U.S. patent application number 11/502309 was filed with the patent office on 2006-12-07 for binding peptides specific for the extracellular domain of erbb2 and uses therefor.
This patent application is currently assigned to University of Vermont and State Agricultural College. Invention is credited to David N. Krag, Lyn Oligino, Stephanie C. Pero.
Application Number | 20060276379 11/502309 |
Document ID | / |
Family ID | 27613187 |
Filed Date | 2006-12-07 |
United States Patent
Application |
20060276379 |
Kind Code |
A1 |
Krag; David N. ; et
al. |
December 7, 2006 |
Binding peptides specific for the extracellular domain of ErbB2 and
uses therefor
Abstract
The invention provides methods and compositions for diagnosing
and treating subjects using EBPs. Specifically disclosed are
peptides and peptidomimetics that bind selectively to the
extracellular domain of ErbB2. These compositions are useful in the
prevention and treatment of disorders characterized by ErbB2
overexpression (e.g., breast cancer).
Inventors: |
Krag; David N.; (Shelburne,
VT) ; Pero; Stephanie C.; (Essex Junction, VT)
; Oligino; Lyn; (Burlington, VT) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, PC
FEDERAL RESERVE PLAZA
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
University of Vermont and State
Agricultural College
Burlington
VT
|
Family ID: |
27613187 |
Appl. No.: |
11/502309 |
Filed: |
August 10, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10272437 |
Oct 15, 2002 |
7098302 |
|
|
11502309 |
Aug 10, 2006 |
|
|
|
60329183 |
Oct 12, 2001 |
|
|
|
Current U.S.
Class: |
424/1.69 ;
435/7.1; 514/19.4; 514/21.1 |
Current CPC
Class: |
C07K 14/475 20130101;
A61K 38/00 20130101; A61K 47/64 20170801 |
Class at
Publication: |
514/009 ;
514/013; 514/014; 514/015; 435/007.1 |
International
Class: |
A61K 38/12 20060101
A61K038/12; A61K 38/10 20060101 A61K038/10; C40B 40/10 20060101
C40B040/10 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This work was funded in part by grant number 1 R01
CA80790-01, from the National Institutes of Health, and grant
number DAMD17-94-J-4373 from the Department of Defense.
Accordingly, the United States Government may have certain rights
to this invention.
Claims
1. A composition comprising a peptide comprising an ErbB2 binding
peptide (EBP) that binds specifically to the extracellular domain
of ErbB2, wherein the ErbB2 binding peptide is between 11 and 20
amino acids in length; comprises at least two cysteines separated
by 9 or 10 amino acids, and wherein the sequence of the 9 or 10
amino acids comprises the amino acid sequence set forth as STWGF
(SEQ ID NO:48).
2. The composition of claim 1, wherein the peptide comprises an
amino acid sequence set forth as SEQ ID NO:1, SEQ ID NO:33, SEQ ID
NO:34, SEQ ID NO:35, or SEQ ID NO:36.
3. The composition of claim 1, wherein the peptide is cyclic.
4. The composition of claim 1, wherein the peptide is conjugated to
an agent.
5. The composition of claim 4, wherein the agent is selected from
the group consisting of a toxin, a radioactive molecule, a
detectable label, an imaging agent, a chemotherapeutic agent, a
diagnostic agent, an anti-cancer agent, an anti-angiogenic agent,
an apoptosis agent, a translocating agent, and an immunomodulatory
agent.
6. The composition of claim 1, wherein the peptide is selected from
the group consisting of a phage display peptide library member, a
synthetic peptide library member, a combinatorial chemistry library
member and a peptidomimetic.
7. The composition of claim 1, further comprising a
pharmaceutically acceptable carrier.
8. The composition of claim 1, further comprising an anti-cancer
agent.
9. The composition of claim 1, wherein the composition is in a
sustained release vehicle.
10. The composition of claim 1, wherein the ErbB2 binding peptide
inhibits phosphorylation of ErbB2.
11. A method for preventing or treating a disorder characterized by
ErbB2 overexpression, comprising administering to a subject in need
of such treatment a composition of claim 1, in an amount effective
to inhibit the disorder.
12. The method of claim 11, wherein the ErbB2 binding peptide
comprises an amino acid sequence set forth as SEQ ID NO:1, SEQ ID
NO:33, ID NO:34, SEQ ID NO:35, or SEQ ID NO:36.
13. The method of claim 11, wherein the disorder is a cancer.
14. The method of claim 13, wherein the cancer is a primary tumor
or a metastasis.
15. A method for detecting a cell characterized by ErbB2
overexpression comprising contacting a composition of claim 1 with
a cell, and determining the level of binding of the ErbB2 binding
peptide to the cell, wherein a level of binding greater than a
control level is indicative of ErbB2 overexpression by the
cell.
16. The method of claim 15, wherein the ErbB2 binding peptide
comprises an amino acid sequence set forth as SEQ ID NO:1, SEQ ID
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ
ID NO:38, or SEQ ID NO:39.
17. The method of claim 15, wherein the contacting occurs in vivo
and the ErbB2 binding peptide is administered to a subject.
18. The method of claim 15, wherein the ErbB2 binding peptide is
conjugated to a detectable label.
19. The method of claim 18, wherein the detectable label is
selected from the group consisting of a radioisotope, a contrast
agent, and a gaseous agent.
20. The method of claim 15, further comprising removing the cell
characterized by ErbB2 overexpression from a tissue or cell
population in which it exists.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of application Ser. No.
10/272,437, filed on Oct. 15, 2002, now allowed, which claims
priority under 35 U.S.C. .sctn.119 to U.S. provisional application
Ser. No. 60/329,183 filed Oct. 12, 2001, the entire contents of
each is incorporated by reference herein.
FIELD OF THE INVENTION
[0003] The invention relates to peptides and peptidomimetics that
bind to the extracellular domain of ErbB2, and their use in
diagnosis, prevention and treatment of disorders associated with
overexpression of ErbB2 (e.g., breast cancer).
BACKGROUND OF THE INVENTION
[0004] One major drawback of most cancer therapeutics is the lack
of specificity and associated toxicity to normal tissues. A
significant advance in therapeutic effectiveness would be likely
achieved if cytotoxic agents could be delivered specifically to
tumor cells, with minimal delivery to normal tissue. Conjugation of
cytotoxic agents to molecules that bind specifically to a tumor
target found would then enable tumor-specific delivery and would
reduce non-specific toxicity.
