U.S. patent application number 11/063101 was filed with the patent office on 2005-08-04 for epha2 as a therapeutic target for cancer.
This patent application is currently assigned to Purdue Research Foundation. Invention is credited to Hein, Patrick W., Kinch, Michael S., Zantek, Nicole D..
Application Number | 20050169931 11/063101 |
Document ID | / |
Family ID | 34810895 |
Filed Date | 2005-08-04 |
United States Patent
Application |
20050169931 |
Kind Code |
A1 |
Kinch, Michael S. ; et
al. |
August 4, 2005 |
EphA2 as a therapeutic target for cancer
Abstract
The present invention is directed to compounds and methods for
the treatment of metastatic disease. The compounds of this
invention have specificity for EphA2, an epithelial cell tyrosine
kinase that is overexpressed in metastatic tumor-cells. The
compounds used in accordance with this invention may be provided in
a pharmaceutical composition for treatment of metastatic
disease.
Inventors: |
Kinch, Michael S.;
(Laytonsville, MD) ; Zantek, Nicole D.; (Silver
Spring, MD) ; Hein, Patrick W.; (Columbia,
IL) |
Correspondence
Address: |
MUETING, RAASCH & GEBHARDT, P.A.
P.O. BOX 581415
MINNEAPOLIS
MN
55458
US
|
Assignee: |
Purdue Research Foundation
West Lafayette
IN
|
Family ID: |
34810895 |
Appl. No.: |
11/063101 |
Filed: |
February 22, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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11063101 |
Feb 22, 2005 |
|
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|
09640935 |
Aug 17, 2000 |
|
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60149258 |
Aug 17, 1999 |
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Current U.S.
Class: |
424/155.1 ;
424/185.1; 514/19.1; 514/19.4; 514/19.5; 514/19.8; 514/7.5 |
Current CPC
Class: |
C07K 16/3069 20130101;
A61P 35/00 20180101; A61K 2039/505 20130101; C07K 16/2866 20130101;
A61K 38/19 20130101 |
Class at
Publication: |
424/155.1 ;
514/002; 424/185.1 |
International
Class: |
A61K 039/395; A61K
039/00; A61K 031/495 |
Claims
What is claimed is:
1. A method for treatment of a patient having a tumor comprising
cells that express EphA2, said method comprising administering to
the patient a therapeutically effective amount of an EphA2 agonist
that increases the phosphotyrosine content of EphA2 in said tumor
cells as compared to untreated tumor cells.
2. The method of claim 1 wherein the tumor comprises a primary
tumor.
3. The method of claim 1 wherein the tumor comprises a metastatic
tumor.
4. The method of claim 1 wherein the tumor has metastatic
potential.
5. The method of claim 1 wherein the tumor comprises a breast
cancer cell.
6. The method of claim 1 wherein the tumor comprises a prostate
cancer cell.
7. The method of claim 1 wherein the tumor comprises a lung cancer
cell.
8. The method of claim 1 wherein the tumor comprises a colon cancer
cell.
9. The method of claim 1 wherein the EphA2 agonist comprises an
antibody.
10. The method of claim 1 wherein the EphA2 agonist comprises a
natural EphA2 ligand.
11. The method of claim 1 wherein the EphA2 agonist comprises an
artificial EphA2 ligand.
12. The method of claim 1 wherein the EphA2 agonist comprises a
peptide.
13. The method of claim 1 wherein the EphA2 agonist comprises a
small molecule.
Description
[0001] This application claims priority under 35 U.S.C. .sctn.
119(e) to U.S. Provisional Application No. 60/149,258, filed Aug.
17, 1999, which is expressly incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to diagnosis and treatment of
metastatic disease. More particularly, this invention relates to
the use of an extracellular epitope of an epithelial cell tyrosine
kinase that is overexpressed in metastatic tumor cells as the
target for the diagnosis and treatment of metastatic disease. Most
particularly, this invention relates to the use of compounds that
interact with and alter expression of the epithelial cell tyrosine
kinase.
BACKGROUND AND SUMMARY OF THE INVENTION
[0003] Cancer is a disease of aberrant signal transduction. The
most dangerous forms of cancer are malignant cells that metastasize
to distant sites in a body. Metastatic cells have acquired the
ability to break away from the primary tumor, translocate to
distant sites, and colonize distant and foreign microenvironments.
Cancer cell metastasis requires cellular capacity to 1) detach from
a primary tumor, 2) migrate and invade through local tissues, 3)
translocate to distant sites in the body (via lymph or blood), 4)
colonize a foreign site, and 5) grow and survive in this foreign
environment. All of these behaviors are linked to cell adhesions.
Cell adhesions control the physical interactions of cells with
their microenvironment. Cell adhesions also initiate signals that
dictate tumor cell growth, death, and differentiation. At the
cellular level, metastatic cells have overcome restraints upon cell
growth and migration that result from physical linkages and signals
conveyed by cell-cell contacts. Malignant cells often have
increased interactions with surrounding extracellular matrix (ECM)
proteins that provide linkages and signals that promote several
aspects of metastasis.
