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Cancer Patent Abstract
The present invention concerns compositions and methods for the
treatment of disorders characterized by the over-expression of an
LIV-1. More specifically, the compositions include DNA and amino
acid sequences of an LIV-1, antibodies to an LIV-1, and methods
for the treatment of a mammal susceptible to or diagnosed with cancer
wherein an LIV-1 is overexpressed.
Cancer Patent Claims
What is claimed is:
1. A method of diagnosing tumor in a mammal, comprising detecting
and quantifying the level of expression of a nucleic acid sequence
comprising a sequence 100% identical to the sequence from nucleotide
412 to and including nucleotide 477 of SEQ ID NO: 3 or to its fully
complementary sequence in a test sample obtained from the mammal,
and in a control sample, wherein a higher expression level in the
test sample indicates the presence of tumor in the mammal from which
the test sample was obtained.
2. The method of claim 1, wherein said test sample is obtained
from an individual suspected to have neoplastic cell growth or proliferation.
3. The method of claim 2, wherein the sample is from breast tissue
of the mammal.
Cancer Patent Description
FIELD OF THE INVENTION
The present invention concerns compositions and methods for the
treatment of disorders characterized by the overexpression of a
LIV-1 gene product in tumors. The compositions comprise a nucleic
acid, a polypeptide encoded by the nucleic acid, and a compound,
preferably an antibody or fragment thereof, that binds to the polypeptide,
preferably binding to the extracellular domain of LIV-1 polypeptide.
BACKGROUND OF THE INVENTION
Breast cancer is a common and devastating form of cancer, affecting
millions of women per year throughout the world. Many breast tumors
are estrogen sensitive and frequently treatable with compounds that
interfere with estrogen binding to estrogen receptors (ERs) expressed
on the breast tumor tissue. Detecting the level of ER expression
and sensitivity to estrogen stimulation is useful for determining
that antihormone-type chemotherapy may succeed in a particular patient.
The overexpression of estrogen-inducible genes, pLIV-1 and pLIV2
(also designated pS2), occurs in some breast tumors which also express
the estrogen receptor, (Manning D. L. et al. European J. Cancer
29A(10): 1462-1468 (1993): Manning. D. L. et al., European J. Cancer
30A(5):675-678 (1994); Manning. D. L. et al. Acta Oncologica 34
(5):641-646 (1995); Manning, D. L. et al., U.S. Pat. No. 5,693,465).
Expression of pLIV-1, but not pS2, is associated with metastasis
of breast cancer cells to regional lymph nodes (Manning et al. U.S.
Pat. No. 5,692,465).
In addition, the pathogenesis of various human malignancies, including
breast cancer, is affected by proto-oncogenes that encode growth
factors and growth factor receptors. Human ErbB2 gene (erbB2, also
known as her2, or c-erbB-2), which encodes a 185-kd transmembrane
glycoprotein receptor (ErbB2, also known as HER2 or p185.sup.HER2)
related to the epidermal growth factor receptor (EGFR), is overexpressed
in about 25% to 30% of human breast cancer (Slamon et al., Science
235:177-182 [1987]: Slamon et al. Science 244:707-712 [1989]).
Several lines of evidence support a direct role for ErbB2 in the
pathogenesis and clinical aggressiveness of ErbB2-overexpressing
tumors. The introduction of ErbB2 into non-neoplastic cells causes
their malignant transformation (Hudziak et al. Proc. Natl. Acad.
Sci. USA 84:7159-7163 [1987]; DiFiore et al., Science 237:78-182
[1987]). Transgenic mice that express ErbB2 develop mammary tumors
(Guy et al., Proc. Natl. Acad. Sci. USA 89:10578-10582 [1992]).
ErbB2 overexpression is commonly regarded as a predictor of a poor
prognosis in humans, especially in patients with primary disease
that involves axillary lymph nodes (Slamon et al. [1987] and [1989],
supra: Ravdin and Chamness, Gene 159:19-27 [1995]; and Hynes and
Stern, Biochim Biophys Acta 1198:165-184 [1994]).
Antibodies directed against human erbB2 protein products (anti-ErbB2
antibodies) and against proteins encoded by the rat equivalent of
the erbB2 gene (neu) (anti-neu protein antibodies) down-modulate
cell surface expression of p185 on B104-1-1 cells (NIH-3T3 cells
transfected with the neu proto-oncogene) and inhibit colony formation
of these cells, Drebin et al., Cell 41:695-706 (1985). Biological
effects of anti-neu protein antibodies are reviewed in Myers et
al., Meth. Enzym. 198:277-290 (1991). See also WO94/22478 published
Oct. 13, 1994.
The anti-ErbB2 antibody, 4D5, exhibited anti proliferative effects
on the SKDR3 human breast tumor cell line, inhibiting cellular proliferation
by approximately 56%, and sensitizing p185.sup.erbB2 -overexpressing
breast tumor cell lines to the cytotoxic effects of TNF-.alpha..
See Hudziak et al., Mol. Cell. Biol. 9(3):1165-1172(1989). See also
WO89/06692 published Jul. 27, 1989. The anti-ErbB2 antibodies discussed
in Hudziak et al. are further characterized in Fendly et al. Cancer
Research 50:1550-1558 (1990); Kotts et al. In Vitro 26(3):59A (1990):
Sarup et al. Growth Regulation 1:72-82 (1991): Shepard et al. J.
Clin. Immunol. 11(3): 117-127 (1991): Kumar et al. Mol. Cell. Biol.
11(2):979-986 (1991); Lewis et al. Cancer Immunol. Immunother. 37:255-263
(1993); Pietras et al. Oncogene 9:1829-1838 (1994); Vitetta et al.
Cancer Research 54:5301-5309 (1994); Sliwkowski et al. J. Biol.
Chem. 269(20):14661-14665 (1994): Scott et al. J. Biol. Chem. 266:14300-5
(1991); and D'souza et al. Proc. Natl. Acad. Sci. 91:7202-7206 (1994).
ErbB2 overexpression is also linked to sensitivity and/or resistance
to hormone therapy and chemotherapeutic regimens, including CMF
(cyclophosphamide, methotrexate, and fluoruracil) and anthracyclines
(Baselga et al., Oncology 11(3 Suppl 1):43-48 [1997]). Despite the
association of ErbB2 overexpression with poor prognosis, the odds
of HER2-positive patients responding clinically to treatment with
taxanes were greater than three times those of HER2-negative patients
(Ibid), rhuMab HER2 was shown to enhance the activity of paclitaxel
(TAXOL.RTM.) and doxorubicin against breast cancer xenografts in
nude mice injected with BT-474 human breast adenocarcinoma cells,
which express high levels of HER2 (Baselga et al. Breast Cancer,
Proceedings of ASCO, Vol. 13. Abstract 53 [1994]).
Because breast and other cancers pose constant threats to health,
there is a continuing need to develop treatments for cancers by
using methods that target cancer cells without simultaneously harming
large numbers of non-cancerous cells, thereby limiting adverse side
effects associated with traditional cancer chemotherapy.
SUMMARY OF THE INVENTION
The present invention relates to the discovery of a unique protein.
LIV-1-164647, that is overexpressed in some tumor tissues, such
as in prostate, colon, lung, breast, and a population of breast
tumors that overexpress LIV-1-164647, but do not overexpress ErbB2.
The present invention further relates to nucleic acid sequences
and amino acid sequences having homology to herein disclosed LIV-1
gene sequence (designated DNA 164647) and the amino acid sequence
of LIV-1 protein encoded by DNA 164647. Applicants' discovery that
LIV-1-164647 is overexpressed in tumor cells led to the additional
discoveries of compositions for detection and treatment of tumor
cells and methods of carrying out such detection and treatment.
In one aspect, the present invention relates to a nucleic acid
sequence having homology to the nucleic acid sequence of DNA 164647
(SEQ ID NO:3 (coding strand)), or a portion thereof. Preferably
the homology is at least approximately 80% homology, more preferably
at least approximately 90%, still more preferably at least approximately
95%, and most preferably at least approximately 97% homology. Preferably,
the nucleic acid of the invention encodes an aqueous soluble extracellular
domain (ECD) that is at least 80% homologous to the DNA 164647 (SEQ
ID NO:3) from approximately nucleic acid 73 to approximately 1060.
Preferably, the homologous nucleic acid of the invention hybridizes
under stringent conditions to a 30 nucleic acid or longer portion
of the nucleic acid sequence of DNA 164647 (SEQ ID NO:3) or its
complementary sequence, preferably hybridizing under stringent conditions
to a 30 nucleotide regions from nucleotide 440 to and including
nucleotide 470 of SEQ ID NO:3, or its complementary sequence. In
a related embodiment, the homologous nucleic acid of the invention
comprises a nucleic acid sequence comprising a sequence that is
at least 50%, preferably at least 80%, more preferably at least
90% homologous to the sequence from nucleotide 446 to and including
nucleotide 463 of SEQ ID NO:3, or a sequence from nucleotide 2297
to and including 2337 of SEQ ID NO:3, or both sequences. Most preferably,
the isolated nucleic acid comprises a sequence from nucleotide 446
to and including nucleotide 464 and/or from nucleotide 2297 to and
including 2337. According to the presently disclosed invention,
the isolated nucleic acid of the invention comprises a sequence
having at least 65%, preferably at least 75%, more preferably at
least 85%, still more preferably at least 90%, and most preferably
at least 96% homologous to a sequence from approximately nucleotide
412 to and including nucleotide 477 of SEQ ID NO:3, or its complementary
sequence. Preferably, the sequence encodes a histidine-rich region
of an antigenic polypeptide, preferably an ECD.
In another aspect, the present invention relates to an isolated
polypeptide comprising an amino acid sequence having homology to
the amino acid sequence (SEQ ID NO:4), or a fragment thereof, encoded
by or within DNA 164647, designated herein as LIV-1-164647. Preferably
the homology is at least approximately 80% homology, more preferably
at least approximately 90%, still more preferably at least approximately
95%, and most preferably at least approximately 97% homology. Preferably,
a LIV-1-164647 amino acid sequence of the invention is an aqueous
soluble ECD homologous to amino acid 1 to approximately amino acid
327 or a fragment thereof comprising at least 10 amino acids. The
region of the ECD is readily determined from a standard hydropathy
plot indicating the relatively more hydrophilic region N-terminal
of a hydrophobic transmembrane region. In a related embodiment,
the homologous amino acid sequence of the invention comprises a
sequence from amino acid 126 to and including amino acid 132 of
SEQ ID NO:4 (specifically, the amino acid sequence HDHHSHH (SEQ
ID NO:17)), or a sequence from amino acid 743 to and including amino
acid 755 of SEQ ID NO:4 (specifically, the amino acid sequence SIFEHKIVFRINF
(SEQ ID NO:18), or both sequences. The present invention further
includes an isolated polypeptide comprising an amino acid sequence
having at least 50%, preferably at least 80%, more preferably at
least 90% homologous to SEQ ID NO:17. The present invention still
further includes an isolated polypeptide comprising an amino acid
sequence having at least 20%, more preferably at least 50%, still
more preferably at least 80%, and most preferably at least 90% homology
to SEQ ID NO:18. In still another embodiment, the invention includes
an isolated nucleic acid of SEQ ID NO:3 and an isolated polypeptide
of SEQ ID NO:4. The present invention further includes an isolated
polypeptide comprising an amino acid sequence having at least 65%,
preferably at least 75%, more preferably at least 85%, still more
preferably at least 90%, and most preferably at least 96% to a sequence
from amino acid 114 to and including amino acid 135 of SEQ ID NO:4.