[0005] Molecules that are found specifically on the surface of
cancer cells are especially promising targets for tumor-specific
homing molecules. An example of such a cell surface molecule is
ErbB2 (also known as HER2 or neu). ErbB2 is a member of the ErbB
family of growth factor receptors, which includes ErbB1 (also known
as epidermal growth factor receptor). ErbB2 is a membrane protein
containing a cysteine-rich extracellular domain (ECD), a
transmembrane domain, and an intracellular tyrosine kinase domain.
It is overexpressed on the surface of breast cancer cells in
approximately 30% of newly diagnosed patients and is associated
with a poor prognosis. Importantly, metastatic tumor cells in the
bone marrow of 60-70% of breast cancer patients overexpress ErbB2
on their surface (Pantel et al., J Natl Cancer Inst 85:1419; Braun
et al., Cancer Research, 61:1890). Therefore, ErbB2 is an extremely
promising target molecule for some forms of cancer.
SUMMARY OF THE INVENTION
[0006] The invention relates to the identification of peptides that
specifically bind to the extracellular domain of ErbB2. ErbB2
overexpression is a hallmark of many forms of cancer, including
most notably breast cancer, ovarian cancer, stomach cancer, lung
cancer and bladder cancer, among others. Accordingly, the discovery
of small, preferably peptide, molecules that bind to ErbB2
facilitates detection, prevention and treatment of disorders
characterized by overexpression of ErbB2 (including those that
overexpress ErbB2), such as those listed above. Prior to the
invention, an antibody to ErbB2 (i.e., Herceptin) had been
identified, and tested clinically. The small peptides of the
present invention have pharmacokinetic properties superior to
larger molecules such as antibodies.
[0007] Accordingly, in one aspect, the invention provides ErbB2
binding peptides, referred to herein as EBP. The invention provides
at least 20 EBP, the sequences of which are provided below. Some of
the EBP of the invention share common sequence elements. In
preferred embodiments, the EBP of the invention bind to the
extracellular domain of ErbB2. The EBP include both the peptides
described herein as well as their functional equivalents. In
preferred embodiments, the functional equivalents are peptides that
have at least 50%, at least 60%, at least 70%, at least 80%, at
least 90%, or at least 95% identity with the peptides described
herein. The functional equivalents may be different from the
peptides described herein at one, two, three, four, or more amino
acid positions. In the most common instances, such differences will
involve conservative amino acid substitutions.
[0008] Thus, in one aspect, the invention provides a composition
comprising a peptide comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3,
SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,
SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID
NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ
ID NO:38 and SEQ ID NO:39, and functional equivalents thereof,
including functionally equivalent fragments thereof. These peptides
are referred to herein as ErbB2-binding peptides (i.e., EBP), and
each is designated with a unique number (e.g., EBP-1, EBP-2, EBP-3,
EBP-4, EBP-5, EBP-6, EBP-7, EBP-8, EBP-9, EBP-10, EBP-11, EBP-12,
EBP-13, etc.). As indicated in the Brief Description of the
Sequence Listing, SEQ ID NO:1 through to SEQ ID NO:13 and SEQ ID
NO:33 through to SEQ ID NO:39 represent the amino acid sequences of
EBP-1 through to EBP-13 and EBP-14 through to EBP-20,
respectively.
[0009] In one embodiment, the EBP is cyclic or is capable of being
cyclized via, for example, a disulfide bond, a thio-ether linkage
or a peptide bond. In another embodiment, the peptide is conjugated
to an agent. The agent may be selected from the group consisting of
a toxin, a radioactive molecule, a detectable label, an imaging
agent, a diagnostic agent, a chemotherapeutic agent, an
anti-angiogenic agent, an anti-cancer agent, an immunomodulatory
agent, an antigen or antigenic moiety, an apoptosis agent, and a
translocating agent. The translocating agent can be used to
translocate the peptide or preferably a therapeutic agent attached
to the peptide into the cell in order to deliver the therapeutic
agent to the cell. In another embodiment, the peptide is used
together with an agent that functions in the cytoplasmic
compartment of a cell, such as for example an agent that inhibits
the cytoskeleton, or inhibits spindle formation. Several of these
latter types of agents are known to be chemotherapeutic agents. In
yet another embodiment, the peptide is conjugated to another
peptide such as one with binding specificity for EGFR, ErbB3, or
ErbB4. In another embodiment, the composition comprises the peptide
with a liposome or viral particle (e.g., for delivery in gene
therapy).
[0010] The functional equivalents of EBP can be comprised of amino
acids or peptidomimetics. In one embodiment, the functional
equivalent is selected from the group consisting of a phage library
member, a synthetic peptide library member, a combinatorial
chemical library member, and a peptidomimetic.
[0011] The foregoing embodiments relating to the peptides and
functional equivalents of the invention apply equally to all
aspects of the invention.
[0012] In one embodiment, the composition further comprises a
pharmaceutically acceptable carrier, and optionally, the peptide or
functional equivalent thereof is present in an effective amount. In
other embodiments, the composition further comprises another
therapeutic agent including but not limited to an anti-cancer
agent. The composition may be provided in a sustained release
vehicle. In various embodiments of the invention, the ErB2 binding
peptides bind the extracellular domain of ErbB2 and, importantly,
also inhibit the phosphorylation of ErbB2 (e.g., at particular
tyrosine or serine residues, as described herein).
[0013] The invention also provides for isolated nucleic acid
molecules that code for ErbB2 binding peptides. Thus, in yet
another aspect, an isolated nucleic acid molecule is provided
comprising (a) a nucleic acid molecule which codes for a peptide
comprising an amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ
ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID
NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ
ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37,
SEQ ID NO:38 and SEQ ID NO:39 (i.e., SEQ ID NO:1 through to SEQ ID
NO:13 inclusive and SEQ ID NO:33 through to SEQ ID NO:39 inclusive)
or functionally equivalent fragments thereof; (b) degenerates of
(a); and (c) complements of (a) and (b).
[0014] In some embodiments, the isolated nucleic acid molecule
comprises a nucleotide sequence selected from the group consisting
of SEQ ID NO:14 through to SEQ ID NO:26 and SEQ ID NO:40 through to
SEQ ID NO:46, inclusive, and degenerates thereof. Using the nucleic
acid codons provided herein, one of ordinary skill in the art will
readily determine the nucleic acid sequences that are degenerates
thereof. The invention similarly embraces these latter nucleic acid
sequences.
