ ID NO: 4.
[0032] A method for determining anti-metastatic activity of a test
compound in a mammal is provided which comprises administering to
the mammal a tumor cell variant with a metastatic homing propensity
to a target tissue, administering the test compound to the mammal,
measuring anti-metastatic activity of the test compound in the
mammal compared to anti-metastatic activity of a control compound
in a control mammal. In a further embodiment the method comprises
measuring metastatic foci in the target tissue of the mammal,
wherein a reduction in metastatic foci in the mammal in response to
the test compound compared to metastatic foci in a control animal
in response to a control compound indicates the anti-metastatic
activity of the test compound. In one aspect, the tumor cell
variant and the test compound are administered to the peripheral
blood circulation of the mammal. In a further aspect, the tumor
cell variant and the test compound are administered orthotopically
into the mammary fat pad of the mammal. In a detailed aspect, the
tumor cell variant is BCM-1, BCM-2 or BMS. In a further aspect, the
test compound is an antibody, a single chain Fv antibody, a small
molecule, an antisense oligonucleotide, double stranded RNA
molecule, short interfering RNA (siRNA,) or short hairpin RNA
(shRNA). In a further embodiment, the mammal is a non-human mammal,
for example, a rodent, rabbit, canine, feline, or non-human
primate. In a further detailed embodiment the mammal is a mouse or
an immune deficient mouse.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] FIGS. 1A, 1B, 1C. Patient derived scFvs Bc-12 and Bc-15
recognize tumor cell integrin .alpha.v.beta.3 in a cation and
activation dependent manner. Flow cytometric analyses in TBS with
or without 1 mM Ca.sup.2+, 1 mM Mg.sup.2+, or 0.2 mM Mn.sup.2+ as
indicated. Binding of soluble scFv detected after incubation with
anti-Flag Mg2 murine mAb followed by FITC-anti mouse F(ab=).sub.2.
(A) scFv binding to human melanoma cells (M21: .alpha.v.beta.3 plus
other .alpha.v integrins) (M21-LIIb: .alpha.IIb.beta.3, no .alpha.v
integrins) or lung adenocarcinoma cells (UCLA-P3: .alpha.v
integrins, no .beta.3 integrin). (B) scFv binding to M21 melanoma
cells. (C) scFv binding to variants of MDA-MB 435 human breast
cancer cells that lack (.beta.3.sup.-) or express .alpha.v.beta.3
either in a non-activated (.beta.3.sub.WT, ParentCo) or activated
form (.beta.3.sub.D723R, Bone, Lung, and BMS metastatic cancer
cells isolated from breast cancer patient blood). Similar results
were obtained in several independent experiments for Bc-12 and
Bc-15 on each of these cell types.
[0034] FIGS. 2A, 2B, 2C, 2D. Translation of scFv DNA sequence
analyses. (A) Consensus amino acid sequence (SEQ ID NO: 7) of Bc-12
(SEQ ID NO: 2) and Bc-15 (SEQ ID NO: 4) (specific for activated
.alpha.v.beta.3) compared to Bc-20 (specific for .alpha.v) (SEQ ID
NO: 8). (B) scFv binding to BMS human breast cancer cells by flow
cytometry, indicating loss of Bc-12 and Bc-15 binding when CDR-H3
RGD is mutated to RGE (Mut-12 and Mut-15). Mut-15 signal is
equivalent to negative control. (C) cDNA sequences for scFv Bc-12
(SEQ ID NO: 1) and scFv Bc-15 (SEQ ID NO: 3). The cDNA encoding
scFv Bc-12 has an ATCC accession number PTA-6303, date of deposit:
Nov. 12, 2004. The cDNA encoding scFv Bc-15 has an ATCC accession
number PTA-6304, date of deposit: Nov. 12, 2004. (D) cDNA sequences
for scFv Mut-12 (SEQ ID NO: 5) and scFv Mut-15 (SEQ ID NO: 6). scFv
Mut-12 is the RGE containing mutant version of scFv Bc-12. scFv
Mut-15 is the RGE containing mutant version of scFv Bc-15.
[0035] FIGS. 3A, 3B, 3C, 3D, 3E. scFv Bc-12 and Bc-15 inhibit
.alpha.v.beta.3 mediated adhesive breast cancer cell functions. (A)
Adhesion of BMS human breast cancer cells to fibrinogen (Fg),
vitronectin (VN) or type I collagen (Col I) in the absence or
presence of 3 .mu.M Bc-12, Bc-15 or their RGE mutants Mut-12,
Mut-15, compared to 200 .mu.M GRGDSPK peptide. Protein coating
concentrations: 10 .mu.g/ml (VN, Col I) or 20 .mu.g/ml (Fg).
Adhesion time: 30 min at 37 EC. (B) Effect of scFvs on haptotactic
BMS cell migration toward a fibrinogen substrate (16 hrs at 37 EC
in transwell chambers with or without 2 .mu.M Bc-12, Bc-15 or their
RGE mutants Mut-12 or Mut-15, compared to 200 .mu.M GRGDSPK
peptide). (C) Effect of scFvs on breast cancer cell arrest during
blood flow. Metastatic BMS cells, labeled with hydroethidine, were
suspended in human blood (anticoagulated with 50 nM PPACK) and
perfused over a collagen I matrix at a venous wall shear rate of 50
s.sup.-1. Under these conditions, breast cancer cells arrest by
.alpha.v.beta.3 mediated binding to adherent, thrombus forming
platelets. Tumor cell adhesion was quantified by image acquisition
at 30 predefined positions during blood flow. Left: representative
images of platelet signal (thrombi, green fluorescence) and tumor
cell signal (red fluorescence) at identical x,y positions. Right:
Number of arrested tumor cells in the presence of 3 .mu.M
non-function blocking anti .alpha.v scFv Bc-20 or function blocking
anti .alpha.v.beta.3 scFvs Bc-12 or Bc-15. (D) scFv Bc-15 is
internalized by human breast cancer cells and reduces cell
proliferation. Left: confocal images of BMS cells incubated for 4
hrs with FITC-Bc-15 at 4 EC (binding) versus 37 EC (allowing
internalization). Right: Phase contrast images of BMS cell cultures
4 days after seeding in the presence of 2 .mu.M RGE containing scFv
Mut-15, or RGD containing Bc-15.
(E) Effect of scFv Bc-12 on the viability of matrix deprived BMS
breast cancer cells in ultra-low-adhesion plates in the presence or
absence of 3.7 .mu.M scFv, or 14.36 .mu.M camptothecin as apoptosis
inducing control. Measurement of apoptosis was based on cytoplasmic
histone-associated DNA fragments after 20 hrs.
[0036] FIG. 4. Patient derived ligand mimetic scFvs Bc-12 and Bc-15
against activated .alpha.v.beta.3 are internalized and bind to
breast cancer cells in human plasma and affect breast cancer
survival. Flow cytometric analysis with human metastatic breast
cancer cells (BMS) isolated from a patient blood sample. All
binding and washing steps were done in fresh human plasma prepared
from blood anticoagulated with 50 nM PPACK.
[0037] FIGS. 5A, 5B, 5C. ScFvs Bc-12 and Bc-15 prevent hematogenous
breast cancer metastasis. (A) Left: Lungs of female SCID mice 32
days after i.v. injection with 1.times.10.sup.5 BMS human breast
cancer cells. The mice were treated with 50 .mu.g Bc-12 or Bc-15 in
100 .mu.l PBS (i.v.) on days 1, 2, 3 and 4. Controls received PBS
only. Right: Numbers of lung surface metastases for each animal,
horizontal lines indicate the median number of metastases per
group. ScFv treated mice had significantly fewer metastases
(P<0.001 by the Kruskal Wallis Test). (B) Left: Histological
sections of the above mouse lungs, stained with hematoxylin/eosin.
Metastases were counted in six sets of three consecutive sections
separated by 140 .mu.m for each lung. Right: Number of metastases
counted in sections of individual lungs, horizontal lines indicate
median number of metastases per animal group. ScFv treated mice had
significantly fewer detectable metastases (P<0.005 by the
Kruskal Wallis Test). (C) Effect of Bc-15 treatment on established
breast cancer metastasis in the lungs. Female SCID mice were
injected i.v. with 5.times.10.sup.5 DsRed2-tagged MDA-MB 435 breast
cancer cells expressing constitutively activated integrin
.alpha.v.beta.3.sub.D723R. Mice were treated on day 7, 9, 11 and
14, 16, 18 by i.v. injections of scFv Bc-15 or its RGE mutant,
Mut-15 (40 .mu.g/dose). Metastatic foci were enumerated by
fluorescence microscopy on day 19. Left: Images of typical tumor
foci within the lung tissue in Mut-15 (top) or Bc-15 treated mice
(bottom) (bar: 50 .mu.m). Right: Number of metastatic foci within
the lung tissue of each animal, horizontal line: median number of
metastases per group. Bc-15 treated mice had significantly fewer
metastases than mice treated with Mut-15 (P<0.001 by the
two-sided Mann-Whitney rank sum Test).
[0038] FIG. 6. .alpha.v.beta.3 integrin receptor expression in
parental cell and tumor cell variants.
[0039] FIG. 7. scFvs Bc-12 and Bc-15 react with tumor cells
expressing gain-of-function mutants of .beta.3 found in metastatic
breast cancer cells.
[0040] FIG. 8. scFvs Bc-12 and Bc-15 inhibit platelet mediated
breast cancer cell arrest during blood flow.
DETAILED DESCRIPTION
[0041] The invention is generally related to methods of producing
an antibody phage population having affinity for a tumor cell
target which is a tumor cell expressing a metastatic phenotype. The
tumor cell expressing the metastatic phenotype can be a cell line
expressing an activated cell surface receptor, for example, an
activated integrin receptor or an .alpha.v.beta.3 integrin
receptor. The invention further relates to an antibody composition
that specifically binds to a cell surface receptor on a metastatic
cell. The antibody composition specifically binds to a activated
cell surface receptor on a metastatic cell, for example, an
activated integrin receptor or an .alpha.v.beta.3 integrin
receptor. The invention further relates to methods for alleviating
a disease state in a mammal by treatment with a cancer therapeutic
comprising the step of administering to the mammal a therapeutic
amount of said pharmaceutical composition of the antibody
composition. The invention further relates to methods of detecting
an activated cell surface receptor on a metastatic tumor cell
surface in a mammalian tissue sample and to methods of identifying
cells liable to undergo metastasis associated with a disease state
comprising contacting a patient suspected of being at risk for
metastasis which includes contacting a patient suspected of being
at risk for metastasis with the antibody composition, said antibody
having associated therewith an imaging moiety.
[0042] It has now been discovered that an activated functional form
of .alpha.v.beta.3 integrin can be used as a target in a
therapeutic regime for the inhibition of cancer metastasis,
especially in breast cancer metastasis. Human single-chain Fv
antibody libraries of cancer patient immune repertoire has been
developed containing antibodies that can recognize .alpha.v.beta.3
integrin specifically in its activated form. The present invention
provides isolation, characterization and in vivo use of these
antibodies to disrupt the activated form of .alpha.v.beta.3 and
prevent breast cancer metastasis.
[0043] Although breast tumors can be detected at ever smaller size,
one cannot presently predict when these will begin to metastasize
and to inhibit this process effectively. To improve the therapeutic
potential of surgery and anti-cancer treatment, new molecular
targets are needed to identify and inhibit metastatic cells.
Studies, in vitro and in vivo, with human breast cancer cell models
and metastatic cells from breast cancer patients demonstrate that
expression of activated cell surface receptors on metastatic cells,
e.g., the adhesion receptor integrin .alpha.v.beta.3 in a
functionally activated form, strongly promotes metastatic activity.
To exploit this metastasis related expression, selection of cancer
patient derived human single-chain Fv antibody libraries yielded
antibodies that specifically recognize the activated form of
.alpha.v.beta.3 and block critical functions of this receptor. Two
of these antibodies, Bc-12 and Bc-15, were found to be natural
ligand mimetics that bind .alpha.v.beta.3 in a cation dependent
manner via an Arg-Gly-Asp integrin recognition motif within CDR-H3.
These antibodies, but not their Arg-Gly-Glu mutants, interfered
with .alpha.v.beta.3 mediated tumor cell adhesion and migration,
specifically recognized metastatic breast cancer cells in blood,
and inhibited platelet supported tumor cell arrest during blood
flow. Importantly, scFvs Bc-12 and Bc-15 prevented lung
colonization by human breast cancer cells in immune deficient mice.
These data imply that disrupting the functions of activated
.alpha.v.beta.3 can inactivate tumor cells in the circulation and
thus prevent breast cancer metastasis.
[0044] There is an absence of reagents available which recognize
murine .alpha.v.beta.3 integrin receptor. Having access to such a
reagent would offer the scientific community a valuable research
reagent. Based on the nature of the scFvs disclosed herein, it is
anticipated that Bc-12 and Bc-15 could react with murine
.alpha.v.beta.3 integrin receptor. If they do, these reagents would
be very helpful for use in vivo studies utilizing mouse models of
human disease states. Murine .alpha.v.beta.3 integrin receptor
reagents would be of particular use for angiogenesis-based studies
which are performed to a large degree in mouse models. It is
expected that the scFvs of the invention react with activated
murine .alpha.v.beta.3 integrin receptor on angiogenic endothelial
cells, possibly in a murine model and very likely in humans.
[0045] ScFvs Bc-12 and Bc-15 are novel reagents because they
selectively bind to the activated conformation of .alpha.v.beta.3
integrin receptor and do not bind to the non-activated conformation
of .alpha.v.beta.3 integrin receptor. Amongst tumor cells
expressing the .alpha.v.beta.3 integrin receptor that have been
tested so far, only cells with a metastatic phenotype express
.alpha.v.beta.3 integrin receptor in a constitutively activated
conformation.
[0046] Surprisingly, scFvs Bc-12 and Bc-15 utilize an RGD ligand
motif (CDR-H3) combined with high specificity for .alpha.v.beta.3
integrin receptor. Other known antibodies containing the RGD motif
react with multiple integrins that recognize the RGD sequence. As
demonstrated herein, scFv Bc-12 and Bc-15 do not bind to other
integrins known as major receptors for the RGD motif, including,
but not limited to, integrin .alpha.v.beta.5, .alpha.v.beta.1,
alpha IIb beta 3, and .alpha.5.beta.1.
[0047] Breast cancers are known to be extremely heterogeneous. A
subset of human breast cancer cells can be identified based on
expression of an adhesion receptor, the integrin .alpha.v.beta.3,
in its constitutively activated functional form. This activated
integrin promotes platelet binding and tumor cells arrest in the
vasculature. In this way, activation of integrin .alpha.v.beta.3
endows metastatic cells with key properties likely to be critical
for successful dissemination and colonization of target organs. The
combined immune repertoire of a number of cancer patients has been
mined using antibody phage display technology by subtractive
panning on poorly versus strongly metastatic variants of a human
breast cancer cell line. This approach yielded single chain Fv
(scFv) antibodies that specifically recognize the activated
functional conformation of the tumor cell adhesion receptor,
integrin .alpha.v.beta.3. The antibodies react selectively with
metastatic variants of the breast cancer cell models and with
metastatic cells isolated from blood samples of stage IV breast
cancer patients. Importantly, these antibodies inhibit colonization
of the lungs by human breast cancer cells in immune deficient
mice.
[0048] Antibody compositions and methods of the present invention
are useful to investigate the ability of human single chain Fv
(scFv) antibodies to report the activated form of integrin
.alpha.v.beta.3 as a diagnostic marker of metastatic cancer cells,
e.g., metastatic breast cancer cells. These scFv antibodies and
their derivatives can specifically detect metastatic breast cancer
cells and report the localization of metastatic disease. Antibody
compositions and methods of the present invention are further
useful to identify therapeutic antibody compositions for treatment
of cancer metastasis, e.g., metastatic breast cancer.
[0049] Antibody compositions and methods of the present invention
are useful to investigate the ability of human single chain Fv
(scFv) antibodies to detect and report the activated form of
integrin .alpha.v.beta.3 as a prognostic marker of metastatic
breast cancer. These scFv antibodies and their derivatives can
specifically detect breast cancer cells that have a propensity to
metastasize.
[0050] Antibody compositions and methods of the present invention
are useful to analyze effects of human scFv antibodies and their
derivatives against constitutively activated integrin
.alpha.v.beta.3 on breast cancer metastasis. Targeted inhibition of
cells expressing the activated form of integrin .alpha.v.beta.3 can
prevent breast cancer metastasis and interfere with established
metastatic disease.
[0051] Imaging and mammography technology can detect very early
breast tumors. However, current prognostic criteria for breast
cancer do not accurately indicate how aggressive a tumor is,
whether it has already begun to spread, and which treatment options
should be chosen to achieve the best possible outcome in each
individual case. Complications from metastatic disease are the
primary cause of death in breast cancer. Breast cancer metastasis
to major target organs, such as lungs, bone, liver, and brain
involves tumor cell dissemination via the blood stream. An
important requirement for successful target organ colonization in
this environment is the ability of the tumor cells to arrest within
the vasculature of their target organs, despite shear forces
generated by blood flow which physically opposed cell attachment.
One mechanism supporting the arrest process has been identified as
an interaction between the tumor cell adhesion receptor, integrin
.alpha.v.beta.3, and platelet integrin .alpha.IIb.beta.3, connected
to each other by di- or multivalent plasma proteins as bridging
ligands. Felding-Habermann et al., J. Biol. Chem., 271: 5892-5900,
1996; Pilch et al., J. Biol. Chem., 277: 21930-21938, 2002;
Felding-Habermann et al, Proc. Natl. Acad. Sci. U.S.A, 98:
1853-1858, 2001; Bakewell et al., Proc. Natl. Acad. Sci. U.S.A,
100: 14205-14210, 2003; Biggerstaff et al., Clin. Exp. Metastasis,
17: 723-730, 1999.
[0052] Integrins are a family of transmembrane cell adhesion
receptors that are composed of .alpha. and .beta. subunits and
mediate cell attachment to proteins within the extracellular
matrix. In addition to recognizing ligand proteins immobilized
within a matrix, the receptors may also react with soluble ligand
proteins, for instance certain plasma proteins, but only if the
receptor molecules are present in an activated functional
conformation. Liddington et al., J. Cell Biol., 158: 833-839, 2002;
Woodside et al., Thromb. Haemost., 86: 316-323, 2001. Thus,
recognition of soluble ligands by integrins strictly depends on
specific changes in receptor conformation. This provides a
molecular switch that controls the ability of cells to aggregate in
an integrin dependent manner and to arrest under the dynamic flow
conditions of the vasculature. This mechanism is well established
for leukocytes and platelets, that circulate within the blood
stream in a resting state while expressing non-activated integrins.
Upon stimulation through proinflammatory or prothrombotic agonists,
these cell types promptly respond with a number of molecular
changes including the switch of key integrins, .beta.2 integrins
for leucocytes and .alpha.v.beta.3 for platelets, from `resting` to
`activated` conformations. This enables these cell types to arrest
within the vasculature, promoting cell cohesion and leading to
thrombus formation. Savage et al., Curr. Opin. Hematol., 8:
270-276, 2001. It has demonstrated that a metastatic subset of
human breast cancer cells expresses integrin .alpha.v.beta.3 in a
constitutively activated form. Rolli et al., Proc. Natl. Acad. Sci.
U.S.A, 100: 9482-9487, 2003. Aberrant expression of .alpha.v.beta.3
plays a role in metastasis of breast cancer as well as prostate
cancer, melanoma, and neuroblastic tumors, but it is important to
understand that it is specifically the activated functional
conformation of the receptor that promotes metastatic activity.
Felding-Habermann et al, Proc. Natl. Acad. Sci. U.S.A, 98:
1853-1858, 2001; Felding-Habermann et al., Clin. Exp. Metastasis,
19: 427-436, 2002; Gladson et al., Am. J. Pathol., 148: 1423-1434,
1996; Zheng et al., Cancer Res., 59: 1655-1664, 1999; Zheng et al.,
J. Biol. Chem., 275: 24565-24574, 2000; Van Belle et al., Hum.
Pathol., 30: 562-567, 1999; Gui et al., Surgery, 117: 102-108,
1995. The activated receptor strongly promotes breast cancer cell
migration and enables the cells to arrest under blood flow
conditions. In this way, activation of .alpha.v.beta.3 endows
metastatic cells with key properties likely to be critical for
successful dissemination and colonization of target organs.
Integrin mediated tumor cell-platelet interactions have been
implicated in metastasis to lung and bone and it has been suggested
that, in addition to enabling arrest in the circulation, platelet
coating of tumor cells may prevent immune recognition by cloaking
tumor antigens, or may supply the tumor cells with growth factors
such as epidermal growth factor or platelet derived growth factor.
Biggerstaff et al., Clin. Exp. Metastasis, 17: 723-730, 1999;
Felding-Habermann et al., Clin. Exp. Metastasis, 19: 427-436, 2002;
Amirkhosravi et al., Thromb. Haemost., 90: 549-554, 2003; Siddiqui
et al., Platelets, 13: 247-253, 2002; Furger, K. A., et al., Mol.
Cancer Res., 1: 810-819, 2003. Tumor cells that have successfully
entered a target organ may further utilize .alpha.v.beta.3 to
thrive in the new environment, as .alpha.v.beta.3 matrix
interactions can promote cell survival and proliferation. For
example, .alpha.v.beta.3 binding to osteopontin, a bone matrix
protein, promotes malignancy and elevated levels of osteopontin
correlate with a poor prognosis in breast cancer. Zheng et al., J.
iol. Chem., 275: 24565-24574, 2000; Singhal et al., Clin. Cancer
Res., 3: 605-611, 1997; Tuck et al., Int. J. Cancer, 79: 502-508,
1998; Rudland et al., Cancer Res., 62: 3417-3427, 2002; Ding et
al., Cancer Res., 62: 5336-5343, 2002; Wang et al., Oncogene, 19:
5801-5809, 2000; Gladson et al., J. Neuropathol. Exp. Neurol., 58:
1029-1040, 1999.
[0053] For all of these reasons, and its established role in
angiogenesis the .alpha.v.beta.3 integrin is one of the most widely
studied integrins and antagonists of this molecule have significant
potential to serve as diagnostic imaging agents and for use in
targeted drug delivery. Bakewell et al., Proc. Natl. Acad. Sci.
U.S.A, 100: 14205-14210, 2003; Varner et al., Important. Adv.
Oncol., 69-87: 69-87, 1996; Tucker et al., Curr. Opin. Investig.
Drugs, 4: 722-731, 2003. Two separate approaches have been used to
target this molecule. One of these uses the high binding
specificity to .alpha.v.beta.3 of peptides containing the
Arg-Gly-Asp (RGD) sequence. This tripeptide, naturally present in
extracellular matrix proteins, is the primary binding site of the
.alpha.v.beta.3 integrin. Ruoslahti et al., Annu. Rev. Cell Dev.
Biol., 12: 697-715, 1996. Initial problems with RGD based reporter
probes are due to fast blood clearance, high kidney and liver
uptake and fast tumor washout, currently being addressed by
chemically modifying cyclised RGD peptides to increase their
stability and valency. Menard et al., Oncogene, 22: 6570-6578,
2003; Chen et al., Bioconjug. Chem., 15: 41-49, 2004; Thumshim et
al., Chemistry., 9: 2717-2725, 2003; Su et al., Nucl. Med. Biol.,
30: 141-149, 2003. These modified peptides are then coupled to
radio-isotpes and used either for tumor imaging or to inhibit tumor
growth. One such molecule, Cilengitide, is currently in Phase II
clinical trials to inhibit tumor progression. The other approach
uses antibodies, either alone or as immunoconjugates. Attempts to
develop monoclonal antibodies (mAbs) as therapeutic agents for
cancer patients have intensified the search for cancer-related
antigens as molecular targets. Function blocking antibodies against
integrin .alpha.v.beta.3, especially mAb LM609 and its derivatives,
have been shown to interfere with critical adhesive tumor cell
functions and neoangiogenesis. Brooks et al., Cell, 79: 1157-1164,
1994; Brooks et al., Science, 264: 569-571, 1994. It was earlier
found that LM609 antibody also inhibited metastatic activity of
human melanoma cells in a mouse model. Felding-Habermann et al.,
Clin. Exp. Metastasis, 19: 427-436, 2002. Antibodies recognizing
tumor cells or supporting host cells have been used in
immunoradiotherapy and radioimmunolocalization, as well as toxin
and chemotherapeutic agent delivery. Goldenberg, Cancer Immunol.
Immunother., 52: 281-296, 2003; Power et al., Methods Mol. Biol.,
207: 335-350, 2003; Kortt et al., Biomol. Eng, 18: 95-108, 2001;
Garnett Adv. Drug Deliv. Rev., 53: 171-216, 2001. Advances in
immunoconjugate technology, together with the availability of fully
human antibodies have revitalized the "magic bullet" promise of
immunotherapy for cancer treatment. In the past few years, several
mAbs received FDA approval for cancer treatment. These include
Rituximab, a chimeric antibody against CD20 for the treatment of
non-Hodgkin's lymphoma, and Trastuzumab (Herceptin), a murine and
now humanized antibody against the Her-2 proto oncogene protein for
the treatment of metastatic breast cancer. Menard et al., Oncogene,
22: 6570-6578, 2003; Smith, Oncogene, 22: 7359-7368, 2003; Perez.
et al., J. Clin. Oncol., 22: 322-329, 2004; Spigel et al., Clin.
Breast Cancer, 4: 329-337, 2003; Ali, Clin. Orthop., S132-S137,
2003. A list of therapeutic mAbs currently `in the pipeline` for
treatment of breast cancer, and mAbs selected for testing in new
clinical trials in combination with chemotherapeutic or other
regimens are summarized in tables presented in the appendix.
Results of clinical trials with single agent Trastuzumab
(anti-Her-2) and in combination with paclitaxel, docetaxel,
vinorelbine, gemcitabine and platinum salts have been encouraging,
and durable remissions (>5 years) have been reported
occasionally. However, none of the current therapies, including
chemo- and antibody based treatments, could effectively stop or
prevent metastasis. Thus, enhancement of existing treatment options
by improved antibody therapy, specifically identifying or targeting
metastatic cells would have a major impact on breast cancer
management and diagnosis.
[0054] To identify target molecules associated with metastatic
human breast cancer cells, and to isolate antibodies of human
origin directed against such markers, the combined immune
repertoire of a number of cancer patients was mined using antibody
phage display technology. This approach yielded single chain Fv
(scFv) antibodies that recognize the tumor cell adhesion receptor,
integrin .alpha.v.beta.3, but only in its activated functional
conformation. The antibodies react selectively with metastatic
variants of the breast cancer cell models and with metastatic cells
isolated from blood samples of stage IV breast cancer patients.