[0004] Levels of protein tyrosine phosphorylation regulate a
balance between cell-cell and cell-ECM adhesions in epithelial
cells. Elevated tyrosine kinase activity weakens cell-cell contacts
and promotes ECM adhesions. Alteration in levels of tyrosine
phosphorylation is believed to be important for tumor cell
invasiveness. Tyrosine phosphorylation is controlled by cell
membrane tyrosine kinases, and increased expression of tyrosine
kinases is known to occur in metastatic cancer cells.
[0005] EphA2 is a 130 kDa receptor tyrosine kinase that is
expressed on adult epithelia. A member of the Eph family of
tyrosine kinases known as Ephrins, EphA2 is a transmembrane
receptor tyrosine kinase with a cell-bound ligand. EphA2 expression
has been found to be altered in many metastatic cells, including
lung, breast, colon, and prostate tumors. Additionally, the
distribution and/or phosphorylation of EphA2 is altered in
metastatic cells. Moreover, cells that have been transformed to
overexpress EphA2 demonstrate malignant growth, and stimulation of
EphA2 is sufficient to reverse malignant growth and invasiveness.
EphA2 is a powerful oncoprotein. The present invention is directed
to compounds and methods that target EphA2 for the diagnosis and
treatment of metastatic cancers.
[0006] One approach to cancer therapy is the administration of
preformed antibodies to predetermined tumor antigens. This process
is known as passive antibody treatment. An example of passive
antibody treatment is the use of Herceptin6 for the treatment of
breast cancer. Herceptin.RTM. is a humanized form of a murine
monoclonal antibody specific for the extracellular domain of
Her2/Neu. The basis for treatment with Herceptin.RTM. is that
25-30% of metastatic breast cancers overexpress the Her2/Neu
receptor tyrosine kinase. Herceptin.RTM. has been well tolerated in
clinical trials and shows much promise for the maintenance and
regression of metastatic breast cancer.
[0007] Effective passive immunotherapy for treatment of tumors
requires isolation and preparation of an antibody that: 1) targets
an antigen that is overexpressed in metastatic tumors; 2) targets
an extracellular epitope of said antigen; 3) is not cross-reactive
with any other antigen in a patient's circulation; and 4) exhibits
tumoricidal or tumoristatic activity.
[0008] In a preferred embodiment, this invention relates to the
selection and use of antibodies that are specific to an
extracellular epitope of EphA2. The methods of this invention
include the preparation, selection, and use of EphA2 specific
antibodies for cancer therapy.
[0009] Another approach to cancer treatment is to use agonists to
stimulate expression. For example, EphrinA1-F.sub.c, the
extracellular domain of ephrinA1 linked to immunoglobulin heavy
chain, (see Miao, H., et al., EphA2 kinase associates with focal
adhesion kinase and upon activation, inhibits integrin-mediated
cell adhesion and migration, Nature Cell Biol 2, 62-69 (2000),
hereby incorporated by reference) can be used to increase the
phosphotyrosine content of EphA2. Thus, in another preferred
embodiment, this invention relates to the use of agonists or
antagonists to alter the expression of EphA2 in metastatic
cells.
[0010] Thus, this invention is directed to the use of agonists and
antagonists to alter the expression of EphA2. EphA2 may be targeted
by use of artificial or hybrid forms of the protein, protein
inhibitors, antisense oligonucleotides, or small molecule
inhibitors. Also, while a preferred embodiment is directed to use
of monoclonal antibodies, polyclonal, artificial, and hybrid
antibodies are known in the art. It should be understood that use
of techniques known in the art to target EphA2 are within the scope
of this invention.
[0011] One aspect of this invention is a pharmaceutical composition
for the treatment of mammalian metastatic tumors which overexpress
EphA2, comprising a compound that specifically interacts with an
extracellular epitope of EphA2 in an amount effective to reduce
metastatic disease and a pharmaceutically acceptable carrier. In
the preferred embodiment, the pharmaceutical composition comprises
the antibody B2D6, an antibody that specifically binds to an
extracellular epitope of EphA2
[0012] Another aspect of this invention is a method of treating a
patient having a metastatic tumor which overexpresses EphA2. The
method comprises administering to the patient a therapeutic amount
of a compound that binds to an extracellular epitope of EphA2. In a
preferred embodiment, the compound is an antibody.
[0013] A third aspect of this invention is a method for detecting
the presence of metastatic cells. The method includes use of a
labeled antibody specific to an extracellular epitope of EphA2. A
cell sample is incubated with the antibody, unbound antibody is
removed, and the bound labeled antibody is detected.
[0014] An additional aspect of this invention is a method for
producing antibodies which inhibit metastatic tumor proliferation
by specifically binding to an extracellular epitope of EphA2. This
method includes injecting tyrosine phosphorylated proteins into the
lymph nodes of a mammal, harvesting the lymph nodes, fusing the
lymph node cells with myeloma cells to form hybridomas, and
selecting hybridomas which produce antibodies specific for
EphA2.
[0015] In still another aspect of this invention, a pharmaceutical
composition for treatment of a mammalian metastatic tumor is
provided, the composition comprising a compound that alters
expression of EphA2 in an amount effective to reduce metastatic
proliferation of said tumor, and a pharmaceutically acceptable
carrier therefor. In a preferred embodiment, the composition
comprises an ephrin.