[0015] The invention further provides in another aspect an
expression vector comprising the afore-mentioned isolated nucleic
acid molecule, preferably operably linked to a promoter, and host
cells transformed or transfected with the expression vectors.
[0016] In another aspect, the invention provides a method for
preventing or treating a disorder characterized by ErbB2
overexpression. The method can be used to prevent the disorder in a
subject at risk of developing the disorder or, alternatively, to
treat the disorder in a subject having the disorder. In embodiments
of either, the methods further comprise first selecting a subject
to be treated (e.g., a subject having the disorder or a subject at
risk of developing the disorder).
[0017] In another aspect, a pharmaceutical preparation is provided
comprising one or a combination of the afore-mentioned compositions
and a pharmaceutically acceptable carrier. The pharmaceutical
preparation and compositions may be in a sustained release
vehicle.
[0018] The method comprises administering to a subject in need of
such treatment an ErbB2 binding peptide that binds to an
extracellular domain of ErbB2, and preferably inhibits
phosphorylation of ErbB2. In some important embodiments, the ErbB2
binding peptide comprises an amino acid sequence selected from the
group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID
NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID
NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:33, SEQ
ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38
and SEQ ID NO:39, inclusive, or functional equivalents thereof.
Functional equivalents thereof include fragments of the peptide
that are capable of binding specifically to the extracellular
domain of ErbB2. In some embodiments, the EBP or functional
equivalent thereof is administered in an amount effective to
inhibit the disorder. In other embodiments, the method involves
co-administering an anti-cancer agent to the subject. In these
latter embodiments, the peptide and the anti-cancer agent are
co-administered in a combined effective amount to inhibit the
disorder. In related aspects of the foregoing methods non-peptide
small molecules that functionally and/or structurally mimic the EBP
of the invention can also be used in place of the EBPs.
[0019] In one embodiment, the disorder is in or is likely to be in
a tissue selected from the group consisting of the breast, ovary,
uterus, cervix, thyroid gland, gastrointestinal tissue, colon,
stomach, lung and bladder. In important embodiments, the disorder
is a cancer. The cancer may be a primary tumor or a metastasis. The
cancer may be selected from the group consisting of breast cancer,
ovarian cancer (including endometrioid carcinoma), Ewing's sarcoma,
cervical cancer, colorectal cancer (e.g., colorectal adenomas and
adenocarcinomas), thyroid cancer, lung cancer, prostate cancer,
stomach cancer, and bladder cancer.
[0020] In one embodiment, the peptide is administered systemically.
In another embodiment, the peptide is administered locally. In yet
another embodiment, the peptide is administered in a plurality of
administrations. In another embodiment, the method further
comprises administering to the subject an anti-cancer agent.
[0021] The invention further provides a method for inhibiting a
metastasis (e.g., preventing tumor cell metastasis) by
administering to a subject in need of such treatment one or a
combination of any of the above-identified peptides or functional
equivalents in an amount effective to prevent the formation or
development of a metastasis. The metastasis may be present in bone
marrow, lung, brain, and liver, but is not so limited.
[0022] In another aspect, the invention provides a method for
detecting a cell characterized by ErbB2 overexpression comprising
contacting an ErbB2 binding peptide, that in some embodiments
comprises an amino acid sequence selected from the group consisting
of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6,
SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,
SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:34, SEQ ID
NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39,
or functional equivalent thereof, to a cell, and determining the
level of binding of the peptide to the cell, wherein a level of
binding greater than a control level is indicative of ErbB2
overexpression by the cell.
[0023] In one embodiment, the contacting occurs in vivo and the
peptide is administered to a subject. In a related embodiments, the
peptide may be administered systemically or locally. In important
embodiments, the peptide is conjugated to a detectable label. The
detectable label may be selected from the group consisting of a
radioisotope, a contrast agent, and a gaseous agent, but is not so
limited.
[0024] In one embodiment, the cell is a breast tissue cell. In
another embodiment, the cell is present in a population selected
from the group consisting of bone marrow cells, lung cells, brain
cells, and liver cells. In a related embodiment, the cell is
harvested from a subject having a disorder characterized by ErbB2
overexpression, prior to treating the subject with radiation or
chemotherapy. The disorder characterized by ErbB2 overexpression
may be breast cancer, but is not so limited.
[0025] In one embodiment, the method further comprises removing the
cell characterized by ErbB2 overexpression from a tissue or cell
population in which it exists. In one embodiment, the cell is
removed from the tissue or cell population using flow cytometry. In
some embodiments, solid matrix (e.g., agarose beads or magnetic
particles) affinity methods are used in combination with the
peptides or functional equivalents thereof. In another embodiment,
the cell is removed from the tissue or cell population using a
cytotoxic agent. The cytotoxic agent may be conjugated to the
peptide, or functional equivalent thereof.
[0026] In another aspect, a method is provided for identifying a
compound that binds to ErbB2 and inhibits interaction between ErbB2
and an ErbB2 binding peptide (that preferably comprises an amino
acid sequence selected from the group consisting of SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7,
SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,
SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID
NO:36, SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39, or functional
equivalent thereof). The method comprises (1) performing a first
assay between ErbB2 and the peptide or functional equivalent
thereof to obtain a first assay result; (2) performing a second
assay between ErbB2 and the peptide or functional equivalent
thereof in the presence of a compound to obtain a second assay
result; and (3) comparing the first and second assay results to
determine whether the compound inhibits interaction between ErbB2
and the peptide or functional equivalent thereof. In some important
embodiments, the ErbB2 molecule is the extracellular domain of
ErbB2. The method may also include a negative pre-screen in which
compounds are initially tested and negatively selected based on
their ability to bind to other ErbB family members.
[0027] In one embodiment, the compound is a molecular library
member. The molecular library may be selected from the group
consisting of a peptide library such as a phage display peptide
library, a peptidomimetic library, a combinatorial chemistry
library, a synthetic peptide library, and a natural compound
library. The screening method may further comprise selecting a
molecular library that is suspected of containing a library member
that modulates the interaction of ErbB2 and an ErbB2 ligand. The
molecular library may contain from two to 10.sup.15 molecules and
any integer number therebetween. In an important embodiment, the
compound is a phage display library member. The phage display
library member may be cyclized.