Importantly, these antibodies inhibit colonization of the lungs by
human breast cancer cells in immune deficient mice. These
antibodies act as ligand mimetics. They contain the RGD integrin
recognition sequence which contributes to their affinity. However,
they also demonstrate the ligand specificity of an antibody-antigen
interaction. In this way, they offer the possibility of combining
the advantages of both of the above therapeutic approaches.
[0055] ScFv fragments have several distinct advantages over whole
IgG for cancer immunotherapy. First, due to their relatively small
size (27 kDa), scFvs clear from plasma readily and can penetrate
rapidly and deeply into tissue. Garnett, Adv. Drug Deliv. Rev., 53:
171-216, 2001. For example, .sup.125I-labeled anti-CEA (carcino
embryonic antigen) scFv showed superior tumor localization compared
to whole IgG. Wu et al., Immunotechnology., 2: 21-36, 1996; Mayer
et al., Clin. Cancer Res., 6: 1711-1719, 2000. Second, potential
side effects may be reduced, since lack of the constant region
ensures that scFvs are not retained in organs with high density of
cells expressing Fc receptors, like the liver and/or kidney. Third,
scFvs can be re-engineered with PCR based approaches into several
different formats such as diabody, triabody, or bispecific antibody
so that the size, flexibility and valency of each antibody fragment
can be tailored to suit specific applications for in vivo imaging
and therapy. Kortt et al., Biomol. Eng, 18: 95-108, 2001. These
scFv antibodies are often internalized by tumors following antigen
engagement and can be conjugated to highly toxic, small molecules.
In addition these antibody fragments have been reported to be
capable of crossing the blood-brain barrier. Frenkel et al., Proc.
Nat. Acad. Sci. U.S.A, 99: 5675-5679, 2002. Brain metastases
present a particularly intractable problem in breast and other
cancers and therapeutic advances in this area would have a very
significant impact.
[0056] Another application addresses the concept that breast tumors
contain a minor, distinct, sub-population of progenitor- or
stem-like cells that are responsible for the initiation of the
tumor. Al Hajj, Proc. Natl. Acad. Sci. U.S.A, 100: 3983-3988,
2003.
[0057] A library of scFv antibodies to integrins can be used to
define the characteristics that would allow one to prospectively
identify the putative tumor initiating population in human breast
cancer. In other organ systems, stem cells occupy a basal
compartment, adhering to the underlying basement membrane via
integrins. Integrin expression patterns have been found to differ
between the stem cells and their differentiated progeny. In
prostate epithelium, spermatogenesis and epidermal keratinocytes,
.beta.1 integrin expression has been used to identify and isolate
populations of putative stem cells. Collins et al., J. Cell Sci.,
114: 3865-3872, 2001; Shinohara et al., Proc. Natl. Acad. Sci.
U.S.A, 96: 5504-5509, 1999; Evans et al., J. Cell Biol., 160:
589-596, 2003. Integrin expression can be used as a marker for
breast tumor initiating populations. The library of scFv antibodies
contains several antibodies against distinct activation states of
different integrins. In an alternative approach, the library of
scFv antibodies to integrins can be used to define integrin
expression and other markers and ultimately to target this cell
population.
scFV PHAGE LIBRARIES
[0058] One approach for a phage display library to identify an
antibody composition that specifically binds to a cell surface
receptor on a metastatic cell, for example, an activated integrin
receptor, has been the use of scFv phage-libraries (see, e.g.,
Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85: 5879-5883, 1988;
Chaudhary et al., Proc. Natl. Acad. Sci. U.S.A., 87: 1066-1070,
1990. Various embodiments of scFv libraries displayed on
bacteriophage coat proteins have been described. Refinements of
phage display approaches are also known, for example as described
in WO96/06213 and WO92/01047 (Medical Research Council et al.) and
WO97/08320 (Morphosys), which are incorporated herein by reference.
The display of Fab libraries is also known, for instance as
described in WO92/01047 (CAT/MRC) and WO91/17271 (Affymax).
[0059] Hybrid antibodies or hybrid antibody fragments that are
cloned into a display vector can be selected against the
appropriate antigen associated with a metastatic cell, e.g., a cell
surface receptor or an activated cell surface receptor on a
metastatic tumor cell, in order to identify variants that
maintained good binding activity because the antibody or antibody
fragment will be present on the surface of the phage or phagemid
particle. See for example Barbas III et al., Phage Display, A
Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 2001, the contents of which are incorporated herein
by reference. For example, in the case of Fab fragments, the light
chain and heavy chain Fd products are under the control of a lac
promoter, and each chain has a leader signal fused to it in order
to be directed to the periplasmic space of the bacterial host. It
is in this space that the antibody fragments will be able to
properly assemble. The heavy chain fragments are expressed as a
fusion with a phage coat protein domain which allows the assembled
antibody fragment to be incorporated into the coat of a newly made
phage or phagemid particle. Generation of new phagemid particles
requires the addition of helper phage which contain all the
necessary phage genes. Once a library of antibody fragments is
presented on the phage or phagemid surface, a process termed
panning follows. This is a method whereby) the antibodies displayed
on the surface of phage or phagemid particles are bound to the
desired antigen, ii) non-binders are washed away, iii) bound
particles are eluted from the antigen, and iv) eluted particles are
exposed to fresh bacterial hosts in order to amplify the enriched
pool for an additional round of selection. Typically three or four
rounds of panning are performed prior to screening antibody clones
for specific binding. In this way phage/phagemid particles allow
the linkage of binding phenotype (antibody) with the genotype (DNA)
making the use of antibody display technology very successful.
However, other vector formats could be used for this humanization
process, such as cloning the antibody fragment library into a lytic
phage vector (modified T7 or Lambda Zap systems) for selection
and/or screening.
[0060] After selection of desired hybrid antibodies and/or hybrid
antibody fragments, it is contemplated that they can be produced in
large volume by any technique known to those skilled in the art,
e.g., prokaryotic or eukaryotic cell expression and the like. For
example, hybrid antibodies or fragments may be produced by using
conventional techniques to construct an expression vector that
encodes an antibody heavy chain in which the CDRs and, if
necessary, a minimal portion of the variable region framework, that
are required to retain original species antibody binding
specificity (as engineered according to the techniques described
herein) are derived from the originating species antibody and the
remainder of the antibody is derived from a target species
immunoglobulin which may be manipulated as described herein,
thereby producing a vector for the expression of a hybrid antibody
heavy chain.
[0061] In a detailed embodiment, a single-chain Fv (scFv) antibody
library can be prepared from the peripheral blood lymphocytes of 5,
10, 15, or 20 or more patients with various cancer diseases.
Completely human high-affinity scFv antibodies can then be selected
by using synthetic sialyl Lewis.sup.x and Lewis.sup.x BSA
conjugates. In one study, these human scFv antibodies were specific
for sialyl Lewis.sup.x and Lewis.sup.x, as demonstrated by ELISA,
BIAcore, and flow cytometry binding to the cell surface of
pancreatic adenocarcinoma cells. Nucleotide sequencing revealed
that at least four unique scFv genes were obtained. The K.sub.d
values ranged from 1.1 to 6.2.times.10.sup.-7 M that were
comparable to the affinities of mAbs derived from the secondary
immune response. These antibodies could be valuable reagents for
probing the structure and function of carbohydrate antigens and in
the treatment of human tumor diseases. Mao et al., Proc. Natl.
Acad. Sci. U.S.A. 96: 6953-6958, 1999.
[0062] In a further detailed embodiment, phage displayed
combinatorial antibody libraries can be used to generate and select
a wide variety of antibodies to an appropriate antigen associated
with a metastatic cell, e.g., a cell surface receptor or an
activated cell surface receptor on a metastatic tumor cell. The
phage coat proteins pVII and pIX can be used to display the
heterodimeric structure of the antibody Fv region. Aspects of this
technology have been extended to construct a large, human
single-chain Fv (scFv) library of 4.5.times.10.sup.9 members
displayed on pIX of filamentous bacteriophage. Furthermore, the
diversity, quality, and utility of the library were demonstrated by
the selection of scFv clones against six different protein
antigens. Notably, more than 90% of the selected clones showed
positive binding for their respective antigens after as few as
three rounds of panning. Analyzed scFvs were also found to be of
high affinity. For example, kinetic analysis (BIAcore) revealed
that scFvs against staphylococcal enterotoxin B and cholera toxin B
subunit had a nanomolar and subnanomolar dissociation constant,
respectively, affording affinities comparable to, or exceeding
that, of mAbs obtained from immunization. High specificity was also
attained, not only between very distinct proteins, but also in the
case of more closely related proteins, e.g., Ricinus communis
("ricin") agglutinins (RCA.sub.60 and RCA.sub.120), despite >80%
sequence homology between the two. The results suggested that the
performance of pIX-display libraries can potentially exceed that of
the pIII-display format and make it ideally suited for panning a
wide variety of target antigens. Gao et al., Proc. Natl. Acad. Sci.
U.S.A. 99: 12612-12616, 2001.
[0063] Specific binding between an antibody or other binding agent
and an antigen means a binding affinity of at least 10.sup.-6 M.
Preferred binding agents bind with affinities of at least about
10.sup.-7 M, and preferably 10.sup.-8 M to 10.sup.-9 M, 10.sup.-10
M, 10.sup.-11 M, or 10.sup.-12 M. The term epitope means an
antigenic determinant capable of specific binding to an antibody.
Epitopes usually consist of chemically active surface groupings of
molecules such as amino acids or sugar side chains and usually have
specific three dimensional structural characteristics, as well as
specific charge characteristics. Conformational and
nonconformational epitopes are distinguished in that the binding to
the former but not the latter is lost in the presence of denaturing
solvents.
[0064] "Patient", "subject" or "mammal" are used interchangeably
and refer to mammals such as human patients and non-human primates,
as well as experimental animals such as rabbits, rats, and mice,
and other animals. Animals include all vertebrates, e.g., mammals
and non-mammals, such as sheep, dogs, cows, chickens, amphibians,
and reptiles.
[0065] "Treating" or "treatment" includes the administration of the
antibody compositions, compounds or agents of the present invention
to prevent or delay the onset of the symptoms, complications, or
biochemical indicia of a disease, alleviating the symptoms or
arresting or inhibiting further development of the disease,
condition, or disorder (e.g., cancer, metastatic cancer, or
metastatic breast cancer). Treatment can be prophylactic (to
prevent or delay the onset of the disease, or to prevent the
manifestation of clinical or subclinical symptoms thereof) or
therapeutic suppression or alleviation of symptoms after the
manifestation of the disease.
[0066] "Cancer" or "malignancy" are used as synonymous terms and
refer to any of a number of diseases that are characterized by
uncontrolled, abnormal proliferation of cells, the ability of
affected cells to spread locally or through the bloodstream and
lymphatic system to other parts of the body (i.e., metastasize) as
well as any of a number of characteristic structural and/or
molecular features. A "cancerous" or "malignant cell" is understood
as a cell having specific structural properties, lacking
differentiation and being capable of invasion and metastasis.
Examples of cancers are, breast, lung, brain, bone, liver, kidney,
colon, and prostate cancer. (see DeVita et al., Eds., Cancer
Principles and Practice of Oncology, 6th. Ed., Lippincott Williams
& Wilkins, Philadelphia, Pa., 2001; this reference is herein
incorporated by reference in its entirety for all purposes).
[0067] "Cancer-associated" refers to the relationship of a nucleic
acid and its expression, or lack thereof, or a protein and its
level or activity, or lack thereof, to the onset of malignancy in a
subject cell. For example, cancer can be associated with expression
of a particular gene that is not expressed, or is expressed at a
lower level, in a normal healthy cell. Conversely, a
cancer-associated gene can be one that is not expressed in a
malignant cell (or in a cell undergoing transformation), or is
expressed at a lower level in the malignant cell than it is
expressed in a normal healthy cell.
[0068] In the context of the cancer, the term "transformation"
refers to the change that a normal cell undergoes as it becomes
malignant. In eukaryotes, the term "transformation" can be used to
describe the conversion of normal cells to malignant cells in cell
culture.
[0069] "Proliferating cells" are those which are actively
undergoing cell division and growing exponentially. "Loss of cell
proliferation control" refers to the property of cells that have
lost the cell cycle controls that normally ensure appropriate
restriction of cell division. Cells that have lost such controls
proliferate at a faster than normal rate, without stimulatory
signals, and do not respond to inhibitory signals.
[0070] "Advanced cancer" means cancer that is no longer localized
to the primary tumor site, or a cancer that is Stage III or IV
according to the American Joint Committee on Cancer (AJCC).
[0071] "Well tolerated" refers to the absence of adverse changes in
health status that occur as a result of the treatment and would
affect treatment decisions.
[0072] "Metastatic" refers to tumor cells, e.g., human breast
cancer cells, that are able to establish secondary tumor lesions in
the lungs, liver, bone or brain of immune deficient mice upon
injection into the mammary fat pad and/or the circulation of the
immune deficient mouse.
[0073] "Non-metastatic" refers to tumor cells, e.g., human breast
cancer cells, that are unable to establish secondary tumor lesions
in the lungs, liver, bone or brain or other target organs of breast
cancer metastasis in immune deficient mice upon injection into the
mammary fat pad and/or the circulation. The human tumor cells used
herein and addressed herein as non-metastatic are able to establish
primary tumors upon injection into the mammary fat pad of the
immune deficient mouse, but they are unable to disseminate from
those primary tumors.
[0074] "Lymphocyte" as used herein has the normal meaning in the
art, and refers to any of the mononuclear, nonphagocytic
leukocytes, found in the blood, lymph, and lymphoid tissues, e.g.,
B and T lymphocytes.
[0075] "Epitope" refers to a protein determinant capable of
specific binding to an antibody. Epitopes usually consist of
chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. Conformational and nonconformational epitopes are
distinguished in that the binding to the former but not the latter
is lost in the presence of denaturing solvents.
[0076] An intact "antibody" comprises at least two heavy (H) chains
and two light (L) chains inter-connected by disulfide bonds. Each
heavy chain is comprised of a heavy chain variable region
(abbreviated herein as HCVR or VH) and a heavy chain constant
region. The heavy chain constant region is comprised of three
domains, CH.sub.1, CH.sub.2 and CH.sub.3. Each light chain is
comprised of a light chain variable region (abbreviated herein as
LCVR or V.sub.L) and a light chain constant region. The light chain
constant region is comprised of one domain, C.sub.L. The V.sub.H
and V.sub.L regions can be further subdivided into regions of
hypervariability, termed complementarity determining regions (CDR),
interspersed with regions that are more conserved, termed framework
regions (FR). Each V.sub.H and V.sub.L is composed of three CDRs
and four FRs, arranged from amino-terminus to carboxyl-terminus in
the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The
variable regions of the heavy and light chains contain a binding
domain that interacts with an antigen. The constant regions of the
antibodies can mediate the binding of the immunoglobulin to host
tissues or factors, including various cells of the immune system
(e.g., effector cells) and the first component (Clq) of the
classical complement system. The term antibody includes
antigen-binding portions of an intact antibody that retain capacity
to bind activated integrin receptor. Examples of binding include
(i) a Fab fragment, a monovalent fragment consisting of the
V.sub.L, V.sub.H, C.sub.L and CH1 domains; (ii) a F(ab').sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the VH and CH1 domains; (iv) a Fv fragment consisting
of the V.sub.L and V.sub.H domains of a single arm of an antibody,
(v) a dAb fragment (Ward et al., Nature 341: 544-546, 1989), which
consists of a VH domain; and (vi) an isolated complementarity
determining region (CDR).
[0077] "Single chain antibodies" or "single chain Fv (scFv)" refers
to an antibody fusion molecule of the two domains of the Fv
fragment, V.sub.L and V.sub.H. Although the two domains of the Fv
fragment, V.sub.L and V.sub.H, are coded for by separate genes,
they can be joined, using recombinant methods, by a synthetic
linker that enables them to be made as a single protein chain in
which the V.sub.L and V.sub.H regions pair to form monovalent
molecules (known as single chain Fv (scFv); see, e.g., Bird et al.,
Science 242: 423-426, 1988; and Huston et al., Proc. Natl. Acad.
Sci. USA, 85: 5879-5883, 1988). Such single chain antibodies are
included by reference to the term "antibody" fragments can be
prepared by recombinant techniques or enzymatic or chemical
cleavage of intact antibodies.
[0078] "Human sequence antibody" includes antibodies having
variable and constant regions (if present) derived from human
germline immunoglobulin sequences. The human sequence antibodies of
the invention can include amino acid residues not encoded by human
germline immunoglobulin sequences (e.g., mutations introduced by
random or site-specific mutagenesis in vitro or by somatic mutation
in vivo). Such antibodies can be generated in non-human transgenic
animals, e.g., as described in PCT Publication Nos. WO 01/14424 and
WO 00/37504. However, the term "human sequence antibody", as used
herein, is not intended to include antibodies in which CDR
sequences derived from the germline of another mammalian species,
such as a mouse, have been grafted onto human framework sequences
(e.g., humanized antibodies).
[0079] Also, recombinant immunoglobulins may be produced. See,
Cabilly, U.S. Pat. No. 4,816,567, incorporated herein by reference
in its entirety and for all purposes; and Queen et al., Proc. Nat'l
Acad. Sci. USA 86: 10029-10033, 1989.
[0080] "Monoclonal antibody" refer to a preparation of antibody
molecules of single molecular composition. A monoclonal antibody
composition displays a single binding specificity and affinity for
a particular epitope. Accordingly, the term "human monoclonal
antibody" refers to antibodies displaying a single binding
specificity which have variable and constant regions (if present)
derived from human germline immunoglobulin sequences. In one
embodiment, the human monoclonal antibodies are produced by a
hybridoma which includes a B cell obtained from a transgenic
non-human animal, e.g., a transgenic mouse, having a genome
comprising a human heavy chain transgene and a light chain
transgene fused to an immortalized cell.
[0081] "Polyclonal antibody" refers to a preparation of more than 1
(two or more) different antibodies to a cell surface receptor,
e.g., human activated integrin receptor. Such a preparation
includes antibodies binding to a range of different epitopes.
Antibodies to activated integrin receptor can bind to an epitope on
human activated integrin receptor so as to inhibit activated
integrin receptor from interacting with a counterreceptor or
co-receptor. These and other antibodies suitable for use in the
present invention can be prepared according to methods that are
well known in the art and/or are described in the references cited
here. In preferred embodiments, anti-activated integrin receptor
antibodies used in the invention are "human antibodies"--e.g.,
antibodies isolated from a human--or they are "human sequence
antibodies" (defined supra).
[0082] "Immune cell response" refers to the response of immune
system cells to external or internal stimuli (e.g., antigen, cell
surface receptors, activated integrin receptors, cytokines,
chemokines, and other cells) producing biochemical changes in the
immune cells that result in immune cell migration, killing of
target cells, phagocytosis, production of antibodies, other soluble
effectors of the immune response, and the like.
[0083] "Immune response" refers to the concerted action of
lymphocytes, antigen presenting cells, phagocytic cells,
granulocytes, and soluble macromolecules produced by the above
cells or the liver (including antibodies, cytokines, and
complement) that results in selective damage to, destruction of, or
elimination from the human body of cancerous cells, metastatic
tumor cells, metastatic breast cancer cells, invading pathogens,
cells or tissues infected with pathogens, or, in cases of
autoimmunity or pathological inflammation, normal human cells or
tissues.
[0084] "T lymphocyte response" and "T lymphocyte activity" are used
here interchangeably to refer to the component of immune response
dependent on T lymphocytes (e.g., the proliferation and/or
differentiation of T lymphocytes into helper, cytotoxic killer, or
suppressor T lymphocytes, the provision of signals by helper T
lymphocytes to B lymphocytes that cause or prevent antibody
production, the killing of specific target cells by cytotoxic T
lymphocytes, and the release of soluble factors such as cytokines
that modulate the function of other immune cells).
[0085] Components of an immune response can be detected in vitro by
various methods that are well known to those of ordinary skill in
the art. For example, (1) cytotoxic T lymphocytes can be incubated
with radioactively labeled target cells and the lysis of these
target cells detected by the release of radioactivity; (2) helper T
lymphocytes can be incubated with antigens and antigen presenting
cells and the synthesis and secretion of cytokines measured by
standard methods (Windhagen et al., Immunity, 2: 373-80, 1995); (3)
antigen presenting cells can be incubated with whole protein
antigen and the presentation of that antigen on MHC detected by
either T lymphocyte activation assays or biophysical methods
(Harding et al., Proc. Natl. Acad. Sci., 86: 4230-4, 1989); (4)
mast cells can be incubated with reagents that cross-link their
Fc-epsilon receptors and histamine release measured by enzyme
immunoassay (Siraganian et al., TIPS, 4: 432-437, 1983).
[0086] Similarly, products of an immune response in either a model
organism (e.g., mouse) or a human patient can also be detected by
various methods that are well known to those of ordinary skill in
the art. For example, (1) the production of antibodies in response
to vaccination can be readily detected by standard methods
currently used in clinical laboratories, e.g., an ELISA; (2) the
migration of immune cells to sites of inflammation can be detected
by scratching the surface of skin and placing a sterile container
to capture the migrating cells over scratch site (Peters et al.,
Blood, 72: 1310-5, 1988); (3) the proliferation of peripheral blood
mononuclear cells in response to mitogens or mixed lymphocyte
reaction can be measured using .sup.3H-thymidine; (4) the
phagocitic capacity of granulocytes, macrophages, and other
phagocytes in PBMCs can be measured by placing PMBCs in wells
together with labeled particles (Peters et al., Blood, 72: 1310-5,
1988); and (5) the differentiation of immune system cells can be
measured by labeling PBMCs with antibodies to CD molecules such as
CD4 and CD8 and measuring the fraction of the PBMCs expressing
these markers.
[0087] For convenience, immune responses are often described in the
present invention as being either "primary" or "secondary" immune
responses. A primary immune response, which is also described as a
"protective" immune response, refers to an immune response produced
in an individual as a result of some initial exposure (e.g. the
initial "immunization") to a particular antigen, e.g., cell surface
receptor, or activated integrin receptor. Such an immunization can
occur, for example, as the result of some natural exposure to the
antigen (for example, from initial infection by some pathogen that
exhibits or presents the antigen) or from antigen presented by
cancer cells of some tumor in the individual (for example, a
metastatic breast cancer cell). Alternatively, the immunization can
occur as a result of vaccinating the individual with a vaccine
containing the antigen. For example, the vaccine can be a cancer
vaccine comprising one or more antigens from a cancer cell e.g., a
metastatic breast cancer cell.
[0088] A primary immune response can become weakened or attenuated
over time and can even disappear or at least become so attenuated
that it cannot be detected. Accordingly, the present invention also
relates to a "secondary" immune response, which is also described
here as a "memory immune response." The term secondary immune
response refers to an immune response elicited in an individual
after a primary immune response has already been produced. Thus, a
secondary or immune response can be elicited, e.g., to enhance an
existing immune response that has become weakened or attenuated, or
to recreate a previous immune response that has either disappeared
or can no longer be detected. An agent that can be administrated to
elicit a secondary immune response is after referred to as a
"booster" since the agent can be said to "boost" the primary immune
response.
[0089] As an example, and not by way of limitation, a secondary
immune response can be elicited by re-introducing to the individual
an antigen that elicited the primary immune response (for example,
by re-administrating a vaccine). However, a secondary immune
response to an antigen can also be elicited by administrating other
agents that can not contain the actual antigen. For example, the
present invention provides methods for potentiating a secondary
immune response by administrating an antibody to activated integrin
receptor to an individual. In such methods the actual antigen need
not necessarily be administered with the antibody to activated
integrin receptor and the composition containing the antibody need
not necessarily contain the antigen. The secondary or memory immune
response can be either a humoral (antibody) response or a cellular
response. A secondary or memory humoral response occurs upon
stimulation of memory B cells that were generated at the first
presentation of the antigen. Delayed type hypersensitivity (DTH)
reactions are a type of cellular secondary or memory immune
response that are mediated by CD4.sup.+ cells. A first exposure to
an antigen primes the immune system and additional exposure(s)
results in a DTH.
[0090] "Immunologically cross-reactive" or "immunologically
reactive" refers to an antigen which is specifically reactive with
an antibody which was generated using the same ("immunologically
reactive") or different ("immunologically cross-reactive") antigen.
Generally, the antigen is activated integrin receptor, or more
typically an .alpha.v.beta.3 integrin receptor or subsequence
thereof.
[0091] "Immunologically reactive conditions" refers to conditions
which allow an antibody, generated to a particular epitope of an
antigen, to bind to that epitope to a detectably greater degree
than the antibody binds to substantially all other epitopes,
generally at least two times above background binding, preferably
at least five times above background. Immunologically reactive
conditions are dependent upon the format of the antibody binding
reaction and typically are those utilized in immunoassay protocols.
See, Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring
Harbor Publications, New York, 1988 for a description of
immunoassay formats and conditions.
[0092] "Cell surface receptor" refers to molecules and complexes of
molecules capable of receiving a signal and the transmission of
such a signal across the plasma membrane of a cell. An example of a
"cell surface receptor" of the present invention is an activated
integrin receptor, for example, an activated .alpha.v.beta.3
integrin receptor on a metastatic cell.
[0093] "Nonspecific T cell activation" refers to the stimulation of
T cells independent of their antigenic specificity.
[0094] "Effector cell" refers to an immune cell which is involved
in the effector phase of an immune response, as opposed to the
cognitive and activation phases of an immune response. Exemplary
immune cells include a cell of a myeloid or lymphoid origin, e.g.,
lymphocytes (e.g., B cells and T cells including cytolytic T cells
(CTLs)), killer cells, natural killer cells, macrophages,
monocytes, eosinophils, neutrophils, polymorphonuclear cells,
granulocytes, mast cells, and basophils. Effector cells express
specific Fe receptors and carry out specific immune functions. An
effector cell can induce antibody-dependent cell-mediated
cytotoxicity (ADCC), e.g., a neutrophil capable of inducing ADCC.
For example, monocytes, macrophages, neutrophils, eosinophils, and
lymphocytes which express Fc.alpha.R are involved in specific
killing of target cells and presenting antigens to other components
of the immune system, or binding to cells that present antigens. An
effector cell can also phagocytose a target antigen, target cell,
metastatic cancer cell, or microorganism.
[0095] "Target cell" refers to any undesirable cell in a subject
(e.g., a human or animal) that can be targeted by the Ab or Ab
composition of the invention. The target cell can be a cell
expressing or overexpressing human activated integrin receptor.
Cells expressing human activated integrin receptor can include
tumor cells, e.g. breast cancer cells or metastatic breast cancer
cells.