[0016] Additional features of the present invention will become
apparent to those skilled in the art upon consideration of the
following detailed description of preferred embodiments
exemplifying the best mode of carrying out the invention as
presently perceived.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 is an overview of the RIMMS procedure, through which
the antibodies of this invention are generated;
[0018] FIG. 2A-C show a series of western blots showing EphA2
expression in human cell lines;
[0019] FIG. 2A is a western blot showing EphA2 expression in
various human prostate cancer cell lines;
[0020] FIG. 2B is similar to FIG. 2A, except showing EphA2
expression in a human prostatic epithelial cell line and expression
in the cell line after transformation by oncogenic K-Ras or
X-irradiation;
[0021] FIG. 2C is similar to FIG. 2B, except showing EphA2
expression in another human prostatic epithelial cell line and
expression in the cell line after transformation by oncogenic K-Ras
or X-irradiation;
[0022] FIG. 3 is similar to FIG. 2, except showing EphA2 expression
in canine prostatic cancer cells; and
[0023] FIG. 4 shows predicted antibody binding plotted against cell
density in a screening procedure for antibodies which are specific
for an extracellular epitope of EphA2.
DETAILED DESCRIPTION OF THE INVENTION
[0024] EphA2 is expressed differently in normal and metastatic
cells. In normal breast and prostate epithelial cells, EphA2 is
enriched in within cites of cell adhesion. Conversely, in
metastatic prostate cells EphA2 is diffusely distributed, and in
metastatic breast cancer cells EphA2 is redistributed into the
membrane ruffles. EphA2 expression is also known to be altered in
lung and colon malignancies, and it is believed that EphA2 altered
expression occurs in other types of metastasis, particularly
epithelial malignancies. Thus, techniques designed to alter EphA2
expression can be exploited to diagnose and treat metastatic
disease.
[0025] In a preferred embodiment, antibodies specific for tyrosine
phosphorylated proteins in cancer cells have been isolated and used
to target cancer cells in passive immunotherapy. This approach is
based upon the fact that many tyrosine kinases, e.g. Her2/Neu, are
expressed by oncogenes and are therefore overexpressed in cancer
cells. The present invention is directed to the production and use
of antibodies capable of recognition of and specific binding to
extracellular epitopes of the tyrosine kinase EphA2. The antibodies
are produced by selected hybridomas, themselves the product of
fusion of myeloma cells with lymph node cells harvested from
animals subjected to a specific inoculation protocol designed for
increased sensitivity and diversity of responding hybridomas.
[0026] To produce these hybridomas, tyrosine phosphorylated
proteins from Ras-transformed human epithelial cells were isolated
by affinity chromatography using existing phosphotyrosine specific
antibodies. The tyrosine phosphorylated proteins are then used as
an immunogen for producing monoclonal antibodies according to the
procedure illustrated in FIG. 1. Low-dose amounts of tyrosine
phosphorylated proteins are injected proximal to lymph nodes of a
mammal, every other day, over a ten day period (the RIMMS
strategy). B cells from engorged lymph nodes are then isolated and
fused with Bcl-2-overexpressing myeloma cells, to minimize
apoptosis after fusion. This method results in increased diversity,
specificity, and cost-effectiveness of hybridoma production. The
hybridomas are screened to identify those hybridomas producing
antibodies that distinguish malignant from normal cancer cells.
[0027] Hybridomas producing antibodies specific to EphA2 have been
selected. Use of the RIMMS technique has resulted in the production
of a multiplicity of hybridomas producing monoclonal antibodies
that specifically bind EphA2. To date, at least 450 hybridomas have
been identified which produce antibodies capable of distinguishing
malignant from normal cancer cells. Of the first four such
hybridomas to be characterized, two recognize independent epitopes
on EphA2. The first, D7, produces an antibody recognizing an
intracellular epitope. The second, B2D6, produces an antibody that
specifically binds an extracellular epitope of EphA2, a
characteristic that enables its effective use for the diagnosis and
treatment of selected metastatic tumors.
[0028] While the RIMMS strategy has proven to be valuable in the
production of EphA2 specific antibodies, other techniques are known
in the art for producing antibodies to a specific antigen, and
these techniques are within the scope of this invention.