[0028] In one embodiment, the assay is a binding assay which
detects binding of ErbB2 to the peptide or functional equivalent
thereof. In another embodiment, the assay is a signaling assay
which detects signaling events following ErbB2 binding to the EBP.
In yet a further embodiment, the method further involves
introducing the molecular library member, and in some instances
conjugates of a library member with a therapeutic agent, into an
animal model of a condition characterized by overexpression of
ErbB2 and determining whether the molecular library member
ameliorates symptoms of the condition.
[0029] In one embodiment, the peptide may be cyclized. In another
embodiment, ErbB2 or the peptide or functional equivalent thereof
may be immobilized onto a solid support such as for example an
agarose bead or a magnetic particle. According to one embodiment,
ErbB2 is present in the context of a cell. The cell may be selected
from the group consisting of breast cancer cell and an ovarian
cancer cell.
[0030] These and other aspects of the invention will be described
in greater detail herein.
[0031] Each of the aspects of the invention can encompass various
embodiments of the invention. It is therefore anticipated that each
of the embodiments of the invention involving any one element or
combinations of elements can be included in each aspect of the
invention.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
[0032] SEQ ID NO:1 is the amino acid sequence of ErbB2-binding
peptide 1 (EBP-1; EC-1).
[0033] SEQ ID NO:2 is the amino acid sequence of ErbB2-binding
peptide 2 (EBP-2).
[0034] SEQ ID NO:3 is the amino acid sequence of ErbB2-binding
peptide 3 (EBP-3).
[0035] SEQ ID NO:4 is the amino acid sequence of ErbB2-binding
peptide 4 (EBP-4).
[0036] SEQ ID NO:5 is the amino acid sequence of ErbB2-binding
peptide 5 (EBP-5).
[0037] SEQ ID NO:6 is the amino acid sequence of ErbB2-binding
peptide 6 (EBP-6).
[0038] SEQ ID NO:7 is the amino acid sequence of ErbB2-binding
peptide 7 (EBP-7).
[0039] SEQ ID NO:8 is the amino acid sequence of ErbB2-binding
peptide 8 (EBP-8).
[0040] SEQ ID NO:9 is the amino acid sequence of ErbB2-binding
peptide 9 (EBP-9).
[0041] SEQ ID NO:10 is the amino acid sequence of ErbB2-binding
peptide 10 (EBP-10).
[0042] SEQ ID NO:11 is the amino acid sequence of ErbB2-binding
peptide 11 (EBP-11).
[0043] SEQ ID NO:12 is the amino acid sequence of ErbB2-binding
peptide 12 (EBP-12).
[0044] SEQ ID NO:13 is the amino acid sequence of ErbB2-binding
peptide 13 (EBP-13).
[0045] SEQ ID NO:14 is a putative nucleic acid sequence coding for
EBP-1.
[0046] SEQ ID NO:15 is a putative nucleic acid sequence coding for
EBP-2.
[0047] SEQ ID NO:16 is a putative nucleic acid sequence coding for
EBP-3.
[0048] SEQ ID NO:17 is a putative nucleic acid sequence coding for
EBP-4.
[0049] SEQ ID NO:18 is a putative nucleic acid sequence coding for
EBP-5.
[0050] SEQ ID NO:19 is a putative nucleic acid sequence coding for
EBP-6.
[0051] SEQ ID NO:20 is a putative nucleic acid sequence coding for
EBP-7.
[0052] SEQ ID NO:21 is a putative nucleic acid sequence coding for
EBP-8.
[0053] SEQ ID NO:22 is a putative nucleic acid sequence coding for
EBP-9.
[0054] SEQ ID NO:23 is a putative nucleic acid sequence coding for
EBP-10.
[0055] SEQ ID NO:24 is a putative nucleic acid sequence coding for
EBP-11.
[0056] SEQ ID NO:25 is a putative nucleic acid sequence coding for
EBP-12.
[0057] SEQ ID NO:26 is a putative nucleic acid sequence coding for
EBP-13.
[0058] SEQ ID NO:27 is the nucleic acid sequence of ErbB2 mRNA
(GenBank Accession Number M11730). See Appendix A for the GenBank
submission.
[0059] SEQ ID NO:28 is the amino acid sequence of ErbB2 protein
(GenBank Accession Number M11730). See Appendix A for the GenBank
submission.
[0060] SEQ ID NO:29 is the amino acid sequence of a biased peptide
phage library.
[0061] SEQ ID NO:30 is the amino acid sequence of a biased peptide
phage library.
[0062] SEQ ID NO:31 is the amino acid sequence of a biased peptide
phage library.
[0063] SEQ ID NO:32 is the amino acid sequence of a biased peptide
phage library.
[0064] SEQ ID NO:33 is the amino acid sequence of ErbB2-binding
peptide 14 (EBP-14; 02-124) derived from a biased peptide phage
library.
[0065] SEQ ID NO:34 is the amino acid sequence of ErbB2-binding
peptide 15 (EBP-15; 02-137) derived from a biased peptide phage
library.
[0066] SEQ ID NO:35 is the amino acid sequence of ErbB2-binding
peptide 16 (EBP-16; 02-140) derived from a biased peptide phage
library.
[0067] SEQ ID NO:36 is the amino acid sequence of ErbB2-binding
peptide 17 (EBP-17; 02-135) derived from a biased peptide phage
library.
[0068] SEQ ID NO:37 is the amino acid sequence of ErbB2-binding
peptide 18 (EBP-18; E-20) derived from a random peptide phage
library.
[0069] SEQ ID NO:38 is the amino acid sequence of ErbB2-binding
peptide 19 (EBP-19; C-19) derived from a random peptide phage
library.
[0070] SEQ ID NO:39 is the amino acid sequence of ErbB2-binding
peptide 20 (EBP-20; C-25) derived from a random peptide phage
library.
[0071] SEQ ID NO:40 is a putative nucleic acid sequence coding for
EBP-14.
[0072] SEQ ID NO:41 is a putative nucleic acid sequence coding for
EBP-15.
[0073] SEQ ID NO:42 is a putative nucleic acid sequence coding for
EBP-16.
[0074] SEQ ID NO:43 is a putative nucleic acid sequence coding for
EBP-17.