[0096] Targets of interest for antibody compositions metastatic
cancer cells, e.g., metastatic breast cancer cells, include, but
are not limited to, cell surface receptors, growth factor
receptors, antibodies, including anti-idiotypic antibodies and
autoantibodies present in cancer, such as metastatic cancer and
metastatic breast cancer. Other targets are adhesion proteins such
as integrins, selecting, and immunoglobulin superfamily members.
Springer, Nature, 346: 425-433, 1990; Osborn, Cell, 62: 3, 1990;
Hynes, Cell, 69: 11, 1992. Other targets of interest are growth
factor receptors (e.g., FGFR, PDGFR, EGF, her/neu, NGFR, and VEGF)
and their ligands. Other targets are G-protein receptors and
include substance K receptor, the angiotensin receptor, the
.alpha.- and .beta.-adrenergic receptors, the serotonin receptors,
and PAF receptor. See, e.g., Gilman, Ann. Rev. Biochem. 56:
625-649, 1987. Other targets include ion channels (e.g., calcium,
sodium, potassium channels, channel proteins that mediate multidrug
resistance), muscarinic receptors, acetylcholine receptors, GABA
receptors, glutamate receptors, and dopamine receptors (see
Harpold, U.S. Pat. No. 5,401,629 and U.S. Pat. No. 5,436,128).
Other targets are cytokines, such as interleukins IL-1 through
IL-13, tumor necrosis factors .alpha.- and .beta., interferons
.alpha.-, .beta.- and .gamma., tumor growth factor Beta
(TGF-.beta.), colony stimulating factor (CSF) and granulocyte
monocyte colony stimulating factor (GM-CSF). See Aggrawal et al.,
eds., Human Cytokines: Handbook for Basic & Clinical Research,
Blackwell Scientific, Boston, Mass., 1991. Other targets are
hormones, enzymes, and intracellular and intercellular messengers,
such as adenyl cyclase, guanyl cyclase, and phospholipase C. Drugs
are also targets of interest. Target molecules can be human,
mammalian or bacterial. Other targets are antigens, such as
proteins, glycoproteins and carbohydrates from microbial pathogens,
both viral and bacterial, and tumors. Still other targets are
described in U.S. Pat. No. 4,366,241, incorporated herein by
reference in its entirety and for all purposes. Some agents
screened by the target merely bind to a target. Other agents
agonize or antagonize the target.
Cancer Treatment
[0097] Blockade of activated integrin receptor by antibody
compositions can enhance the memory or secondary immune response to
cancerous cells in the patient. Antibodies to activated integrin
receptor can be combined with an immunogenic agent, such as
cancerous cells, purified tumor antigens (including recombinant
proteins, peptides, and carbohydrate molecules), cells, and cells
transfected with genes encoding immune stimulating cytokines and
cell surface antigens, or used alone, to stimulate immunity.
[0098] Antibodies to activated integrin receptor is effective when
following a vaccination protocol. Many experimental strategies for
vaccination against tumors have been devised (see Rosenberg, ASCO
Educational Book Spring: 60-62, 2000; Logothetis, ASCO Educational
Book Spring: 300-302, 2000; Khayat, ASCO Educational Book Spring:
414-428, 2000; Foon, ASCO Educational Book Spring: 730-738, 2000;
see also Restifo et al., Cancer: Principles and Practice of
Oncology, 61: 3023-3043, 1997. In one of these strategies, a
vaccine is prepared using autologous or allogeneic tumor cells.
These cellular vaccines have been shown to be most effective when
the tumor cells are transduced to express GM-CSF. GM-CSF has been
shown to be a potent activator of antigen presentation for tumor
vaccination. Dranoff et al., Proc. Natl. Acad. Sci. U.S.A., 90:
3539-43, 1993.
[0099] Antibodies to activated integrin receptor can boost
GMCSF-modified tumor cell vaccines improves efficacy of vaccines in
a number of experimental tumor models such as mammary carcinoma
(Hurwitz et al., 1998, supra), primary prostate cancer (Hurwitz et
al., Cancer Research, 60: 2444-8, 2000) and melanoma (van Elsas et
al., J. Exp. Med., 190: 355-66, 1999). In these instances,
non-immunogenic tumors, such as the B 16 melanoma, have been
rendered susceptible to destruction by the immune system. The tumor
cell vaccine can also be modified to express other immune
activators such as IL2, and costimulatory molecules, among
others.
[0100] The study of gene expression and large scale gene expression
patterns in various tumors has led to the definition of so called
"tumor specific antigens" (Rosenberg, Immunity, 10: 281-7, 1999).
In many cases, these tumor specific antigens are differentiation
antigens expressed in the tumors and in the cell from which the
tumor arose, for example melanocyte antigens gp100, MAGE antigens,
Trp-2. More importantly, many of these antigens can be shown to be
the targets of tumor specific T cells found in the host. Antibodies
to activated integrin receptor can be used as a boosting agent in
conjunction with vaccines based on recombinant versions of proteins
and/or peptides found to be expressed in a tumor in order to
potentiate a secondary or memory immune response to these proteins.
These proteins are normally viewed by the immune system as self
antigens and are therefore tolerant to them. The tumor antigen can
also include the protein telomerase, which is required for the
synthesis of telomeres of chromosomes and which is expressed in
more than 85% of human cancers and in only a limited number of
somatic tissues (Kim et al., Science, 266: 2011-2013, 1994). These
somatic tissues can be protected from immune attack by various
means. Tumor antigen can also be "neo-antigens" expressed in cancer
cells because of somatic mutations that alter protein sequence or
create fusion proteins between two unrelated sequences (e.g.
bcr-abl in the Philadelphia chromosome), or idiotype from B cell
tumors. Other tumor vaccines can include the proteins from viruses
implicated in human cancers such a Human Papilloma Viruses (HPV),
Hepatitis Viruses (HBV and HCV) and Kaposi's Herpes Sarcoma Virus
(KHSV). Another form of tumor specific antigen which can be used in
conjunction with antibodies to activated integrin receptor is
purified heat shock proteins (HSP) isolated from the tumor tissue
itself. These heat shock proteins contain fragments of proteins
from the tumor cells and these HSPs are highly efficient at
delivery to antigen presenting cells for eliciting tumor immunity
(Suot et al., Science, 269: 1585-1588, 1995; Tamura et al.,
Science, 278: 117-120, 1997.
[0101] Dendritic cells (DC) are potent antigen presenting cells
that can be used to prime antigen-specific responses to activated
integrin receptors on metastatic tumor cells. DC's can be produced
ex vivo and loaded with various protein and peptide antigens as
well as tumor cell extracts (Nestle et al., Nature Medicine, 4:
328-332, 1998). DCs can also be transduced by genetic means to
express these tumor antigens as well. DCs have also been fused
directly to tumor cells for the purposes of immunization (Kugler et
al., Nature Medicine, 6: 332-336, 2000). As a method of
vaccination, DC immunization can be effectively boosted with
antibodies to activated integrin receptor to activate more potent
anti-tumor responses.
[0102] Another type of melanoma vaccine that can be combined with
antibodies to activated integrin receptor is a vaccine prepared
from a melanoma cell line lysate, in conjunction with an
immunological adjuvant, such as the MELACINE.TM. vaccine, a mixture
of lysates from two human melanoma cell lines plus DETOX.TM.
immunological adjuvant. Vaccine treatment can be boosted with
anti-activated integrin receptor, with or without additional
chemotherapeutic treatment.
[0103] Antibodies to activated integrin receptor can also be used
to boost immunity induced through standard cancer treatments. In
these instances, it can be possible to reduce the dose of
chemotherapeutic reagent administered (Mokyr et al., Cancer
Research, 58: 5301-5304, 1998). The scientific rationale behind the
combined use of antibodies to activated integrin receptor and
chemotherapy is that cell death, that is a consequence of the
cytotoxic action of most chemotherapeutic compounds, should result
in increased levels of tumor antigen in the antigen presentation
pathway. Thus, antibodies to activated integrin receptor can boost
an immune response primed to chemotherapy release of tumor cells.
Examples of chemotherapeutic agents combined with treatment with
antibodies to activated integrin receptor can include, but are not
limited to, aldesleukin, altretamine, amifostine, asparaginase,
bleomycin, capecitabine, carboplatin, carmustine, cladribine,
cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine
(DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol,
duocarmycin, epoetin alpha, etoposide, filgrastim, fludarabine,
fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin,
ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole,
leucovorin, megestrol, mesna, methotrexate, metoclopramide,
mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron,
paclitaxel (Taxol.TM.), pilocarpine, prochloroperazine, rituximab,
saproin, tamoxifen, taxol, topotecan hydrochloride, trastuzumab,
vinblastine, vincristine and vinorelbine tartrate. For prostate
cancer treatment, a preferred chemotherapeutic agent with which
anti-activated integrin receptor can be combined is paclitaxel
(Taxol.TM.). For melanoma cancer treatment, a preferred
chemotherapeutic agent with which anti-activated integrin receptor
can be combined is dacarbazine (DTIC).
[0104] Other combination therapies that can result in immune system
priming through cell death are radiation, surgery, and hormone
deprivation (Kwon et al., Proc. Natl. Acad. Sci. U.S.A., 96:
15074-9, 1999. Each of these protocols creates a source of tumor
antigen in the host. For example, any manipulation of the tumor at
the time of surgery can greatly increase the number of cancer cells
in the blood (Schwartz et al., Principles of Surgery. 4.sup.th ed.,
p. 338, 1984). Angiogenesis inhibitors can also be combined with
antibodies to activated integrin receptor. Inhibition of
angiogenesis leads to tumor cell death which can feed tumor antigen
into host antigen presentation pathways. All of these cause tumor
release and possible immune system priming that antibodies to
activated integrin receptor can boost.
Antibody Therapeutics
[0105] As is well understood in the art, biospecific capture
reagents include antibodies, binding fragments of antibodies which
bind to activated integrin receptors on metastatic cells (e.g.,
single chain antibodies, Fab' fragments, F(ab)'2 fragments, and
scFv proteins and affibodies (Affibody, Teknikringen 30, floor 6,
Box 700 04, Stockholm SE-10044, Sweden; See U.S. Pat. No.
5,831,012, incorporated herein by reference in its entirety and for
all purposes)). Depending on intended use, they also may include
receptors and other proteins that specifically bind another
biomolecule.
[0106] The hybrid antibodies and hybrid antibody fragments include
complete antibody molecules having full length heavy and light
chains, or any fragment thereof, such as Fab, Fab', F(ab').sub.2,
Fd, scFv, antibody light chains and antibody heavy chains. Chimeric
antibodies which have variable regions as described herein and
constant regions from various species are also suitable. See, for
example, U.S. Application No. 20030022244.
[0107] Initially, a predetermined target object is chosen to which
an antibody may be raised. Techniques for generating monoclonal
antibodies directed to target objects are well known to those
skilled in the art. Examples of such techniques include, but are
not limited to, those involving display libraries, xeno or humab
mice, hybridomas, and the like Target objects include any substance
which is capable of exhibiting antigenicity and are usually
proteins or protein polysaccharides. Examples include receptors,
enzymes, hormones, growth factors, peptides and the like. It should
be understood that not only are naturally occurring antibodies
suitable for use in accordance with the present disclosure, but
engineered antibodies and antibody fragments which are directed to
a predetermined object are also suitable.
[0108] Antibodies (Abs) that can be subjected to the techniques set
forth herein include monoclonal and polyclonal Abs, and antibody
fragments such as Fab, Fab', F(ab').sub.2, Fd, scFv, diabodies,
antibody light chains, antibody heavy chains and/or antibody
fragments derived from phage or phagemid display technologies. To
begin with, an initial antibody is obtained from an originating
species. More particularly, the nucleic acid or amino acid sequence
of the variable portion of the light chain, heavy chain or both, of
an originating species antibody having specificity for a target
antigen is needed. The originating species is any species which was
used to generate the antibodies or antibody libraries, e.g., rat,
mice, rabbit, chicken, monkey, human, and the like Techniques for
generating and cloning monoclonal antibodies are well known to
those skilled in the art. After a desired antibody is obtained, the
variable regions (V.sub.H and V.sub.L) are separated into component
parts (i.e., frameworks (FRs) and CDRs) using any possible
definition of CDRs (e.g., Kabat alone, Chothia alone, Kabat and
Chothia combined, and any others known to those skilled in the
art). Once that has been obtained, the selection of appropriate
target species frameworks is necessary. One embodiment involves
alignment of each individual framework region from the originating
species antibody sequence with variable amino acid sequences or
gene sequences from the target species. Programs for searching for
alignments are well known in the art, e.g., BLAST and the like. For
example, if the target species is human, a source of such amino
acid sequences or gene sequences (germline or rearranged antibody
sequences) may be found in any suitable reference database such as
Genbank, the NCBI protein databank
(http://ncbi.nlm.nih.gov/BLAST/), VBASE, a database of human
antibody genes (http://www.mrc-cpe.cam.ac.uk/imt-doc), and the
Kabat database of immunoglobulins (http://www.immuno.bme.nwu.edu)
or translated products thereof. If the alignments are done based on
the nucleotide sequences, then the selected genes should be
analyzed to determine which genes of that subset have the closest
amino acid homology to the originating species antibody. It is
contemplated that amino acid sequences or gene sequences which
approach a higher degree homology as compared to other sequences in
the database can be utilized and manipulated in accordance with the
procedures described herein. Moreover, amino acid sequences or
genes which have lesser homology can be utilized when they encode
products which, when manipulated and selected in accordance with
the procedures described herein, exhibit specificity for the
predetermined target antigen. In certain embodiments, an acceptable
range of homology is greater than about 50%. It should be
understood that target species may be other than human.
[0109] The term "treating" refers to any indicia of success in the
treatment or amelioration or prevention of an cancer, including any
objective or subjective parameter such as abatement; remission;
diminishing of symptoms or making the disease condition more
tolerable to the patient; slowing in the rate of degeneration or
decline; or making the final point of degeneration less
debilitating. The treatment or amelioration of symptoms can be
based on objective or subjective parameters; including the results
of an examination by a physician. Accordingly, the term "treating"
includes the administration of the compounds or agents of the
present invention to prevent or delay, to alleviate, or to arrest
or inhibit development of the symptoms or conditions associated
with ocular disease. The term "therapeutic effect" refers to the
reduction, elimination, or prevention of the disease, symptoms of
the disease, or side effects of the disease in the subject.
[0110] "In combination with", "combination therapy" and
"combination products" refer, in certain embodiments, to the
concurrent administration to a patient of a first therapeutic and
the compounds as used herein. When administered in combination,
each component can be administered at the same time or sequentially
in any order at different points in time. Thus, each component can
be administered separately but sufficiently closely in time so as
to provide the desired therapeutic effect.
[0111] "Treating" or "treatment" of cancer or metastatic cancer
using the methods of the present invention includes preventing the
onset of symptoms in a subject that may be at increased risk of
ocular infection but does not yet experience or exhibit symptoms of
infection, inhibiting the symptoms of infection (slowing or
arresting its development), providing relief from the symptoms or
side-effects of infection (including palliative treatment), and
relieving the symptoms of infection (causing regression).
[0112] "Dosage unit" refers to physically discrete units suited as
unitary dosages for the particular individual to be treated. Each
unit can contain a predetermined quantity of active compound(s)
calculated to produce the desired therapeutic effect(s) in
association with the required pharmaceutical carrier. The
specification for the dosage unit forms can be dictated by (a) the
unique characteristics of the active compound(s) and the particular
therapeutic effect(s) to be achieved, and (b) the limitations
inherent in the art of compounding such active compound(s).
[0113] The terms "identical" or percent "identity," in the context
of two or more nucleic acids or polypeptide sequences, refers to
two or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., about 60% identity, preferably 65%, 70%, 75%, 80%,
85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher
identity over a specified region (e.g., nucleotide sequence
encoding a collectin described herein or amino acid sequence of a
collectin described herein), when compared and aligned for maximum
correspondence over a comparison window or designated region) as
measured using a BLAST or BLAST 2.0 sequence comparison algorithms
with default parameters described below, or by manual alignment and
visual inspection (see, e.g., NCBI web site). Such sequences are
then said to be "substantially identical." This term also refers
to, or can be applied to, the compliment of a test sequence. The
term also includes sequences that have deletions and/or additions,
as well as those that have substitutions. As described below, the
preferred algorithms can account for gaps and the like. Preferably,
identity exists over a region that is at least about 25 amino acids
or nucleotides in length, or more preferably over a region that is
50-100 amino acids or nucleotides in length.
[0114] For sequence comparison, typically one sequence acts as a
reference sequence, to which test sequences are compared. When
using a sequence comparison algorithm, test and reference sequences
are entered into a computer, subsequence coordinates are
designated, if necessary, and sequence algorithm program parameters
are designated. Preferably, default program parameters can be used,
or alternative parameters can be designated. The sequence
comparison algorithm then calculates the percent sequence
identities for the test sequences relative to the reference
sequence, based on the program parameters.
[0115] A "comparison window," as used herein, includes reference to
a segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith and
Waterman, Adv. Appl. Math, 2: 482, 1981, by the homology alignment
algorithm of Needleman and Wunsch, J. Mol. Biol, 48:443, 1970, by
the search for similarity method of Pearson and Lipman, Proc.
Nat'l. Acad. Sci. USA, 85:2444, 1988, by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Ausubel et al., eds.,
Current Protocols in Molecular Biology. 1995 supplement).
[0116] A preferred example of algorithm that is suitable for
determining percent sequence identity and sequence similarity are
the BLAST and BLAST 2.0 algorithms, which are described in Altschul
et al., Nuc. Acids Res, 25:3389-3402, 1977 and Altschul et al., J.
Mol. Biol, 215:403-410, 1990, respectively. BLAST and BLAST 2.0 are
used, with the parameters described herein, to determine percent
sequence identity for the nucleic acids and proteins of the
invention. Software for performing BLAST analyses is publicly
available through the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). This algorithm involves first
identifying high scoring sequence pairs (HSPs) by identifying short
words of length W in the query sequence, which either match or
satisfy some positive-valued threshold score T when aligned with a
word of the same length in a database sequence. T is referred to as
the neighborhood word score threshold (Altschul et al., supra).
These initial neighborhood word hits act as seeds for initiating
searches to find longer HSPs containing them. The word hits are
extended in both directions along each sequence for as far as the
cumulative alignment score can be increased. Cumulative scores are
calculated using, for nucleotide sequences, the parameters M
(reward score for a pair of matching residues; always >0) and N
(penalty score for mismatching residues; always <0). For amino
acid sequences, a scoring matrix is used to calculate the
cumulative score. Extension of the word hits in each direction are
halted when: the cumulative alignment score falls off by the
quantity X from its maximum achieved value; the cumulative score
goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached. The BLAST algorithm parameters W, T, and X determine
the sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, M=5, N=-4 and a comparison of both strands.
For amino acid sequences, the BLASTP program uses as defaults a
wordlength of 3, and expectation (E) of 10, and the BLOSUM62
scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci.
USA, 89:10915, 1989) alignments (B) of 50, expectation (E) of 10,
M=5, N=-4, and a comparison of both strands.
[0117] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
[0118] The term "amino acid" refers to naturally occurring and
synthetic amino acids, as well as amino acid analogs and amino acid
mimetics that function in a manner similar to the naturally
occurring amino acids. Naturally occurring amino acids are those
encoded by the genetic code, as well as those amino acids that are
later modified, e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an .alpha. carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
[0119] Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes.
[0120] "Conservatively modified variants" applies to both amino
acid and nucleic acid sequences. With respect to particular nucleic
acid sequences, conservatively modified variants refers to those
nucleic acids which encode identical or essentially identical amino
acid sequences, or where the nucleic acid does not encode an amino
acid sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence with respect to the expression product, but not with
respect to actual probe sequences.
[0121] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles of the invention.
[0122] The following eight groups each contain amino acids that are
conservative substitutions for one another: 1) Alanine (A), Glycine
(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),
Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)).
[0123] Macromolecular structures such as polypeptide structures can
be described in terms of various levels of organization. For a
general discussion of this organization, see, e.g., Alberts et al.,
Molecular Biology of the Cell, 3rd ed., 1994) and Cantor and
Schimmel, Biophysical Chemistry Part I. The Conformation of
Biological Macromolecules, 1980. "Primary structure" refers to the
amino acid sequence of a particular peptide. "Secondary structure"
refers to locally ordered, three dimensional structures within a
polypeptide. These structures are commonly known as domains, e.g.,
enzymatic domains, extracellular domains, transmembrane domains,
pore domains, and cytoplasmic tail domains. Domains are portions of
a polypeptide that form a compact unit of the polypeptide and are
typically 15 to 350 amino acids long. Exemplary domains include
domains with enzymatic activity, e.g., a kinase domain. Typical
domains are made up of sections of lesser organization such as
stretches of .beta.-sheet and .alpha.-helices. "Tertiary structure"
refers to the complete three dimensional structure of a polypeptide
monomer. "Quaternary structure" refers to the three dimensional
structure formed by the noncovalent association of independent
tertiary units. Anisotropic terms are also known as energy
terms.
[0124] A particular nucleic acid sequence also implicitly
encompasses "splice variants." Similarly, a particular protein
encoded by a nucleic acid implicitly encompasses any protein
encoded by a splice variant of that nucleic acid. "Splice
variants," as the name suggests, are products of alternative
splicing of a gene. After transcription, an initial nucleic acid
transcript can be spliced such that different (alternate) nucleic
acid splice products encode different polypeptides. Mechanisms for
the production of splice variants vary, but include alternate
splicing of exons. Alternate polypeptides derived from the same
nucleic acid by read-through transcription are also encompassed by
this definition. Any products of a splicing reaction, including
recombinant forms of the splice products, are included in this
definition.
[0125] The term "recombinant" when used with reference, e.g., to a
cell, or nucleic acid, protein, or vector, indicates that the cell,
nucleic acid, protein or vector, has been modified by the
introduction of a heterologous nucleic acid or protein or the
alteration of a native nucleic acid or protein, or that the cell is
derived from a cell so modified. Thus, for example, recombinant
cells express genes that are not found within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all.
[0126] The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acids, but
to no other sequences. Stringent conditions are sequence-dependent
and will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
"Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes," Overview of principles of hybridization and
the strategy of nucleic acid assays, 1993. Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions can also be achieved with the addition of
destabilizing agents such as formamide. For selective or specific
hybridization, a positive signal is at least two times background,
preferably 10 times background hybridization. Exemplary stringent
hybridization conditions can be as following: 50% formamide,
5.times.SSC, and 1% SDS, incubating at 42.degree. C., or,
5.times.SSC, 1% SDS, incubating at 65.degree. C., with wash in
0.2.times.SSC, and 0.1% SDS at 65.degree. C.
[0127] Nucleic acids that do not hybridize to each other under
stringent conditions are still substantially identical if the
polypeptides which they encode are substantially identical. This
occurs, for example, when a copy of a nucleic acid is created using
the maximum codon degeneracy permitted by the genetic code. In such
cases, the nucleic acids typically hybridize under moderately
stringent hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar stringency. Additional guidelines
for determining hybridization parameters are provided in numerous
reference, e.g., Ausubel et al, supra.
[0128] For PCR, a temperature of about 36.degree. C. is typical for
low stringency amplification, although annealing temperatures can
vary between about 32.degree. C. and 48.degree. C. depending on
primer length. For high stringency PCR amplification, a temperature
of about 62.degree. C. is typical, although high stringency
annealing temperatures can range from about 50.degree. C. to about
65.degree. C., depending on the primer length and specificity.
Typical cycle conditions for both high and low stringency
amplifications include a denaturation phase of 90.degree.
C.-95.degree. C. for 30 sec-2 min., an annealing phase lasting 30
sec.-2 min., and an extension phase of about 72.degree. C. for 1-2
min. Protocols and guidelines for low and high stringency
amplification reactions are provided, e.g., in Innis et al., PCR
Protocols, A Guide to Methods and Applications, Academic Press,
Inc. N.Y., 1990.
[0129] "Antibody" refers to a polypeptide comprising a framework
region from an immunoglobulin gene or fragments thereof that
specifically binds and recognizes an antigen. The recognized
immunoglobulin genes include the kappa, lambda, alpha, gamma,
delta, epsilon, and mu constant region genes, as well as the myriad
immunoglobulin variable region genes. Light chains are classified
as either kappa or lambda. Heavy chains are classified as gamma,
mu, alpha, delta, or epsilon, which in turn define the
immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
Typically, the antigen-binding region of an antibody will be most
critical in specificity and affinity of binding.
[0130] "Pharmaceutically acceptable excipient" means an excipient
that is useful in preparing a pharmaceutical composition that is
generally safe, non-toxic, and desirable, and includes excipients
that are acceptable for veterinary use as well as for human
pharmaceutical use. Such excipients can be solid, liquid,
semisolid, or, in the case of an aerosol composition, gaseous.
[0131] "Pharmaceutically acceptable salts and esters" means salts
and esters that are pharmaceutically acceptable and have the
desired pharmacological properties. Such salts include salts that
can be formed where acidic protons present in the compounds are
capable of reacting with inorganic or organic bases. Suitable
inorganic salts include those formed with the alkali metals, e.g.
sodium and potassium, magnesium, calcium, and aluminum. Suitable
organic salts include those formed with organic bases such as the
amine bases, e.g. ethanolamine, diethanolamine, triethanolamine,
tromethamine, N methylglucamine, and the like. Such salts also
include acid addition salts formed with inorganic acids (e.g.,
hydrochloric and hydrobromic acids) and organic acids (e.g., acetic
acid, citric acid, maleic acid, and the alkane- and arene-sulfonic
acids such as methanesulfonic acid and benzenesulfonic acid).
Pharmaceutically acceptable esters include esters formed from
carboxy, sulfonyloxy, and phosphonoxy groups present in the
compounds, e.g. C.sub.1-6 alkyl esters. When there are two acidic
groups present, a pharmaceutically acceptable salt or ester can be
a mono-acid-mono-salt or ester or a di-salt or ester; and similarly
where there are more than two acidic groups present, some or all of
such groups can be salified or esterified. Compounds named in this
invention can be present in unsalified or unesterified form, or in
salified and/or esterified form, and the naming of such compounds
is intended to include both the original (unsalified and
unesterified) compound and its pharmaceutically acceptable salts
and esters. Also, certain compounds named in this invention may be
present in more than one stereoisomeric form, and the naming of
such compounds is intended to include all single stereoisomers and
all mixtures (whether racemic or otherwise) of such
stereoisomers.
[0132] The terms "pharmaceutically acceptable", "physiologically
tolerable" and grammatical variations thereof, as they refer to
compositions, carriers, diluents and reagents, are used
interchangeably and represent that the materials are capable of
administration to or upon a human without the production of
undesirable physiological effects to a degree that would prohibit
administration of the composition.