The amino acid sequence from amino acid 114 to 135 is designated
SEQ ID NO:19. A still further embodiment includes an isolated polypeptide
comprising SEQ ID NO:17 and/or SEQ ID NO:18.
In still another embodiment, the invention includes an isolated
polypeptide comprising an amino acid sequence wherein the sequence
is at least 98% homologous to the sequence from amino acid 1 to
and including amino acid 327 of SEQ ID NO:4, more preferably comprising
the ECD of LIV-1-164647. Most preferably, the sequence comprises
SEQ ID NO:17, forms a portion of an extracellular domain (ECD),
preferably the ECD of LIV-1-164647. The term "portion"
as used herein refers to a sequence that comprises at least 7 amino
acids of the ECD of LIV-1-164647 from amino acid 1 to and including
amino acid 327 of SEQ ID NO:4.
In another embodiment, the present invention concerns an antibody
which specifically binds a LIV-1 polypeptide. Preferably, the antibody
is a monoclonal antibody. More preferably, the antibody is a human
antibody or a humanized antibody. In one embodiment, the antibody
reduces activity of overexpressed LIV-1 polypeptide in a cell. In
another aspect, the antibody is a monoclonal antibody, which preferably
has nonhuman complementarity determining region (CDR) residues and
human framework region (FR) residues. The antibody may be labeled
and may be immobilized on a solid support. In a further aspect,
the antibody is an antibody fragment, a single-chain antibody, or
an anti-idiotypic antibody. Preferably, a LIV-1-binding antibody
of the invention binds specifically to a polypeptide having at least
approximately 80% homology, more preferably at least approximately
90% homology, still more preferably at least approximately 95% homology,
and most preferably at least approximately 97% homology to the LIV-1
ECD nucleic acid sequence, or a fragment thereof, encoded within
the ECD coding region (nucleotides 1-1000) of DNA 164647 (SEQ ID
NO:3). More preferably, a LIV-1-binding antibody of the invention
binds specifically to a polypeptide having at least 80% homology,
more preferably at least approximately 90% homology, still more
preferably at least approximately 95% homology, and most preferably
at least approximately 97% homology to the amino acid sequence of
LIV-1 ECD (amino acids 1-327 of SEQ ID NO:4). In a preferred embodiment,
the present invention concerns an isolated antibody which specifically
binds a LIV-1 polypeptide encoded by a nucleic acid sequence comprising
a nucleic acid sequence having at least 65%, preferably at least
75%, more preferably at least 85%, still more preferably at least
90%, and most preferably at least 96%, homology to a sequence from
nucleotide 446 to and including nucleotide 463 of SEQ ID NO:3, or
the nucleic acid sequence from 2297 to and including 2337 of SEQ
ID NO:3, or both nucleic acid sequences. In another preferred embodiment,
the present invention concerns an isolated antibody which specifically
binds a LIV-1 polypeptide comprising the amino acid sequence having
at least 65%, preferably at least 75%, more preferably at least
85%, still more preferably at least 90%, and most preferably at
least 96% homology to a sequence from amino acid 126 to and including
amino acid 132 of SEQ ID NO:4, or the amino acid sequence from amino
acid 743 to and including amino acid 755 of SEQ ID NO:4, or both
amino acid sequences.
In still another embodiment, the invention concerns an antibody,
preferably a monoclonal antibody, that specifically binds the same
epitope of LIV-1, preferably LIV-1-1164647, that is bound by any
one of the monoclonal antibodies produced by the hybridoma cell
lines deposited with the American Type Culture Collection (ATCC)
as disclosed herein.
In a further embodiment, the invention includes an antibody that
binds to a polypeptide comprising a sequence from amino acid 1 to
and including amino acid 147 of SEQ ID NO:4. In another embodiment,
the antibody binds a polypeptide comprising amino acid 148 to and
including amino acid 298 of SEQ ID NO:4. Preferably, the antibodies
are monoclonal antibodies. More preferably, the monoclonal antibodies
are human antibodies or humanized antibodies.
In another aspect, the invention concerns a composition comprising
an antibody which binds LIV-1 polypeptide in an admixture with a
pharmaceutically acceptable carrier. In one aspect, the composition
comprises a therapeutically effective amount of the antibody. In
another aspect, the composition comprises a further active ingredient,
which may, for example, be a further antibody or a cytotoxic or
chemotherapeutic agent. Preferably, the composition is sterile.
In a further embodiment, the invention concerns a nucleic acid
encoding an anti-LIV-1 antibody according to the invention, and
vectors and recombinant host cells comprising such nucleic acid.
In a still further embodiment, the invention concerns a method
for producing an anti-LIV-1 antibody by culturing a host cell transformed
with nucleic acid encoding the antibody under conditions such that
the antibody is expressed, and recovering the antibody from the
cell culture.
The invention further concerns antagonists and agonists of a LIV-1
polypeptide, which antagonists inhibit one or more of the functions
or activities of the LIV-1 polypeptide and which agonists mimic
one or more of the functions or activities of the LIV-1 polypeptide.
Preferably the LIV-1 polypeptide is the LIV-1-164647 polypeptide
whose functions or activities are antagonized or agonized.
In another embodiment, the invention concerns a method for determining
the presence of a LIV-1 polypeptide or fragment thereof comprising
exposing a cell suspected of containing the LIV-1 polypeptide to
an anti-LIV-1 antibody of the invention and determining binding
of the antibody to the cell.
In yet another embodiment, the present invention concerns a method
of diagnosing tumor in a mammal, comprising detecting the level
of expression of a gene encoding a LIV-1 polypeptide in a test sample
of tissue cells obtained from the mammal, and in a control sample
of known normal tissue cells of the same cell type, wherein a higher
expression level in the test sample indicates the presence of tumor
in the mammal from which the test tissue cells were obtained.
In another embodiment, the present invention concerns a method
of diagnosing tumor in a mammal, comprising contacting an anti-LIV-1
antibody with a test sample of tissue cells obtained from the mammal,
and detecting the formation of a complex between the anti-LIV-1
antibody and the LIV-1 polypeptide in the test sample. The detection
may be qualitative or quantitative, and may be performed in comparison
with monitoring the complex formation in a control sample of known
normal tissue cells of the same cell type. A larger quantity of
complexes formed in the test sample indicates the presence of tumor
in the mammal from which the test tissue cells were obtained. The
antibody preferably carries a detectable label. Complex formation
can be monitored, for example, by light microscopy, flow cytometry,
fluorimetry, or other techniques known in the art.
For the methods of diagnosing, the test sample is usually obtained
from an individual suspected to have neoplastic cell growth or proliferation
(e.g. cancerous cells).
In another embodiment, the invention involves a method of diagnosing
breast tumor tissue as tissue that overexpresses LIV-1 protein and
expresses normal levels of ErbB2. The method comprises providing
a test sample of tissue suspected of being cancerous, contacting
the test sample with an antibody to the naturally occurring form
of the LIV-1 gene product, contacting the same or duplicate test
sample with an anti-ErbB2 antibody, detecting the relative binding
of the antibodies to the test sample compared to a control sample,
where the control may be a positive control, a negative control,
or both. A test sample that overexpresses LIV-1 gene product (relative
to normal tissue), but does not overexpress ErbB2 protein (relative
to normal tissue), is diagnosed as a member of the population of
breast tumors to be treated by the compositions and methods of the
invention. Useful in the diagnostic assay method of the invention
are anti-ErbB2 antibodies that bind the extracellular domain of
the ErbB2 receptor, and preferably bind to the epitope 4D5 or 3H4
within the ErbB2 extracellular domain sequence. Mole preferably,
the antibody is the antibody 4D5. Other preferred ErbB2-binding
antibodies include, but are not limited to, antibodies 7C2, 7F3,
and 2C4. Information regarding anti-ErbB2 antibodies is found, for
example, in Hudziak, R. M. et al. U.S. Pat. No. 5,772,997, incorporated
herein by reference in its entirety.
In another aspect, the present invention concerns a cancer diagnostic
kit, comprising an anti-LIV-1-164647 antibody and a carrier (e.g.
a buffer) in suitable packaging. The kit preferably contains instructions
for using the antibody to detect the LIV-1 polypeptide.
In yet another aspect, the invention concerns a method for inhibiting
the growth of tumor cells comprising exposing a cell which overexpresses
a LIV-1 polypeptide to an effective amount of an agent inhibiting
the expression and/or activity of the LIV-1 polypeptide. The agent
preferably is an anti-LIV-1-164647 antibody, a small organic and
inorganic molecule, peptide, phosphopeptide, antisense or ribozyme
molecule, or a triple helix molecule. In a specific aspect, the
agent, e.g. anti-LIV-1-164647 antibody induces cell death, or at
least slows cell growth sufficiently to allow other methods of cancer
treatment to be administered. In a further aspect, the tumor cells
are further exposed to radiation treatment and/or a cytotoxic or
chemotherapeutic agent.
In yet another aspect, the invention concerns a method for the
treatment of a human patient susceptible to or diagnosed with a
disorder characterized by overexpression of LIV-1 gene product without
overexpression of ErbB2 receptor. In an embodiment, the method comprises
administering a therapeutically effective amount of an anti-LIV-1
polypeptide antibody, where administration may be intravenous, subcutaneous,
or other pharmaceutically acceptable method of antibody delivery.
Preferably the antibody specifically binds the naturally occurring
form of the LIV-1-164647 polypeptide, wherein the binding is preferably
to the extracellular domain or a fragment thereof. Preferably, the
initial dose (or doses) as well as the subsequent maintenance dose
or doses are administered subcutaneously. Optionally, where the
patient's tolerance of the anti-LIV-1 antibody is unknown, the initial
dose is administered by intravenous infusion, followed by subcutaneous
administration of the maintenance doses it the patient's tolerance
for the antibody is acceptable. According to the embodiment of the
invention, the initial dose or doses is followed by subsequent doses
of equal or smaller amounts of antibody at intervals sufficiently
close to maintain the trough serum concentration of antibody at
or above an efficacious target level. Preferably, an initial dose
or individual subsequent dose does not exceed 100 mg/kg, and each
subsequent dose is at least 1 .mu.g/kg. The choice of delivery method
for the initial and maintenance doses is made according to the ability
of the animal or human patient to tolerate introduction of the antibody
into the body. Where the antibody is well-tolerated, the time of
infusion may be reduced. The choice of delivery method as disclosed
for this embodiment applies to all drug delivery regimens contemplated
according to the invention.