[0075] SEQ ID NO:44 is a putative nucleic acid sequence coding for
EBP-18.
[0076] SEQ ID NO:45 is a putative nucleic acid sequence coding for
EBP-19.
[0077] SEQ ID NO:46 is a putative nucleic acid sequence coding for
EBP-20.
BRIEF DESCRIPTION OF THE FIGURES
[0078] FIG. 1A is a bar graph showing ELISA results using the E12
and CB1 peptide phage clones.
[0079] FIG. 1B is a bar graph showing ELISA results using EC-1 and
new clones isolated from EC-1 biased phage display libraries. The
graph shows binding to the extracellular domain of ErbB2 (CBECD)
and the control binding to BSA.
[0080] FIG. 2A is a bar graph showing ELISA results using membrane
lysates and the CB1 peptide phage clone.
[0081] FIG. 2B is a bar graph showing ELISA results for EC-1 and
new clones isolated from EC-1 biased phage display library showing
binding to SKBR3 (which overexpress ErbB2) but not to MCF7 (which
minimally express ErbB2) lysates. Buffer alone (no lysate) is the
control. Additional negative controls include a negative phage
clone and no phage (blocker only).
[0082] FIG. 3 is a series of photographs showing the ability of the
peptide phage clones to bind to BT474 cells.
[0083] FIG. 4 is a series of photographs showing the ability of the
peptide phage clones to bind to SKBR3 cells.
[0084] FIG. 5 is a series of photographs showing lack of peptide
phage clone binding to MCF7 cells, which express only low levels of
ErbB2.
[0085] FIG. 6A is a Western blot analysis of SKBR3 cell lysates
made at various times following 15 minute treatment with 25 .mu.M
EC-1 peptide. Blots were probed with pY1248 phospho ErbB2 antibody,
pY877 phospho ErbB2 antibody and total ErbB2 antibody.
[0086] FIG. 6B is a graph showing the densitometric analysis of the
Western blots shown in FIG. 6A. The graph shows that EC-1 peptide
exposure inhibits phosphorylation of ErbB2 on tyrosine residues
pY877 and pY1248 but has no effect on the total expression level of
ErbB2.
[0087] It is to be understood that the drawings are not required
for enablement of the claimed invention.
DETAILED DESCRIPTION OF THE INVENTION
[0088] The invention relates in part to the identification and use
of peptides that bind specifically to ErbB2, and more specifically
to the extracellular domain of ErbB2. These peptides are referred
to herein as ErbB2 binding peptides (i.e., EBP). In addition to
binding ErbB2, the EBP can also interfere with the functioning of
ErbB2 (particularly in cancer cells) by interfering with the
ability of ErbB2 to become phosphorylated, to interact with other
compounds (e.g., polypeptides such as ErbB1 (EGFR), ErbB3, ErbB4 or
mucins such as mucin 4), and/or to transduce a signal into a cell.
The peptides, and their functional equivalents are useful in the
diagnosis and treatment of disorders characterized by ErbB2
overexpression. They are also useful in the isolation and,
optionally, removal of cells that overexpress ErbB2 (e.g., tumor
cells). These peptides can also be used to identify further EBP.
These and other aspects will be described in greater detail
herein.
[0089] ErbB2 is the human homolog of the protein encoded by the neu
oncogene and is a receptor-like tyrosine kinase. As used herein,
ErbB2 is referred to as HER-2 and c-neu protein interchangeably.
The nucleotide and amino acid sequence of the human c-erbB2 mRNA
and ErbB2 protein are provided herein as SEQ ID NO:27 and SEQ ID
NO:28 (from GenBank Accession Number X03363). ErbB2 mRNA is
approximately 4.8 kb in length and the protein it encodes is 1255
amino acids in length and approximately 185 kilodalton (kD). ErbB2
has an extracellular domain having two cysteine rich repeat
clusters, a transmembrane domain, and an intracellular kinase
domain. The polypeptide can be glycosylated at a number of sites.
The extracellular domain corresponds to nucleotides 151 to 2109
relative to SEQ ID NO:27 and amino acid residues 1 to 653 relative
to SEQ ID NO:28 (GenBank Accession Number M11730). (Coussens et
al., Science 230:1132.)
[0090] ErbB2 is expressed at low or negligible levels in most
normal adult tissues, with the possible exception of kidney. (Mori
et al., Laboratory Investigation, 61:93.) ErbB2 is expressed fetal
tissues including fetal renal tubules and fetal epithelium. (Natali
et al., Int. J. Cancer, 1990 45(3):457-461.) ErbB2 gene
amplification has been observed in a number of primary cancers,
metastatic lesions, and cancer cell lines. This amplification
results in ErbB2 overexpression in several tumor types including
breast carcinoma, glioblastoma, lung cancer, prostate cancer,
salivary gland adenocarcinoma, gastric and colon adenocarcinomas,
renal adenocarcinoma, mammary gland carcinoma, ovarian cancer,
cervical cancer, colorectal carcinomas and adenocarcinomas, thyroid
tumors, Ewing's sarcoma, and squamous carcinomas. Moreover, ErbB2
overexpression is found in tumors of 30% of breast cancer patients
and this increase in expression correlates with poor patient
prognosis. In several breast cancer cell lines, ErbB2 is
co-amplified with Grb7 by virtue of the fact that erbB2 and the
gene that codes for Grb7 are located close to each other on
chromosome 17. Combined overexpression of ErbB2 and Grb7 proteins
in these tumors likely up-regulates a signaling pathway which plays
an important role in tumor pathogenesis. (Janes et al., 1997, J
Biol Chem 272: 8490-8497; Tanaka et al., 1997 Cancer Research
57:28-31.) ErbB2 overexpression has also been reported to be
associated with early stages of colorectal cancer, as it has been
demonstrated that pre-neoplastic lesions express higher levels of
ErbB2 than do neoplastic lesions.
[0091] The invention involves, in various related and
interconnected aspects, isolated ErbB2-binding peptides (i.e.,
EBP), functional equivalents and modifications and variants
thereof, unique fragments thereof, nucleic acid molecules encoding
the foregoing, as well as diagnostics and therapeutics relating
thereto.