[0133] A "therapeutically effective amount" means the amount that,
when administered to a subject for treating a disease, is
sufficient to effect treatment for that disease.
[0134] Except when noted, the terms "subject" or "patient" are used
interchangeably and refer to mammals such as human patients and
non-human primates, as well as experimental animals such as
rabbits, rats, and mice, and other animals. Accordingly, the term
"subject" or "patient" as used herein means any mammalian patient
or subject to which the compositions of the invention can be
administered. In some embodiments of the present invention, the
patient will be suffering from a condition that causes lowered
resistance to disease, e.g., HIV. In an exemplary embodiment of the
present invention, to identify subject patients for treatment with
a pharmaceutical composition comprising one or more collectins
and/or surfactant proteins according to the methods of the
invention, accepted screening methods are employed to determine the
status of an existing disease or condition in a subject or risk
factors associated with a targeted or suspected disease or
condition. These screening methods include, for example, ocular
examinations to determine whether a subject is suffering from an
ocular disease. These and other routine methods allow the clinician
to select subjects in need of therapy. In certain embodiments of
the present invention, ophthalmic compositions for storing,
cleaning, re-wetting and/or disinfecting a contact lens, as well as
artificial tear compositions and/or contact lenses will contain one
or more collectins and/or surfactant proteins thereby inhibiting
the development of ocular disease in contact-lens wearers.
[0135] "Concomitant administration" of a known cancer therapeutic
drug with a pharmaceutical composition of the present invention
means administration of the drug and the collectin and/or
surfactant protein composition at such time that both the known
drug and the composition of the present invention will have a
therapeutic effect. Such concomitant administration may involve
concurrent (i.e. at the same time), prior, or subsequent
administration of the antimicrobial drug with respect to the
administration of a compound of the present invention. A person of
ordinary skill in the art, would have no difficulty determining the
appropriate timing, sequence and dosages of administration for
particular drugs and compositions of the present invention.
[0136] After selecting suitable frame work region candidates from
the same family and/or the same family member, either or both the
heavy and light chain variable regions are produced by grafting the
CDRs from the originating species into the hybrid framework
regions. Assembly of hybrid antibodies or hybrid antibody fragments
having hybrid variable chain regions with regard to either of the
above aspects can be accomplished using conventional methods known
to those skilled in the art. For example, DNA sequences encoding
the hybrid variable domains described herein (i.e., frameworks
based on the target species and CDRs from the originating species)
may be produced by oligonucleotide synthesis and/or PCR. The
nucleic acid encoding CDR regions may also be isolated from the
originating species antibodies using suitable restriction enzymes
and ligated into the target species framework by ligating with
suitable ligation enzymes. Alternatively, the framework regions of
the variable chains of the originating species antibody may be
changed by site-directed mutagenesis.
[0137] Since the hybrids are constructed from choices among
multiple candidates corresponding to each framework region, there
exist many combinations of sequences which are amenable to
construction in accordance with the principles described herein.
Accordingly, libraries of hybrids can be assembled having members
with different combinations of individual framework regions. Such
libraries can be electronic database collections of sequences or
physical collections of hybrids.
[0138] Assembly of a physical antibody or antibody fragment library
is preferably accomplished using synthetic oligonucleotides. In one
example, oligonucleotides are designed to have overlapping regions
so that they could anneal and be filled in by a polymerase, such as
with polymerase chain reaction (PCR). Multiple steps of overlap
extension are performed in order to generate the V.sub.L and
V.sub.H gene inserts. Those fragments are designed with regions of
overlap with human constant domains so that they could be fused by
overlap extension to produce full length light chains and Fd heavy
chain fragments. The light and heavy Fd chain regions may be linked
together by overlap extension to create a single Fab library insert
to be cloned into a display vector. Alternative methods for the
assembly of the humanized library genes can also be used. For
example, the library may be assembled from overlapping
oligonucleotides using a Ligase Chain Reaction (LCR) approach.
Chalmers et al., Biotechniques, 30-2: 249-252, 2001.
[0139] Various forms of antibody fragments may be generated and
cloned into an appropriate vector to create a hybrid antibody
library or hybrid antibody fragment library. For example variable
genes can be cloned into a vector that contains, in-frame, the
remaining portion of the necessary constant domain. Examples of
additional fragments that can be cloned include whole light chains,
the Fd portion of heavy chains, or fragments that contain both
light chain and heavy chain Fd coding sequence. Alternatively, the
antibody fragments used for humanization may be single chain
antibodies (scFv).
[0140] Any selection display system may be used in conjunction with
a library according to the present disclosure. Selection protocols
for isolating desired members of large libraries are known in the
art, as typified by phage display techniques. Such systems, in
which diverse peptide sequences are displayed on the surface of
filamentous bacteriophage have proven useful for creating libraries
of antibody fragments (and the nucleotide sequences that encode
them) for the in vitro selection and amplification of specific
antibody fragments that bind a target antigen. Scott et al.,
Science, 249: 386, 1990. The nucleotide sequences encoding the VH
and VL regions are linked to gene fragments which encode leader
signals that direct them to the periplasmic space of E. coli and as
a result the resultant antibody fragments are displayed on the
surface of the bacteriophage, typically as fusions to bacteriophage
coat proteins (e.g., pIII or pVIII). Alternatively, antibody
fragments are displayed externally on lambda phage or T7 capsids
(phagebodies). An advantage of phage-based display systems is that,
because they are biological systems, selected library members can
be amplified simply by growing the phage containing the selected
library member in bacterial cells. Furthermore, since the
nucleotide sequence that encode the polypeptide library member is
contained on a phage or phagemid vector, sequencing, expression and
subsequent genetic manipulation is relatively straightforward.
Methods for the construction of bacteriophage antibody display
libraries and lambda phage expression libraries are well known in
the art. McCafferty et al., Nature, 348: 552, 1990; Kang et al.,
Proc. Natl. Acad. Sci. U.S.A., 88: 4363, 1991.
[0141] The present invention further relates to antibodies and
T-cell antigen receptors (TCR) which specifically bind the
polypeptides of the present invention. The antibodies of the
present invention include IgG (including IgG1, IgG2, IgG3, and
IgG4), IgA (including IgA1 and IgA2), IgD, IgE, or IgM, and IgY. As
used herein, the term "antibody" (Ab) is meant to include whole
antibodies, including single-chain whole antibodies, and
antigen-binding fragments thereof. Most preferably the antibodies
are human antigen binding antibody fragments of the present
invention and include, but are not limited to, Fab, Fab' and
F(ab').sub.2, Fd, single-chain Fvs (scFv), single-chain antibodies,
disulfide-linked Fvs (sdFv) and fragments comprising either a
V.sub.L or V.sub.H domain. The antibodies may be from any animal
origin including birds and mammals. Preferably, the antibodies are
human, murine, rabbit, goat, guinea pig, camel, horse, or
chicken.
[0142] Antigen-binding antibody fragments, including single-chain
antibodies, may comprise the variable region(s) alone or in
combination with the entire or partial of the following: hinge
region, CH.sub.1, CH.sub.2, and CH.sub.3 domains. Also included in
the invention are any combinations of variable region(s) and hinge
region, CH.sub.1, CH.sub.2, and CH.sub.3 domains. The present
invention further includes monoclonal, polyclonal, chimeric,
humanized, and human monoclonal and human polyclonal antibodies
which specifically bind the polypeptides of the present invention.
The present invention further includes antibodies which are
anti-idiotypic to the antibodies of the present invention.
[0143] The antibodies of the present invention may be monospecific,
bispecific, trispecific or of greater multispecificity.
Multispecific antibodies may be specific for different epitopes of
a polypeptide of the present invention or may be specific for both
a polypeptide of the present invention as well as for heterologous
compositions, such as a heterologous polypeptide or solid support
material. See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO
92/05793; Tutt et al., J. Immunol. 147: 60-69, 1991; U.S. Pat. Nos.
5,573,920; 4,474,893; 5,601,819; 4,714,681; 4,925,648, each
incorporated herein by reference in their entirety and for all
purposes; Kostelny et al., J. Immunol. 148: 1547-1553, 1992.
[0144] Antibodies of the present invention may be described or
specified in terms of the epitope(s) or portion(s) of a polypeptide
of the present invention which are recognized or specifically bound
by the antibody. The epitope(s) or polypeptide portion(s) may be
specified as described herein, e.g., by N-terminal and C-terminal
positions, by size in contiguous amino acid residues. Antibodies
which specifically bind any epitope or polypeptide of the present
invention may also be excluded. Therefore, the present invention
includes antibodies that specifically bind polypeptides of the
present invention, and allows for the exclusion of the same.
[0145] Antibodies of the present invention may also be described or
specified in terms of their cross-reactivity. Antibodies that do
not bind any other analog, ortholog, or homolog of the polypeptides
of the present invention are included. Antibodies that do not bind
polypeptides with less than 95%, less than 90%, less than 85%, less
than 80%, less than 75%, less than 70%, less than 65%, less than
60%, less than 55%, and less than 50% identity (as calculated using
methods known in the art and described herein) to a polypeptide of
the present invention are also included in the present invention.
Further included in the present invention are antibodies which only
bind polypeptides encoded by polynucleotides which hybridize to a
polynucleotide of the present invention under stringent
hybridization conditions (as described herein). Antibodies of the
present invention may also be described or specified in terms of
their binding affinity. Preferred binding affinities include those
with a dissociation constant or K.sub.d less than
5.times.10.sup.-6M, 10.sup.-6M, 5.times.10.sup.-7M, 10.sup.-7M,
5.times.10.sup.-8M, 10.sup.-8M, 5.times.10.sup.-9M, 10.sup.-9M,
5.times.10.sup.-10M, 10.sup.-10M, 5.times.10.sup.-11M, 10.sup.-11M,
5.times.10.sup.-12M, 10.sup.-12M, 5.times.10.sup.-13M, 10.sup.-13M,
5.times.10.sup.14M, 10.sup.14M, 5.times.10.sup.15M, and
10.sup.15M.
[0146] Antibodies to activated integrin receptors vention have uses
that include, but are not limited to, methods known in the art to
purify, detect, and target the polypeptides of the present
invention including both in vitro and in vivo diagnostic and
therapeutic methods. For example, the antibodies have use in
immunoassays for qualitatively and quantitatively measuring levels
of the polypeptides of the present invention in biological samples.
See, e.g., Harlow and Lane, supra, incorporated herein by reference
in its entirety and for all purposes.
[0147] The antibodies of the present invention may be used either
alone or in combination with other compositions. The antibodies may
further be recombinantly fused to a heterologous polypeptide at the
N- or C-terminus or chemically conjugated (including covalently and
non-covalently conjugations) to polypeptides or other compositions.
For example, antibodies of the present invention may be
recombinantly fused or conjugated to molecules useful as labels in
detection assays and effector molecules such as heterologous
polypeptides, drugs, or toxins. See, e.g., WO 92/08495; WO
91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 0 396 387,
each incorporated herein by reference in their entirety and for all
purposes.
[0148] The antibodies of the present invention may be prepared by
any suitable method known in the art. For example, a polypeptide of
the present invention or an antigenic fragment thereof can be
administered to an animal in order to induce the production of sera
containing polyclonal antibodies. The term "monoclonal antibody" is
not a limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
Monoclonal antibodies can be prepared using a wide variety of
techniques known in the art including the use of hybridoma,
recombinant, and phage display technology.
[0149] Hybridoma techniques include those known in the art and
taught in Harlow and Lane, supra; Hammerling et al., Monoclonal
Antibodies and T-Cell Hybridomas, 563-681, 1981, said references
incorporated by reference in their entireties. Fab and F(ab').sub.2
fragments may be produced by proteolytic cleavage, using enzymes
such as papain (to produce Fab fragments) or pepsin (to produce
F(ab').sub.2 fragments).
[0150] Alternatively, antibodies to activated integrin receptor can
be produced through the application of recombinant DNA and phage
display technology or through synthetic chemistry using methods
known in the art. For example, the antibodies of the present
invention can be prepared using various phage display methods known
in the art. In phage display methods, functional antibody domains
are displayed on the surface of a phage particle which carries
polynucleotide sequences encoding them. Phage with a desired
binding property are selected from a repertoire or combinatorial
antibody library (e.g. human or murine) by selecting directly with
antigen, typically antigen bound or captured to a solid surface or
bead. Phage used in these methods are typically filamentous phage
including fd and M13 with Fab, Fv or disulfide stabilized Fv
antibody domains recombinantly fused to either the phage gene III
or gene VIII protein. Examples of phage display methods that can be
used to make the antibodies of the present invention include those
disclosed in Brinkman et al., J. Immunol. Methods 182: 41-50, 1995;
Ames et al., J. Immunol. Methods 184: 177-186, 1995; Kettleborough
et al., Eur. J. Immunol. 24: 952-958, 1994; Persic et al., Gene
187: 9-18, 1997; Burton et al., Advances in Immunology 57:191-280,
1994; PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO
92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and U.S. Pat. Nos.
5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753;
5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727
and 5,733,743, each incorporated herein by reference in their
entirety and for all purposes.
[0151] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired antigen binding fragment, and expressed in any
desired host including mammalian cells, insect cells, plant cells,
yeast, and bacteria. For example, techniques to recombinantly
produce Fab, Fab' and F(ab')2 fragments can also be employed using
methods known in the art such as those disclosed in WO 92/22324;
Mullinax et al., BioTechniques 12: 864-869, 1992; and Sawai et al.,
AJRI 34: 26-34, 1995; and Better et al., Science 240: 1041-1043,
1988.
[0152] Examples of techniques which can be used to produce
single-chain Fvs and antibodies include those described in U.S.
Pat. Nos. 4,946,778 and 5,258,498, each incorporated herein by
reference in their entirety and for all purposes; Huston et al.,
Methods in Enzymology, 203: 46-88, 1991; Shu, L. et al., PNAS 90:
7995-7999, 1993; and Skerra et al., Science 240: 1038-1040, 1988.
For some uses, including in vivo use of antibodies in humans and in
vitro detection assays, it may be preferable to use chimeric,
humanized, or human antibodies. Methods for producing chimeric
antibodies are known in the art. See e.g., Morrison, Science 229:
1202, 1985; Oi et al., BioTechniques 4: 214, 1986; Gillies et al.,
J. Immunol. Methods, 125: 191-202, 1989; and U.S. Pat. No.
5,807,715. Antibodies can be humanized using a variety of
techniques including CDR-grafting (EP 0 239 400; WO 91/09967; and
U.S. Pat. Nos. 5,530,101 and 5,585,089), veneering or resurfacing
(EP 0 592 106; EP 0 519 596; Padlan E. A., Molecular Immunology,
28: 489-498, 1991; Studnicka et al., Protein Engineering 7:
805-814, 1994; Roguska et al., PNAS 91: 969-973, 1994), and chain
shuffling (U.S. Pat. No. 5,565,332). Human antibodies can be made
by a variety of methods known in the art including phage display
methods described above. See also, U.S. Pat. Nos. 4,444,887;
4,716,111; 5,545,806; and 5,814,318; and WO 98/46645; WO 98/50433;
WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO
91/10741, each incorporated herein by reference in their entirety
and for all purposes.
[0153] Further included in the present invention are antibodies
recombinantly fused or chemically conjugated (including both
covalently and non-covalently conjugations) to a polypeptide of the
present invention. The antibodies may be specific for antigens
other than polypeptides of the present invention. For example,
antibodies may be used to target the polypeptides of the present
invention to particular cell types, either in vitro or in vivo, by
fusing or conjugating the polypeptides of the present invention to
antibodies specific for particular cell surface receptors.
Antibodies fused or conjugated to the polypeptides of the present
invention may also be used in in vitro immunoassays and
purification methods using methods known in the art. See e.g.,
Harbor et al., supra, and WO 93/21232; EP 0 439 095; Naramura et
al., Immunol. Lett. 39: 91-99, 1994; U.S. Pat. No. 5,474,981,
incorporated herein by reference in its entirety and for all
purposes; Gillies et al., PNAS 89: 1428-1432, 1992; Fell et al., J.
Immunol. 146: 2446-2452, 1991.
[0154] The present invention further includes compositions
comprising the polypeptides of the present invention fused or
conjugated to antibody domains other than the variable regions. For
example, the polypeptides of the present invention may be fused or
conjugated to an antibody Fc region, or portion thereof. The
antibody portion fused to a polypeptide of the present invention
may comprise the hinge region, CH.sub.1 domain, CH.sub.2 domain,
and CH.sub.3 domain or any combination of whole domains or portions
thereof. The polypeptides of the present invention may be fused or
conjugated to the above antibody portions to increase the in vivo
half life of the polypeptides or for use in immunoassays using
methods known in the art. The polypeptides may also be fused or
conjugated to the above antibody portions to form multimers. For
example, Fc portions fused to the polypeptides of the present
invention can form dimers through disulfide bonding between the Fc
portions. Higher multimeric forms can be made by fusing the
polypeptides to portions of IgA and IgM. Methods for fusing or
conjugating the polypeptides of the present invention to antibody
portions are known in the art. See, e.g., U.S. Pat. Nos. 5,336,603;
5,622,929; 5,359,046; 5,349,053; 5,447,851; 5,112,946; EP 0 307
434, EP 0 367 166; WO 96/04388; and WO 91/06570, each incorporated
herein by reference in their entirety and for all purposes;
Ashkenazi et al., PNAS, 88: 10535-10539, 1991; Zheng et al., J.
Immunol., 154: 5590-5600, 1995; and Vil et al., PNAS, 89:
11337-11341, 1992.
[0155] The invention further relates to antibodies which act as
agonists or antagonists of the polypeptides of the present
invention. For example, the present invention includes antibodies
which disrupt the receptor/ligand interactions with the
polypeptides of the invention either partially or fully. Included
are both receptor-specific antibodies and ligand-specific
antibodies. Included are receptor-specific antibodies which do not
prevent ligand binding but prevent receptor activation. Receptor
activation (i.e., signaling) may be determined by techniques
described herein or otherwise known in the art. Also include are
receptor-specific antibodies which both prevent ligand binding and
receptor activation. Likewise, included are neutralizing antibodies
which bind the ligand and prevent binding of the ligand to the
receptor, as well as antibodies which bind the ligand, thereby
preventing receptor activation, but do not prevent the ligand from
binding the receptor. Further included are antibodies which
activate the receptor. These antibodies may act as agonists for
either all or less than all of the biological activities affected
by ligand-mediated receptor activation. The antibodies may be
specified as agonists or antagonists for biological activities
comprising specific activities disclosed herein. The above antibody
agonists can be made using methods known in the art. See e.g., WO
96/40281; U.S. Pat. No. 5,811,097, each incorporated herein by
reference in their entirety and for all purposes; Deng et al.,
Blood 92: 1981-1988, 1998; Chen, et al., Cancer Res., 58:
3668-3678, 1998; Harrop et al., J. Immunol. 161: 1786-1794, 1998;
Zhu et al., Cancer Res., 58: 3209-3214, 1998; Yoon, et al., J.
Immunol., 160: 3170-3179, 1998; Prat et al., J. Cell. Sci., 111:
237-247, 1998; Pitard et al., J. Immunol. Methods, 205: 177-190,
1997; Liautard et al., Cytokinde, 9: 233-241, 1997; Carlson et al.,
J. Biol. Chem., 272: 11295-11301, 1997; Taryman et al., Neuron, 14:
755-762, 1995; Muller et al., Structure, 6: 1153-1167, 1998;
Bartunek et al., Cytokinem, 8: 14-20, 1996. As discussed above,
antibodies to activated integrin receptors on metatstatic cells
can, in turn, be utilized to generate anti-idiotype antibodies that
"mimic" polypeptides of the invention using techniques well known
to those skilled in the art. (See, e.g., Greenspan et al., FASEB J.
7: 437-444, 1989 and Nissinoff, J. Immunol. 147: 2429-2438, 1991).
For example, antibodies which bind to and competitively inhibit
polypeptide multimerization and/or binding of a polypeptide of the
invention to ligand can be used to generate anti-idiotypes that
"mimic" the polypeptide multimerization and/or binding domain and,
as a consequence, bind to and neutralize polypeptide and/or its
ligand. Such neutralizing anti-idiotypes or Fab fragments of such
anti-idiotypes can be used in therapeutic regimens to neutralize
polypeptide ligand. For example, such anti-idiotypic antibodies can
be used to bind a polypeptide of the invention and/or to bind its
ligands/receptors, and thereby block its biological activity.
[0156] "Inhibitors," "activators," and "modulators" of activated
integrin receptor on metastatic cells are used to refer to
inhibitory, activating, or modulating molecules, respectively,
identified using in vitro and in vivo assays for integrin receptor
binding or signaling, e.g., ligands, agonists, antagonists, and
their homologs and mimetics.
[0157] The term "modulator" includes inhibitors and activators.
Inhibitors are agents that, e.g., bind to, partially or totally
block stimulation, decrease, prevent, delay activation, inactivate,
desensitize, or down regulate the activity of activated integrin
receptors, e.g., antagonists. Activators are agents that, e.g.,
bind to, stimulate, increase, open, activate, facilitate, enhance
activation, sensitize or up regulate the activity of activated
integrin receptors, e.g., agonists. Modulators include agents that,
e.g., alter the interaction of activated integrin receptor with:
proteins that bind activators or inhibitors, receptors, including
proteins, peptides, lipids, carbohydrates, polysaccharides, or
combinations of the above, e.g., lipoproteins, glycoproteins, and
the like. Modulators include genetically modified versions of
naturally-occurring activated integrin receptor ligands, e.g., with
altered activity, as well as naturally occurring and synthetic
ligands, antagonists, agonists, small chemical molecules and the
like. Such assays for inhibitors and activators include, e.g.,
applying putative modulator compounds to a cell expressing an
activated integrin receptor and then determining the functional
effects on integrin receptor signaling, as described herein.
Samples or assays comprising activated integrin receptor that are
treated with a potential activator, inhibitor, or modulator are
compared to control samples without the inhibitor, activator, or
modulator to examine the extent of inhibition. Control samples
(untreated with inhibitors) can be assigned a relative integrin
receptor activity value of 100%. Inhibition of activated integrin
receptor is achieved when the integrin receptor activity value
relative to the control is about 80%, optionally 50% or 25-0%.
Activation of integrin receptor is achieved when the integrin
receptor activity value relative to the control is 110%, optionally
150%, optionally 200-500%, or 1000-3000% higher.
[0158] The ability of a molecule to bind to activated integrin
receptor can be determined, for example, by the ability of the
putative ligand to bind to activated integrin receptor
immunoadhesin coated on an assay plate. Specificity of binding can
be determined by comparing binding to non-activated integrin
receptor.
[0159] In one embodiment, antibody binding to activated integrin
receptor can be assayed by either immobilizing the ligand or the
receptor. For example, the assay can include immobilizing activated
integrin receptor fused to a His tag onto Ni-activated NTA resin
beads. Antibody can be added in an appropriate buffer and the beads
incubated for a period of time at a given temperature. After washes
to remove unbound material, the bound protein can be released with,
for example, SDS, buffers with a high pH, and the like and
analyzed.
Fusion Proteins
[0160] Antibodies to activated integrin receptor can be used to
generate fusion proteins. For example, the antibodies of the
present invention, when fused to a second protein, can be used as
an antigenic tag. Antibodies raised against activated integrin
receptor can be used to indirectly detect the second protein by
binding to the polypeptide. Moreover, because secreted proteins
target cellular locations based on trafficking signals, the
integrin receptor can be used as a targeting molecule once fused to
other proteins.
[0161] Examples of domains that can be fused to polypeptides
include not only heterologous signal sequences, but also other
heterologous functional regions. The fusion does not necessarily
need to be direct, but may occur through linker sequences.
[0162] Moreover, fusion proteins may also be engineered to improve
characteristics of the polypeptide. For instance, a region of
additional amino acids, particularly charged amino acids, may be
added to the N-terminus of the polypeptide to improve stability and
persistence during purification from the host cell or subsequent
handling and storage. Also, peptide moieties may be added to the
polypeptide to facilitate purification. Such regions may be removed
prior to final preparation of the polypeptide. The addition of
peptide moieties to facilitate handling of polypeptides are
familiar and routine techniques in the art.
[0163] Moreover, antibody compositions or cell surface receptors,
or integrin receptors, including fragments, and specifically
epitopes, can be combined with parts of the constant domain of
immunoglobulins (IgG), resulting in chimeric polypeptides. These
fusion proteins facilitate purification and show an increased
half-life in vivo. One reported example describes chimeric proteins
consisting of the first two domains of the human CD4-polypeptide
and various domains of the constant regions of the heavy or light
chains of mammalian immunoglobulins. EP A 394,827; Traunecker et
al., Nature, 331: 84-86, 1988. Fusion proteins having
disulfide-linked dimeric structures (due to the IgG) can also be
more efficient in binding and neutralizing other molecules, than
the monomeric secreted protein or protein fragment alone.
Fountoulakis et al., J. Biochem. 270: 3958-3964, 1995.
[0164] Similarly, EP-A-O 464 533 (Canadian counterpart 2045869)
discloses fusion proteins comprising various portions of constant
region of immunoglobulin molecules together with another human
protein or part thereof. In many cases, the Fc part in a fusion
protein is beneficial in therapy and diagnosis, and thus can result
in, for example, improved pharmacokinetic properties. (EP-A 0232
262.) Alternatively, deleting the Fc part after the fusion protein
has been expressed, detected, and purified, would be desired. For
example, the Fc portion may hinder therapy and diagnosis if the
fusion protein is used as an antigen for immunizations. In drug
discovery, for example, human proteins, such as hIL-5, have been
fused with Fc portions for the purpose of high-throughput screening
assays to identify antagonists of hIL-5. Bennett et al., J.
Molecular Recognition 8: 52-58, 1995; K. Johanson et al., J. Biol.
Chem., 270: 9459-9471 1995.
[0165] Moreover, the polypeptides can be fused to marker sequences,
such as a peptide which facilitates purification of the fused
polypeptide. In preferred embodiments, the marker amino acid
sequence is a hexa-histidine peptide, such as the tag provided in a
pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,
91311), among others, many of which are commercially available. As
described in Gentz et al., Proc. Natl. Acad. Sci. USA 86: 821-824,
1989, for instance, hexa-histidine provides for convenient
purification of the fusion protein. Another peptide tag useful for
purification, the "HA" tag, corresponds to an epitope derived from
the influenza hemagglutinin protein. Wilson et al., Cell 37: 767,
1984.
[0166] Thus, any of these above fusions can be engineered using the
polynucleotides or the polypeptides of the present invention.