In a further aspect, the invention provides a method of treating
LIV-1 gene product-overexpressing cancer (lacking overexpression
of ErbB2) in a human patient comprising administering to the patient
effective amounts of an anti-LIV-1 antibody (which antibody preferably
binds the extracellular domain of LIV-1 gene product) and a chemotherapeutic
agent. In one embodiment of the invention, the chemotherapeutic
agent in a toxoid including, but not limited to, paclitaxel and
doxetaxel. In another embodiment, the chemotherapeutic agent is
an anthracyline derivative including, but not limited to, doxorubicin
and epirubicin. In still another embodiment, the chemotherapeutic
agent is not administered to the patient simultaneously with the
anti-LIV-1 antibody. One or more additional chemotherapeutic agents
may also be administered to the patient.
The disorder to be treated by a method of the invention preferably
is a benign or malignant tumor characterized by the overexpression
of the LIV-1 gene product. Preferably, the malignant cells of the
tumor express approximately the same level of ErbB2 (or less) as
a non-cancerous cell of the same type. For example, the disorder
to be treated is a cancer, such as breast cancer, lung cancer, and
prostate cancer.
Accordingly, one aspect of the invention involves compounds that
bind to the LIV-1 protein and inhibit its activity. Preferably the
compounds bind to the extracellular region of the LIV-1 protein
and inhibit its activity. In an embodiment of the invention, the
inhibiting compounds are antibodies specific to the LIV-1 gene product
or fragments thereof. Preferably, the inhibiting compounds of the
invention bind specifically to the extracellular region of the LIV-1
protein.
In another aspect, the invention involves compounds that block
the binding of an activating ligand of LIV-1 protein. Such ligand-blocking
compounds include, but are not limited to polypeptides, proteins,
antibodies and the like. Preferably the ligand-blocking compounds
specifically block the activity of a LIV-1 activating ligand. More
preferably, the ligand-blocking compounds of the invention inhibit
growth of a LIV-1-expressing cell.
In another aspect, the invention involves a method for identifying
a compound capable of inhibiting the expression and/or activity
of a LIV-1 polypeptide, comprising contacting a candidate compound
with a LIV-1 polypeptide under conditions and for a time sufficient
to allow these two components to interact. In a specific aspect,
either the candidate compound or the LIV-1 polypeptide is immobilized
on a solid support. In another aspect, the non-immobilized component
carries a detectable label.
In yet another aspect, the invention concerns an article of manufacture,
comprising a container; a composition within the container comprising
an anti-LIV-1 antibody that binds the LIV-1 protein (preferably
binding to the extracellular domain or a fragment thereof) or binds
an activating ligand of the LIV-1 protein; and optionally a label
on or associated with the container that indicates that the composition
can be used for treating a condition characterized by overexpression
of LIV-1 without overexpression of ErbB2. According to another embodiment
of the invention, the article of manufacture further includes a
package insert comprising instructions to administer the anti-LIV-1
antibody subcutaneously for at least one of the doses, preferably
for all of the subsequent doses following the initial dose, most
preferably for all doses.
Where the methods and compositions of the invention comprise an
anti-LIV-1 antibody, which specifically and preferably binds the
extracellular domain of a LIV-1 gene product, or a fragment of the
extracellular domain. The compositions of the invention preferably
include a humanized LIV-1 antibody. Thus, the invention further
pertains to a composition comprising an antibody that specifically
and preferably binds the extracellular domain of LIV-1 gene product,
and pertains to the use of the antibody for treating LIV-1+/ErbB2-expressing
cancer in a human, e.g., LIV-1 overexpressing cancer that does not
coexpress high levels of ErbB2. Preferably the antibody is a monoclonal
antibody, e.g., humanized anti-LIV-1 monoclonal antibody that binds
to the extracellular domain (or a portion of the extracellular domain)
of LIV-1 (hereinafter anti-LIV-1) The antibody may be an intact
antibody (e.g., an intact IgG.sub.1 antibody) or an antibody fragment
(e.g., a Fab, F(Ab).sub.2, diabody, and the like). The variable
light chain and variable heavy chain regions of humanized anti-LIV-1
antibody.
These and other advantages and features of the present invention
will become apparent to those persons skilled in the art upon reading
the details of the invention as more fully set forth below and in
the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1C are the nucleic acid sequence (SEQ ID NO:1 (coding
sequence). FIGS. 1A-1 to 1A-2) and the amino acid sequence (SEQ
ID NO:2, FIG. 1B) of the LIV-1 protein. The dashed overlined portion
is predicted to be a signal sequence (approximately amino acids
1 to 20). The predicted extracellular domain of LIV-1 protein is
that portion of the LIV-1 amino acid sequence underscored by a dashed
line (approximately amino acids 24 to 312). The predicted transmembrane
domain extends from approximately amino acid 318 to approximately
amino acid 367. The approximate positions of the domains were predicted
using a standard hydropathy analysis program. A nucleic acid sequence
(SEQ ID NO:5) encoding a portion of LIV-1 and useful in microarray
analysis is shown in FIG. 1C.
FIGS. 2A-2B are the nucleic acid sequence and amino acid sequence,
respectively, corresponding to DNA 164647. FIGS. 2A1-2A5 is the
nucleotide sequence of DNA 164647 that is a cDNA encoding a native
sequence LIV-1 protein. SEQ ID NO:3 is the coding strand of DNA
164647. FIG. 2B is the derived amino acid sequence (SEQ ID NO:4)
of a native LIV-1 polypeptide encoded by DNA 164647.
FIGS. 3A-1-3A-9 is an alignment of SEQ ID NO:1 and SEQ ID NO:3
nucleic acid sequences.
FIGS. 4-1-4-3 is an alignment of SEQ ID NO:2 and SEQ ID NO:4 amino
acid sequences. The sequences differ in the ECD (near amino acid
130 of SEQ ID NO:4) and C-terminal region beyond about amino acid
740 of SEQ ID NO:4. A single amino acid difference was found at
amino acid 651 of SEQ ID NO:4.
FIG. 5 is a flow chart illustrating the FLIP cloning method up
to the restriction digestion selection step, as described herein.
The shaded boxes flanking the vector sequence represent the target
gene sequences.
FIG. 6 is a graphical representation of a fluorescent activated
cell sorting (FACS) analysis in which an anti-LIV-1-164647 monoclonal
antibody was shown to bind primarily to the surface of 3T3 cells
transfected with DNA 164647. The term "pRK5" refers cells
transfected with vector lacking a LIV-1-164647 insert. The term
"pRK5-LIV-1-164647" refers to cells transfected with vector
expressing full length LIV-1-164647.
FIG. 7 is a bar graph demonstrating that the LIV-1-164647 extracellular
domain is expressed on the surface of cells transfected with DNA
encoding the full-length LIV-1-164647 protein.
DESCRIPTION OF THE EMBODIMENTS
1. Definitions
As used herein, the term "LIV-1" refers to a gene or
its encoded protein, which gene transcript is detected in above
normal levels in some cancer cells. More specifically, a LIV-1 gene
or protein of the present invention is one which is encoded by DNA
164647 (SEQ ID NO:3) and has the deduced amino acid sequence of
SEQ ID NO:4. As used herein, the term LIV-1 refers to LIV-1-164647
where the disclosure refers to a nucleic acid comprising at least
21 nucleotides of SEQ ID NO:3, or where the disclosure refers to
an amino acid sequence comprising at least 7 amino acids of SEQ
ID NO:4 as disclosed herein. According to the invention. LIV-1-164647
is expressed in higher than normal amounts in a cell, while the
gene encoding ErbB2 receptor is not expressed in higher than normal
amounts. Such higher than normal expression is termed "overexpression"
of a gene or protein. The term "LIV-1" or "LIV-1-164647"
may be used to refer to a LIV-1 gene or its encoded protein. Generally,
where a protein or peptide is contemplated, the term "LIV-1
protein" will be used.
The term "LIV-1 gene product" or "LIV-1 protein"
refers to the expressed protein product of the gene, preferably
a polypeptide or protein form of the gene product. According to
the invention, a polypeptide or protein form of the LIV-1 gene product
includes a soluble form of the gene product (i.e. the extracellular
domain (ECD) of the LIV-1 gone product), which soluble form is useful
as an antigen for raising anti-LIV-1 gene product antibodies that
bind the extracellular domain of full length LIV-1 gene product
and inhibit its activation. It is understood that LIV-1 gene product
may also refer to the messenger RNA (mRNA) gene product and, where
appropriate, the distinction between protein and mRNA is made. A
LIV-1 protein according to the invention is encoded by a nucleic
acid of the invention comprising a sequence at least 80% homologous
to the DNA 164647 (SEQ ID NO:3 or its complement; FIG. 2A), preferably
at least approximately 90% homologous, more preferably at least
approximately 95%, and most preferably at least approximately 97%
homologous to SEQ ID NO:3 or its complement), or a portion thereof.
A LIV-1 nucleic acid of the invention hybridizes under stringent
conditions to SEQ ID NO:3 or its complement) or a portion thereof.
A LIV-1 protein of the invention is at least 80% homologous to the
amino acid sequence encoded by DNA 164647 (LIV-1 polypeptide SEQ
ID NO:4; FIG. 2B), preferably at least approximately 90% homologous,
more preferably at least approximately 95%, and most preferably
at least approximately 97% homologous to SEQ ID NO:4, or a fragment
thereof.
The terms "anti-LIV-1 antibody," "LIV-1 antibody,"
and grammatically analogous terms refer to an antibody which binds
specifically to at least a portion of the extracellular domain of
the LIV-1-164647 protein having a predicted amino acid sequence
of SEQ ID NO:4 (the predicted full length amino acid sequence of
LIV-1-164647 gene). Preferably, the antibody binds to the extracellular
domain of LIV-1 gene product, more preferably binding to the same
epitope as epitopes A, B, or C to which the monoclonal antibodies
disclosed herein bind. Even more preferably, the anti-LIV-1-164647
antibody binds to a polypeptide having at least 65% homology to
a sequence from amino acid 114 to and including amino acid 135 of
SEQ ID NO:4. Preferably, an anti-LIV-1 antibody of the invention
is human or humanized when the antibody is to be used to treat a
human patient.
The antibody of the invention is preferably one which binds specifically
to human LIV-1-164647, meaning that it does not significantly cross-react
with other proteins. In such embodiments, the extent of binding
of the antibody to proteins other than LIV-1 gene product will be
less than about 10% as determined by fluorescence activated cell
sorting (FACS) analysis or radioimmunoprecipitation (RIA).
The term "antibody" is used in the broadest sense and
specifically covers, for example, single anti-LIV-1 monoclonal antibodies
(including agonist, antagonist, and neutralizing antibodies), anti-LIV-1
antibody compositions with polyepitopic specificity, single chain
anti-LIV-1 antibodies. The term "monoclonal antibody"
as used herein refers to an antibody obtained from a population
of substantially homogeneous antibodies, i.e., the individual antibodies
comprising the population are identical except for possible naturally-occurring
mutations that may be present in minor amounts.