[0029] It is known in the art to use antibodies to detect the
presence or overexpression of a specific protein. Because EphA2 is
overexpressed in metastatic cells, EphA2-specific antibodies of
this invention may be used to detect this overexpression and, thus,
to detect metastatic disease. Such techniques include but are not
limited to western blotting, precipitation, agglutination, and
ELISA assays. These techniques are well known in the art. Also, the
extracellular epitope specificity of EphA2-specific antibodies of
this invention can be exploited to detect changes in EphA2
localization which are associated with metastasis. In normal breast
and prostate epithelial cells, EphA2 is enriched in within cites of
cell adhesion, whereas in metastatic cells, EphA2 distribution is
altered. In metastatic prostate cells EphA2 is diffusely
distributed, and in metastatic breast cancer cells EphA2 is
redistributed into the membrane ruffles. EphA2 expression is also
known to be altered in lung and colon malignancies, and it is
believed that EphA2 altered expression occurs in other types of
metastasis, particularly epithelial malignancies Techniques such as
immunohistological staining or immunofluorescent microscopy are
well known in the art and may be used to visualize EphA2
distribution. See, for example, U.S. Pat. No. 5,514,554, hereby
incorporated by reference. In order to detect overexpression or
altered distribution of EphA2, the EphA2-specific antibodies may be
labeled covalently or non-covalently with any of a number of known
detectable labels, such fluorescent or radioactive substances, as
is known in the art. Alternatively, a secondary antibody specific
for the antibodies of this invention is labeled with a known
detectable label and used to detect the EphA2-specific antibodies
in the above techniques. Thus, the antibodies of this invention
provide methods to detect metastatic transformation.
[0030] The present invention also employs antibodies specific for
an extracellular epitope of EphA2 in therapeutic compositions and
methods for use. When used for in vivo therapy, a pharmaceutical
composition administered to a patient comprises EphA2-specific
antibodies in therapeutically effective amounts in a
pharmaceutically acceptable carrier. In a preferred embodiment, the
EphA2-specific antibodies have been "humanized." Humanized
antibodies include "chimeric antibodies" made by splicing genes
from a mouse (or other mammal) antibody of appropriate antigen
specificity together with genes from a human antibody molecule of
appropriate biological activity. Such techniques are known in the
art. See, for example, U.S. Pat. No. 5,811,098, hereby incorporated
by reference. In addition to antibodies, natural or artificial
ligands, peptides, anti-sense, ATP analogies, or other small
molecules capable of specifically targeting EphA2 may be
employed.
[0031] An example of another way to target EphA2 is the use of
ephrins to activate or inhibit EphA2. For example,
EphrinA1-F.sub.c, the extracellular domain of ephrinA1 linked to
immunoglobulin heavy chain, increases the phosphotyrosine content
of EphA2. EphrinA1-F.sub.c reverses the malignant behavior of EphA2
transformed cells. Thus, another preferred embodiment of this
invention is a pharmaceutical composition comprising an ephrin or a
hybrid form of ephrin administered in a therapeutic amount.
[0032] Therapeutic amounts are amounts which eliminate or reduce
the patient's tumor burden, or which prevent or reduce the
proliferation of metastatic cells. The dosage will depend on many
parameters, including the nature of the tumor, patient history,
patient condition, the possible co-use of other oncolytic agents,
and methods of administration. Methods of administration include
injection (e.g., parenteral, subcutaneous, intravenous,
intraperitoneal, etc.) for which the antibodies are provided in a
nontoxic pharmaceutically acceptable carrier such as water, saline,
Ringer's solution, dextrose solution, 5% human serum albumin, fixed
oils, ethyl oleate, or liposomes. Typical dosages may range from
about 0.01 to about 20 mg/kg, and more particularly from about 0.1
to about 10 mg/kg. Other methods of administration include oral and
transdermal. Acceptable carriers for oral ingestion in accordance
with the present invention can be formulated using art-recognized
techniques into pharmaceutically acceptable liquid carriers or in
combination with pharmaceutically acceptable solid carriers in the
form of tablets, capsules, caplets, or gel-seals. Other effective
methods of administration and dosages may be determined by routine
experimentation and are within the scope of this invention.
[0033] Therapeutic methods employing EphA2-specific antibodies may
be combined with chemotherapy, surgery, and radiation therapy,
depending on type of the tumor, patient condition, other health
issues, and a variety of factors. The methods may also include
immunoconjugats for targeted immunotoxin-mediated therapy, wherein
antibodies of this invention are covalently or non-covalently
conjugated to various cytotoxic agents, further enhancing toxicity
to targeted cells. See, for example, U.S. Pat. No. 5,872,223,
hereby incorporated by reference. Such agents, including various
bacterial toxins (e.g., Pseudomonas exotoxin), ricin A-chain,
daunorubicin, methotrexate, and ribosome inhibitors (e.g.,
trichosantin). Also, the antibodies of this invention may be
labeled with alpha, beta, or Auger electron emitters, resulting in
immunoconjugates for targeted radiotherapy.
[0034] Thus, EphA2-specific antibodies may be used in a variety of
methods and compositions for detecting and treating metastatic
disease.
EXAMPLE 1
Characterization of EphA2 Expression in Metastatic Cells
[0035] Following the RIMMS strategy using tyrosine phosphorylated
proteins from Ras-transformed human epithelial cells, hybridomas
were screened, and an antibody specific for EphA2 has been
isolated. This antibody, was used to assess the levels of EphA2
expression in nontransformed prostatic epithelial cells and
prostatic tumor cells. Low levels of EphA2 expression were found in
non-transformed prostatic epithelial cells, but this EphA2
expression was enriched within sites of cell-cell contact and
interacted with cell-bound ligand. Compared to non-transformed
cells, two features distinguish EphA2 in metastatic prostate cancer
cells: 1) EphA2 is overexpressed; 2) EphA2 is diffusely distributed
and does not appear to interact with ligand. To confirm these data,
western blots were performed using the EphA2 specific antibodies.