Expression of Recombinant Antibodies
[0167] Chimeric, humanized and human antibodies to cell surface
receptor, e.g., activated integrin receptor on metastatic cells,
are typically produced by recombinant expression. Recombinant
polynucleotide constructs typically include an expression control
sequence operably linked to the coding sequences of antibody
chains, including naturally-associated or heterologous promoter
regions. Preferably, the expression control sequences are
eukaryotic promoter systems in vectors capable of transforming or
transfecting eukaryotic host cells. Once the vector has been
incorporated into the appropriate host, the host is maintained
under conditions suitable for high level expression of the
nucleotide sequences, and the collection and purification of the
crossreacting antibodies. See U.S. Application No. 20020199213
incorporated herein by reference in its entirety and for all
purposes.
[0168] These expression vectors are typically replicable in the
host organisms either as episomes or as an integral part of the
host chromosomal DNA. Commonly, expression vectors contain
selection markers, e.g., ampicillin-resistance or
hygromycin-resistance, to permit detection of those cells
transformed with the desired DNA sequences.
[0169] E. coli is one prokaryotic host particularly useful for
cloning the DNA sequences of the present invention. Microbes, such
as yeast are also useful for expression. Saccharomyces is a
preferred yeast host, with suitable vectors having expression
control sequences, an origin of replication, termination sequences
and the like as desired. Typical promoters include
3-phosphoglycerate kinase and other glycolytic enzymes. Inducible
yeast promoters include, among others, promoters from alcohol
dehydrogenase, isocytochrome C, and enzymes responsible for maltose
and galactose utilization.
[0170] Mammalian cells are a preferred host for expressing
nucleotide segments encoding immunoglobulins or fragments thereof.
See Winnacker, From Genes To Clones, VCH Publishers, NY, 1987. A
number of suitable host cell lines capable of secreting intact
heterologous proteins have been developed in the art, and include
Chinese hamster ovary (CHO) cell lines, various COS cell lines,
HeLa cells, L cells and myeloma cell lines. Preferably, the cells
are nonhuman. Expression vectors for these cells can include
expression control sequences, such as an origin of replication, a
promoter, an enhancer, and necessary processing information sites,
such as ribosome binding sites, RNA splice sites, polyadenylation
sites, and transcriptional terminator sequences. Queen et al.,
Immunol. Rev. 89: 49, 1986. Preferred expression control sequences
are promoters derived from endogenous genes, cytomegalovirus, SV40,
adenovirus, bovine papillomavirus, and the like. Co, et al., J.
Immunol. 148: 1149, 1992.
[0171] Alternatively, antibody coding sequences can be incorporated
in transgenes for introduction into the genome of a transgenic
animal and subsequent expression in the milk of the transgenic
animal. See, e.g., U.S. Pat. Nos. 5,741,957; 5,304,489; and
5,849,992, each incorporated herein by reference in their entirety
and for all purposes. Suitable transgenes include coding sequences
for light and/or heavy chains in operable linkage with a promoter
and enhancer from a mammary gland specific gene, such as casein or
beta lactoglobulin.
[0172] The vectors containing the DNA segments of interest can be
transferred into the host cell by well-known methods, depending on
the type of cellular host. For example, calcium chloride
transfection is commonly utilized for prokaryotic cells, whereas
calcium phosphate treatment, electroporation, lipofection,
biolistics or viral-based transfection can be used for other
cellular hosts. Other methods used to transform mammalian cells
include the use of polybrene, protoplast fusion, liposomes,
electroporation, and microinjection (see generally, Sambrook et
al., Molecular Cloning). For production of transgenic animals,
transgenes can be microinjected into fertilized oocytes, or can be
incorporated into the genome of embryonic stem cells, and the
nuclei of such cells transferred into enucleated oocytes.
[0173] Once expressed, collections of antibodies are purified from
culture media and host cells. Antibodies can be purified according
to standard procedures of the art, including HPLC purification,
column chromatography, gel electrophoresis and the like. Usually,
antibody chains are expressed with signal sequences and are thus
released to the culture media. However, if antibody chains are not
naturally secreted by host cells, the antibody chains can be
released by treatment with mild detergent. Antibody chains can then
be purified by conventional methods including ammonium sulfate
precipitation, affinity chromatography to immobilized target,
column chromatography, gel electrophoresis and the like (see
generally Scopes, Protein Purification, Springer-Verlag, N.Y.,
1982).
[0174] The above methods result in libraries of nucleic acid
sequences encoding antibody chains having specific affinity for a
chosen target. The libraries of nucleic acids typically have at
least 5, 10, 20, 50, 100, 1000, 10.sup.4, 10.sup.5, 10.sup.6,
10.sup.7, 10.sup.8, or 10.sup.9 different members. Usually, no
single member constitutes more than 25 or 50% of the total
sequences in the library. Typically, at least 25, 50%, 75, 90, 95,
99 or 99.9% of library members encode antibody chains with specific
affinity for the target molecules. In the case of double chain
antibody libraries, a pair of nucleic acid segments encoding heavy
and light chains respectively is considered a library member. The
nucleic acid libraries can exist in free form, as components of any
vector or transfected as a component of a vector into host
cells.
[0175] The nucleic acid libraries can be expressed to generate
polyclonal libraries of antibodies having specific affinity for a
target. The composition of such libraries is determined from the
composition of the nucleotide libraries. Thus, such libraries
typically have at least 5, 10, 20, 50, 100, 1000, 10.sup.4,
10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, or 10.sup.9 members with
different amino acid composition. Usually, no single member
constitutes more than 25 or 50% of the total polypeptides in the
library. The percentage of antibody chains in an antibody chain
library having specific affinity for a target is typically lower
than the percentage of corresponding nucleic acids encoding the
antibody chains. The difference is due to the fact that not all
polypeptides fold into a structure appropriate for binding despite
having the appropriate primary amino acid sequence to support
appropriate folding. In some libraries, at least 25, 50, 75, 90,
95, 99 or 99.9% of antibody chains have specific affinity for the
target molecules. Again, in libraries of multi-chain antibodies,
each antibody (such as a Fab or intact antibody) is considered a
library member. The different antibody chains differ from each
other in terms of fine binding specificity and affinity for the
target. Some such libraries comprise members binding to different
epitopes on the same antigen. Some such libraries comprises at
least two members that bind to the same antigen without competing
with each other.
[0176] Polyclonal libraries of human antibodies resulting from the
above methods are distinguished from natural populations of human
antibodies both by the high percentages of high affinity binders in
the present libraries, and in that the present libraries typically
do not show the same diversity of antibodies present in natural
populations. The reduced diversity in the present libraries is due
to the nonhuman transgenic animals that provide the source
materials not including all human immunoglobulin genes. For
example, some polyclonal antibody libraries are free of antibodies
having lambda light chains. Some polyclonal antibody libraries of
the invention have antibody heavy chains encoded by fewer than 10,
20, 30 or 40 V.sub.H genes. Some polyclonal antibody libraries of
the invention have antibody light chains encoded by fewer than 10,
20, 30 or 40 V.sub.L genes.
Modified Antibodies
[0177] Also included in the invention are modified antibodies to
cell surface receptors, e.g., activated integrin receptors, on
metastatic cells.
[0178] "Modified antibody" refers to antibodies and derivatives of
human single chain Fv (scFv) antibody fragments optimized
chemically or by molecular engineering into different formats,
including but not limited to diabodies, triabodies or bispecific
antibodies, pegylated derivatives, variants derived from molecular
evolution to enhance affinity, stability, or valency. Modified
antibodies also include formats such as monoclonal antibodies,
chimeric antibodies, and humanized antibodies which have been
modified by, e.g., deleting, adding, or substituting portions of
the antibody. For example, an antibody can be modified by deleting
the constant region and replacing it with a constant region meant
to increase half-life, e.g., serum half-life, stability or affinity
of the antibody.
[0179] The antibody conjugates of the invention can be used to
modify a given biological response or create a biological response
(e.g., to recruit effector cells). The drug moiety is not to be
construed as limited to classical chemical therapeutic agents. For
example, the drug moiety can be a protein or polypeptide possessing
a desired biological activity. Such proteins can include, for
example, an enzymatically active toxin, or active fragment thereof,
such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;
a protein such as tumor necrosis factor or interferon-alpha; or,
biological response modifiers such as, for example, lymphokines,
interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophage colony stimulating factor
("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or
other growth factors. Other derivatives can include antibody fusion
proteins with apoptosis inducing moieties such as TRIAL, tumor
necrosis factor-related apoptosis-inducing ligand, and reporter
molecules such as luciferase or fluorescent probes and
nano-particles for non-invasive imaging or targeted delivery of
pay-load molecules to sites with tumor burden and micro- and
macro-metastases.
[0180] In certain preferred embodiments of the invention, the
antibodies and antibody compositions of the invention, for example,
can be coupled or conjugated to one or more therapeutic or
cytotoxic moieties. As used herein, "cytotoxic moiety" simply means
a moiety that inhibits cell growth or promotes cell death when
proximate to or absorbed by a cell. Suitable cytotoxic moieties in
this regard include radioactive agents or isotopes (radionuclides),
chemotoxic agents such as differentiation inducers, inhibitors and
small chemotoxic drugs, toxin proteins and derivatives thereof, as
well as nucleotide sequences (or their antisense sequence).
Therefore, the cytotoxic moiety can be, by way of non-limiting
example, a chemotherapeutic agent, a photoactivated toxin or a
radioactive agent.
[0181] In general, therapeutic agents can be conjugated to the
antibodies and antibody compositions of the invention, for example,
by any suitable technique, with appropriate consideration of the
need for pharmokinetic stability and reduced overall toxicity to
the patient. A therapeutic agent can be coupled to a suitable
antibody moiety either directly or indirectly (e.g. via a linker
group). A direct reaction between an agent and an antibody is
possible when each possesses a functional group capable of reacting
with the other. For example, a nucleophilic group, such as an amino
or sulfhydryl group, can be capable of reacting with a
carbonyl-containing group, such as an anhydride or an acid halide,
or with an alkyl group containing a good leaving group (e.g., a
halide). Alternatively, a suitable chemical linker group can be
used. A linker group can function as a spacer to distance an
antibody from an agent in order to .alpha.void interference with
binding capabilities. A linker group can also serve to increase the
chemical reactivity of a substituent on a moiety or an antibody,
and thus increase the coupling efficiency. An increase in chemical
reactivity can also facilitate the use of moieties, or functional
groups on moieties, which otherwise would not be possible.
[0182] Suitable linkage chemistries include maleimidyl linkers and
alkyl halide linkers (which react with a sulfhydryl on the antibody
moiety) and succinimidyl linkers (which react with a primary amine
on the antibody moiety). Several primary amine and sulfhydryl
groups are present on immunoglobulins, and additional groups can be
designed into recombinant immunoglobulin molecules. It will be
evident to those skilled in the art that a variety of bifunctional
or polyfunctional reagents, both homo- and hetero-functional (such
as those described in the catalog of the Pierce Chemical Co.,
Rockford, Ill.), can be employed as a linker group. Coupling can be
effected, for example, through amino groups, carboxyl groups,
sulfhydryl groups or oxidized carbohydrate residues (see, e.g.,
U.S. Pat. No. 4,671,958).
[0183] As an alternative coupling method, cytotoxic agents can be
coupled to the antibodies and antibody compositions of the
invention, for example, through an oxidized carbohydrate group at a
glycosylation site, as described in U.S. Pat. Nos. 5,057,313 and
5,156,840. Yet another alternative method of coupling the antibody
and antibody compositions to the cytotoxic or imaging moiety is by
the use of a non-covalent binding pair, such as
streptavidin/biotin, or avidin/biotin. In these embodiments, one
member of the pair is covalently coupled to the antibody moiety and
the other member of the binding pair is covalently coupled to the
cytotoxic or imaging moiety.
[0184] Where a cytotoxic moiety is more potent when free from the
antibody portion of the immunoconjugates of the present invention,
it can be desirable to use a linker group which is cleavable during
or upon internalization into a cell, or which is gradually
cleavable over time in the extracellular environment. A number of
different cleavable linker groups have been described. The
mechanisms for the intracellular release of a cytotoxic moiety
agent from these linker groups include cleavage by reduction of a
disulfide bond (e.g., U.S. Pat. No. 4,489,710), by irradiation of a
photolabile bond (e.g., U.S. Pat. No. 4,625,014), by hydrolysis of
derivatized amino acid side chains (e.g., U.S. Pat. No. 4,638,045),
by serum complement-mediated hydrolysis (e.g., U.S. Pat. No.
4,671,958), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.
4,569,789).
[0185] It can be desirable to couple more than one therapeutic,
cytotoxic and/or imaging moiety to an antibody or antibody
composition of the invention. By poly-derivatizing the antibodies
of the invention, several cytotoxic strategies can be
simultaneously implemented, an antibody can be made useful as a
contrasting agent for several visualization techniques, or a
therapeutic antibody can be labeled for tracking by a visualization
technique. In one embodiment, multiple molecules of a cytotoxic
moiety are coupled to one antibody molecule. In another embodiment,
more than one type of moiety can be coupled to one antibody. For
instance, a therapeutic moiety, such as an polynucleotide or
antisense sequence, can be conjugated to an antibody in conjunction
with a chemotoxic or radiotoxic moiety, to increase the
effectiveness of the chemo- or radiotoxic therapy, as well as
lowering the required dosage necessary to obtain the desired
therapeutic effect. Regardless of the particular embodiment,
immunoconjugates with more than one moiety can be prepared in a
variety of ways. For example, more than one moiety can be coupled
directly to an antibody molecule, or linkers that provide multiple
sites for attachment (e.g., dendrimers) can be used. Alternatively,
a carrier with the capacity to hold more than one cytotoxic moiety
can be used.
[0186] As explained above, a carrier can bear the agents in a
variety of ways, including covalent bonding either directly or via
a linker group, and non-covalent associations. Suitable
covalent-bond carriers include proteins such as albumins (e.g.,
U.S. Pat. No. 4,507,234), peptides, and polysaccharides such as
aminodextran (e.g., U.S. Pat. No. 4,699,784), each of which have
multiple sites for the attachment of moieties. A carrier can also
bear an agent by non-covalent associations, such as non-covalent
bonding or by encapsulation, such as within a liposome vesicle
(e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088). Encapsulation
carriers are especially useful in chemotoxic therapeutic
embodiments, as they can allow the therapeutic compositions to
gradually release a chemotoxic moiety over time while concentrating
it in the vicinity of the target cells.
[0187] Preferred radionuclides for use as cytotoxic moieties are
radionulcides which are suitable for pharmacological
administration. Such radionuclides include .sup.123I, .sup.125I,
.sup.131I, .sup.90Y, .sup.211At, .sup.67Cu, .sup.186Re, .sup.188Re,
.sup.212Pb, and .sup.212Bi. Iodine and astatine isotopes are more
preferred radionuclides for use in the therapeutic compositions of
the present invention, as a large body of literature has been
accumulated regarding their use. .sup.131I is particularly
preferred, as are other .beta.-radiation emitting nuclides, which
have an effective range of several millimeters. .sup.123I,
.sup.125I, .sup.131I, or .sup.211At can be conjugated to antibody
moieties for use in the compositions and methods utilizing any of
several known conjugation reagents, including lodogen,
N-succinimidyl 3-[.sup.211At]astatobenzoate, N-succinimidyl
3-[.sup.131I]iodobenzoate (SIB), and, N-succinimidyl
5-[.sup.131I]iodob-3-pyridinecarboxylate (SIPC). Any iodine isotope
can be utilized in the recited iodo-reagents. Other radionuclides
can be conjugated to the antibody or antibody compositions of the
invention by suitable chelation agents known to those of skill in
the nuclear medicine arts.
[0188] Preferred chemotoxic agents include small-molecule drugs
such as methotrexate, and pyrimidine and purine analogs. Preferred
chemotoxin differentiation inducers include phorbol esters and
butyric acid. Chemotoxic moieties can be directly conjugated to the
antibody or antibody compositions of the invention via a chemical
linker, or can encapsulated in a carrier, which is in turn coupled
to the antibody or antibody compositions of the invention.
[0189] Preferred toxin proteins for use as cytotoxic moieties
include ricin, abrin, diphtheria toxin, cholera toxin, gelonin,
Pseudomonas exotoxin, Shigella toxin, pokeweed antiviral protein,
and other toxin proteins known in the medicinal biochemistry arts.
As these toxin agents can elicit undesirable immune responses in
the patient, especially if injected intravascularly, it is
preferred that they be encapsulated in a carrier for coupling to
the antibody and antibody compositions of the invention.
[0190] The cytotoxic moiety of the immunotoxin may be a cytotoxic
drug or an enzymatically active toxin of bacterial or plant origin,
or an enzymatically active fragment ("A chain") of such a toxin.
Enzymatically active toxins and fragments thereof used are
diphtheria A chain, nonbinding active fragments of diphtheria
toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A
chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites
fordii proteins, dianthin proteins, Phytolacca americana proteins
(PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin,
crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin,
restrictocin, phenomycin, and enomycin. In another embodiment, the
antibodies are conjugated to small molecule anticancer drugs.
Conjugates of the monoclonal antibody and such cytotoxic moieties
are made using a variety of bifunctional protein coupling agents.
Examples of such reagents are SPDP, IT, bifunctional derivatives of
imidoesters such a dimethyl adipimidate HCl, active esters such as
disuccinimidyl suberate, aldehydes such as glutaraldehyde,
bis-azido compounds such as bis (p-azidobenzoyl) hexanediamine,
bis-diazonium derivatives such as
bis-(p-diazoniumbenzoyl)-ethylenediamine, diisocyanates such as
tolylene 2,6-diisocyanate, and bis-active fluorine compounds such
as 1,5-difluoro-2,4-dinitrobenzene. The lysing portion of a toxin
may be joined to the Fab fragment of antibodies.
[0191] Advantageously, the antibodies and antibody compositions of
the invention specifically binding the external domain of the
target receptor, e.g. the activated .alpha..beta.3 integrin
receptor, can be conjugated to ricin A chain. Most advantageously
the ricin A chain is deglycosylated and produced through
recombinant means. An advantageous method of making the ricin
immunotoxin is described in Vitetta et al., Science 238, 1098,
1987, which is incorporated by reference in its entirety.
[0192] The term "contacted" when applied to a cell is used herein
to describe the process by which an antibody, antibody composition,
cytotoxic agent or moiety, gene, protein and/or antisense sequence,
is delivered to a target cell or is placed in direct proximity with
the target cell. This delivery may be in vitro or in vivo and may
involve the use of a recombinant vector system.
[0193] In another aspect, the present invention features an
antibody or antibody composition of the invention, or a fragment
thereof, conjugated to a therapeutic moiety, such as a cytotoxin, a
drug (e.g., an immunosuppressant) or a radiotoxin. Such conjugates
are referred to herein as "immunoconjugates". Immunoconjugates
which include one or more cytotoxins are referred to as
"immunotoxins." A cytotoxin or cytotoxic agent includes any agent
that is detrimental to (e.g., kills) cells. Examples include taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, duocarmycin, saporin, dihydroxy
anthracin didne, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof.
[0194] Suitable therapeutic agents for forming immunoconjugates of
the invention include, but are not limited to, antimetabolites
(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU)
and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,
streptozotocin, mitomycin C, and cis-dichlorodiamine platinum
(II)(DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine). In a preferred embodiment, the therapeutic agent is a
cytotoxic agent or a radiotoxic agent. In another embodiment, the
therapeutic agent is an immunosuppressant. In yet another
embodiment, the therapeutic agent is GM-CSF. In a preferred
embodiment, the therapeutic agent is doxorubicin (adriamycin),
cisplatin bleomycin sulfate, carmustine, chlorambucil,
cyclophosphamide hydroxyurea or ricin A.
[0195] Antibodies and antibody compositions of the invention also
can be conjugated to a radiotoxin, e.g., radioactive iodine, to
generate cytotoxic radiopharmaceuticals for treating, for example,
a cancer. The antibody conjugates of the invention can be used to
modify a given biological response, and the drug moiety is not to
be construed as limited to classical chemical therapeutic agents.
For example, the drug moiety may be a protein or polypeptide
possessing a desired biological activity. Such proteins may
include, for example, an enzymatically active toxin, or active
fragment thereof, such as abrin, ricin A, pseudomonas exotoxin, or
diphtheria toxin; a protein such as tumor necrosis factor or
interferon-.gamma.; or, biological response modifiers such as, for
example, lymphokines, interleukin-1 ("IL-1"), interleukin-2
("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony
stimulating factor ("GM-CSF"), granulocyte colony stimulating
factor ("G-CSF"), or other growth factors.
[0196] Techniques for conjugating such therapeutic moiety to
antibodies are well known. See, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Reisfeld et al., eds., Monoclonal Antibodies And Cancer Therapy,
Alan R. Liss, Inc., pp. 243-56, 1985); Hellstrom et al.,
"Antibodies For Drug Delivery", in Controlled Drug Delivery 2nd
Ed., Marcel Dekker, Inc., Robinson et al., eds., pp. 623-53, 1987;
Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al., eds., pp. 475-506, 1985; "Analysis,
Results, And Future Prospective Of The Therapeutic Use Of
Radiolabeled Antibody In Cancer Therapy", in Monoclonal Antibodies
For Cancer Detection And Therapy, Baldwin et al., eds., Academic
Press, pp. 303-16 1985, and Thorpe et al., "The Preparation And
Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol. Rev.,
62: 119-58, 1982.
Uses of Polypeptides or Antibody Compositions
[0197] Each of the polypeptides or antibody compositions, e.g.,
antibodies to cell surface receptors, cell surface receptor, such
as, activated integrin receptor on a metastatic cell, identified
herein can be used in numerous ways. The following description
should be considered exemplary and utilizes known techniques.
[0198] A polypeptide or antibody composition of the present
invention can be used to assay protein levels in a biological
sample using antibody-based techniques. For example, protein
expression in tissues can be studied with classical
immunohistological methods. Jalkanen et al., J. Cell. Biol. 101:
976-985, 1985; Jalkanen et al., J. Cell. Biol. 105: 3087-3096,
1987. Other antibody-based methods useful for detecting protein
gene expression include immunoassays, such as the enzyme linked
immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
Suitable antibody assay labels are known in the art and include
enzyme labels, such as, glucose oxidase, and radioisotopes or other
radioactive agent, such as iodine (.sup.125I, .sup.121I), carbon
(.sup.14C), sulfur (.sup.35S), tritium (.sup.3H), indium
(.sup.112In), and technetium (.sup.99mTc), and fluorescent labels,
such as fluorescein and rhodamine, and biotin.
[0199] In addition to assaying secreted protein levels in a
biological sample, proteins or antibody compositions can also be
detected in vivo by imaging. Antibody labels or markers for in vivo
imaging of protein 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, which may be incorporated into the antibody by labeling
of nutrients for the relevant scFv clone.
[0200] A protein-specific 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.99 mTc), a
radio-opaque substance, or a material detectable by nuclear
magnetic resonance, is introduced (for example, parenterally,
subcutaneously, or intraperitoneally) into the mammal. 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 radioisotope moiety,
for a human subject, the quantity of radioactivity injected will
normally range from about 5 to 20 millicuries of .sup.99 mTc. The
labeled antibody or antibody fragment will then preferentially
accumulate at the location of cells which contain the specific
protein. In vivo tumor imaging is described in Burchiel et al.,
Tumor Imaging: The Radiochemical Detection of Cancer 13, 1982.
[0201] Thus, the invention provides a diagnostic method of a
disorder, which involves (a) assaying the expression of a
polypeptide by measuring binding of an antibody composition of the
present invention in cells or body fluid of an individual; (b)
comparing the level of gene expression with a standard gene
expression level, whereby an increase or decrease in the assayed
polypeptide gene expression level compared to the standard
expression level is indicative of a disorder.
[0202] Moreover, polypeptides or antibody compositions of the
present invention can be used to treat disease. For example,
patients can be administered a polypeptide or antibody compositions
of the present invention in an effort to replace absent or
decreased levels of the polypeptide (e.g., insulin), to supplement
absent or decreased levels of a different polypeptide (e.g.,
hemoglobin S for hemoglobin B), to inhibit the activity of a
polypeptide (e.g., an oncogene), to activate the activity of a
polypeptide (e.g., by binding to a receptor), to reduce the
activity of a membrane bound receptor by competing with it for free
ligand (e.g., soluble TNF receptors used in reducing inflammation),
or to bring about a desired response (e.g., blood vessel
growth).
[0203] Similarly, antibody compositions of the present invention
can also be used to treat disease. For example, administration of
an antibody directed to a polypeptide of the present invention can
bind and reduce overproduction of the polypeptide. Similarly,
administration of an antibody can activate the polypeptide, such as
by binding to a polypeptide bound to a membrane receptor.
Pharmaceutical Compositions
[0204] Antibody compositions that specifically binds to an
activated integrin receptor on a metastatic tumor cell, ligand
mimetics, derivatives and analogs thereof, useful in the present
compositions and methods can be administered to a human patient per
se, in the form of a stereoisomer, prodrug, pharmaceutically
acceptable salt, hydrate, solvate, acid salt hydrate, N-oxide or
isomorphic crystalline form thereof, or in the form of a
pharmaceutical composition where the compound is mixed with
suitable carriers or excipient(s) in a therapeutically effective
amount, for example, cancer or metastatic cancer.
[0205] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions for administering the antibody compositions (see,
e.g., Remington's Pharmaceutical Sciences, Mack Publishing Co.,
Easton, Pa. 18.sup.th ed., 1990, incorporated herein by reference).
The pharmaceutical compositions generally comprise a differentially
expressed protein, agonist or antagonist in a form suitable for
administration to a patient. The pharmaceutical compositions are
generally formulated as sterile, substantially isotonic and in full
compliance with all Good Manufacturing Practice (GMP) regulations
of the U.S. Food and Drug Administration.
Diagnostic Uses
[0206] Characteristics of Antibodies and Antibody Compositions of
the invention for Use as Diagnostic Reagents. Human antibodies for
use in diagnostic methods to identify metastatic tumor cells, e.g.,
metastatic breast cancer cells, are preferably produced using the
methods described above. The methods result in virtually unlimited
numbers of antibodies and antibody compositions of the invention of
any epitope binding specificity and very high binding affinity to
any desired antigen. In general, the higher the binding affinity of
an antibody for its target, the more stringent wash conditions can
be performed in an immunoassay to remove nonspecifically bound
material without removing target antigen. Accordingly, antibodies
and antibody compositions of the invention used in the above assays
usually have binding affinities of at least 10.sup.8, 10.sup.9,
10.sup.10, 10.sup.11 or 10.sup.12 M.sup.-1. Further, it is
desirable that antibodies used as diagnostic reagents have a
sufficient on-rate to reach equilibrium under standard conditions
in at least 12 hours, preferably at least five hours and more
preferably at least one hour.