"Antibodies" (Abs) and "immunoglobulins" (Igs)
are glycoproteins having the same structural characteristics. While
antibodies exhibit binding specificity to a specific antigen, immunoglobulins
include both antibodies and other antibody-like molecules which
lack antigen specificity. Polypeptides of the latter kiln are, for
example, produced at low levels by the lymph system and at increased
levels by myelomas. The term "antibody" is used in the
broadest sense and specifically covers, without limitation, intact
monoclonal antibodies, polyclonal antibodies, multispecific antibodies
(e.g. bispecific antibodies) formed from at least two intact antibodies,
and antibody fragments so long as they exhibit the desired biological
activity.
"Native antibodies" and "native immunoglobulins"
are usually heterotetrameric glycoproteins of about 150,000 daltons
composed of two identical light (L) chains and two identical heavy
(H) chains. Each light chain is linked to a heavy chain by one covalent
disulfide bond, while the number of disulfide linkages varies among
the heavy chains of different immunoglobulin isotypes. Each heavy
and light chain also has regularly spaced intrachain disulfide bridges.
Each heavy chain has at one end a variable domain (V.sub.H) followed
by a number of constant domains. Each light chain has a variable
domain at one end (V.sub.L) and a constant domain at its other end;
the constant domain of the light chain is aliened with the first
constant domain of the heavy chain, and the light-chain variable
domain is aligned with the variable domain of the heavy chain. Particular
amino acid residues are believed to form an interface between the
light- and heavy-chain variable domains.
The term "variable" refers to the fact that certain portions
of the variable domains differ extensively in sequence among antibodies
and are used in the binding and specificity of each particular antibody
for its particular antigen. However, the variability is not evenly
distributed throughout the variable domains of antibodies. It is
concentrated in three segments called complementarity-determining
regions (CDRs) or hypervariable regions both in the light-chain
and the heavy-chain variable domains. The more highly conserved
portions of variable domains are called the framework (FR). The
variable domains of native heavy and light chains each comprise
four FR regions, largely adopting a -sheet configuration, connected
by three CDRs, which form loops connecting, and in some cases forming
part of, the -sheet structure. The CDRs in each chain are held together
in close proximity by the FR regions and, with the CDRs from the
other chain, contribute to the formation of the antigen-binding
site of antibodies (see Kabat et al. NIH Publ. No.91-3242, Vol.
1 pages 647-669 (1991)). The constant domains are not involved directly
in binding an antibody to an antigen, but exhibit various effector
functions, such as participation of the antibody in antibody-dependent
cellular toxicity.
The term "hypervariable region" when used herein refers
to the amino acid residues of an antibody which are responsible
for antigen-binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" to
"CDR" (i.e., residues in the light chain variable domain
and residues in the heavy chain variable domain; Kabat et al., Sequences
of Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institute of Health, Bethesda, Md. [1991]) and/or those
residues from a "hypervariable loop" (i.e., residues in
the light chain variable domain and residues in the heavy chain
variable domain; Clothia and specificity, the monoclonal antibodies
are advantageous in that they are synthesized by the hybridoma culture,
uncontaminated by other immunoglobulins. The modifier "monoclonal"
indicates the character of the antibody as being obtained from a
substantially homogeneous population of antibodies, and is not to
be construed as requiring production of the antibody by any particular
method. For example, the monoclonal antibodies to be used in accordance
with the present invention may be made by the hybridoma method first
described by Kohler et al., Nature, 256:495 [1975], or may be made
by recombinant DNA methods (see, e.g. U.S. Pat. No. 4,816,567).
The monoclonal antibodies may also be isolated from phage antibody
libraries using the techniques described in Clackson et al., Nature,
352:624-628 [1991] and Marks et al., J. Mol. Biol. 222:581-597 (1991),
for example.
The monoclonal antibodies herein specifically include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in antibodies
derived from a particular species or belonging to a particular antibody
class or subclass, while the remainder of the chain(s) is identical
with or homologous to corresponding sequences in antibodies derived
from another species or belonging to another antibody class or subclass,
as well as fragments of such antibodies, so long as they exhibit
the desired biological activity (U.S. Pat. No. 4,816,567; Morrison
et al., Proc. Natl. Acad. Sci. USA. 81:6851-6855 [1984]). Chimeric
antibodies of interest herein include human constant region sequences
together with antigen bindings regions of rodent (e.g. murine) origin,
or "primatized" antibodies comprising variable domain
antigen-binding sequences derived from a non-human primate (e.g.
Old World Monkey, Ape, macaque, etc.), or antigen binding regions
derived from antibodies generated in other non-human species that
have been immunized with the antigen of interest.
"Humanized" forms of non-human (e.g., rodent) antibodies
are chimeric immunoglobulins, immunoglobulin chains or fragments
thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived
from non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by residues
from a hypervariable region of a non-human species (donor antibody)
such as mouse, rat or rabbit having the desired specificity, affinity,
and capacity. In some instances, framework region (FR) residues
of the human immunoglobulin are replaced by corresponding non-human
residues. Furthermore, humanized antibodies may comprise residues
which are found neither in the recipient antibody nor in the donor
antibody. These modifications are made to further refine and maximize
antibody performance. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable domains,
in which all or substantially all of the hypervariable loops correspond
to those of a non-human immunoglobulin and all or substantially
all of the FRs are those of a human immunoglobulin sequence. The
humanized antibody optionally also will comprise at least a portion
of an immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see, Jones et al., Nature,
311:522-525 (1986); Reichmann et al., Nature, 332:323-329 [1988];
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
"Single-chain Fv" or "sFv" antibody fragments
comprise the V.sub.H and V.sub.L domains of antibody, wherein these
domains are present in a single polypeptide chain. Preferably, the
Fv polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the desired
structure for antigen binding. For a review of sFv see Pluckthun
in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenhurg
and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).
The term "diabodies" refers to small antibody fragments
with two antigen-binding sites, which fragments comprise a heavy-chain
variable domain (V.sub.H) connected to a light-chain variable domain
(V.sub.L) in the same polypeptide chain (V.sub.H-V.sub.L). By using
a linker that is too short to allow pairing between the two domains
on the same chain, the domains are forced to pair with the complementary
domains of another chain and create two antigen-binding sites. Diabodies
are described more fully in, for example, EP 404,097; WO 93/11161;
and Hollinger et al., Proc. Natl. Acad. Sci. USA. 90:6444-6448 (1993).
An "isolated" antibody is one which has been identified
and separated and/or recovered from a component of its natural environment.
Contaminant components of its natural environment are materials
which would interfere with diagnostic or therapeutic uses for the
antibody, and may include enzymes, hormones, and other proteinaceous
or nonproteinaceous solutes. In preferred embodiments, the antibody
will be purified (1) to greater than 95% (by weight of antibody
as determined by the Lowry method, and most preferably more than
99% by weight, (2) to a degree sufficient to obtain at least 15
residues of N-terminal or internal amino acid sequence by use of
a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under
reducing or nonreducing conditions using Coomassie blue or, preferably,
silver stain. Isolated antibody includes the antibody in situ within
recombinant cells since at least one component of the antihody's
natural environment will not be present. Ordinarily, however, isolated
antibody will be prepared by at least one purification step.
The term "overexpression," as used herein refers to overexpression
of a gene and/or its encoded protein in a cell, such as a cancer
cell. A cancer cell that "overexpresses" a protein is
one that has significantly higher levels of that protein compared
to a noncancerous cell of the same tissue type. For example, according
to the present invention, the overexpression of a protein LIV-1
protein may be caused by gene amplification or by increased transcription
or translation.
Overexpression of a LIV-1 protein may be determined in a diagnostic
or prognostic assay by evaluating increased levels of a LIV-1 mRNA
in a cell or tissue (e.g. via a quantitative PCR method) or by detecting
a LIV-1 protein present on the surface of a cell (e.g. via an immunohistochemistry
assay). Alternatively, or additionally, one may measure levels of
LIV-1-encoding nucleic acid in the cell, e.g. via fluorescent in
situ hybridization (FISH; see WO98/45479 published October, 1998),
southern blotting, or polymerase chain reaction (PCR) techniques,
such as real time quantitative PCR (RT-PCR). One may also study
LIV-1 overexpression by measuring shed antigen (e.g., LIV-1 extracellular
domain) in a biological fluid such as serum by contacting the fluid
or other sample with an antibody that binds to a LIV-1 protein or
fragment thereof. Various in vitro and in vivo assays are available
to the skilled practitioner. For example, one may expose a fluid
or tissue comprising a LIV-1 protein or fragment thereof, or cells
within the body of the patient to an antibody which is optionally
labeled with a detectable label, e.g. a radioactive isotope, and
binding of the antibody to cells in a sample or the patient can
be evaluated for overexpression, e.g. by external scanning for radioactivity
or by analyzing a biopsy taken from a patient previously exposed
to the antibody.
A cell that "overexpresses" LIV-1 has significantly higher
than normal LIV-1 nucleic acid levels compared to a noncancerous
cell of the same tissue type. Typically, the cell is a cancer cell,
e.g. a breast, ovarian, prostate, stomach, endometrial, salivary
gland, lung, kidney, colon, thyroid, pancreatic or bladder cell.
The cell may also be a cell line such as SKBR3, BT474, Calu 3, MDA-MB-453,
MDA-MB-361 or SKOV3.
Conversely, a cancer that is "not characterized by overexpression
of a LIV-1 protein or a LIV-1 gene is one which, in a diagnostic
assay, does not express higher than normal levels of LIV-1 gene
or LIV-1 protein compared to a noncancerous cell of the same tissue
type.
The terms "cancer" and "cancerous" refer to
or describe the physiological condition in mammals that is typically
characterized by unregulated cell growth. Examples of cancer include
but are not limited to, carcinoma, lymphoma, blastoma, sarcoma,
and leukemia. More particular examples of such cancers include highest
cancer, prostate cancer, colon cancer, squamous cell cancer, small-cell
lunge cancer, non-small cell lunar cancer, gastrointestinal cancer,
pancreatic cancer, glioblastoma cervical cancer, ovarian cancer,
liver cancer, bladder cancer, hepatoma, colorectal cancer, endometrial
carcinoma, salivary gland carcinoma kidney cancer, liver cancer,
vulval cancer, thyroid cancer, hepatic carcinoma and various types
of head and neck cancer.
The phrases "gene amplification" and "gene duplication"
are used interchangeably and refer to a process by which multiple
copies of a gene or gene fragment are formed in a particular cell
or cell line. The duplicated region (a stretch of amplified DNA)
is often referred to as "amplicon." Usually, the amount
of the messenger RNA (mRNA) produced, i.e., the level of gene expression,
also increases in the proportion of the number of copies made of
the particular gene expressed.
"Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all pre-cancerous
and cancerous cells and tissues.
"Treatment" is an intervention performed with the intention
of preventing the development or altering the pathology of a disorder.