EphA2 overexpression in human prostate cancer cells (LNCAP, DU145,
PC3) directly correlates with their invasiveness in vitro and in
vivo. Of the three lines tested, LNCAP is the least aggressive,
DU145 is more aggressive, and PC3 is the most aggressive. As seen
in FIG. 2, DU145 cells exhibit higher levels of EphA2 expression
than LNCAP, and PC3 cells exhibit even higher levels of EphA2
expression. Similarly, as shown in FIGS. 2B and 2C, EphA2
expression is elevated in variants of human prostatic epithelial
cells transformed by oncogenic K-Ras or X-irradiation. The three
lanes in FIG. 2B show "normal" MCL prostatic epithelial cells, and
K-Ras and X-ray transformed cell lines derived therefrom.
Similarly, the three lanes of FIG. 2C show "normal" 267B1 prostatic
epithelial cells, and K-Ras and X-ray transformed cell lines
derived therefrom. As seen in FIGS. 2B and 2C, the transformed
cells all exhibited elevated EphA2 levels. FIG. 3 shows similar
western blots, except using prostate cancer cell lines from dogs.
As shown in FIG. 3, consistent with the results from human cells,
EphA2 is overexpressed in metastatic prostatic carcinoma cells
derived from dogs with spontaneous prostate cancer.
[0036] The metastatic prostate cell lines can be subdivided into
three categories: 1) cells derived from primary prostate tumors; 2)
cells derived from metastases that are poorly metastatic in vivo;
3) cells derived from metastases that are highly metastatic in
vivo. The western blots using EphA2-specific antibodies have
revealed that EphA2 expression is elevated in all cells derived
from metastases, with highest EphA2 expression in cells that retain
metastatic potential in vivo (as assessed using athymic mouse
models). Interestingly, B2D6 studies have shown that EphA2 is
overexpressed in cells from prostate cancer metastases compared to
lines established from the primary tumor of same patient. Taken
together, these results all reveal EphA2 overexpression in
metastatic prostate tumor cells.
[0037] Similar EphA2 expression patterns have been found with
breast cancer cells. In normal mammary epithelial cells, EphA2 is
enriched within the cell-cell junctions. By contrast,
non-metastatic breast cancer cells do not express EphA2, while
metastatic breast cancer cells overexpress EphA2. In metastatic
breast cancer cells, EphA2 is redistributed into the membrane
ruffles and, thus, is available for antibody binding.
EXAMPLE 2
In Vitro Targeting of Metastatic Cells
[0038] EphA2 overexpression renders metastatic cells susceptible to
antibody-mediated selected killing with the present antibodies
specific for an extracellular epitope of EphA2. While normal cells
express EphA2, it is believed that ligand binding or clustering
within sites of cell-cell contact occludes extracellular epitopes
in normal cells and renders them inaccessible to antibodies
specific for an extracellular epitope of EphA2. The tumor
selectivity of the antibodies of the present invention is believed
to rival or exceed that of Herceptin.RTM. for targeting metastatic
cancer.
[0039] EphA2 overexpression provides a basis for targeting
metastatic cancer cells with EphA2-specific antibodies. Antibodies
specific for an extracellular epitope of EphA2, such as those
produced by hybridoma B2D6, may be used to alter selectively
(versus normal cells) the proliferative or invasive behaviors of
metastatic cancer cells. In both metastasis-derived and
laboratory-induced transformation, EphA2 overexpression correlates
with invasiveness, whereas non-invasive cells have lower levels of
EphA2 expression.
[0040] To measure the effect of B2D6 on cell growth, cells are
incubated with purified B2D6, and cell proliferation is measured by
counting cells microscopically (using a hemacytometer) and by
measuring DNA synthesis. For example, normal growth media is
supplemented with B2D6 and BrdU, and BrdU incorporation is measured
over the following four hours. To measure the effects of B2D6 over
longer times, samples are counted at 24 hour intervals, with BrdU
added to the culture media for the final four hours of incubation.
As a third measure of cell growth, the effect of B2D6 on the growth
of metastatic cells in soft agar is determined. Soft agar plating
assays are used, wherein 2.times.10.sup.4 metastatic cells are
plated atop agar, in the presence or absence of B2D6 (0-10 nM), and
colony growth is evaluated at three-day intervals thereafter.
[0041] It is believed that B2D6 decreases the growth of metastatic
cells. Preliminary results reveal that B2D6 aggregates EphA2 and
blocks about 50% of growth of metastatic breast cancer cells (which
also overexpress EphA2) over the first four hours of incubation.
Although EphA2 is not tyrosine phosphorylated in metastatic breast
cancer cells, tyrosine phosphorylation is restored these B2D6
treated cells. Thus, B2D6 is believed to restore normal EpbA2
function.