[0207] Antibodies and antibody compositions of the invention used
in the claimed methods preferably have a high immunoreactivity,
that is, percentages of antibodies molecules that are correctly
folded so that they can specifically bind their target antigen.
Such can be achieved by expression of sequences encoding the
antibodies in E. coli as described above. Such expression usually
results in immunoreactivity of at least 80%, 90%, 95% or 99%.
[0208] Some methods of the invention employ polyclonal preparations
of antibodies and antibody compositions of the invention as
diagnostic reagents, and other methods employ monoclonal isolates.
The use of polyclonal mixtures has a number of advantages with
respect to compositions made of one monoclonal antibody. By binding
to multiple sites on a target, polyclonal antibodies or other
polypeptides can generate a stronger signal (for diagnostics) than
a monoclonal that binds to a single site. Further, a polyclonal
preparation can bind to numerous variants of a prototypical target
sequence (e.g., allelic variants, species variants, strain
variants, drug-induced escape variants) whereas a monoclonal
antibody may bind only to the prototypical sequence or a narrower
range of variants thereto. However, monoclonal antibodies are
advantageous for detecting a single antigen in the presence or
potential presence of closely related antigens.
[0209] In methods employing polyclonal human antibodies prepared in
accordance with the methods described above, the preparation
typically contains an assortment of antibodies with different
epitope specificities to the intended target antigen. In some
methods employing monoclonal antibodies, it is desirable to have
two antibodies of different epitope binding specificities. A
difference in epitope binding specificities can be determined by a
competition assay.
[0210] Samples and Target. Although human antibodies can be used as
diagnostic reagents for any kind of sample, they are most useful as
diagnostic reagents for human samples. Samples can be obtained from
any tissue or body fluid of a patient. Preferred sources of samples
include, whole blood, plasma, semen, saliva, tears, urine, fecal
material, sweat, buccal, skin and hair. Samples can also be
obtained from biopsies of internal organs or from cancers. Samples
can be obtained from clinical patients for diagnosis or research or
can be obtained from undiseased individuals, as controls or for
basic research.
[0211] The methods can be used for detecting any type of target
antigen. Exemplary target antigens including bacterial, fungal and
viral pathogens that cause human disease, such as. HIV, hepatitis
(A, B, & C), influenza, herpes, Giardia, malaria, Leishmania,
Staphylococcus aureus, Pseudomonas aeruginosa. Other target
antigens are human proteins whose expression levels or compositions
have been correlated with human disease or other phenotype.
Examples of such antigens include adhesion proteins, hormones,
growth factors, cellular receptors, autoantigens, autoantibodies,
and amyloid deposits. Other targets of interest include tumor cell
antigens, such as carcinoembryonic antigen. Other antigens of
interest are class I and class II MHC antigens.
[0212] Formats for Diagnostic Assays. Human antibodies can be used
to detect a given target in a variety of standard assay formats.
Such formats include immunoprecipitation, Western blotting, ELISA,
radioimmunoassay, and immunometric assays. See Harlow & Lane,
supra; U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,879,262;
4,034,074; 3,791,932; 3,817,837; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; and 4,098,876, each incorporated herein by
reference in their entirety and for all purposes.
[0213] Immunometric or sandwich assays are a preferred format. See
U.S. Pat. Nos. 4,376,110; 4,486,530; 5,914,241; and 5,965,375, each
incorporated herein by reference in their entirety and for all
purposes. Such assays use one antibody or population of antibodies
immobilized to a solid phase, and another antibody or population of
antibodies in solution. Typically, the solution antibody or
population of antibodies is labelled. If an antibody population is
used, the population typically contains antibodies binding to
different epitope specificities within the target antigen.
Accordingly, the same population can be used for both solid phase
and solution antibody. If monoclonal antibodies are used, first and
second monoclonal antibodies having different binding specificities
are used for the solid and solution phase. Solid phase and solution
antibodies can be contacted with target antigen in either order or
simultaneously. If the solid phase antibody is contacted first, the
assay is referred to as being a forward assay. Conversely, if the
solution antibody is contacted first, the assay is referred to as
being a reverse assay. If target is contacted with both antibodies
simultaneously, the assay is referred to as a simultaneous assay.
After contacting the target with antibody, a sample is incubated
for a period that usually varies from about 10 min to about 24 hr
and is usually about 1 hr. A wash step is then performed to remove
components of the sample not specifically bound to the antibody
being used as a diagnostic reagent. When solid phase and solution
antibodies are bound in separate steps, a wash can be performed
after either or both binding steps. After washing, binding is
quantified, typically by detecting label linked to the solid phase
through binding of labelled solution antibody. Usually for a given
pair of antibodies or populations of antibodies and given reaction
conditions, a calibration curve is prepared from samples containing
known concentrations of target antigen. Concentrations of antigen
in samples being tested are then read by interpolation from the
calibration curve. Analyte can be measured either from the amount
of labelled solution antibody bound at equilibrium or by kinetic
measurements of bound labelled solution antibody at a series of
time points before equilibrium is reached. The slope of such a
curve is a measure of the concentration of target in a sample
[0214] Suitable supports for use in the above methods include, for
example, nitrocellulose membranes, nylon membranes, and derivatized
nylon membranes, and also particles, such as agarose, a
dextran-based gel, dipsticks, particulates, microspheres, magnetic
particles, test tubes, microtiter wells, SEPHADEX.TM.. (Amersham
Pharmacia Biotech, Piscataway N.J., and the like. Immobilization
can be by absorption or by covalent attachment. Optionally,
antibodies can be joined to a linker molecule, such as biotin for
attachment to a surface bound linker, such as avidin.
Labels
[0215] The particular label or detectable group used in the assay
is not a critical aspect of the invention, so long as it does not
significantly interfere with the specific binding of the antibody
used in the assay. The detectable group can be any material having
a detectable physical or chemical property. Such detectable labels
have been well-developed in the field of immunoassays and, in
general, most any label useful in such methods can be applied to
the present invention. Thus, a label is any composition detectable
by spectroscopic, photochemical, biochemical, immunochemical,
electrical, optical or chemical means. Useful labels in the present
invention include magnetic beads (e.g., Dynabeads.TM.), fluorescent
dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, and
the like), radiolabels (e.g., .sup.3H, .sup.14C, .sup.35S,
.sup.125I, .sup.121I, .sup.112In, .sup.99mTc), other imaging agents
such as microbubbles (for ultrasound imaging), .sup.18F, .sup.11C,
.sup.15O, (for Positron emission tomography), .sup.99mTC,
.sup.111In (for Single photon emission tomography), enzymes (e.g.,
horse radish peroxidase, alkaline phosphatase and others commonly
used in an ELISA), and calorimetric labels such as colloidal gold
or colored glass or plastic (e.g. polystyrene, polypropylene,
latex, and the like) beads. Patents that described the use of such
labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,277,437; 4,275,149; and 4,366,241, each incorporated
herein by reference in their entirety and for all purposes. See
also Handbook of Fluorescent Probes and Research Chemicals, 6th
Ed., Molecular Probes, Inc., Eugene Oreg.).
[0216] The label may be coupled directly or indirectly to the
desired component of the assay according to methods well known in
the art. As indicated above, a wide variety of labels may be used,
with the choice of label depending on sensitivity required, ease of
conjugation with the compound, stability requirements, available
instrumentation, and disposal provisions.
[0217] Non-radioactive labels are often attached by indirect means.
Generally, a ligand molecule (e.g., biotin) is covalently bound to
the molecule. The ligand then binds to an anti-ligand (e.g.,
streptavidin) molecule which is either inherently detectable or
covalently bound to a signal system, such as a detectable enzyme, a
fluorescent compound, or a chemiluminescent compound. A number of
ligands and anti-ligands can be used. Where a ligand has a natural
anti-ligand, for example, biotin, thyroxine, and cortisol, it can
be used in conjunction with the labeled, naturally occurring
anti-ligands. Alternatively, any haptenic or antigenic compound can
be used in combination with an antibody.
[0218] The molecules can also be conjugated directly to signal
generating compounds, e.g., by conjugation with an enzyme or
fluorophore. Enzymes of interest as labels will primarily be
hydrolases, particularly phosphatases, esterases and glycosidases,
or oxidoreductases, particularly peroxidases. Fluorescent compounds
include fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, umbelliferone, and the like Chemiluminescent
compounds include luciferin, and 2,3-dihydrophthalazinediones,
e.g., luminol. For a review of various labeling or signal producing
systems which may be used, see, U.S. Pat. No. 4,391,904,
incorporated herein by reference in its entirety and for all
purposes.
[0219] Means of detecting labels are well known to those of skill
in the art. Thus, for example, where the label is a radioactive
label, means for detection include a scintillation counter or
photographic film as in autoradiography. Where the label is a
fluorescent label, it may be detected by exciting the fluorochrome
with the appropriate wavelength of light and detecting the
resulting fluorescence. The fluorescence may be detected visually,
by means of photographic film, by the use of electronic detectors
such as charge coupled devices (CCDs) or photomultipliers and the
like. Similarly, enzymatic labels may be detected by providing the
appropriate substrates for the enzyme and detecting the resulting
reaction product. Finally simple calorimetric labels may be
detected simply by observing the color associated with the label.
Thus, in various dipstick assays, conjugated gold often appears
pink, while various conjugated beads appear the color of the
bead.
[0220] Some assay formats do not require the use of labeled
components. For instance, agglutination assays can be used to
detect the presence of the target antibodies. In this case,
antigen-coated particles are agglutinated by samples comprising the
target antibodies. In this format, none of the components need be
labeled and the presence of the target antibody is detected by
simple visual inspection.
[0221] Frequently, the activated integrin receptor or
.alpha.v.beta.3 integrin receptor proteins and antibodies to
activated integrin receptor will be labeled by joining, either
covalently or non-covalently, a substance which provides for a
detectable signal.
Treatment Regimes
[0222] The invention provides pharmaceutical compositions
comprising one or a combination of antibodies, e.g., antibodies to
activated integrin receptor (monoclonal, polyclonal or single chain
Fv; intact or binding fragments thereof) formulated together with a
pharmaceutically acceptable carrier. Some compositions include a
combination of multiple (e.g., two or more) monoclonal antibodies
or antigen-binding portions thereof of the invention. In some
compositions, each of the antibodies or antigen-binding portions
thereof of the composition is a monoclonal antibody or a human
sequence antibody that binds to a distinct, pre-selected epitope of
an antigen.
[0223] In prophylactic applications, pharmaceutical compositions or
medicaments are administered to a patient susceptible to, or
otherwise at risk of a disease or condition (i.e., an immune
disease) in an amount sufficient to eliminate or reduce the risk,
lessen the severity, or delay the outset of the disease, including
biochemical, histologic and/or behavioral symptoms of the disease,
its complications and intermediate pathological phenotypes
presenting during development of the disease. In therapeutic
applications, compositions or medicants are administered to a
patient suspected of, or already suffering from such a disease in
an amount sufficient to cure, or at least partially arrest, the
symptoms of the disease (biochemical, histologic and/or
behavioral), including its complications and intermediate
pathological phenotypes in development of the disease. An amount
adequate to accomplish therapeutic or prophylactic treatment is
defined as a therapeutically- or prophylactically-effective dose.
In both prophylactic and therapeutic regimes, agents are usually
administered in several dosages until a sufficient immune response
has been achieved. Typically, the immune response is monitored and
repeated dosages are given if the immune response starts to
wane.
Effective Dosages
[0224] Effective doses of the antibody compositions of the present
invention, e.g., antibodies to activated integrin receptor, for the
treatment of immune-related conditions and diseases, e.g., metastic
cancer, described herein vary depending upon many different
factors, including means of administration, target site,
physiological state of the patient, whether the patient is human or
an animal, other medications administered, and whether treatment is
prophylactic or therapeutic. Usually, the patient is a human but
nonhuman mammals including transgenic mammals can also be treated.
Treatment dosages need to be titrated to optimize safety and
efficacy.
[0225] For administration with an antibody, the dosage ranges from
about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the
host body weight. For example dosages can be 1 mg/kg body weight or
10 mg/kg body weight or within the range of 1-10 mg/kg. An
exemplary treatment regime entails administration once per every
two weeks or once a month or once every 3 to 6 months. In some
methods, two or more monoclonal antibodies with different binding
specificities are administered simultaneously, in which case the
dosage of each antibody administered falls within the ranges
indicated. Antibody is usually administered on multiple occasions.
Intervals between single dosages can be weekly, monthly or yearly.
Intervals can also be irregular as indicated by measuring blood
levels of antibody in the patient. In some methods, dosage is
adjusted to achieve a plasma antibody concentration of 1-1000
.mu.g/ml and in some methods 25-300 .mu.g/ml. Alternatively,
antibody can be administered as a sustained release formulation, in
which case less frequent administration is required. Dosage and
frequency vary depending on the half-life of the antibody in the
patient. In general, human antibodies show the longest half life,
followed by humanized antibodies, chimeric antibodies, and nonhuman
antibodies. The dosage and frequency of administration can vary
depending on whether the treatment is prophylactic or therapeutic.
In prophylactic applications, a relatively low dosage is
administered at relatively infrequent intervals over a long period
of time. Some patients continue to receive treatment for the rest
of their lives. In therapeutic applications, a relatively high
dosage at relatively short intervals is sometimes required until
progression of the disease is reduced or terminated, and preferably
until the patient shows partial or complete amelioration of
symptoms of disease. Thereafter, the patent can be administered a
prophylactic regime.
[0226] Doses for nucleic acids encoding immunogens range from about
10 ng to 1 g, 100 ng to 100 mg, 1 .mu.g to 10 mg, or 30-300 .mu.g
DNA per patient. Doses for infectious viral vectors vary from
10-100, or more, virions per dose.
Routes of Administration
[0227] Antibody compositions for inducing an immune response, e.g.,
antibodies to activated integrin receptor, for the treatment of
immune-related conditions and diseases, e.g., metastic cancer, can
be administered by parenteral, topical, intravenous, oral,
subcutaneous, intraarterial, intracranial, intraperitoneal,
intranasal or intramuscular means for prophylactic as inhalants for
antibody preparations targeting brain lesions, and/or therapeutic
treatment. The most typical route of administration of an
immunogenic agent is subcutaneous although other routes can be
equally effective. The next most common route is intramuscular
injection. This type of injection is most typically performed in
the arm or leg muscles. In some methods, agents are injected
directly into a particular tissue where deposits have accumulated,
for example intracranial injection. Intramuscular injection on
intravenous infusion are preferred for administration of antibody.
In some methods, particular therapeutic antibodies are injected
directly into the cranium. In some methods, antibodies are
administered as a sustained release composition or device, such as
a Medipad.TM. device.
[0228] Agents of the invention can optionally be administered in
combination with other agents that are at least partly effective in
treating various diseases including various immune-related
diseases. In the case of tumor metastasis to the brain, agents of
the invention can also be administered in conjunction with other
agents that increase passage of the agents of the invention across
the blood-brain barrier (BBB).
Formulation
[0229] Antibody compositions for inducing an immune response, e.g.,
antibodies to activated integrin receptor, for the treatment of
immune-related conditions and diseases, e.g., metastic cancer, are
often administered as pharmaceutical compositions comprising an
active therapeutic agent, i.e., and a variety of other
pharmaceutically acceptable components. (See Remington's
Pharmaceutical Science, 15.sup.th ed., Mack Publishing Company,
Easton, Pa., 1980). The preferred form depends on the intended mode
of administration and therapeutic application. The compositions can
also include, depending on the formulation desired,
pharmaceutically-acceptable, non-toxic carriers or diluents, which
are defined as vehicles commonly used to formulate pharmaceutical
compositions for animal or human administration. The diluent is
selected so as not to affect the biological activity of the
combination. Examples of such diluents are distilled water,
physiological phosphate-buffered saline, Ringer's solutions,
dextrose solution, and Hank's solution. In addition, the
pharmaceutical composition or formulation may also include other
carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic
stabilizers and the like.
[0230] Pharmaceutical compositions can also include large, slowly
metabolized macromolecules such as proteins, polysaccharides such
as chitosan, polylactic acids, polyglycolic acids and copolymers
(such as latex functionalized Sepharose.TM., agarose, cellulose,
and the like), polymeric amino acids, amino acid copolymers, and
lipid aggregates (such as oil droplets or liposomes). Additionally,
these carriers can function as immunostimulating agents (i.e.,
adjuvants).
[0231] For parenteral administration, compositions of the invention
can be administered as injectable dosages of a solution or
suspension of the substance in a physiologically acceptable diluent
with a pharmaceutical carrier that can be a sterile liquid such as
water oils, saline, glycerol, or ethanol. Additionally, auxiliary
substances, such as wetting or emulsifying agents, surfactants, pH
buffering substances and the like can be present in compositions.
Other components of pharmaceutical compositions are those of
petroleum, animal, vegetable, or synthetic origin, for example,
peanut oil, soybean oil, and mineral oil. In general, glycols such
as propylene glycol or polyethylene glycol are preferred liquid
carriers, particularly for injectable solutions. Antibodies can be
administered in the form of a depot injection or implant
preparation which can be formulated in such a manner as to permit a
sustained release of the active ingredient. An exemplary
composition comprises monoclonal antibody at 5 mg/mL, formulated in
aqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl,
adjusted to pH 6.0 with HCl.
[0232] Typically, compositions are prepared as injectables, either
as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid vehicles prior to injection
can also be prepared. The preparation also can be emulsified or
encapsulated in liposomes or micro particles such as polylactide,
polyglycolide, or copolymer for enhanced adjuvant effect, as
discussed above. Langer, Science 249: 1527, 1990 and Hanes,
Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of this
invention can be administered in the form of a depot injection or
implant preparation which can be formulated in such a manner as to
permit a sustained or pulsatile release of the active
ingredient.
[0233] Additional formulations suitable for other modes of
administration include oral, intranasal, and pulmonary
formulations, suppositories, and transdermal applications.
[0234] For suppositories, binders and carriers include, for
example, polyalkylene glycols or triglycerides; such suppositories
can be formed from mixtures containing the active ingredient in the
range of 0.5% to 10%, preferably 1%-2%. Oral formulations include
excipients, such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, and
magnesium carbonate. These compositions take the form of solutions,
suspensions, tablets, pills, capsules, sustained release
formulations or powders and contain 10%-95% of active ingredient,
preferably 25%-70%.
[0235] Topical application can result in transdermal or intradermal
delivery. Topical administration can be facilitated by
co-administration of the agent with cholera toxin or detoxified
derivatives or subunits thereof or other similar bacterial toxins.
Glenn et al., Nature 391: 851, 1998. Co-administration can be
achieved by using the components as a mixture or as linked
molecules obtained by chemical crosslinking or expression as a
fusion protein.
[0236] Alternatively, transdermal delivery can be achieved using a
skin patch or using transferosomes. Paul et al., Eur. J. Immunol.
25: 3521-24, 1995; Cevc et al., Biochem. Biophys. Acta 1368:
201-15, 1998.
[0237] The pharmaceutical compositions are generally formulated as
sterile, substantially isotonic and in full compliance with all
Good Manufacturing Practice (GMP) regulations of the U.S. Food and
Drug Administration.
Toxicity
[0238] Preferably, a therapeutically effective dose of the antibody
compositions described herein will provide therapeutic benefit
without causing substantial toxicity.
[0239] Toxicity of the proteins described herein can be determined
by standard pharmaceutical procedures in cell cultures or
experimental animals, e.g., by determining the LD.sub.50 (the dose
lethal to 50% of the population) or the LD.sub.100 (the dose lethal
to 100% of the population). The dose ratio between toxic and
therapeutic effect is the therapeutic index. The data obtained from
these cell culture assays and animal studies can be used in
formulating a dosage range that is not toxic for use in human. The
dosage of the proteins described herein lies preferably within a
range of circulating concentrations that include the effective dose
with little or no toxicity. The dosage can vary within this range
depending upon the dosage form employed and the route of
administration utilized. The exact formulation, route of
administration and dosage can be chosen by the individual physician
in view of the patient's condition. (See, e.g., Fingl et al., 1975,
In: The Pharmacological Basis of Therapeutics, Ch. 1,
Kits
[0240] Also within the scope of the invention are kits comprising
the compositions (e.g., monoclonal antibodies, human sequence
antibodies, human antibodies, multispecific and bispecific
molecules) of the invention and instructions for use. The kit can
further contain a least one additional reagent, or one or more
additional human antibodies of the invention (e.g., a human
antibody having a complementary activity which binds to an epitope
in the antigen distinct from the first human antibody). Kits
typically include a label indicating the intended use of the
contents of the kit. The term label includes any writing, or
recorded material supplied on or with the kit, or which otherwise
accompanies the kit.
[0241] The following cDNA clones described in the specification and
further described in the examples below have been deposited with
the American Type Culture Collection, 10801 University Boulevard,
Manassas, Va. 20110-2209 under the Budapest Treaty on Nov. 12,
2004. The cDNA clone for scFv Bc-12 has been given the ATCC
Accession No. indicated: PTA-6303. The cDNA clone for scFv Bc-15
has been given the ATCC Accession No. indicated: PTA-6304.
EXEMPLARY EMBODIMENTS
Example 1
Cell Lines
[0242] Human M21 melanoma and UCLA-P3 lung adenocarcinoma cells
were from Dr. D. L. Morton, John Wayne Cancer Center, Santa Monica,
Calif. M21-L cells were from Dr. D. A. Cheresh, The Scripps
Research Institute. M21-L4 and M21-L12 cells were described in
Felding-Habermann et al., Clin. Exp. Metastasis 19: 427-436, 2002.
MDA-MB 435 human breast cancer cells were from Dr. J. E. Price,
M.D. Anderson Cancer Center, Houston, Tex. Variants from this cell
line were selected in vivo from lung (Lung) or bone (Bone)
metastases upon injecting the parental cells into the mammary fat
pad of immune deficient mice. A .beta.3 integrin negative variant
(.beta.3-) was selected in vitro by treating the parental cells
with an anti-.beta.3 saporin conjugate. .beta.3- cells were
transfected either with .beta.3 wild type (.beta.3.sub.WT) or
mutant .beta.3.sub.D723R (.beta.3.sub.D723R) cDNA.
Felding-Habermann, et al., Proc. Natl. Acad. Sci. U.S.A 98:
1853-1858, 2001. Primary metastatic cells from blood samples of
stage IV breast cancer patients (BCM1, BCM2, BMS) were isolated by
immuno magnetic bead sorting with antiepithelial antibody BerEP4
(Dynal). These cells express integrin .alpha.v.beta.3 at levels
comparable to those of the MDA-MB 435 cell variants. Rolli et al.,
Proc. Natl. Acad. Sci. U. S. A 100: 9482-9487, 2003. Cells were
cultured in EMEM, 10% FBS, pyruvate, L-glutamine, vitamins, and
nonessential amino acids.
Example 2
scFv Antibody Library and Phage Display
[0243] A single-chain Fv antibody library was generated from total
RNA of peripheral blood lymphocytes from 20 cancer patients, 5 of
whom had breast cancer. From this library, phage displaying scFvs
on gene III were rescued as detailed. Mao et al., Proc. Natl. Acad.
Sci. U.S.A. 96: 6953-6958, 1999.
[0244] Proteins. Recombinant tissue necrosis factor-.alpha.
(TNF-.alpha.) (trimer, 51 kDa) was kindly provided by Siliang Hu
(Shanghai Research Center of Biotechnology, Chinese Academy of
Sciences, Shanghai, China). BSA (66 kDa), staphylococcal
enterotoxin B (SEB) (28.5 kDa), cholera toxin B subunit (CTB)
(pentamer, 58 kDa), Ricinus communis agglutinin (RCA.sub.120, 120
kDa; "ricin" RCA.sub.60, 60 kDa) were purchased from Sigma.
[0245] Construction of Phage-Display Vector pCGMT9. The vector
pCGMT9 was derived from pCGMT. The gene IX (gIX) was amplified by
PCR from single-stranded DNA of helper phage VCSM13 as the template
by using primers P9 (5'-AAA TAG ACT AGT GGA GGC GGT GGC TCT ATG AGT
GTT TTA GTG TAT TCT-3'), and P9rev (5'-GAT TTA GCT AGC TTA TTA TGA
GGA AGT TTC CAT TAA ACG-3'). The PCR product was digested by SpeI
and NheI and inserted into the pCGMT vector, which was cut with the
same restriction enzymes. SpeI digestion and further DNA sequencing
was used to characterize the orientation of gIX in the vector.
[0246] Preparation of the cDNA Template. Total RNA was Prepared
from 10 different samples of human peripheral blood lymphocytes
(PBLs) by using a RNA Purification kit (Stratagene). First-strand
cDNA was synthesized from total RNA by using a First-Strand cDNA
Synthesis kit (Amersham Pharmacia) with random hexamers.
[0247] Amplification of Antibody Variable Region Genes. Both the
V.sub.H and V.sub.L gene repertoires were PCR amplified by using
the cDNA and a previously constructed scFv-phage library plasmid as
templates. To amplify the V.sub.H and V.sub.L genes from the cDNA
and plasmid template, the primers were designed based on those
published previously and the most recent gene segments entered in
the V-Base sequence directory. All primary PCR reactions were
carried out with separate backward primers and combined forward
primers. For the amplification of the V.sub.H gene repertoires, 12
separate PCR reactions were set up by using one of 12 different
human V.sub.H (HV.sub.H) back primers and an equimolar mixture of
four human heavy chain J region (HJ.sub.H) forward primers. For the
.kappa. and .lamda. V.sub.L genes, the same approach was used with
13 separate reactions defined by individual
HV.kappa./HV.sub..lamda. back primers and a mixture of
HJ.kappa./HJ.sub..lamda. forward primers. PCRs were performed in
100 .mu.l volumes containing 2 .mu.l of cDNA reaction mixture, 2
.mu.M of primer solutions, 200 .mu.M of dNTPs, 5% DMSO, and 10
.mu.l of Pfu polymerase reaction buffer (Stratagene). After 5 min
of denaturation at 94.degree. C., 5 units of Pfu polymerase was
added, followed by 30 cycles of 1 min at 94.degree. C., 1 min at
57.degree. C., and 1 min at 72.degree. C., and at the end of
cycling an incubation of 10 min at 72.degree. C. After PCR, the
various reactions afforded V.sub.H, V.kappa., and V.sub..lamda.
subpools from each of the 10 different PBL samples and scFv-phage
library plasmid that were mixed to give three final V.sub.H,
V.kappa., and V.sub..lamda. pools ready for purification and
assembly.