Accordingly, "treatment" refers to both therapeutic treatment
and prophylactic or preventative measures. Those in need of treatment
include those already with the disorder as well as those in which
the disorder is to be prevented. In tumor (e.g., cancer) treatment,
a therapeutic agent may directly decrease the pathology of tumor
cells, or render the tumor cells more susceptible to treatment by
other therapeutic agents, e.g., radiation and/or chemotherapy.
The "pathology" of cancer includes all phenomena that
compromise the well-being of the patient. This includes, without
limitation, abnormal or uncontrollable cell growth, metastasis,
interference with the normal functioning of neighboring cells, release
of cytokines or other secretory products at abnormal levels, suppression
or aggravation of inflammatory or immunological response, etc.
"Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic and farm animals,
and zoo, sports, or pet animals, such as dogs, horses, cats, cattle,
pigs, sheep, etc. Preferably, the mammal is human.
"Carriers" as used herein include pharmaceutically acceptable
carriers, excipients, or stabilizers which are nontoxic to the cell
or mammal being exposed thereto at the dosages and concentrations
employed. Often the physiologically acceptable carrier is an aqueous
pH buffered solution. Examples of physiologically acceptable carriers
include buffers such as phosphate, citrate, and other organic acids,
antioxidants including ascorbic acid; low molecular weight (less
than about 10 residues) polypeptide; proteins, such as serum albumin,
gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming,
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.TM., polyethylene glycol (PEG), and PLURONICS.TM..
Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and consecutive
administration in any order.
The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include radioactive
isotopes (e.g., I.sup.131, I.sup.125, Y.sup.90 and Re.sup.186),
chemotherapeutic agents, and toxins such as enzymatically active
toxins of bacterial, fungal, plant or animal origin, or fragments
thereof.
A "chemotherapeutic agent" is a chemical compound useful
in the treatment of cancer. Examples of chemotherapeutic agents
include adriamycin, doxorubicin, epirubicin, 5-fluorouracil, cytosine
arabinoside ("Ara-C"), cyclophosphamide, thiotepa, busulfan,
cytoxin, taxoids, e.g., paclitaxel (Taxol, Bristol-Myers Squibb
Oncology, Princeton, N.J.), and doxetaxel (Taxotere, Rhone-Poulenc
Rorer, Antony, Rnace), toxotere, methotrexate, cisplatin, melphalan,
vinblastine, bleomycin, etoposide, ifosfamide, mitomycin C, mitoxantrone,
vincristine, vinorelbine, carboplatin, teniposide, daunomycin, carminomycin,
aminopterin, dactinomycin, mitomycins, esperamicins (see U.S. Pat.
No. 4,675,187), 5-FU, 6-thioguanine, 6-mercaptopurine, actinomycin
D, VP-16, chlorambucil, melphalan, and other related nitrogen mustards.
Also included in this definition are hormonal agents that act to
regulate or inhibit hormone action on tumors such as tamoxifen and
onapristone.
A "growth inhibitory agent" when used herein refers to
a compound or composition which inhibits growth of a cell, especially
cancer cell overexpressing any of the genes identified herein, either
in vitro or in vivo. Thus, the growth inhibitory agent is one which
significantly reduces the percentage of cells overexpressing such
genes in S phase. Examples of growth inhibitory agents include agents
that block cell cycle progression (at a place other than S phase),
such as agents that induce G1 arrest and M-phase arrest. Classical
M-phase blockers include the vincas (vincristine and vinblastine),
taxol, and topo II inhibitors such as doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin. Those agents that arrest GI also spill
over into S-phase arrest, for example. DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,
methotrexate, 5-fluorouracil, and ara-C. Further information can
be found in The Molecular Basis of Cancer, Mendelsohn and Israel,
eds., Chapter 1, entitled "Cell cycle regulation, oncogens,
and antineoplastic drugs" by Murakami et al., (WB Saunders;
Philadelphia, 1995), especially p. 13.
"Doxorubicin" is an athracycline antibiotic. The full
chemical name of doxorubicin is (85-cis)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexopyranosyl)oxy]-7,-
8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-napht-
hacenedione.
The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid stimulating
hormone (TSH), and luteinizing hormone (LH); hepatic growth factor;
fibroblast growth factor; prolactin; placental lactogen; tumor necrosis
factor-.alpha. and -.beta.; mullerian-inhibiting substance; mouse
gonadotropin-associatcd peptide; inhibin; activin; vascular endothelial
growth factor; integrin; thrombopoietin (TPO); nerve growth factors
such as NGF-.beta.; platelet-growth factor; transforming growth
factors (TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like
growth factor-I and -II; erythropoietin (EPO); osteoinductive factors;
interferons such as interferon-.alpha., -.beta., and -.gamma.; colony
stimulating factors (CSFs) such as macrophage-CSF (M-CSF); granulocyte-macrophage-CSF
(GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as
IL-1, IL-1.alpha., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL9,
IL11, IL12; a tumor necrosis factor such as TNF-.alpha. or TNF-.beta.;
and other polypeptide factors including LIF and kit ligand (KL).
As used herein, the term cytokine includes proteins from natural
sources or from recombinant cell culture and biologically active
equivalents of the native sequence cytokines.
The term "prodrug" as used in this application refers
to a precursor or derivative form of a pharmaceutically active substance
that is less cytotoxic to tumor cells compared to the parent drug
and is capable of being enzymatically activated or converted into
the more active parent form. See, e.g., Wilman, "Prodrugs in
Cancer Chemotherapy" Biochemical Society Transactions, 14,
pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs:
A Chemical Approach to Targeted Drug Delivery." Directed Drug
Delivery, Borchardt et al., (ed.), pp. 247-267. Humana Press (1985).
The prodrugs of this invention include, but are not limited to,
phosphate-containing prodrugs, thiophosphate-containing prodrugs,
sulfate-containing prodrugs, peptide-containing prodrugs. D-amino
acid-modified prodrugs, glycosylated prodrugs, .beta.-lactam-containing
prodrugs, optionally substituted phenoxyacetamide-containing prodrugs
or optionally substituted phenylacetamide-containing prodrugs. 5-fluorocytosing
and other 5-fluorouridine prodrugs which can be converted into the
more active cytotoxic free drug. Examples of cytotoxic drugs that
can be derivatized into a prodrug form for use in this invention
include, but are not limited to, those chemotherapeutic agents described
above.
An "effective amount" of a polypeptide disclosed herein
or an antagonist thereof, in reference to inhibition of neoplastic
cell growth, tumor growth or cancer cell growth, is an amount capable
of inhibiting to some extent, the growth of target cells. The term
includes an amount capable of invoking a growth inhibitory, cytostatic
and/or cytotoxic effect and/or apoptosis of the target cells. An
"effective amount" of a LIV-1 polypeptide antagonist for
purposes of inhibiting neoplastic cell growth, tumor growth or cancer
cell (growth, may be determined empirically and in a routine manner.
A "therapeutically effective amount", in reference to
the treatment of tumor, refers to an amount capable of invoking
one or more of the following effects: (1) inhibition, to some extent,
of tumor growth, including, slowing down and complete growth arrest;
(2) reduction in the number of tumor cells; (3) reduction in tumor
size; (4) inhibition (i.e., reduction, slowing down or complete
stopping) of tumor cell infiltration into peripheral organs; (5)
inhibition (i.e., reduction, slowing down or complete stopping)
of metastasis; (6) enhancement of anti-tumor immune response, which
may, but does not have to, result in the regression or rejection
of the tumor; and/or (7) relief, to some extent, of one or more
symptoms associated with the disorder. A "therapeutically effective
amount" of a LIV-1 polypeptide antagonist for purposes of treatment
of tumor may be determined empirically and in a routine manner.
A "growth inhibitory amount" of a LIV-1 antagonist is
an amount capable of inhibiting the growth of a cell, especially
tumor, e.g., cancer cell, either in vitro or in vivo. A "growth
inhibitory amount" of a LIV-1 antagonist for purposes of inhibiting
neoplastic cell growth may be determined empirically and in a routine
manner.
A "cytotoxic amount" of a LIV-1 antagonist is an amount
capable of causing the destruction of a cell, especially tumor,
e.g., cancer cell, either in vitro or in vivo. A "cytotoxic
amount" of a LIV-1 antagonist for purposes of inhibiting neoplastic
cell growth may be determined empirically and in a routine manner.
"Percent (%) amino acid sequence homology or identity"
with respect to the LIV-1 polypeptide sequences identified herein
is defined as the percentage of amino acid residues in a candidate
sequence that are identical with the amino acid residues in a LIV-1
sequence, after aligning the sequences and introducing gaps, if
necessary, to achieve the maximum percent sequence identity, and
not considering any conservative substitutions as part of the sequence
identity. Alignment for purposes of determining percent amino acid
sequence identity can be achieved in various ways that are within
the skill in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate parameters
for measuring alignment, including any algorithms needed to achieve
maximal alignment over the full-length of the sequences being compared.
For purposes herein, however, % amino acid sequence identity values
are obtained as described below by using the sequence comparison
computer program ALIGN-2, wherein the complete source code for the
ALIGN-2 program was authored by Genentech, Inc., and the source
code shown in FIGS. 20A-Q has been filed with user documentation
in the U.S. Copyright Office. Washington D.C., 20559, where it is
registered under U.S. Copyright Registration No. TXU510087, and
is provided in Table 1. The ALIGN-2 program is publicly available
through Genentech, Inc. South San Francisco, Calif. The ALIGN-2
program should be compiled for use on a UNIX operating system, preferably
digital UNIX V4.0D. All sequence comparison parameters are set by
the ALIGN-2 program and do not vary.
For purposes herein, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid sequence
B (which can alternatively be phrased as a given amino acid sequence
A that has or comprises a certain % amino acid sequence identity
to, with or against a given amino acid sequence B) is calculated
as follows: 100 times the fraction X/Y where X is the number of
amino acid residues scored as identical matches by the sequence
alignment program ALIGN-2 in that program's alignment of A and B,
and where Y is the total number of amino acid residues in B. It
will be appreciated that where the length of amino acid sequence
A is not equal to the length of amino acid sequence B, the % amino
acid sequence identity of A to B will not equal the % amino acid
sequence identity of B to A.
Unless specifically stated otherwise, all % amino acid sequence
homology or identity values used herein are obtained as described
above using the ALIGN-2 sequence comparison computer program. However,
% amino acid sequence identity may also be determined using the
sequence comparison program NCBI-BLAST2 (Altschul et al., Nucleic
Acids Res., 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison
program may be downloaded from (www.)ncbi.nlm.nih.gov. NCBI-BLAST2
uses several search parameters, wherein all of those search parameters
are set to default values including, for example, unmask=yes, strand=all,
expected occurrences=10, minimum low complexity length=15/5, multi-pass
e-value=0.01, constant for multi-pass=25, dropoff for final gapped
alignment=25 and scoring matrix=BLOSUM62.
In situations where NCBI-BLAST2 is employed for amino acid sequence
comparisons, the % amino acid sequence identity of a given amino
acid sequence A to, with, or against a given amino acid sequence
B (which can alternatively be phrased as a given amino acid sequence
A that has or comprises a certain % amino acid sequence identity
to, with, or against a given amino acid sequence B) is calculated
as follows: 100 times the fraction X/Y where X is the number of
amino acid residues scored as identical matches by the sequence
alignment program NCBI-BLAST2 in that program's alignment of A and
B, and where Y is the total number of amino acid residues in B.