[0042] Additional studies with prostate cancer cells are being
performed to determine if longer incubations with B2D6 further
inhibit metastatic cell growth. Non-transformed epithelial cells
express some EphA2, albeit much less than metastatic cells, and
some toxicity to non-transformed cells is possible. The minimum
effective and maximum non-toxic dosage levels of antibodies in
accordance with one aspect of this invention can be identified by
routine experimentation, but preferably, typical doses will range
from about 0.1 to about 20 mg/kg of patient body weight. The
preferred dose will depend on many parameters, including the nature
of the tumor, patient history, patient condition, the possible
co-use of other oncolytic agents, and methods of administration.
Antibody levels that best discriminate between normal and
metastatic cells will be used in treatment of metastatic tumors
overexpressing EphA2 proteins.
EXAMPLE 3
In Vitro Antibody Mediated Cytotoxicity
[0043] Preliminary results demonstrate that EphA2 antibodies impede
metastatic cell growth. To measure antibody-directed cytotoxicity,
preferably, non-radioactive versions of .sup.51Cr-release assays
using target cells (normal or metastatic prostatic epithelial
cells) labeled with europium chloride (EuCl.sub.3) and
diethylenetriamine-pentaacetic acid (DTPA) are performed. After
washing away unincorporated Eu.sup.3+, naphthoyltrifluoroacetone
(NTA) and trioctylphosphine oxide are incubated with the cytolytic
agents and assay supernatants. The luminescence of resultant
ternary complex (Eu.sup.3+/NTA/trioctylphosphine oxide) is measured
using a fluorescence microplate reader. The sensitivity of this
Eu.sup.3+-release assay for complement-mediated cytolysis has been
reported to be fivefold better than .sup.51Cr-release assays. To
determine specific lysis, parallel samples are hyponically lysed by
adding distilled water. Untreated samples and isotype matched
antibodies serve as negative controls.
[0044] To model complement-mediated death in vitro, cells are
labeled with B2D6 and exposed to sera that has not been
heat-inactivated. For antibody dependent cellular cytotoxicity
("ADCC"), both control and B2D6-treated prostate cells are
incubated with peripheral blood mononuclear cells (PBMC; 10:1 E/T
ratio).
[0045] It is believed that complement and ADCC both promote
specific killing of the metastases. Antibody dose can be varied in
order to establish LD.sub.50 measurements for metastatic and normal
cells. Antibody concentrations that maximize specific killing of
metastatic cells (PC3, 267-Ras, 267-X) while minimizing the death
to non-transformed prostatic epithelia (267, MLC) are determined by
routine experimentation. Also, ADCC can be combined with complement
to further enhance tumor killing by treating the samples with tumor
necrosis factor (TNF). There is evidence that TNF potentiates the
killing of tumor cells by EGFR and Her2-specific antibodies. It is
expected that humanizing the antibodies of this invention would
provide for better complement or ADCC results.
[0046] It is believed that B2D6 kills tumor cells through
complement cascade or ADCC. However, covalent or non-covalent
conjugation of the present antibodies to art-recognized cytotoxic
agents can further enhance toxicity to targeted cells. Examples of
toxins appropriate for immunoconjugation include Pseudomonas
exotoxin or ricin A-chain.
EXAMPLE 4
In Vivo Targeting of Metastatic Cells
[0047] The present EphA2 antibodies, particularly those produced by
hybridoma B2D6 are effective in blocking the growth and
invasiveness of prostate cancer cells in vivo. The efficacy of B2D6
in blocking the growth of primary prostate tumors using
subcutaneous implantation of PC3 tumor cells in mice is determined
by use of subcutaneous models. The primary advantages of
subcutaneous models are the ease of implantation and subsequent
monitoring of tumor size. 5.times.10.sup.5 PC3 cells are inoculated
subcutaneously into the right craniolateral thorax (axilla) using
aseptic technique. Tumors are measured every 3-4 days using vernier
calipers until they reach a volume of 0.2-0.3 cm.sup.3. At that
time, the mice are divided into four groups (8-10 animals each):
Group 1 (vehicle control), Groups 2-4 are treated with 0.1, 1.0, or
10 mg/kg B2D6, administered intraperitoneally, twice a week. The
mice are then monitored every 3 days to measure tumor volume (with
vernier calipers), body weight, and life span. After no greater
than 60 days past implantation, the animals are sacrificed and
postmortem evaluations of tumorigenesis, including measurement and
weight of implanted tumors and proximal lymph nodes, macroscopic
evaluation of soft tissues for tumors (lymph nodes and lung), and
formalin fixation of the primary tumor and tissues, are performed.
The tissues are evaluated by immunohistochemistry using D7 (another
EphA2 specific antibody that is amenable to immunohistochemistry)
to determine the level of EphA2 expression in the tumors. In
particular, tumor cells that escape B2D6 treatment are studied to
determine whether they have low levels of EphA2 expression. Also,
EphA2 expression in the individual animals is correlated with tumor
invasiveness.
[0048] As target-negative controls for specificity, parallel
studies are performed using DU145 cells, which express very low
levels of EphA2. Whereas the growth of PC3 tumors are believed to
be sensitive to B2D6, tumors caused by DU145 are believed be
insensitive. Statistical significance of B2D6 inhibition of
tumorigenicity, overall metastatic frequency, and frequency of
distant metastases is tested using computerized statistical
packages (PC/SAS Ver. 6.04), with differences considered
significant if p<0.05.