[0248] Construction of the scFv Library. The amplified V.sub.H and
V.sub.L genes were gel-purified on agarose, and the scFv genes were
assembled by overlap PCR using V.sub.H and V.sub.L fragments as
templates. First, approximately 20 ng each of V.sub.H and V.sub.L
were assembled with a linker by PCR without primers in which the
short regions of complementarity built into the ends of the linker
promoted hybridization of the various fragments. An initial
denaturation step for 5 min at 94.degree. C. was followed by five
cycles of 1 min at 94.degree. C., 1 min at 60.degree. C., and 1.5
min at 72.degree. C. in the absence of primers. After adding the
outer primers HVH (SfiI) and HJL (SfiI), 30 cycles of 30 s at
94.degree. C., 30 s at 60.degree. C., and 1.5 min at 72.degree. C.
were performed. The scFv genes were digested with SfiI, agarose
gel-purified, and ligated into the phage-display vector pCGMT9 that
had been cut with the same restriction enzyme. The ligated products
were electroporated into Escherichia coli XL1-Blue competent cells
to yield a diversity of .apprxeq.4.5.times.10.sup.9 independent
transformants. After electroporation, cells were plated on LB agar
containing 2% glucose, 50 .mu.g/ml carbenicillin, and 20 .mu.g/ml
tetracycline in 40 dishes (150 mm.times.10 mm; Nunc) and incubated
overnight at 30.degree. C. The clones were scraped off the plates
into 300 ml of superbroth (SB) medium with 10% glycerol and
subsequently stored at -70.degree. C.
[0249] The phage display library, generated from cancer patient
blood lymphocyte cDNA libraries, contained approximately
2.times.10.sup.8 clones. In the subtractive panning strategy,
clones were isolated that bound specifically to metastatic variants
of the human breast cancer cell model and failed to bind to a
non-metastatic variant of the same cell model. From these isolated
clones, in each of three approaches, 20 clones were arbitrarily
picked for further analysis and characterization. For each of these
20 clones, selective binding specificity was verified, comparing
the metastatic versus non-metastatic variants of the breast cancer
cell model. On average, 2 of the picked 20 clones strictly
distinguished between the metastatic versus non-metastatic cell
variants, binding only to the metastatic cells. Two clones were
further analyzed and characterized in detail (scFvs Bc-12 and
Bc-15). The remaining scFvs with specificity for metastatic breast
cancer variants indicate that the subtractive panning strategy
yields antibodies that react specifically with cells that have
established metastatic activity. These antibodies will be
characterized further in ongoing studies.
[0250] Rescue of scFv-Phage. To rescue the scFv-phage, 1 L of SB
medium containing 2% glucose, 50 .mu.g/ml carbenicillin, and 20
.mu.g/ml tetracycline was inoculated overnight with
.apprxeq.5.times.10.sup.10 cells from the library glycerol stock.
The culture was shaken at 37.degree. C. until
OD.sub.600.apprxeq.0.5-0.7 was obtained. Then,
.apprxeq.4.times.10.sup.13 plaque forming units of helper phage
VCSM13 and 2 ml of 0.5 M isopropyl .sup.3-D-thiogalactopyranoside
(IPTG) were added. After 30-min incubation at room temperature, the
culture was diluted into 5 liters of SB medium containing 50
.mu.g/ml carbenicillin, 20 .mu.g/ml tetracycline, and 0.5 mM IPTG
and grown for 2 h at 30.degree. C. Kanamycin was then added to a
final concentration of 70 .mu.g/ml, and the culture was grown
overnight at 30.degree. C. Phage were prepared by polyethylene
glycol (PEG)/NaCl precipitation.
[0251] Panning of scFv-Phage. The library was subjected to three or
four rounds of panning. Specific scFv-phage were affinity selected
by using proteins adsorbed to immunotubes (Maxisorb, Nunc). For
selection of BSA, TNF-.alpha., SEB, CTB, RCA.sub.60, and
RCA.sub.120, immunotubes were coated with the individual proteins
overnight at room temperature by using 1 ml of 50 .mu.g/ml protein
in PBS (10 mM phosphate/150 mM NaCl, pH 7.4) for the first round,
10 .mu.g/ml for the second round, and 5 .mu.g/ml for the third and
fourth rounds of panning. The immunotubes were blocked with Blotto
(4% skimmed milk in PBS) for 1 h at room temperature and then
.apprxeq.10.sup.13 cfu scFv-phage were added into the immunotube in
2% skimmed milk/2% BSA in PBS (BSA was omitted when panning against
BSA). After 2 h of incubation with rocking at room temperature, the
unbound and nonspecifically bound scFv-phage were eluted by using
10 washes with PBS/0.1% Tween-20 and 10 washes with PBS. The
specifically bound scFv-phage was eluted with 1 ml elution buffer
(100 mM HCl, adjusted to pH 2.2 with solid glycine and containing
0.1% BSA) for 10 min at room temperature. The eluate was
neutralized with 60 .mu.l of 2 M Tris base and was used to infect
freshly prepared E. coli XL1-Blue cells. The scFv-phage were then
amplified and rescued as outlined above and entered into the next
round of panning.
[0252] ELISA of scFv-Phage Binding. Relative affinity and
specificity of scFv-phage and soluble scFvs was assessed against
the six protein antigens. BSA, TNF-.alpha., SEB, CTB, RCA.sub.60,
and RCA.sub.120 solutions at 10 .mu.g/ml were coated on a
microtiter plate at room temperature overnight. Any remaining
binding sites were blocked with Blotto. Approximately 25 .mu.l per
well of scFv-phage or soluble scFv supernatant from overnight cell
cultures was added and incubated for 1 h at 37.degree. C. For
scFv-phage ELISA, after washing, 25 .mu.l of anti-M13 mAb
horseradish peroxidase (HRP) conjugate (Amersham Pharmacia) diluted
1:1000 in Blotto was added for 30 min at 37.degree. C. For ELISA
using soluble scFv, anti-Flag M2 mAb HRP conjugate (Sigma) in
Blotto was added and incubated for 30 min at 37.degree. C.
Detection was accomplished by adding 50 .mu.l of
tetramethylbenzidine substrate (Pierce) and the absorbance was read
at 450 nm.
[0253] Purification of scFvs and Affinity Measurements. The scFv
genes were subcloned into expression vector pETFlag, expressed and
purified to homogeneity. Dissociation constants (K.sub.d) were
calculated from the measured association (k.sub.on) and
dissociation (k.sub.off) rate constants by using the BIAcore
instrumentation and software (Amersham Pharmacia). For BIAcore
experiments, protein antigens were immobilized on CM5 chips. After
scFv binding measurements, chips were regenerated with 75 mM
HCl.
Example 3
Subtractive Panning of Phage Antibodies
[0254] Phage displaying antibodies reactive with integrin
.alpha.v.beta.3 were isolated by five rounds of subtractive panning
on live breast cancer cells. In each round, the library
(.about.5.times.10.sup.12 cfu) was first subtracted on
3.times.10.sup.7 MDA-MB 435 variant cells expressing non activated
.alpha.v.beta.3 suspended in serum free EMEM, 1% BSA, for 45 min at
RT. The depleted library was then panned on 1.times.10.sup.7 MDA-MB
435 variant cells expressing activated .alpha.v.beta.3. After 30
min at RT, the cells were centrifuged and washed 10 times in EMEM.
Bound scFv phage was eluted with 100 mM glycine/HCl, 1% BSA, 150 mM
NaCl, pH 2.4, neutralized with 1M Tris/HCl, pH 7.4, amplified in E.
coli SL-1 Blue, and precipitated with PEG/NaCl for further
subtraction, selection, purification or cell binding analysis by
flow cytometry.
[0255] scFv purification. scFv gene fragments were subcloned into
pETFlag (derived from pET-15b, Novagen) and transformed into E.
coli B834(DE3). Expression was induced with 0.5 mM IPTG, and
FLAG-tagged scFv fragments were purified on anti-Flag mAb M2
affinity agarose (Sigma) as described. Mao, S., et al., Proc. Natl.
Acad. Sci. U.S.A 96: 6953-6958, 1999; Gao, C., et al., J. Immunol.
Methods 274: 185-197, 2003. Monomeric scFv was purified by
Sephacryl-100 FPLC (Amersham Pharmacia).
[0256] Flow cytometry. scFv binding to human tumor cells was
analyzed by flow cytometry, initially with cloned scFv phage and
then with purified scFv protein. Per sample, 2.times.10.sup.5 tumor
cells were blocked with goat serum and incubated either with scFv
phage (2-5.times.10.sup.10) (1 h at RT) followed by mouse anti-M13
mAb (Serotec) (30 min on ice) and goat FITC-anti mouse (Pierce) (30
min on ice), or with purified scFv (1.25 to 40 .mu.g/ml, routinely
10 to 15 .mu.g/ml) (45 min on ice), followed by mouse anti-FLAG mAb
M2 (Sigma) (30 min on ice) and goat FITC-anti mouse.
Binding/washing buffer was TBS with or without divalent cations (1
mM Ca.sup.2+, 1 mM Mg.sup.2+, 0.2 mM Mn.sup.2+). Alternatively,
tumor cells were washed in 10 ml of plasma (prepared freshly from
human blood anticoagulated with 50 nM D-Phe-D-Pro-D-arginyl
chloromethyl ketone, PPACK, Felding-Habermann et al., J. Biol.
Chem. 271: 5892-5900, 1996), resuspended in plasma and then
incubated sequentially, without washing, with purified scFv (45 min
on ice), followed by anti-FLAG M2 (30 min on ice) and FITC-anti
mouse (30 min on ice). All samples were counter stained with
propidium iodide (PI) and analyzed on a Becton-Dickinson FACScan,
with live gate set to exclude PI positive cells.
[0257] Sequence analysis of scFvs. Nucleic acid sequencing of
selected clones was carried out on a 373-A DNA sequencer (Applied
Biosystems). All sequences were searched in the Kabat database
(http://www.nci.nlm.nih.gov) to compare them with previously
sequenced V.sub.H and V.sub.L chains, and in the International
Immunogenetics database (http://www.Genetik.uni-Koeln.de/dnaplot)
to propose correlations of scFvs with potential germline gene
sequences and assess V-segment usage. The GCG Wisconsin Package was
used for alignments.
[0258] Cell adhesion and migration. Tumor cell adhesion under
stationary conditions was analyzed as detailed earlier (19).
Adhesion buffer was Hanks balanced salt solution (HBSS) pH 7.4,
0.5% BSA, 1 mM MgCl.sub.2, 0.2 mM MnCl.sub.2. For inhibition, cells
were incubated for 5 min at RT either with 3 .mu.M scFv or 200
.mu.M GRGDSPK peptide, then plated in the presence of inhibitor and
allowed to attach for 30 min at 37EC. Haptotactic tumor cell
migration toward fibrinogen was analyzed in transwell chambers as
detailed earlier. Rolli, M., et al., Proc. Natl. Acad. Sci. U.S.A
100: 9482-9487, 2003. Before the assay, tumor cells were starved
for 16 hrs at 37EC in serum free EMEM, washed and then allowed to
migrate in serum free EMEM in the presence or absence of 2 .mu.M
scFv or 200 .mu.M GRGDSPK peptide for 16 hrs at 37EC, 5%
CO.sub.2.
[0259] Tumor cell arrest during blood flow. Breast cancer cell
arrest during blood flow and interaction with platelets was
measured as described. Felding-Habermann, et al., Proc. Natl. Acad.
Sci. U.S.A 98: 1853-1858, 2001. Felding-Habermann et al., J. Biol.
Chem. 271: 5892-5900, 1996. Briefly, tumor cells were suspended in
human blood anticoagulated with 50 nM PPACK and perfused over a
collagen I matrix at a venous wall shear rate of 50 s.sup.-1, 2
dynes/cm.sup.2. Adhesive events and cell interactions were
visualized and recorded by fluorescence video microscopy and
quantified by image acquisition at 30 predefined positions followed
by computerized image analysis (MetaMorph, Universal Imaging).
Tumor cells were stained with hydroethidine (red fluorescence) (20
.mu.g/ml, 30 min, 37EC), washed, and mixed with blood containing 10
.mu.M mepacrine (green fluorescence). Blood cells, tumor cells, and
platelets acquired green fluorescence and were visualized at
488/515 nm (excitation/emission). Tumor cells were identified by
their unique red fluorescence at 543/590 nm. To test inhibition,
tumor cells were incubated with 3 .mu.M scFv for 5 min at 37EC,
then mixed into blood, scFv added to 3 .mu.M final concentration
and perfused immediately.
[0260] Antibody internalization. Breast cancer cells grown in
chamber slide wells were incubated with 20 .mu.g/ml FITC-labeled
scFv in serum free EMEM for 3 h either at 4.degree. C. or
37.degree. C., washed 10 times, fixed and permeabilized with 95%
ethanol, stained with propidium iodide, mounted in antifade
solution, and analyzed with a laser scanning confocal
microscope.
[0261] Experimental metastasis in vivo. 1.times.10.sup.5 BMS human
metastatic breast cancer cells were injected into the lateral tail
veins of 9 week old female C.B17/lcrTac-Prkdc scid mice (Taconic
Farms) (n=8 to 10) together with a 50 .mu.g bolus dose of scFv.
I.v. Injections of 50 .mu.g scFv bolus doses were repeated on days
2, 3 and 4 of the experiment. Control animals received vehicle only
(PBS). For in vivo use, endotoxin was removed from scFv
preparations on Detoxy-Gel resin (Pierce). Remaining traces ranged
from 0.001 to 0.07 EU endotoxin/mg scFv (LAL test, Bio Whittaker).
On day 32, mice were euthanized, dissected, the lungs excised,
fixed in Bouin=s solution, and metastatic foci counted at the lung
surface under a dissecting microscope. The same lungs were embedded
in paraffin, and 10 .mu.m sections were cut and stained with
hematoxylin/eosin. Per lung, 7 sets of three consecutive sections
were collected, separated by 140 .mu.m. The sections were
randomized and coded, and the total number of metastatic foci
counted.
[0262] To treat mice with established metastatic disease,
5.times.10.sup.5 DsRed2 tagged MDA-MB 435 cells expressing
constitutively activated .alpha.v.beta.3.sub.D723R were injected
intravenously. After all mice in a control group had developed lung
metastases after one week, the mice in treatment groups received
intravenous injections of 40 .mu.g scFv Bc-15, or scFv Mut-15 as
control on day 7, 9, 11, 14, 16, and 18 after tumor cell
inoculation. The mice were euthanized on day 19 and lung metastases
counted under a fluorescence microscope. Care and use of the
animals complied with NIH and AAALAC guidelines.
Example 4
[0263] Cancer Patient Derived scFv Antibodies Recognize Tumor Cell
Integrin .alpha.v.beta.3 in an Activation Dependent Manner
[0264] To generate therapeutic reagents that specifically react
with activated .alpha.v.beta.3, a phage display library of single
chain antibody fragments (scFv) was exploited. This library was
derived from cancer patient blood lymphocyte cDNA libraries and
allowed us to test the hypothesis that the expressed immune
repertoire contains antibodies that recognize integrin
.alpha.v.beta.3, and distinguish between its activated and
non-activated forms. Mao et al., Proc. Natl. Acad. Sci. U.S.A 96:
6953-6958, 1999. A subtractive panning approach was designed based
on variants of MDA-MB 435 human breast cancer cells. It has been
demonstrated that the majority of the parental cell population
expresses non-activated .alpha.v.beta.3, but contains a subset of
cells expressing the activated receptor. Apparently, these cells
are selected during metastasis, as variant cells isolated from lung
and bone metastases in immune deficient mice express constitutively
activated .alpha.v.beta.3. To establish a cell population that
uniformly expresses non-activated .alpha.v.beta.3, MDA-MB 435
parental cells were cloned by limiting dilution, and clones tested
for .alpha.v.beta.3 functionality. Twenty clones were identified,
in which the receptor failed to support fibrinogen directed
migration and .alpha.v.beta.3 dependent tumor cell arrest during
blood flow, confirming a non-activated integrin. The pooled clones,
termed Parent Combo, expressed .alpha.v.beta.3 at a level
comparable to the parental population. A cell line, MDA-MB 435
Lung-Lung, was established which expressed .alpha.v.beta.3 in a
constitutively activated form. This variant stems from a lung
metastase of an immune deficient mouse whose mammary fat pad had
been injected with parental MDA-MB 435 cells. Felding-Habermann et
al., Proc. Natl. Acad. Sci. U.S.A 98: 1853-1858, 2001; Rolli et
al., Proc. Natl. Acad. Sci. U.S.A 100: 9482-9487, 2003. To enrich
for the ability to colonize the lung from the blood stream, the
Lung variant was subsequently injected intravenously, and tumor
cells cultured 3 weeks later from the excised lung. Parent Combo
cells were used to subtract the scFv phage library to eliminate
antibodies against antigens shared by the MDA-MB 435 cell variants.
After 5 rounds of subtracting the phage library on Parent Combo
cells and panning on Lung-Lung cells, scFv clones were analyzed by
flow cytometry for their ability to bind integrin .alpha.v.beta.3
on human tumor cells, and to distinguish between the activated and
non-activated forms of the receptor (FIG. 1). Each clone was tested
on a panel of human tumor cells, which either express or lack
.alpha.v.beta.3, but express .alpha.v or .beta.3 in combination
with other integrin subunits. These were M21 melanoma cells
(.alpha.v.beta.3, no other .beta.3 integrin), M21-LIIb cells
(.alpha.IIb.beta.3, no .alpha.v integrin), and UCLA-P3 lung
adenocarcinoma cells (.alpha.v integrins, but no .alpha.v.beta.3).
Two scFv clones, Bc-12 and Bc-15, were identified that reacted only
with cells expressing the .alpha.v.beta.3 heterodimer. A third scFv
clone, Bc-20, reacted with .alpha.v positive cells, regardless of
the associated .beta. subunit (FIG. 1A). Bc-12 and Bc-15 failed to
bind M21-L cells (no .alpha.v integrins)--but did react with M21-L4
cells, in which .alpha.v.beta.3 expression had been restored by
transfection, confirming their reactivity with .alpha.v.beta.3.
Felding-Habermann, B., et al., Clin. Exp. Metastasis 19: 427-436,
2002.
[0265] The recognition of .alpha.v.beta.3 by Bc-12 and Bc-15 showed
a cation dependence. Binding was measurable in the presence of
physiological Ca.sup.2+ levels and greatly enhanced in the presence
of Mn.sup.2+, a metal ion that can activate integrins (FIG. 1A,B).
To test this further, the effects of cation combinations on Bc-12
and Bc-15 binding were examined. Mg.sup.2+ supported Bc-12 and
Bc-15 binding at the same level as Ca.sup.2+, and Ca.sup.2+ reduced
the enhancing effect of Mn.sup.2+ (FIG. 1B). ScFv Bc-20 binding to
.alpha.v-positive cells did not require divalent metal cations.
This indicates that scFv Bc-12 and Bc-15 preferentially recognize
activated integrin .alpha.v.beta.3 and demonstrates binding
requirements reminiscent of natural plasma protein ligands of this
receptor, such as fibrinogen, vitronectin and fibronectin. Smith,
Methods Cell Biol. 69: 247-259, 2002; Hughes et al., J. Biol. Chem.
271: 6571-6574, 1996. To confirm that scFv Bc-12 and Bc-15
selectively recognize .alpha.v.beta.3 in its activated form,
binding of these antibodies to in vitro generated and in vivo
selected variants of the MDA-MB 435 breast cancer cell model was
tested. Bc-12 and Bc-15 failed to bind .beta.3-negative MDA-MB 435
cells (.beta.3.sup.-) and a .beta.3 wild type expressing derivative
of these cells (.beta.3.sub.WT), the latter of which was, however,
recognized when activated with Mn.sup.2+. The same Mn.sup.2+
dependence was true for MDA-MB 435 Parent Combo cells which express
non-activated .alpha.v.beta.3 (FIG. 1C). In contrast, Bc-12 and
Bc-15 were able to bind an MDA-MB 435 variant expressing
constitutively activated mutant .alpha.v.beta.3.sub.D723R, without
exogenous stimulation with Mn.sup.2+. Binding was further enhanced
when Mn.sup.2+ was added. Importantly, scFvs Bc-12 and Bc-15
recognized in vivo selected MDA-MB 435 variants from bone and lung
metastases, as well as metastatic cells isolated from breast cancer
patient blood samples (FIG. 1C). These cells express
.alpha.v.beta.3 in a constitutively activated form.
Felding-Habermann et al., Proc. Natl. Acad. Sci. U.S.A 98:
1853-1858, 2001; Rolli et al., Proc. Natl. Acad. Sci. U.S.A 100:
9482-9487, 2003. Thus, the results indicate that Bc-12 and Bc-15
recognize .alpha.v.beta.3 and require its presentation in an
activated, high affinity state.
[0266] Patient derived scFv antibodies against activated
.alpha.v.beta.3 are natural ligand mimetics. The findings that
patient derived scFv antibodies Bc-12 and Bc-15 require divalent
metal cations for binding to .alpha.v.beta.3 expressing tumor
cells, and that the receptor had to be present in an activated
functional form, indicate that Bc-12 and Bc-15 resemble natural
ligands of .alpha.v.beta.3. To analyze similarities between the
scFv antibodies and natural ligands, the DNA sequences of Bc-12,
Bc-15 and Bc-20 were determined and translated (FIG. 2A). The DNA
sequences of scFv Bc-12 cDNA and scFv Bc-15 cDNA are shown in FIGS.
2C. The protein sequence showed that the third complementarity
determining regions of the heavy chains (CDR-H3) in Bc-12 and Bc-15
contain an RGD ligand recognition motif. This motif, common to
natural .alpha.v.beta.3 ligands, is absent in Bc-20 (FIG. 2). To
examine the contribution of the RGD motif in CDR-H3 of scFv
antibodies Bc-12 and Bc-15 to their specificity for activated
integrin .alpha.v.beta.3, this sequence was changed to RGE by site
directed mutagenesis. The D to E exchange within the RGD motif is
known to reduce or abolish ligand recognition by .alpha.v.beta.3.
Binding of the mutated scFvs to human breast cancer cells was
strongly reduced in Mut-12, the RGE version of Bc-12, and abolished
in Mut-15, the RGE version of Bc-15 (FIG. 2B). This indicates that
RGD binding critically determines antibody-antigen recognition.
However, the antibodies did not react with the
.alpha.v.beta.3-related platelet integrin .alpha.IIb.beta.3, nor
with other .alpha.v integrins or .alpha.5.beta.1 (FIG. 1), which
are known RGD binding receptors. Ruoslahti, Annu. Rev. Cell Dev.
Biol. 12: 697-715, 1996. This implies that a synergistic binding
region may exist, with contributions from both the antibody
tertiary structure and the RGD sequence to the observed selective
recognition of the activated conformation of .alpha.v.beta.3. The
cDNA sequences for scFv Bc-12 and scFv Bc-15 are shown in FIG. 2C.
The cDNA sequences for scFv Mut-12 and scFv Mut-15 are shown in
FIG. 2D.
[0267] Ligand mimetic scFv antibodies inhibit .alpha.v.beta.3
mediated adhesive tumor cell functions. Since the patient derived
scFv antibodies Bc-12 and Bc-15 apparently mimic natural ligands of
integrin .alpha.v.beta.3, it was hypothesized that these antibodies
might interfere with .alpha.v.beta.3 mediated ligand binding and
adhesive tumor cell functions. When applied under stationary
conditions, Bc-12 and Bc-15 efficiently inhibited adhesion of BMS
human breast cancer cells to immobilized fibrinogen, a process that
is mediated exclusively by .alpha.v.beta.3 in these cells. Rolli et
al., Proc. Natl. Acad. Sci. U.S.A 100: 9482-9487, 2003. The extent
of inhibition by 3 .mu.M scFv was similar to that observed with 200
.mu.M GRGDSPK, a fibronectin derived peptide (FIG. 3A). BMS cell
attachment to vitronectin was also inhibited by Bc-12 and Bc-15,
but to a lesser degree. Adhesion to this protein is mediated by
.alpha.v.beta.3 together with other .alpha.v integrins on these
cells. The antibodies had no effect on breast cancer cell adhesion
to collagen type I, which is mediated primarily by BMS cell
integrin .alpha.2.beta.1 with no involvement of .alpha.v.beta.3.
Another function, critical for tumor cell dissemination, is matrix
directed migration. Tumor cells often produce vascular permeability
factors that cause leakage of adhesive plasma proteins into the
tumor area. McDonald et al., Cancer Res. 62: 5381-5385, 2002. Thus,
a gradient of fibrinogen and polymerizing fibrin, the most abundant
of these proteins, may guide metastatic cells toward tumor
supporting blood vessels. In the breast cancer cell model,
migration toward immobilized fibrinogen or fibrin is exclusively
mediated by integrin .alpha.v.beta.3 and requires the activated
state of the receptor. Rolli et al., Proc. Natl. Acad. Sci. U.S.A
100: 9482-9487, 2003. It was therefore examined whether Bc-12 and
Bc-15 can impact fibrinogen directed migration. At a 2 .mu.M
concentration, Bc-12 and Bc-15 inhibited BMS human breast cancer
cell migration almost completely. This effect was similar to that
seen with 200 .mu.M GRGDSPK peptide (FIG. 3B). RGE containing scFv
Mut-15 had no effect and Mut-12 only a minor effect on fibrinogen
directed migration.
[0268] In the circulation, metastatic tumor cells are intensely
exposed to a cancer patient=s immune surveillance, encountering
potential function blocking antibodies like Bc-12 and Bc-15. The
hostility of this environment toward metastatic cells is
intensified by shear forces generated by blood flow which
physically oppose tumor cell arrest within the vasculature, a
process necessary for target organ colonization. It has been
demonstrated that breast cancer cell integrin .alpha.v.beta.3 in
its activated form can support arrest of metastatic cells during
blood flow. Therefore experiments were designed to mimic blood flow
conditions in the vasculature, and to examine whether the ligand
mimetic scFv antibodies could interfere with breast cancer cell
arrest. BMS breast cancer cells were prestained with a red
fluorescent dye and mixed into human blood, which was spiked with a
green fluorescent dye. The mixture was perfused over a thrombogenic
collagen I matrix at a venous wall shear rate of 50 sec.sup.-1. On
this matrix, platelets attach easily and become activated. Ruggeri,
Nat. Med. 8: 1227-1234, 2002. This triggers local thrombus
formation mediated by activated platelet integrin
.alpha.IIb.beta.3. Breast cancer cells that express activated
integrin .alpha.v.beta.3 can utilize this receptor to interact with
platelets during blood flow and attach to thrombi formed at the
adhesive surface. Felding-Habermann et al., Proc. Natl. Acad. Sci.