It will be appreciated that where the length of amino acid sequence
A is not equal to the length of amino acid sequence B, the % amino
acid sequence identity of A to B will not equal the % amino acid
sequence identity of B to A.
In addition, % amino acid sequence identity may also be determined
using the WU-BLAST-2 computer program (Altschul et al., Methods
in Enzymology, 266:460-480 (1996)). Most of the WU-BLAST-2 search
parameters are set to the default values. Those not set to default
values, i.e., the adjustable parameters, are set with the following
values: overlap span=1, overlap fraction=0.125, word threshold (T)=11,
and scoring matrix=BLOSUM62. For purposes herein, a % amino acid
sequence identity value is determined by dividing (a) the number
of matching identical amino acids residues between the amino acid
sequence of the LIV-1 polypeptide of interest having a sequence
derived from the native LIV-1 polypeptide encoded by DNA 164647
and the comparison amino acid sequence of interest (i.e., the sequence
against which the LIV-1 polypeptide of interest is being compared
which may be a LIV-1 variant polypeptide) as determined by WU BLAST-2
by (b) the total number of amino acid residues of the LIV-1 polypeptide
of interest. For example, in the statement "a polypeptide comprising
an amino acid sequence A which has or having at least 80% amino
acid sequence identity to the amino acid sequence B", the amino
acid sequence A is the comparison amino acid sequence of interest
and the amino acid sequence B is the amino acid sequence of the
LIV-1 polypeptide of interest.
"Percent (%) nucleic acid sequence homology or identity"
with respect to the LIV-1 polypeptide-encoding nucleic acid sequences
identified herein is defined as the percentage of nucleotides in
a candidate sequence that are identical with the nucleotides in
a LIV-1 polypeptide-encoding nucleic acid sequence, after aligning
the sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity. Alignment for purposes of determining
percent nucleic acid sequence identity can be achieved in various
ways that are within the skill in the art, for instance, using publicly
available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2
or Megalign (DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any algorithms
needed to achieve maximal alignment over the full-length of the
sequences being compared. For purposes herein, however, % nucleic
acid sequence identity values are obtained as described below by
using the sequence comparison computer program ALIGN-2, wherein
the complete source code for the ALIGN-2 program is has been filed
with user documentation in the U.S. Copyright Office, Washington
D.C., 20559, where it is registered under U.S. Copyright Registration
No. TXU510087 and is provided herein in Table 1 as source code.
The ALIGN-2 program is publicly available through Genentech, Inc.,
South San Francisco, Calif. The ALIGN-2 program should be compiled
for use on a UNIX operating system, preferably digital UNIX V4.0D.
All sequence comparison parameters are set by the ALIGN-2 program
and do not vary
For purposes herein, the % nucleic acid sequence identity of a
given nucleic acid sequence C to, with, or against a given nucleic
acid sequence D (which can alternatively be phrased as a given nucleic
acid sequence C that has or comprises a certain % nucleic acid sequence
identity to with, or against a given nucleic acid sequence D) is
calculated as follows: 100 times the fraction W/Z where W is the
number of nucleotides scored as identical matches by the sequence
alignment program ALIGN-2 in that program's alignment of C and D,
and where Z is the total number of nucleotides in D. It will be
appreciated that where the length of nucleic acid sequence C is
not equal to the length of nucleic acid sequence D, the % nucleic
acid sequence identity of C to D will not equal the % nucleic acid
sequence identity of D to C.
Unless specifically stated otherwise, all % nucleic acid sequence
identity values used herein are obtained as described above using
the ALIGN-2 sequence comparison computer program. However, % nucleic
acid sequence identity may also be determined using the sequence
comparison program NCBI-BLAST2 (Altschul et al., Nucleic Acids Res.,
25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparison program
may be downloaded from (www.)ncbi.nlm.nih.gov. NCBI-BLAST2 uses
several search parameters, wherein all of those search parameters
are set to default values including, for example, unmask=yes, strand=all,
expected occurrences=10, minimum low complexity length=15/5, multi-pass
e-value=0.01, constant for multi-pass=25, dropoff for final gapped
alignment=25 and scoring matrix=BLOSUM62.
In situations where NCBI-BLAST2 is employed for sequence comparisons,
the % nucleic acid sequence identity of a given nucleic acid sequence
C to, with, or against a given nucleic acid sequence D (which can
alternatively be phrased as a given nucleic acid sequence C that
has or comprises a certain % nucleic acid sequence identity to,
with, or against a given nucleic acid sequence D) is calculated
as follows: 100 times the fraction W/Z where W is the number of
nucleotides scored as identical matches by the sequence alignment
program NCBI-BLAST2 in that program's alignment of C and D, and
where Z is the total number of nucleotides in D. It will be appreciated
that where the length of nucleic acid sequence C is not equal to
the length of nucleic acid sequence D, the % nucleic acid sequence
identity of C to D will not equal the % nucleic acid sequence identity
of D to C.
In addition, % nucleic acid sequence identity values may also be
generated using the WU-BLAST-2 computer program (Altschul et al.,
Methods in Enzymology, 266:460-480 (1996)). Most of the WU-BLAST-2
search parameters are set to the default values. Those not set to
default values, i.e., the adjustable parameters, are set with the
following values: overlap span=1, overlap fraction=0.125, word threshold
(T)=11, and scoring matrix=BLOSUM62. For purposes herein, a % nucleic
acid sequence identity value is determined by dividing (a) the number
of matching identical nucleotides between the nucleic acid sequence
of the LIV-1 polypeptide-encoding nucleic acid molecule of interest
having a sequence derived from the native sequence LIV-1 polypeptide-encoding
nucleic acid and the comparison nucleic acid molecule of interest
(i.e., the sequence against which the LIV-1 polypeptide-encoding
nucleic acid molecule of interest is being compared which may be
a variant LIV-1 polynucleotide) as determined by WU-BLAST-2 by (b)
the total number of nucleotides of the LIV-1 polypeptide-encoding
nucleic acid molecule of interest. For example, in the statement
"an isolated nucleic acid molecule comprising a nucleic acid
sequence A which has or having at least 80% nucleic acid sequence
identity to the nucleic acid sequence B", the nucleic acid
sequence A is the comparison nucleic acid molecule of interest and
the nucleic acid sequence B is the nucleic acid sequence of the
LIV-1 polypeptide-encoding nucleic acid molecule of interest.
The term "positives", in the context of the amino acid
sequence identity comparisons performed as described above, includes
amino acid residues in the sequences compared that are not only
identical, but also those that have similar properties. Amino acid
residues that score a positive value to an amino acid residue of
interest are those that are either identical to the amino acid residue
of interest or are a preferred substitution (as defined in Table
2 below) of the amino acid residue of interest.
TABLE-US-00001 TABLE 2 Original Exemplary Preferred Residue Substitutions
Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys
Asn (N) gln: his; lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln
(Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His (H) asn; gln;
lys; arg arg Ile (I) leu; val; met; ala; phe; leu norleucine Leu
(L) norleucine: ile: val; ile met; ala; phe Lys (K) arg; gln; asn
arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu
Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe
tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; leu
ala; norleucine
Substantial modifications in function or immunological identity
of the polypeptide are accomplished by selecting substitutions that
differ significantly in their effect on maintaining (a) the structure
of the polypeptide backbone in the area of the substitution, for
example, as a sheet or helical conformation, (b) the charge or hydrophobicity
of the molecule at the target site, or (c) the bulk of the side
chain. Naturally occurring residues are divided into groups based
on common side-chain properties: (1) hydrophobic: norleucine, met,
ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3)
acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues
that influence chain orientation: gly, pro; and (6) aromatic: trp,
tyr, phe.
Non-conservative substitutions will entail exchanging a member
of one of these classes for another class. Such substituted residues
also may be introduced into the conservative substitution sites
or, more preferably, into the remaining (non-conserved) sites.
The variations can be made using methods known in the art such
as oligonucleotide-mediated (site-directed) mutagenesis, alanine
scanning, and PCR mutagenesis. Site-directed mutagenesis [Carter
et al., Nucl. Acids Res., 13:4331 (1986), Zoller et al., Nucl. Acids
Res. 10:6487 (1987)], cassette mutagenesis [Wells et al., Gene.
34:315 (1985)], restriction selection mutagenesis [Wells et al.,
Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known
techniques can be performed.
For purposes herein, the % value of positives of a given amino
acid sequence A to, with, or against a given amino acid sequence
B (which can alternatively be phrased as a given amino acid sequence
A that has or comprises a certain % positives to, with, or against
a given amino acid sequence B) is calculated as follows: 100 times
the fraction X/Y where X is the number of amino acid residues scoring
a positive value as defined above by the sequence alignment program
ALIGN-2 in that program's alignment of A and B, and where Y is the
total number of amino acid residues in B. It will be appreciated
that where the length of amino acid sequence A is not equal to the
length of amino acid sequence B, the % positives of A to B will
not equal the % positives of B to A.
"Isolated." when used to describe the various polypeptides
disclosed herein, means polypeptide that has been identified and
separated and/or recovered from a component of its natural environment.
Preferably, the isolated polypeptide is free of association with
all components with which it is naturally associated. Contaminant
components of its natural environment are materials that would typically
interfere with diagnostic or therapeutic uses for the polypeptide,
and may include enzymes, hormones, and other proteinaceous or non-proteinaceous
solutes. In preferred embodiments, the polypeptide will be purified
(1) to a degree sufficient to obtain at least 15 residues of N-terminal
or internal amino acid sequence by use of a spinning cup sequenator,
or (2) to homogeneity by SDS-PAGE under non-reducing or reducing
conditions using Coomassie blue or, preferably, silver stain. Isolated
polypeptide includes polypeptide in situ within recombinant cells,
since at least one component of the LIV-1 natural environment will
not be present. Ordinarily, however, isolated polypeptide will be
prepared by at least one purification step.
An "isolated" nucleic acid molecule encoding a LIV-1
polypeptide or an "isolated" nucleic acid encoding an
anti-LIV-1 antibody, is a nucleic acid molecule that is identified
and separated from at least one contaminant nucleic acid molecule
with which it is ordinarily associated in the natural source of
the LIV-1-encoding nucleic acid or the anti-LIV-1-encoding nucleic
acid. Preferably, the isolated nucleic acid is free of association
with all components with which it is naturally associated. An isolated
LIV-1-encoding nucleic acid molecule or an anti-LIV-1-encoding nucleic
acid molecule is other than in the form or setting in which it is
found in nature.