[0049] While subcutaneous implantation is a popular and valuable
method for modeling tumor cell growth, differences between the
microenvironment of the skin and prostate can cause rather dramatic
differences in cell behavior. For example, PC3 cells rarely
metastasize when implanted subcutaneously whereas intraprostatic
implantation (orthotopic) facilitates metastasis. Thus, PC3 cells
are implanted in the prostate by exposing the prostate via
laparotomy and inoculating tumor cells into prostate gland using a
surgical microscope. After seven days, the mice begin receiving
treatment with B2D6, as described above, and the animals are
sacrificed no later than 60 days after implantation (or if the
animals become moribund). The tumors are palpated at 3-5 day
intervals, at which time data on tumor size, animal weight, and
survival are collected. Post-mortem evaluations are also performed
as described above, with emphasis upon the effect of B2D6 upon
metastatic potential (to lungs and regional lymph nodes). B2D6 is
believed to block the primary tumor and metastatic potential of PC3
cells in a dose-dependent manner.
[0050] To minimize identification of strain or clonal-specific
effects, identical analyses using other model systems can be
employed. For example, the effects of B2D6 on the growth or
metastasis of tumors caused by implantation of K-Ras or X-ray
transformed 267B1 can be compared to the effects on MLC human
prostatic epithelial cell lines.
EXAMPLE 5
Development of "Second Generation" EphA2-Based Antibody
Therapeutics
[0051] While EphA2 overexpression in metastatic prostate cancer
cells provides a degree of selectivity comparable to Herceptin.RTM.
in breast cancer, unique properties of EphA2 are believed to allow
for even more selective targeting. In particular, EphA2 at the
surface of non-transformed epithelia is tightly packed into
cell-cell contacts whereas EphA2 on metastatic cells is diffusely
distributed. It is thus likely that some epitopes on EphA2 are
accessible in metastases but protected by ligand in normal
prostatic epithelia. The EphA2 antibodies that provide the optimal
discrimination between normal and metastatic prostatic epithelia
are selected.
[0052] The panel of antibodies generated previously are screened
for epitopes on EphA2 that are found at the cell surface. Using
flow cytometry, EphA2 expression in a variety of "normal" (e.g.,
267B or MLC cells) and metastatic cells (PC3 cells) are compared.
For example, confluent monolayers of normal and metastatic cells
are labeled with B2D6. To insure that antibodies are selected that
are specific for epitopes inaccessible in normal cells but
accessible in metastatic cells, antibodies are selected whose
binding decreases in normal cells with increasing cell density but
whose binding remains constant in metastatic cells, as shown in
FIG. 4. After labeling with fluorescein-secondary antibodies, EphA2
expression is evaluated by using flow cytometry. The antibodies
that best distinguish between normal and metastatic cells are
selected. Specificity for EphA2 is confirmed via western blotting
and immunoprecipitation studies. Antibodies exhibiting the best
selectivity are then humanized using art recognized techniques.
EXAMPLE 6
Altered EphA2 Expression Through Transfection
[0053] To assess the consequences of EphA2 overexpression, MCF-10A
cells were transfected with human EphA2 cDNA (EphA2) or a vector
control (vector). After establishing cultures of MCF-10A cells with
stable overexpression of EphA2, microscopic evaluation revealed
differences in the cell morphology as compared to
vector-transfected control cells (not shown). Non-transformed
MCF-10A cells display an epithelial morphology and interact with
one-another, even at low cell density. In contrast,
EphA2-overexpressing MCF-10A cells (MCF.sup.EphA2) adopt a
fibroblast-like morphology and do not form cell-cell contacts, even
at high cell density. To confirm that the mesenchymal morphology
does not represent clonal variation, a separate sample of MCF-10A
cells transfected with EphA2 cDNAs yielded identical results.
[0054] Cell-ECM adhesions were evaluated by incubating cells on ECM
at 37.degree. C. for 30 minutes before vigorous washing to remove
weakly adherent cells. These assays revealed a 24-fold increase in
ECM attachments in MCF.sup.EphA2 cells relative to
vector-transfected controls (P<4.times.10.sup.-4). Cell-cell
adhesions were assayed by incubating cells in suspension and
counting the average size of cell colonies. Whereas
vector-transfected MCF-10A cells interact with one-another in
colonies with an average size of 4.1 cells, the average colony size
of MCF.sup.EphA2 cells is reduced to 1.3 cells
(P<3.times.10.sup.-5).
[0055] Since stable cell-cell contacts cause EphA2 to become
enriched within sites of cell-cell contact, EphA2 subcellular
localization was assessed by immunostaining with specific
antibodies. The EphA2 on non-transformed MCF-10A cells was
restricted to a narrow line where adjacent cells came into direct
contact, with little staining of membrane that was not in contact
with neighboring cells. In contrast, the pattern of EphA2 staining
on MCF.sup.EphA2 cells was diffuse, with little staining of
cell-cell contacts.