U.S.A 98: 1853-1858, 2001. scFv antibodies Bc-12 and Bc-15
efficiently inhibited this process, while the non-RGD containing
anti .alpha.v antibody Bc-20 had no effect (FIG. 3C). Similar
results were obtained with other metastatic breast cancer cell
lines. The RGE containing scFv mutants Mut-12 and Mut-15 had no
effect on breast cancer cell arrest during blood flow. These
results indicate that the ligand mimetic scFv antibodies, which
recognize integrin .alpha.v.beta.3 in a functionally activated
form, can block breast cancer cell interaction with platelets, and
thereby inhibit cancer cell arrest in flowing blood. Integrin
.alpha.IIb.beta.3 mediated adhesive platelet functions were not
affected by the antibodies (FIG. 3C, left images).
[0269] As ligand mimetics, scFvs Bc-12 and Bc-15 may impact not
only breast cancer cell adhesive functions but also cell survival.
It has been demonstrated that disruption of ligand binding to
.alpha.v.beta.3 can stimulate apoptosis. Stupack et al., J. Cell
Biol. 155: 459-470, 2001. Through an alternative pathway,
internalized RGD containing compounds may directly induce cell
death by activating the pro-apoptotic enzyme caspase-3 through an
alternative pathway. Buckley et al., Nature 397: 534-539, 1999.
scFvs Bc-12 and Bc-15 were efficiently bound and readily
internalized by adherent BMS breast cancer cells at permissive
temperature. The growth of several human breast cancer cell lines,
isolated from patient blood samples, was retarded in the presence
of the RGD containing scFvs Bc-12 and Bc-15, while their RGE mutant
versions, Mut-12 and Mut-15, had no effect (FIG. 3D). Furthermore,
when breast cancer cells were deprived of an adhesive matrix, as
occurs in the circulation, exposure to scFv Bc-12 resulted in
apoptotic cell death (FIG. 3E). Thus, patient derived, ligand
mimetic antibodies can disrupt specific adhesive functions of
activated integrin .alpha.v.beta.3 and affect the growth behavior
of tumor cells bearing this receptor.
[0270] Antimetastatic activity of scFv antibodies against activated
.alpha.v.beta.3 inhibit hematogenous breast cancer metastasis in
vivo. Having demonstrated that the immune repertoire of cancer
patients contains antibodies that specifically recognize the
activated, metastasis supporting form of integrin .alpha.v.beta.3,
the next critical aspect investigated was whether binding of these
antibodies to circulating tumor cells affects metastasis from the
blood stream. To be effective in that microenvironment, the
antibodies must bind tumor cell integrin .alpha.v.beta.3 in blood
or plasma. The blood perfusion studies demonstrated that scFvs
Bc-12 and -15 can inhibited breast cancer cell arrest during blood
flow, indicating that these antibodies recognize tumor cells under
those conditions. To confirm this, BMS breast cancer cells were
incubated with increasing concentrations of Bc-12 or -15 in fresh
human plasma. The antibodies bound the cells in a saturable manner
with half maximal binding measured at 40 nM scFv (1 .mu.g/ml) (FIG.
4). Similar results were obtained with other metastatic breast
cancer cell lines. This demonstrates that RGD containing scFv
antibodies Bc-12 and Bc-15 bind to tumor cell integrin
.alpha.v.beta.3 in the presence of a multitude of RGD containing
plasma proteins, the most abundant of which is fibrinogen at a
physiological plasma concentration of 6-12 .mu.M.
[0271] To test directly whether targeting activated integrin
.alpha.v.beta.3 with the ligand mimetic antibodies Bc-12 and -15
affects target organ colonization by circulating metastatic breast
cancer cells, 1.times.10.sup.5 BMS human breast cancer cells were
injected intravenously into female C.B-17 SCID mice, together with
50 .mu.g sterile, endotoxin free preparations of Bc-12 or Bc-15
(n=8-10 mice per group). At an average blood volume of 2 ml per
mouse, this provides an initial scFv concentration of 1 .mu.M. The
clearance time for scFv antibody fragments in the circulation is
less than 1 h. Kortt et al, Biomol. Eng, 18: 95-108, 2001. Tumor
cells may remain in the circulation for several days. Therefore,
scFv antibody injections (50 .mu.g bolus doses, i.v.) were repeated
on the second, third and fourth day. Antibody treated and control
mice appeared healthy during the experiment. After 32 days, the
mice were euthanized, dissected and analyzed by gross examination.
No obvious abnormalities were observed. The lungs were excised,
fixed, and metastatic foci counted at the lung surface. Each of the
control treated mice had tumor foci on their lungs (range: 8 to 68
foci per lung). In stark contrast, only two animals in the Bc-12
treated group, and 1 animal in the Bc-15 treated group had one
visible nodule at their lung surface (FIG. 5A). Similar results
were obtained in a second experiment under more challenging
conditions, using a higher cell dose and a different breast cancer
cell type (3.times.10.sup.5 BCM1 cells/mouse).
[0272] To analyze whether scFv treatment had reduced or actually
prevented metastatic colonization of the lung tissue, the lungs of
each mouse were embedded in paraffin, sectioned, stained with
hematoxylin/eosin, and examined histologically for evidence of
metastatic colonies within the lung tissue. Per lung, six sets of
three consecutive sections, separated by 140 .mu.m, were collected.
The sections were randomized and coded, and the total number of
metastatic foci counted. All control animals had metastases in
their lung tissues (range: 7 to 48 counts per lung), and those were
of considerable size (FIG. 5B top image). In contrast, only 3 of 10
Bc-12 treated mice and 1 of 9 Bc-15 treated mice had
microscopically detectable metastases in their lung tissue (range:
0 to 5 counts per lung) (FIG. 5B). Thus, injections with scFv Bc-12
or Bc-15 interfered with lung colonization by circulating breast
cancer cells at a statistically significant level (P<0.005).
[0273] Taken together, the data imply that the circulating immune
repertoire of at least some cancer patients contains antibodies
against the activated form of integrin .alpha.v.beta.3. By
amplifying these naturally occurring antibodies in vitro, their
specificity and potential have been demonstrated for disrupting
critical functions of circulating metastatic cells thereby
inhibiting breast cancer metastasis in vivo. The next clinically
relevant question will be whether targeting the activated form of
.alpha.v.beta.3 can interfere with established breast tumors and
ongoing spontaneous metastasis. This seems plausible since
metastatic cells as well as tumor supporting angiogenic endothelial
cells apparently bypass the normal control of adhesive, migratory
and invasive properties by expressing constitutively activated
integrin .alpha.v.beta.3. Felding-Habermann et al., Proc. Natl.
Acad. Sci. U.S.A 98: 1853-1858, 2001; Kiosses et al., Nat. Cell
Biol. 3: 316-320, 2001.
Example 5
Diagnosis, Prognosis and Treatment of Metastatic Cancer in a
Mammalian Subject
[0274] These studies have shown that combinatorial antibody
libraries of cancer patients contain antibodies with disease
fighting potential. The studies have demonstrated that such
antibodies can be isolated in vitro and that these antibodies can
be used to inhibit metastasis in an experimental animal model.
Based on this finding, one might expect a high frequency of similar
antibodies with disease fighting potential in antibody libraries
from patients who are long-term survivors of metastatic cancer.
Such libraries can be generated and mined for the presence of such
antibodies.
[0275] The present invention addresses diagnostic, prognostic and
therapeutic approaches that can prevent breast cancer disease from
becoming systemic. The present invention provides a further
understanding of how to design and test drugs to treat metastases.
Metastases are ultimately responsible for much of the suffering and
mortality from breast cancer. The present invention addresses this
need to identify and target molecular and functional markers that
identify metastatic breast cancer cells and to generate therapeutic
reagents for their specific inhibition.
[0276] Combinatorial antibody libraries can be isolated using
antibody phage display technology and subtractive panning and
screening strategy. Human tumor cell models of metastatic tumor
cells are generated, for example, neoplastic tumors, solid tumor,
breast cancer, hematological malignancy, leukemia, colorectal
cancer, uterine cancer, uterine leiomyomas, ovarian cancer,
endometrial cancer, polycystic ovary syndrome, endometrial polyps,
prostate cancer, prostatic hypertrophy, pituitary cancer,
adenomyosis, adenocarcinomas, meningioma, melanoma, bone cancer,
multiple myeloma, CNS cancer, glioma, or astroblastoma.
[0277] Breast cancers are known to be extremely heterogeneous. The
present invention has demonstrated that a subset of human breast
cancer cells can be identified based on expression of an adhesion
receptor, the integrin .alpha.v.beta.3, in its constitutively
activated functional form. This activated integrin promotes
platelet binding and tumor cells arrest in the vasculature. In this
way, activation of integrin .alpha.v.beta.3 endows metastatic cells
with key properties likely to be critical for successful
dissemination and colonization of target organs. The combined
immune repertoire of a number of cancer patients has been mined
using antibody phage display technology by subtractive panning on
poorly versus strongly metastatic variants of a human breast cancer
cell line. This approach yielded single chain Fv (scFv) antibodies
that specifically recognize the activated functional conformation
of the tumor cell adhesion receptor, integrin .alpha.v.beta.3. The
antibodies react selectively with metastatic variants of the breast
cancer cell models described herein and with metastatic cells
isolated from blood samples of stage IV breast cancer patients.
These antibodies inhibit colonization of the lungs by human breast
cancer cells in immune deficient mice.
[0278] Studies will investigate the ability of human single chain
Fv (scFv) antibodies to report the activated form of integrin
.alpha.v.beta.3 as a diagnostic marker of metastatic breast cancer
cells. The scFv antibodies and their derivatives are useful to
specifically detect metastatic breast cancer cells and report the
localization of metastatic disease.
[0279] Studies will investigate the ability of human single chain
Fv (scFv) antibodies to report the activated form of integrin
.alpha.v.beta.3 as a prognostic marker of metastatic breast cancer.
The scFv antibodies and their derivatives are useful to
specifically detect breast cancer cells that have a propensity to
metastasize.
[0280] Studies will analyze effects of human scFv antibodies and
their derivatives against constitutively activated integrin
.alpha.v.beta.3 on breast cancer metastasis. Targeted inhibition of
cells expressing the activated form of integrin .alpha.v.beta.3 are
useful to prevent breast cancer metastasis and interfere with
established metastatic disease.
[0281] All publications and patent applications cited in this
specification are herein incorporated by reference in their
entirety for all purposes as if each individual publication or
patent application were specifically and individually indicated to
be incorporated by reference for all purposes.
[0282] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to one of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
Sequence CWU 1
1
131786DNAArtificialSynthetic Construct 1atg gca cag gtt cag ctg gta
cag tct gga gct gag gtg aag aag cct 48Met Ala Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro1 5 10 15ggg gcc tca gtg aag gtc
tcc tgc aag gct tct ggt tac acc ttt tcc 96Gly Ala Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser20 25 30aac tat ggt atc acc
tgg gtg cga cag gcc cct gga caa ggg ctt gag 144Asn Tyr Gly Ile Thr
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu35 40 45tgg atg gga tgg
atc aac aat ggt aac aca cac tat gca cag aag ttc 192Trp Met Gly Trp
Ile Asn Asn Gly Asn Thr His Tyr Ala Gln Lys Phe50 55 60cag ggc aga
gtc acc atg acc aca gac aca tcc acg agc aca gcc tac 240Gln Gly Arg
Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80atg
gag ctg agg agc ctt aga tct gac gac acg gcc gtt tat tac tgt 288Met
Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys85 90
95gcg aga gac ccc cgg ggt gac gac gag ccc tac tgg ggc cag gga acc
336Ala Arg Asp Pro Arg Gly Asp Asp Glu Pro Tyr Trp Gly Gln Gly
Thr100 105 110ctg gtc acc gtc tcc tca ggc ggc ggc ggc tct ggc gga
ggt ggc agc 384Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser115 120 125ggc ggt ggc gga tcc gaa att gtg ttg acg cag
tct cca ctc tcc ctg 432Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln
Ser Pro Leu Ser Leu130 135 140ccc gtc acc ctt gga cag ccg gcc tcc
atc tcc tgc cgg tct agt caa 480Pro Val Thr Leu Gly Gln Pro Ala Ser
Ile Ser Cys Arg Ser Ser Gln145 150 155 160aac ctc gta tac agt gat
gga aac acc tac ttg agt tgg ttt cag cag 528Asn Leu Val Tyr Ser Asp
Gly Asn Thr Tyr Leu Ser Trp Phe Gln Gln165 170 175agg cca ggc caa
tct cca agg cgc cta att tat aag gtt tct aac cgg 576Arg Pro Gly Gln
Ser Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg180 185 190gac tct
ggg gtc cca gac aga ttc agt ggc agt ggg tca ggc act gat 624Asp Ser
Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp195 200
205ttc aca ctg aaa atc agc agg gtg gag gct gag gat att ggg gtc tat
672Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Ile Gly Val
Tyr210 215 220tac tgc atg caa ggc aca cac tgg cct ccg cgg acg ttc
ggc caa ggg 720Tyr Cys Met Gln Gly Thr His Trp Pro Pro Arg Thr Phe
Gly Gln Gly225 230 235 240acc aag gtg gag atc aaa cgt ggc ctc ggg
ggc ctg gtc gac tac aaa 768Thr Lys Val Glu Ile Lys Arg Gly Leu Gly
Gly Leu Val Asp Tyr Lys245 250 255gat gac gat gac aaa taa 786Asp
Asp Asp Asp Lys2602261PRTArtificialSynthetic Construct 2Met Ala Gln
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro1 5 10 15Gly Ala
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser20 25 30Asn
Tyr Gly Ile Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu35 40
45Trp Met Gly Trp Ile Asn Asn Gly Asn Thr His Tyr Ala Gln Lys Phe50
55 60Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala
Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys85 90 95Ala Arg Asp Pro Arg Gly Asp Asp Glu Pro Tyr Trp
Gly Gln Gly Thr100 105 110Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser115 120 125Gly Gly Gly Gly Ser Glu Ile Val
Leu Thr Gln Ser Pro Leu Ser Leu130 135 140Pro Val Thr Leu Gly Gln
Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln145 150 155 160Asn Leu Val
Tyr Ser Asp Gly Asn Thr Tyr Leu Ser Trp Phe Gln Gln165 170 175Arg
Pro Gly Gln Ser Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg180 185
190Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp195 200 205Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Ile
Gly Val Tyr210 215 220Tyr Cys Met Gln Gly Thr His Trp Pro Pro Arg
Thr Phe Gly Gln Gly225 230 235 240Thr Lys Val Glu Ile Lys Arg Gly
Leu Gly Gly Leu Val Asp Tyr Lys245 250 255Asp Asp Asp Asp
Lys2603750DNAArtificialSynthetic Construct 3atg gca cag gtg cag ctg
gta cag tct gga gct gag gtg aag gag cct 48Met Ala Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Glu Pro1 5 10 15ggg tcc tcg gtg aag
gtc tcc tgc aag gct tct gga ggc acc ttc agc 96Gly Ser Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser20 25 30agc tat gct atc
tac tgg gtg cga cag gcc cct gga caa ggg ctt gag 144Ser Tyr Ala Ile
Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu35 40 45tgg atg gga
tgg atc aat cct gac agt ggt gac aca aac tct gca cag 192Trp Met Gly
Trp Ile Asn Pro Asp Ser Gly Asp Thr Asn Ser Ala Gln50 55 60cag ttt
cag ggc agg gtc acc atg acc agg gac acg tcc atc agc aca 240Gln Phe
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr65 70 75
80gcc tat atg gag ctg agc agg ctg aga tct gac gac acg gcc atg tat
288Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Met
Tyr85 90 95tac tgt gcg aga ccc ccc cgt ggg gat gga cct gac tac tgg
ggc cag 336Tyr Cys Ala Arg Pro Pro Arg Gly Asp Gly Pro Asp Tyr Trp
Gly Gln100 105 110ggc acc ctg gtc acc gtc tcc tca ggc ggc ggt ggc
gga tcc gaa att 384Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Gly Ser Glu Ile115 120 125gtg ctg act cag tct cca ggc acc ctg tct
ttg tct cca ggg gaa aga 432Val Leu Thr Gln Ser Pro Gly Thr Leu Ser
Leu Ser Pro Gly Glu Arg130 135 140gcc acc ctc tcc tgc agg gcc agt
cag agt gtt agc agc agc tac tta 480Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Val Ser Ser Ser Tyr Leu145 150 155 160gcc tgg tac cag cag
aaa cct ggc cag gct ccc agg ctc ctc atc tat 528Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr165 170 175ggt gca tcc
agc agg gcc act ggc atc cca gac agg ttc agt ggt agt 576Gly Ala Ser
Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser180 185 190ggg
tct ggg aca gac ttc act ctc acc atc agc aga ctg gag cct gaa 624Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu195 200
205gat ttt gca gtg tat tac tgt cag cag tat ggt agc tca cct cgg acg
672Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Arg
Thr210 215 220ttc ggc caa ggg acc aaa gtg gat atc aaa cgt ggc ctc
ggg ggc ctg 720Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Gly Leu
Gly Gly Leu225 230 235 240gtc gac tac aaa gat gac gat gac aaa taa
750Val Asp Tyr Lys Asp Asp Asp Asp Lys2454249PRTArtificialSynthetic
Construct 4Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Glu Pro1 5 10 15Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly
Thr Phe Ser20 25 30Ser Tyr Ala Ile Tyr Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu35 40 45Trp Met Gly Trp Ile Asn Pro Asp Ser Gly Asp
Thr Asn Ser Ala Gln50 55 60Gln Phe Gln Gly Arg Val Thr Met Thr Arg
Asp Thr Ser Ile Ser Thr65 70 75 80Ala Tyr Met Glu Leu Ser Arg Leu
Arg Ser Asp Asp Thr Ala Met Tyr85 90 95Tyr Cys Ala Arg Pro Pro Arg
Gly Asp Gly Pro Asp Tyr Trp Gly Gln100 105 110Gly Thr Leu Val Thr
Val Ser Ser Gly Gly Gly Gly Gly Ser Glu Ile115 120 125Val Leu Thr
Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg130 135 140Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu145 150
155 160Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
Tyr165 170 175Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe
Ser Gly Ser180 185 190Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu Glu Pro Glu195 200 205Asp Phe Ala Val Tyr Tyr Cys Gln Gln
Tyr Gly Ser Ser Pro Arg Thr210 215 220Phe Gly Gln Gly Thr Lys Val
Asp Ile Lys Arg Gly Leu Gly Gly Leu225 230 235 240Val Asp Tyr Lys
Asp Asp Asp Asp Lys2455786DNAArtificialSynthetic Construct
5atggcacagg ttcagctggt acagtctgga gctgaggtga agaagcctgg ggcctcagtg
60aaggtctcct gcaaggcttc tggttacacc ttttccaact atggtatcac ctgggtgcga
120caggcccctg gacaagggct tgagtggatg ggatggatca acaatggtaa
cacacactat 180gcacagaagt tccagggcag agtcaccatg accacagaca
catccacgag cacagcctac 240atggagctga ggagccttag atctgacgac
acggccgttt attactgtgc gagagacccc 300cggggtgagg acgagcccta
ctggggccag ggaaccctgg tcaccgtctc ctcaggcggc 360ggcggctctg
gcggaggtgg cagcggcggt ggcggatccg aaattgtgtt gacgcagtct
420ccactctccc tgcccgtcac ccttggacag ccggcctcca tctcctgccg
gtctagtcaa 480aacctcgtat acagtgatgg aaacacctac ttgagttggt
ttcagcagag gccaggccaa 540tctccaaggc gcctaattta taaggtttct
aaccgggact ctggggtccc agacagattc 600agtggcagtg ggtcaggcac
tgatttcaca ctgaaaatca gcagggtgga ggctgaggat 660attggggtct
attactgcat gcaaggcaca cactggcctc cgcggacgtt cggccaaggg
720accaaggtgg agatcaaacg tggcctcggg ggcctggtcg actacaaaga
tgacgatgac 780aaataa 7866750DNAArtificialSynthetic Construct
6atggcacagg tgcagctggt acagtctgga gctgaggtga aggagcctgg gtcctcggtg
60aaggtctcct gcaaggcttc tggaggcacc ttcagcagct atgctatcta ctgggtgcga
120caggcccctg gacaagggct tgagtggatg ggatggatca atcctgacag
tggtgacaca 180aactctgcac agcagtttca gggcagggtc accatgacca
gggacacgtc catcagcaca 240gcctatatgg agctgagcag gctgagatct
gacgacacgg ccatgtatta ctgtgcgaga 300cccccccgtg gggagggacc
tgactactgg ggccagggca ccctggtcac cgtctcctca 360ggcggcggtg
gcggatccga aattgtgctg actcagtctc caggcaccct gtctttgtct
420ccaggggaaa gagccaccct ctcctgcagg gccagtcaga gtgttagcag
cagctactta 480gcctggtacc agcagaaacc tggccaggct cccaggctcc
tcatctatgg tgcatccagc 540agggccactg gcatcccaga caggttcagt
ggtagtgggt ctgggacaga cttcactctc 600accatcagca gactggagcc
tgaagatttt gcagtgtatt actgtcagca gtatggtagc 660tcacctcgga
cgttcggcca agggaccaaa gtggatatca aacgtggcct cgggggcctg
720gtcgactaca aagatgacga tgacaaatag 7507259PRTArtificialSynthetic
Construct 7Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro1 5 10 15Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Ser20 25 30Asn Tyr Gly Ile Thr Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu35 40 45Trp Met Gly Trp Ile Asn Asn Gly Asn Thr His
Tyr Ala Gln Lys Phe50 55 60Gln Gly Arg Val Thr Met Thr Thr Asp Thr
Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu Arg Ser
Asp Asp Thr Ala Val Tyr Tyr Cys85 90 95Ala Arg Asp Pro Arg Gly Asp
Asp Glu Pro Tyr Trp Gly Gln Gly Thr100 105 110Leu Val Thr Val Ser
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser115 120 125Gly Gly Gly
Gly Ser Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu130 135 140Pro
Val Thr Leu Gly Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln145 150
155 160Asn Leu Val Tyr Ser Asp Gly Asn Thr Tyr Leu Ser Trp Phe Gln
Gln165 170 175Arg Pro Gly Gln Ser Pro Arg Arg Leu Ile Tyr Lys Val
Ser Asn Arg180 185 190Asp Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp195 200 205Phe Thr Leu Lys Ile Ser Arg Val Glu
Arg Glu Asp Ile Gly Val Tyr210 215 220Tyr Cys Met Gln Gly Thr His
Trp Pro Pro Arg Thr Phe Gly Gln Gly225 230 235 240Thr Lys Val Glu
Ile Lys Arg Gly Leu Gly Gly Leu Val Asp Tyr Lys245 250 255Asp Asp
Asp8247PRTArtificialSynthetic Construct 8Met Ala Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Glu Pro1 5 10 15Gly Ser Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser20 25 30Ser Tyr Ala Ile
Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu35 40 45Trp Met Gly
Trp Ile Asn Pro Asp Ser Gly Asp Thr Asn Ser Ala Gln50 55 60Gln Phe
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr65 70 75
80Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Met Tyr85
90 95Tyr Cys Ala Arg Pro Pro Arg Gly Asp Gly Pro Asp Tyr Trp Gly
Gln100 105 110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Gly
Ser Glu Ile115 120 125Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu
Ser Pro Gly Glu Arg130 135 140Ala Thr Leu Ser Cys Arg Ala Ser Gln
Ser Val Ser Ser Ser Tyr Leu145 150 155 160Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr165 170 175Gly Ala Ser Ser
Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly Ser180 185 190Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro Glu195 200
205Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro Arg
Thr210 215 220Phe Gly Gln Gly Thr Lys Val Asp Ile Lys Arg Gly Leu
Gly Gly Leu225 230 235 240Val Asp Tyr Lys Asp Asp
Asp2459189PRTArtificialSynthetic Construct 9Met Ala Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Pro Gly1 5 10 15Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Thr Phe Ser Tyr Ile Trp20 25 30Val Arg Gln Ala
Pro Gly Gln Gly Leu Glu Trp Met Gly Trp Ile Asn35 40 45Gly Thr Ala
Gln Phe Gln Gly Arg Val Thr Met Thr Asp Thr Ser Ser50 55 60Thr Ala
Tyr Met Glu Leu Leu Arg Ser Asp Asp Thr Ala Tyr Tyr Cys65 70 75
80Ala Arg Pro Arg Gly Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val85
90 95Ser Ser Gly Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser
Pro100 105 110Leu Gly Ala Ser Cys Arg Ser Gln Ser Tyr Leu Trp Gln
Gln Pro Gly115 120 125Gln Pro Arg Leu Ile Tyr Ser Arg Gly Pro Asp
Arg Phe Ser Gly Ser130 135 140Gly Ser Gly Thr Asp Phe Thr Leu Ile
Ser Arg Glu Glu Asp Val Tyr145 150 155 160Tyr Cys Gln Gly Pro Arg
Thr Phe Gly Gln Gly Thr Lys Val Ile Lys165 170 175Arg Gly Leu Gly
Gly Leu Val Asp Tyr Lys Asp Asp Asp180
18510257PRTArtificialSynthetic Construct 10Met Ala Gln Val Gln Leu
Val Gln Ser Gly Gly Gly Leu Val Gln Pro1 5 10 15Gly Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser20 25 30Ser Tyr Trp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu35 40 45Trp Val Ala
Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp50 55 60Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser65 70 75
80Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr85
90 95Tyr Cys Ala Arg Asp Gly Gly Phe Ala Gly Trp Ala Phe Asp Ile
Trp100 105 110Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly115 120 125Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile
Gln Met Thr Gln Ser130 135 140Pro Ser Ala Leu Ser Ala Ser Val Gly
Asp Arg Val Thr Ile Thr Cys145 150 155 160Arg Ala Ser Gln Gly Leu
Asp Asn Tyr Leu Ala Trp Tyr Gln Leu Gln165 170 175Pro Gly Lys Ala
Pro Lys Leu Leu Ile Tyr Asp Ala Phe Thr Leu Gln180 185 190Ser Gly
Val Pro Ser Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe195 200
205Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr
Phe210 215 220Cys Gln Gln Leu Gln Ser Tyr Pro Leu Thr Phe Gly Gly
Gly Thr Lys225 230 235 240Val Glu Ile Lys Arg Gly Leu Gly Gly Leu
Val Asp Tyr Lys Asp Asp245 250
255Asp117PRTArtificialSynthetic Construct 11Gly Arg Gly Asp Ser Pro
Lys1 51248DNAArtificialPrimer 12aaatagacta gtggaggcgg tggctctatg
agtgttttag tgtattct 481339DNAArtificialPrimer 13gatttagcta
gcttattatg aggaagtttc cattaaacg 39
* * * * *
References