The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are suitable
for prokaryotes, for example, include a promoter, optionally an
operator sequence, and a ribosome binding site. Eukaryotic cells
are known to utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into
a functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably linked
to DNA for a polypeptide if it is expressed as a preprotein that
participates in the secretion of the polypeptide; a promoter or
enhancer is operably linked to a coding sequence if it affects the
transcription of the sequence; or a ribosome binding site is operably
linked to a coding sequence if it is positioned so as to facilitate
translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However, enhancers
do not have to be contiguous. Linking is accomplished by ligation
at convenient restriction sites. If such sites do not exist, the
synthetic oligonucleotide adaptors or linkers are used in accordance
with conventional practice.
"Stringency" of hybridization reactions is readily determinable
by one of ordinary skill in the art, and generally is an empirical
calculation dependent upon probe length, washing temperature, and
salt concentration. In general, longer probes require higher temperatures
for proper annealing, while shorter probes need lower temperatures.
Hybridization generally depends on the ability of denatured DNA
to reanneal when complementary strands are present in an environment
below their melting temperature. The higher the degree of desired
homology between the probe and hybridizable sequence, the higher
the relative temperature which can be used. As a result, it follows
that higher relative temperatures would tend to make the reaction
conditions more stringent, while lower temperatures less so. For
additional details and explanation of stringency of hybridization
reactions, see Ausubel et al., Current Protocols in Molecular Biology,
Wiley Interscience Publishers. (1995).
"Stringent conditions" or "high stringency conditions",
as defined herein, may be identified by those that: (1) employ low
ionic strength and high temperature for washing, for example 0.015
M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate
at 50.degree. C.; (2) employ during hybridization a denaturing agent,
such as formamide, for example, 50% (v/v) formamide with 0.1% bovine
serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium
citrate at 42.degree. C.; or (3) employ 50% formamide, 5.times.SSC
(0.75 M NaCl, 0.075 M sodium citrate). 50 mM sodium phosphate (pH
6.8), 0.1% sodium pyrophosphate, 5.times. Denhardt's solution, sonicated
salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at
42.degree. C., with washes at 42.degree. C. in 0.2.times.SSC (sodium
chloride/sodium citrate) and 50% formamide at 55.degree. C., followed
by a high-stringency wash consisting of 0.1.times.SSC containing
EDTA at 55.degree. C.
"Moderately stringent conditions" may be identified as
described by Sambrook et al., Molecular Cloning: A Laboratory Manual,
New York: Cold Spring Harbor Press, 1989, and include the use of
washing solution and hybridization conditions (e.g., temperature,
ionic strength and % SDS) less stringent than those described above.
An example of moderately stringent conditions is overnight incubation
at 37.degree. C. in a solution comprising 20% formamide, 5.times.SSC
(150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH
7.6), 5.times. Denhardt's solution, 10% dextran sulfate, and 20
mg/ml denatured sheared salmon sperm DNA, followed by washing the
filters in 1.times.SSC at about 35-50.degree. C. The skilled artisan
will recognize how to adjust the temperature, ionic strength, etc.
as necessary to accommodate factors such as probe length and the
like.
"Active" or "activity" for the purposes herein
refers to form(s) of LIV-1 polypeptides which retain a biological
and/or an immunological activity/property of a native or naturally-occurring
LIV-1 polypeptide, wherein "biological" activity refers
to a function (either inhibitory or stimulatory) caused by a native
or naturally-occurring LIV-1 polypeptide other than the ability
to induce the production of an antibody against an antigenic epitope
possessed by a a native or naturally-occurring LIV-1 polypeptide
and an "immunological" activity refers to the ability
to induce the production of an antibody against an antigenic epitope
possessed by a native or naturally-occurring LIV-1 polypeptide.
"Biological activity" in the context of an antibody or
another antagonist molecule that can be identified by the screening
assays disclosed herein (e.g., an organic or inorganic small molecule,
peptide, etc.) is used to refer to the ability of such molecules
to bind or complex with the polypeptides encoded by the amplified
genes identified herein, or otherwise interfere with the interaction
of the encoded polypeptides with other cellular proteins or otherwise
interfere with the transcription or translation of a LIV-1 polypeptide.
A preferred biological activity is growth inhibition of a target
tumor cell. Another preferred biological activity is cytotoxic activity
resulting in the death of the target tumor cell.
The term "biological activity" in the context of a LIV-1
polypeptide means the ability of a LIV-1 polypeptide to induce neoplastic
cell growth or uncontrolled cell growth or to act as an indication
of a particular form of neoplasm that is particularly metastatic.
The phrase "immunological activity" means immunological
cross-reactivity with at least one epitope of a LIV-1 polypeptide.
"Immunological cross-reactivity" as used herein means
that the candidate polypeptide is capable of competitively inhibiting
the qualitative biological activity of a LIV-1 polypeptide having
this activity with polyclonal antisera raised against the known
active LIV-1 polypeptide. Such antisera are prepared in conventional
fashion by injecting goats or rabbits, for example, subcutaneously
with the known active analogue in complete Freund's adjuvant, followed
by booster intraperitoneal or subcutaneous injection in incomplete
Freunds. The immunological cross-reactivity preferably is "specific",
which means that the binding affinity of the immunologically cross-reactive
molecule (e.g. antibody) identified, to the corresponding LIV-1
polypeptide is significantly higher (preferably at least about 2-times,
more preferably at least about 4-times, even more preferably at
least about 8-times, most preferably at least about 10-times higher)
than the binding affinity of that molecule to any other known native
polypeptide.
The term "antagonist" is used in the broadest sense,
and includes any molecule that partially or fully blocks, inhibits,
or neutralizes a biological activity of a native LIV-1 polypeptide
disclosed herein or the transcription or translation thereof. Suitable
antagonist molecules specifically include antagonist antibodies
or antibody fragments, fragments, peptides, small organic molecules,
anti-sense nucleic acids, etc. Included are methods for identifying
antagonists of a LIV-1 polypeptide with a candidate antagonist molecule
and measuring a detectable chance in one or more biological activities
normally associated with the LIV-1 polypeptide.
A "small molecule" is defined herein to have a molecular
weight below about 500 Daltons.
The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the antibody so as to generate a "labeled" antibody.
The label may be detectable by itself (e.g., radioisotope labels
or fluorescent labels) or, in the case of an enzymatic label, may
catalyze chemical alteration of a substrate compound or composition
which is detectable. Radionuclides that can serve as detectable
labels include, for example, I-131, I-123, I-125, Y-90, Re-188,
Re-186, At-211, Cu-67, Bi-212, and Pd109.
By "solid phase" is meant a non-aqueous matrix to which
the antibody of the present invention can adhere. Examples of solid
phases encompassed herein include those formed partially or entirely
of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose),
polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In
certain embodiments, depending on the context, the solid phase can
comprise the well of an assay plate: in others it is a purification
column (e.g., an affinity chromatography column). This term also
includes a discontinuous solid phase of discrete particles, such
as those described in U.S. Pat. No. 4,275,149.
A "liposome" is a small vesicle composed of various types
of lipids, phospholipids and/or surfactant which is useful for delivery
of a drug (such as a LIV-1 polypeptide or antibody thereto and,
optionally, a chemotherapeutic agent) to a mammal. The components
of the liposome are commonly arranged in a bilayer formation, similar
to the lipid arrangement of biological membranes.
As used herein, the term "immunoadhesin" designates antibody-like
molecules which combine the binding specificity of a heterologous
protein (an "adhesin," for example a receptor, ligand,
or enzyme) with the effector functions of immunoglobulin constant
domains. Structurally, the immunoadhesins comprise a fusion of the
adhesin amino acid sequence with the desired binding specificity
which is other than the antigen recognition and binding site (antigen
combining site) of an antibody (i.e., is "heterologrous"),
and an immunoglobulin constant domain sequence. The adhesin part
of an immunoadhesin molecule typically is a continuous amino acid
sequence comprising at least the binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the immunoadhesin
may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3,
or IgG-4 subtypes. IgA (including IgA-1 and IgA-2), IgE, IgD or
IgM, and any subclass or isotype thereof.
The terms "HER2", "ErbB2" "c-Erb-B2"
are used interchangeably. Unless indicated otherwise, the terms
"ErbB2" "c-Erb-B2" and "HER2" when
used herein refer to the human protein, and "erbB2,",
"c-erb-B2," and "her2" refer to human gene.
The human erbB2 gene and ErbB2 protein are, for example, described
in Semba et al., PNAS (USA) 82:6497-6501 (1985) and Yamamoto et
al., Nature 319:230-234 (1986)(Genebank accession number X03363).
ErbB2 comprises four domains (Domains 1-4).
The "epitope 4D5" is the region in the extracellular
domain of ErbB2 to which the antibody 4D5 (ATCC CRL 10463) binds.
This epitope is close to the transmembrane region of ErbB2. To screen
for antibodies which bind to the 4D5 epitope, a routine cross-blocking
assay such as that described in Antibodies, A Laboratory Manual,
Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988),
can be performed. Alternatively, epitope mapping can be performed
to assess whether the antibody binds to the 4D5 epitope of ErbB2
(i.e. any one or more residues in the region from about residue
529, e.g. about residue 561 to about residue 625, inclusive).
The "epitope 3H4" is the region in the extracellular
domain of ErbB2 to which the antibody 3H4 binds. This epitope includes
residues from about 541 to about 599, inclusive, in the amino acid
sequence of ErbB2 extracellular domain.
The "epitope 7C2/7F3" is the region at the N terminus
of the extracellular domain of ErbB2 to which the 7C2 and/or 7F3
antibodies (ATCC HB-12215 and ATCC HB-12216, respectively) bind.
To screen for antibodies which bind to the 7C2/7F3 epitope, a routine
cross-blocking assay such as that described in Antibodies, A Laboratory
Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane
(1988), can be performed. Alternatively, epitope mapping can be
performed to establish whether the antibody binds to the 7C2/7F3
epitope on ErbB2.
The term "induces cell death" or "capable of inducing
cell death" refers to the ability of the anti-LIV-1 gene product
antibody, alone or in co-treatment with a chemotherapeutic agent,
to make a viable cell become nonviable. The "cell" here
is one which expresses the LIV-1 gene product, especially where
the cell overexpresses the LIV-1 gene product. A cell which "overexpresses"
LIV-1 has significantly higher than normal LIV-1 mRNA and/or LIV-1
protein levels compared to a noncancerous cell of the same tissue
type. A cell to be treated by the method of the invention does not
also overexpress ErbB2 (i.e. the cell expresses ErbB2 at a level
that is approximately the same or less than a normal, non-cancerous
cell of the same cell or tissue type). Preferably, the cell is a
cancer cell, e.g. a breast, lung, or prostate cell, In vitro, the
cell may be from a cell line transformed with LIV-1 DNA, preferably
DNA 164647, to express LIV-1 on the cell surface. Cell death in
vitro may be determined in the absence of complement and immune
effector cells to distinguish cell death induced by antibody dependent
cellular cytotoxicity (ADCC) or complement dependent cytotoxicity
(CDC). Thus, the assay for cell death may be performed using heat
inactivated serum (i.e. in the absence of complement) and in the
absence of immune effector cells. To determine whether the antibody
is able to induce cell death, loss of membrane integrity as evaluated
by uptake of propidium iodide (PI), trypan blue (see Moore et al.,
Cytotechnology 17:1-11 [1995]) or 7AAD can be assessed relative
to untreated cells. Preferred cell death-inducing antibodies are
those which induce PI uptake in the "PI uptake assay in LIV-1
expressing cells".