[0056] The lack of EphA2 within cell-cell contacts in MCF.sup.EphA2
cells was intriguing since EphA2 is stimulated by ligands that are
anchored to the cell membrane. To measure EphA2 stimulation, the
phosphotyrosine content of immunoprecipitated EphA2 was measured by
Western blot analysis with phosphotyrosine specific antibodies.
Whereas the EphA2 in vector-transfected MCF-10 cells was tyrosine
phosphorylated, EphA2 was not tyrosine phosphorylated in
MCF.sup.EphA2 cells. The decreased phosphotyrosine content was
confirmed using multiple EphA2 antibodies for immunoprecipitation
(D7, B2D6) and different phosphotyrosine-specific antibodies (4G10,
PY20) for Western blot analyses.
EXAMPLE 7
Malignancy and Metastasis Through EphA2 Transfection
[0057] Malignant transformation was studied in vitro, and
MCF.sup.EphA2 cells were found to colonize soft agar. Whereas
vector-transfected MCF-10A cells formed 0.3 colonies per high-power
field, while MCF.sup.EphA2 cells displayed increased colony growth
in soft agar, with an average of 3.0 colonies per high-power field
(P<3.times.10.sup.-7). Vector and EphA2 overexpressing MCF-10A
cells were allowed to interact with Matrigel (Collaborative,
Bedford, Mass.). Non-transformed MCF-10A cells rapidly organized
into spherical colonies when cultured on Matrigel whereas
MCF.sup.EphA2 cells adopted a stellate organization that was
indistinguishable from the behavior of metastatic cells (e.g.,
MDA-MB-231, MDA-MB,435).
[0058] Since in vitro analyses of transformation do not always
predict tumorigenic potential in vivo, control or
EphA2-overexpressing MCF-10A cells were implanted into athymic
(nu/nu) mice. Subcutaneous injection of MCF.sup.EphA2 cells caused
the formation of palpable tumors within four days in 19 of 19 mice.
The median volume of resulting tumors related to the number of
implanted cells and reached an average of 300 mm.sup.3 (for samples
injected with 5.times.10.sup.6 cells) within 10 days (Table I).
Necropsy revealed that the tumors were firmly attached to the
underlying axillary muscle and surrounded by fibrous tissue.
Histologically, the neoplastic cells were invasive and associated
with fibrous connective tissue. These neoplastic cells exhibited
moderate cytoplasmic and nuclear pleiomorphism and formed
dysplastic tubular and secreting structures. In control
experiments, cells transfected with vector DNA failed to grow in
athymic mice (0 of 13; Table I) and necropsy failed to identify any
growth or invasion of these cells.
[0059] Since the highest levels of EphA2 were consistently found in
breast cancer cells that are metastatic in vivo, 1.times.10.sup.6
control or MCF.sup.EphA2 cells were injected into the tail vein of
athymic mice. Within seven days, necropsy revealed lung
micrometastases within large vessels in 2 of 4 mice injected with
MCF.sup.EphA2 cells (Table I). The metastases were generally found
to occlude large blood vessels but did not breach the vessel wall.
Immunohistochemical staining with cytokeratin antibodies confirmed
the epithelial nature of the thrombus and a lack of anti-thrombin
staining revealed that the thrombus did not represent an abnormal
or atypical outgrowth of endothelial cells. No lung colonization
was observed in mice that had been injected with control MCF-10A
cells (Table I).
1TABLE I Tumorigenic and Metastatic Potential of EphA2-Transformed
MCF-10A Cells Site of # of Cells Incidence of Tumor Volume Cell
Inoculation Injected Tumorigenicity (mm.sup.3) Ctrl Subcutaneous 1
.times. 10.sup.6 0/9 N/A EphA2 1 .times. 10.sup.6 9/9 66 .+-. 20
Ctrl Subcutaneous 5 .times. 10.sup.6 0/4 N/A EphA2 5 .times.
10.sup.6 10/10 293 .+-. 70 Ctrl Tail Vein 1 .times. 10.sup.6 0/4
EphA2 1 .times. 10.sup.6 2/4
EXAMPLE 8
Metastatic Targeting Using EphA2 Agonists
[0060] To test if EphA2 could be stimulated by an agonist,
MCF.sup.EphA2 cells were suspended in soft agar in the presence or
absence of 0.5 mg/mL EphrinA1-F.sub.c. EphrinA1-F.sub.c increased
the phosphotyrosine content of EphA2, and EphrinA1-F.sub.c-treated
reduced colony formation in soft agar by 49% relative to
vehicle-treated controls (P<5.times.10.sup.-6)- . To test if
EphA2 stimulation could alter cell behavior on Matrigel, the
MCF.sup.EphA2 cells were treated with 0.5 mg/mL EphrilA1-F.sub.c,
which restored a spherical phenotype that was comparable to
non-transformed MCF-10A cells. Thus, EphA2 stimulation reverses the
effects of EphA2 overexpression. EphrinA1-F.sub.c Despite its
inability to interact with its endogenous ligands, the EphA2 in
MCF.sup.EphA2 cells responded to exogenous stimuli.
[0061] Although the invention has been described in detail with
reference to preferred embodiments, variations and modifications
exist within the scope and spirit of the invention as described and
defined in the following claims.
* * * * *