[0092] The invention provides in one aspect peptides comprising an
amino acid sequence selected from the group consisting of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID
NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID
NO:12, SEQ ID NO:13, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ
ID NO:36, SEQ ID NO:37, SEQ ID NO:38 and SEQ ID NO:39, and
functional equivalents thereof. The amino acid sequences of the EBP
of the invention (EBP-1 through to EBP-20, respectively) are listed
in Table 1.
[0093] The peptides can exhibit therapeutic activity by binding to
and inhibiting the activity of ErbB2. Such an activity is not
dependent upon conjugation of the peptide to a cytotoxic agent.
Accordingly, in one aspect, the EBP are ErbB2 antagonists. In this
sense, the peptides may block the interaction of ErbB2 with its
native ligand(s) or with other proteins with which ErbB2 interacts
such as other ErbB family members (e.g., ErbB1, ErbB3 or ErbB4).
The ability to block the interaction of ErbB2 (either in monomeric
or dimeric form) with ErbB1, ErbB3 or ErbB4 may be particularly
important for inhibiting metastatic spread in a subject.
Accordingly, peptides capable of inhibiting such interactions are
particularly suitable to the prevention or treatment of metastasis.
The peptides of the invention can also function by inhibiting the
formation of larger complexes that contain ErbB2. In this latter
instance, the peptides bind directly to ErbB2 yet their effect is
indirect in that they preclude ErbB2 interacting proteins (such as
ErbB1, ErbB3 and ErbB4) from interacting with other peptides or
polypeptides (such as, for example, heregulin).
[0094] To date, few native ErbB2 ligands have been identified. One
such ligand is the membrane mucin MUC4/sialomucin (SMC) complex
which reportedly contacts ErbB2 via an intramembrane domain. As a
result, both ErbB2 and SMC are expressed by the same cell in order
for interaction to occur. Binding of SMC by ErbB2 reportedly
modulates the phosphorylation status of ErbB2 in the presence and
absence of heregulin. It has been reported that expression of SMC
promotes tumor growth in vivo. The peptides of the invention can be
used to preclude the binding of ErbB2 to SMC or to disrupt
pre-formed complexes between ErbB2 and SMC.
[0095] Heterodimers of ErbB2 with ErbB1, ErbB3 or ErbB4 appear to
evade normal cellular controls by, for example, decreasing the rate
of internalization or avoiding degradation once internalized. It
has been postulated that one way in which ErbB2 functions in
cancers is to avoid internalization, leading to continuous cell
surface expression of an activated receptor tyrosine kinase. The
peptides of the invention can be used to induce overall cellular
ErbB2 internalization by, for example, increasing internalization
rates. One way in which the peptides can accomplish this is by
conjugation to internalization sequences such as translocation
sequences. The peptides of the invention can also prolong the
internalization time of ErbB2, for example, by increasing the time
necessary to degrade the peptide/ErbB2 complex and/or stimulating
degradation of ErbB2 once it is internalized. Interference with the
normal trafficking of internalized ErbB2can lead to lower levels of
ErbB2 on the cell surface.
[0096] It is clear that ErbB2 overexpression is a hallmark of many
cancer types, and particularly a hallmark of metastatic cells in
such cancers. Thus, ErbB2 may be involved in the homing of primary
cancer cells to secondary sites within the body. The peptides of
the invention by binding to the extracellular domain of ErbB2 may
block ErbB2 interactions necessary for malignant and metastatic
phenotypes.
[0097] The peptides may also impact upon signaling events
downstream of ErbB2 extracellular engagement. As an example, ErbB2
may signal intracellularly during interaction with its native
ligand or other factors with which it dimerizes or complexes. Such
signaling may determine the malignant and metastatic phenotype of
the ErbB2 expressing cell. The peptide may prevent this by blocking
such interaction yet not mimicking the signal transduction induced
by the native ligand. ErbB2 containing heterodimers can recruit
MAPK and PI3K. Signaling through ErbB2 reportedly affects
activation of cyclin and CDK complexes, thereby inhibiting
apoptosis and stimulating proliferation of such cells. Downstream
targets of ErbB2 signaling include cell cycle regulators such as
p21.sup.wafl.
[0098] EBPs and functional equivalents thereof which function as
ErbB2 antagonists can be used in combination with other therapeutic
agents in order to inhibit a disorder characterized by ErbB2
overexpression. It is expected that such a combination will yield a
synergistic response (i.e., one that is greater than the additive
effects of the therapeutic agents when used alone) because the EBP
and the other therapeutic agents will generally function via
different pathways. Accordingly, the combination can be used to
increase the efficacy of a particular therapeutic agent without the
need for higher doses (and associated systemic toxicity) of the
therapeutic agent.
[0099] Another synergistic combination is the administration of the
peptides of the invention with an anti-estrogen. It has been
reported that estrogen can modulate ErbB2 signaling via the
estrogen receptor. Accordingly, the combination of an EBP with an
anti-estrogen can induce a synergistic response.
[0100] The peptides bind strongly and specifically to the
extracellular domain of ErbB2. As shown in the Examples, peptide
phage displaying a peptide having an amino acid sequence of SEQ ID
NO:1 through to SEQ ID NO:13, and SEQ ID NO:33 through to SEQ ID
NO:39, inclusive, yield a reproducible ErbB2-specific signal by
ELISA and immunofluorescence assay. By binding to the extracellular
domain of ErbB2, the EBP can inhibit the function of ErbB2 by, at a
minimum, preventing its association with extracellular factors such
as its naturally occurring ligand(s). Specific inhibitors of ErbB2
are useful in elucidating the complete function and exact role of
ErbB2 in cancer progression, as well as in the development of
cancer therapeutics which target ErbB2. The peptides bind
specifically to ErbB2 and not to any other member of the ErbB
family of growth factor receptors. In some embodiments, the
peptides bind with greater affinity to ErbB2 than to other proteins
(e.g., more than a five fold greater affinity, more than a ten fold
greater affinity or more than a fifty fold greater affinity).
[0101] Other peptides and polypeptides that bind to ErbB2 have been
identified previously, including Herceptin, a monoclonal antibody
specific for ErbB2 (Genentech, Inc., South San Francisco, Calif.).
Due to their small size, the peptides of the invention can have
improved pharrnacokinetics, including increased tumor targeting and
penetration. Park et al. recently reported the identification of
another peptide that binds to the extracellular domain of ErbB2.