The phrase "induces apoptosis" or "capable of inducing
apoptosis" refers to the ability of the antibody to induce
programmed cell death as determined by binding of annexin V, fragmentation
of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell
fragmentation, and/or formation of membrane vesicles (called apoptotic
bodies). The cell is one which overexpresses the LIV-1 gene product.
Preferably the "cell" is a tumor cell, e.g. a breast,
lung, or prostate cell. In vitro, the cell may be from a cell line
transformed with LIV-1 DNA, such as DNA 164647. Various methods
are available for evaluating the cellular events associated with
apoptosis. For example, phosphatidyl serine (PS) translocation can
be measured by annexin binding; DNA fragmentation can be evaluated
through DNA laddering as disclosed in the example herein; and nuclear/chromatin
condensation along with DNA fragmentation can be evaluated by any
increase in hypodiploid cells. Preferably, the antibody which induces
apoptosis is one which results in about 2 to 50 fold, preferably
about 5 to 50 fold, and most preferably about 10 to 50 fold induction
of annexin binding relative to untreated cell in an "annexin
binding assay using cells" (see below).
As used herein, the term "salvage receptor binding epitope"
refers to an epitope of the Fc region of an IgG molecule (e.g.,
IgG.sub.1, IgG.sub.2, IgG.sub.3, or IgG.sub.4) that is responsible
for increasing the in vitro serum half-life of the IgG molecule.
"Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures. Those in need of treatment
include those already with the disorder as well as those in which
the disorder is to be prevented.
A "disorder" is any condition that would benefit from
treatment with the anti-LIV-1 gene product antibody. This includes
chronic and acute disorders or diseases including those pathological
conditions which predispose the mammal to the disorder in question.
Non-limiting examples of disorders to be treated herein include
benign and malignant tumors of breast, lung, and prostate tissue.
The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic products,
that contain information about the indications, usage, dosage, administration,
contraindications and/or warnings concerning the use of such therapeutic
products.
The term "serum concentration," "serum drug concentration,"
or serum anti-LIV-1 "antibody concentration" refers to
the concentration of a drug in the blood serum of an animal or human
patient being treated with the drugs. Serum concentration of antibody,
for example, is preferably determined by immunoassay. Preferably,
the immunoassay is ELISA according to the procedure disclosed herein.
The term "peak serum concentration" refers to the maximal
serum drug concentration shortly after delivery of the drug into
the animal or human patient, after the drug has been distributed
throughout the blood system, but before significant tissue distribution,
metabolism or excretion of drug by the body has occurred.
The term "trough serum concentration" refers to the serum
drug concentration at a time after delivery of a previous dose and
immediately prior to delivery of the next subsequent dose of drug
in a series of doses. Generally, the trough serum concentration
is a minimum sustained efficacious drug concentration in the series
of drug administrations. Also, the trough serum concentration is
frequently targeted as a minimum serum concentration for efficacy
because it represents the serum concentration at which another dose
of drug is to be administered as part of the treatment regimen.
If the delivery of drug is by intravenous administration, the trough
serum concentration is most preferably attained within 1 day of
a front loading initial drug delivery. It the delivery of drug is
by subcutaneous administration, the peak serum concentration is
preferably attained in 3 days or less. According to the invention,
the trough serum concentration is preferably attained in 4 weeks
or less, preferably 3 weeks or less, more preferably 2 weeks or
less, most preferably in 1 week or less, including 1 day or less
using any of the drug delivery methods disclosed herein.
The term "intravenous infusion" refers to introduction
of a drug into the vein of an animal or human patient over a period
of time greater than approximately 5 minutes, preferably between
approximately 30 to 90 minutes, although, according to the invention,
intravenous infusion is alternatively administered for 10 hours
or less.
The term "intravenous bolus" or "intravenous push"
refers to drug administration into a vein of an animal or human
such that the body receives the drug in approximately 15 minutes
or less, preferably 5 minutes or less.
The term "subcutaneous administration" refers to introduction
of a drug under the skin of an animal or human patient, preferable
within a pocket between the skin and underlying tissue, by relatively
slow, sustained delivery from a drug receptacle. The pocket may
be created by pinching or drawing the skin up and away from underlying
tissue.
The term "subcutaneous infusion" refers to introduction
of a drug under the skin of an animal or human patient, preferably
within a pocket between the skin and underlying tissue, by relatively
slow, sustained delivery from a drug receptacle for a period of
time including, but not limited to, 30 minutes or less, or 90 minutes
or less. Optionally, the infusion may be made by subcutaneous implantation
of a drug deliver, pump implanted under the skin of the animal or
human patient, wherein the pump delivers a predetermined amount
of drug for a predetermined period of time, such as 30 minutes,
90 minutes, or a time period spanning the length of the treatment
regimen.
The term "subcutaneous bolus" refers to drug administration
beneath the skin of an animal or human patient, where bolus drug
delivery is preferably less than approximately 15 minutes, more
preferably less than 5 minutes, and most preferably less than 60
seconds. Administration is preferably within a pocket between the
skin and underlying tissue, where the pocket is created, for example,
by pinching or drawing the skin up and away from underlying tissue.
The term "front loading," when referring to drug administration
is meant to describe an initially higher dose followed by the same
or lower doses at intervals. The initial higher dose or doses are
meant to more rapidly increase the animal or human patient's serum
drug concentration to an efficacious target serum concentration.
Published information related to LIV-1 gene expression and gene
product includes the following issued patents and published applications:
Manning, D. L. et al., U.S. Pat. No. 5,693,465, issued Dec. 2, 1997;
Manning, D. L. et al., European J. Cancer 29A(10):1462-1468 [1993];
Manning, D. L. et al., European J. Cancer, 30A(5):675-678 [1994];
Manning. D. L. et al., Acta Oncologica 34(5):641-646 [1995]; McClelland,
R. A. et al., Breast Cancer Res. & Treatment 41(1):31-41 [1996];
Knowlden, J. M. et al. Clin. Cancer Res. 3(11):2165-2172 [1997];
and McClelland, R. A. et al., British J. Cancer 77(10):1653-1656
[1998].
Published information related to anti-ErbB2 antibody includes the
following issued patents and published applications: PCT/US89/00051,
published Jan. 5, 1989, PCT/US90/02697, published May 18, 1990,
EU 0474727, issued Jul. 23, 1997, DE 69031120.6, issued Jul. 23,
1997, PCT/US97/18385, published Oct. 9, 1997, SA 97/9185, issued
Oct. 14, 1997, U.S. Pat. No. 5,677,171, issued Oct. 14, 1997, U.S.
Pat. No. 5,720,937, issued Feb. 24, 1998. U.S. Pat. No. 5,720,954,
issued Feb. 24, 1998, U.S. Pat. No. 5,725,856, issued Mar. 10, 1998,
U.S. Pat. No. 5,770,195, issued Jun. 23, 1998, U.S. Pat. No. 5,772,997,
issued Jun. 30, 1998, PCT/US98/2626, published Dec. 10, 1998, and
PCT/US99/06673, published Mar. 26, 1999, each of which patents and
publications is herein incorporated by reference in its entirety.
The following examples are offered for illustrative purposes only,
and are not intended to limit the scope of the present invention
in any way.
All patent and literature references cited in the present specification
are hereby incorporated by reference in their entirety.
EXAMPLES
Commercially available reagents referred to in the examples were
used according to manufacturer's instructions unless otherwise indicated.
The source of those cells identified in the following examples,
and throughout the specification, by ATCC accession numbers is the
American Type Culture Collection, Manassas, Va. Unless otherwise
noted, the present invention uses standard procedures of recombinant
DNA technology, such as those described herein and in the following
textbooks: Sambrook et al., Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Press N.Y. 1989; Ausubel et al., Current Protocols
in Molecular Biology, Green Publishing Associates and Wiley Interscience,
N.Y. 1989; Innis et al., PCR Protocols: A Guide to Methods and Applications,
Academic Press, inc., N.Y. 1990; Harlow et al., Antibodies: A Laboratory
Manual, Cold Spring Harbor Press, Cold Spring Harbor, 1988; Gait,
M. J. Oligonucleotide Synthesis, IRL Press, Oxford, 1984, R. I.
Freshney, Animal Cell Culture, 1987 Coligan et al., Current Protocols
in Immunology, 1991.
Example 1
LIV-1 Expression in Tumor Cells Examined by Microarray Analysis
A form of breast cancer in which the cells overexpress the LIV-1
gene product (e.g. LIV-1-164647 mRNA) but do not overexpress ErbB2
has been discovered and is uniquely disclosed herein. Detection
of the tumor type was made using microarray technology. Using nucleic
acid microarrays, test and control mRNA samples from test and control
tissue samples are reverse transcribed and labeled to generate cDNA
probes. The cDNA probes are then hybridized to an array of nucleic
acids immobilized on a solid support. The array is configured such
that the sequence and position of each member of the array is known.
Hybridization of a labeled probe with a particular array member
indicates that the sample from which the probe was derived expresses
that gene. If the hybridization signal of a probe from a test (disease
tissue) sample is greater than hybridization signal of a probe from
a control (normal tissue) sample, the gene or genes overexpressed
in the disease tissue are identified. The implication of this result
is that an overexpressed protein in a diseased tissue is useful
not only as a diagnostic marker for the presence of the disease
condition, but also as a therapeutic target for treatment of the
disease condition.
Tumor cells were crossly dissected from surrounding, non-cancerous
cells in breast tumor tissue. Hematoxylin and eosin staining of
the cells confirmed that the excised cells were from tumor. The
mRNA of the tumor cells (reflecting cell-specific expression of
a variety of genes) was converted to cDNA by RT/PCR methodology,
labeled with fluorescent tags, and allowed to hybridize to the ESTs
arrayed on a class slide. An imaging device detected and measured
the fluorescence of each sample on the slide, where fluorescence
represents a labeled messenger from the test cells identifiable
due to its hybridization with a known nucleic acid sequence (an
EST) at a known position on the slide. Relative fluorescence indicated
relative activity of a gene, with strong flourescence indicating
an active gene expressing a relative large amount of messenger.
Little or no flourescence indicated that no labeled messenger hybridized
to the ESTs. Detection of fluorescently labeled probes hybridized
to sequences on the microarray slide is described, for example,
in U.S. Pat. No. 5,143,854, herein incorporated by reference.
It was found that cDNA of the preparation hybridized to a publicly
available EST sequence (accession no. H29315 (from the Washington
University-Merck EST Project, authors Hillier. L. et al., SEQ ID
NO:5, purchased from Research Genetics (Alabama, USA) in a pattern
suggesting overexpression in breast tumor tissue that did not also
overexpress ErbB2. The cDNA was sequenced and disclosed herein as
LIV-1-DNA 164647. In situ hybridization of a radioactivel |