(Park et al., Nature Biotechnology, 18:194.) The peptide inhibited
in vitro cell proliferation and colony formation, as well as in
vivo tumor formation in nude mice bearing ErbB2-overexpressing
cells. It also increased the sensitivity of ErbB2-overexpressing
cells to radiation and significantly increased the effectiveness of
doxorubicin inhibition of tumor growth when administered
concurrently. Park et al. rationally designed the peptide based on
the structure of CDR regions from a known anti-ErbB2 antibody. The
peptides of the present invention share no homology with that
reported by Park et al.
[0102] As described in the Examples, the EBP of the invention were
generated and identified using random peptide phage display
technology. Using this technology, it was possible to produce and
screen candidate peptides more rapidly and with less cost than
would have been possible with for example antibody technology.
Accordingly, the discovery of the lead compounds described herein
was significantly faster and less expensive than traditional
methods of lead discovery.
[0103] As indicated in Table 1, all the EBP possess at least two
cysteine residues between which there exists a stretch of 9-10
amino acid residues. Additionally, the EBP may also possess
flanking amino acid sequences beyond the cysteines at either or
both their amino or carboxy ends. The length of the EBP ranges from
11-20 amino acid residues.
[0104] Although listed above in linear form, the EBP may be cyclic
as well, or at least capable of being cyclized. One way in which
the EBP may be easily cyclized is through the formation of a
disulfide bond between the two cysteines commonly possessed by each
of the EBP. Alternatively, one or more cysteine residues may be
introduced into the peptides. As indicated by the EBPs of Table 1,
it is not necessary that the cysteine residues be located at the
ends of the peptides (i.e., at the first and last amino acid
positions). Rather, it is only required that the cysteine residues
are spaced far enough apart from each other to allow for a
disulfide bond to be formed (e.g., with 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20 or more residues in between the
two cysteine residues). The peptide having an amino acid sequence
of SEQ ID NO:1 is an example of a peptide in which the disulfide
bonded cysteines may be those already present at positions 5 and
16, or alternatively, additional cysteine residues may be added to
the amino and carboxy termini. In some instances, it may be
preferable to substitute thio-ether linkages for the disulfide bond
produced between cysteine residues. Such a modification is
described in PCT patent application WO 98/02176 (PCT/US97/12501),
and in Oligino et al., 1997, J Mol Chem 272:29046-29052 and Lou et
al., 1999, Arch Biochem Biophys, 372:309-314.
[0105] In still other embodiments, the linkage may be a peptide
linkage between the two arms of the peptide. It is to be understood
that the invention embraces other varieties of linkages known in
the art for the purpose of producing a cyclic peptide. The
proceeding examples of linking molecules are also suitable for the
conjugation of EBP to agents such as diagnostic (e.g., imaging
agents) and therapeutic (e.g., anti-cancer) agents. Examples of
suitable linking molecules which can be used include bifunctional
crosslinker molecules. The crosslinker molecules may be
homobifunctional or heterobifunctional, depending upon the nature
of the molecules to be conjugated. Homobifunctional cross-linkers
have two identical reactive groups. Heterobifunctional
cross-linkers are defined as having two different reactive groups
that allow for sequential conjugation reaction. Various types of
commercially available crosslinkers are reactive with one or more
of the following groups: primary amines, secondary amines,
sulphydryls, carboxyls, carbonyls and carbohydrates. Examples of
amine-specific cross-linkers are bis(sulfosuccinimidyl) suberate,
bis[2-(succinimidooxycarbonyloxy)ethyl] sulfone, disuccinimidyl
suberate, disuccinimidyl tartarate, dimethyl adipimate.2 HCl,
dimethyl pimelimidate.2 HCl, dimethyl suberimidate.2 HCl, and
ethylene glycolbis-[succinimidyl-[succinate]]. Cross-linkers
reactive with sulfhydryl groups include bismaleimidohexane,
1,4-di-[3'-(2'-pyridyldithio)-propionamido)]butane,
1-[p-azidosalicylamido]-4-[iodoacetamido]butane, and
N-[4-(p-azidosalicylamido)
butyl]-3'-[2'-pyridyldithio]propionamide. Crosslinkers
preferentially reactive with carbohydrates include azidobenzoyl
hydrazine. Crosslinkers preferentially reactive with carboxyl
groups include 4-[p-azidosalicylamido]butylamine.
Heterobifunctional cross-linkers that react with amines and
sulfhydryls include N-succinimidyl-3-[2-pyridyldithio]propionate,
succinimidyl[4-iodoacetyl]aminobenzoate, succinimidyl
4-[N-maleimidomethyl] cyclohexane-1-carboxylate,
m-maleimidobenzoyl-N-hydroxysuccinimide ester, sulfosuccinimidyl
6-[3-[2-pyridyldithio] propionamido]hexanoate, and
sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate.
Heterobifunctional cross-linkers that react with carboxyl and amine
groups include 1-ethyl-3-[[3-dimethylaminopropyl] carbodiimide
hydrochloride. Heterobifunctional cross-linkers that react with
carbohydrates and sulhydryls include
4-[N-maleimidomethyl]-cyclohexane-1-carboxylhydrazide.2 HCl,
4-(4-N-maleimidophenyl)-butyric acid hydrazide.2 HCl, and
3-[2-pyridyldithio]propionyl hydrazide. The cross-linkers are
bis-[.beta.-4-azidosalicylamido)ethyl]disulfide and glutaraldehyde.
Amine or thiol groups may be added at any nucleotide of a synthetic
nucleic acid so as to provide a point of attachment for a
bifunctional crosslinker molecule. The nucleic acid may be
synthesized incorporating conjugation-competent reagents such as
Uni-Link AminoModifier, 3'-DMT-C6-Amine-ON CPG, AminoModifier II,
N-TFA-C6-AminoModifier, C6-ThiolModifier, C6-Disulfide
Phosphoramidite and C6-Disulfide CPG (Clontech, Palo Alto, Calif.).
The peptides of the invention can also comprise one or more
non-peptide linkages in their backbones.
[0106] The invention further intends the use of fragments of the
EBPs disclosed herein, including unique fragments and functionally
equivalent fragments of the EBPs in the diagnostic and therapeutic
methods described herein. Unique fragments can be used to prepare
antibodies that are specific for the EBP. Functionally equivalent
fragments are useful as substitutes for the EBPs of the invention.
This is particularly useful when achieving the smallest
ErbB2-binding peptide possible is desired.
[0107] A unique fragment of an EBP, in general, has the features
and characteristics of unique fragments of nucleic acid molecules
as discussed herein. A unique fragment can act as a signature for
identifying peptides or polypeptides that comprise the amino acid
sequences selected from the group consisting of SEQ ID NO:1 through
to SEQ ID NO:13, and SEQ ID NO:33 through to SEQ ID NO:39,
inclusive. Such polypeptides may be native binding partners of
ErbB2, and more preferably, may be native inhibitory binding
partners of ErbB2. Those skilled in the art are well versed in
methods for selecting unique amino acid sequences. A comparison of
the sequence of the fragment to those in known databases is all
that is typically required. Preferably, the unique fragment is
unique in humans, i.e., it is long enough to assure that its
precise sequence is not found in other molecules encoded by the
human genome which have been identified and publicly disclosed as
of the date of invention and/or the filing date of this
application.
[0108] As will be recognized by those skilled in the art, the size
of the unique fragment will depend upon factors such as whether the
fragment constitutes a portion of a conserved motif. Thus, some
regions of SEQ ID NO:1 will require longer segments to be unique
while others will require only short segments, typically between 5
and 12 amino acids (e.g., 5, 6, 7, 8, 9, 10, 11 and 12 amino acids
long or more, including each integer up to the full length).
Virtually any segment of SEQ ID NO:1 through to SEQ ID NO:13, and
SEQ ID NO:33 through to SEQ ID NO:39, inclusive, that is 9 or more
amino acids in length will be unique.
[0109] Unique fragments preferably will have the same functionality
as the full length peptides provided herein, including the ability
to bind to the extracellular domain of ErbB2. The unique fragments
can be used in place of the full length peptides in order to
inhibit or interfere with ErbB2 functioning (e.g., binding to
another compound or signal transduction), or they can be used to
generate binding partners to the full length peptide (e.g.,
antibodies). Unique fragments may be those that 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 amino acids of the full
length peptide. In preferred embodiments, the unique fragments are
at least 5, at least 10, or at least 15 amino acids in length.
[0110] One particularly important subset of EBP fragments are
functionally equivalent fragments of EBP. Generally, a "functional
equivalent of an EBP" is a peptide or small molecule that is able
to function in a similar manner to an EBP disclosed herein. For
example, the functional equivalent would bind to ErbB2 and
interfere with the functioning of ErbB2 and its role in
tumorigenesis and metastasis. The functional equivalent may be
capable of inhibiting ErbB2 interaction with other factors and/or
disrupting pre-formed complexes that contain ErbB2. The ability of
a functional equivalent to bind to ErbB2 specifically can be
determined using the binding assays described herein or known in
the art. The preferred functional equivalents are those that bind
specifically to the extracellular domain of ErbB2 but not to other
ErbB family members. The functional equivalent of the EBP may be
peptide, non-peptide or chimeric in nature. The synthesis of such
functionally equivalent variants is described below. In some
preferred instances, a functional equivalent mimics the EBP of the
invention with respect to size, structure, and charge distribution.
In other embodiments, the functional equivalent mimics the EBP of
the invention by comprising partial sequence from the EBPs
described herein. For example, a functional equivalent can be a
peptide that is identical to an EBP described herein at, for
example, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the
amino acid positions, with the non-identical positions occupied by
conservative amino acids or amino acid analogs. In some
embodiments, non-conservative amino acid substitutions can also be
introduced into the functional equivalents. Accordingly, functional
equivalents of 11 amino acid peptides may have 1, 2, 3, 4, 5, 6, 7,
8, 9, 10 or 11 substitutions, while those functional equivalents of
19 or 20 amino acid peptides may have 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 substitutions.
Preferably, the functional equivalent peptides have substitutions
at fewer than 50% of the amino acid positions, at fewer than 40% of
the amino acid positions, at fewer than 30% of the amino acid
positions, at fewer than 20% of the amino acid positions, or at
fewer than 10% of the amino acid positions.
[0111] In other embodiments, functional equivalents of the peptides
described herein possess a number of amino acids identical in
sequence to the EBPs and such amino acids are arranged in a
contiguous manner. That is, some functional equivalents possess 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18, or 19
contiguous amino acids that are identical in sequence to the EBPs
described herein.
[0112] Functional equivalence refers to an equivalent activity
(e.g., binding to ErbB2), however it also embraces variation in the
level of such activity. For example, a functional equivalent is a
variant that binds to ErbB2 with lesser, equal, or greater affinity
than the EBPs described herein, provided that the variant is still
useful in the invention (e.g., it binds specifically and uniquely
to ErbB2, and optionally interferes with ErbB2 function).
[0113] The skilled artisan will realize that conservative amino
acid substitutions may also be made in the EBP disclosed herein to
provide functional equivalents of the foregoing peptides. As used
herein, a "conservative amino acid substitution" refers to an amino
acid substitution which does not alter the relative charge or size
characteristics of the peptide or protein in which the amino acid
substitution is made. Functional equivalents can be prepared
according to methods for altering peptide sequence known to one of
ordinary skill in the art such as are found in references which
compile such methods, e.g. Molecular Cloning: A Laboratory Manual,
J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Current
Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John
Wiley & Sons, Inc., New York. Exemplary functional equivalents
of the EBPs include conservative amino acid substitutions of SEQ ID
NO:1 through to SEQ ID NO:13, inclusive. Conservative substitutions
of amino acids include substitutions made amongst amino acids
within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R,
H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
[0114] Conservative amino-acid substitutions in the amino acid
sequence of EBPs to produce functionally equivalent variants may be
made by alteration of nucleic acid molecules encoding the EBPs
(e.g., SEQ ID NO:14 through to SEQ ID NO:26, and SEQ ID NO:40
through to SEQ ID NO:46, inclusive, and degenerates thereof). Such
substitutions can be made by a variety of methods known to one of
ordinary skill in the art. For example, amino acid substitutions
may be made by chemical synthesis of a nucleic acid molecule
encoding an EBP using an automated DNA synthesizer, for example.
The activity of functionally equivalent fragments of the EBP can be
tested by cloning the nucleic acid molecule encoding the altered
EBP into a bacterial or mammalian expression vector, introducing
the vector into an appropriate host cell, expressing the altered
EBP, and testing for the functional capability of the EBPs so
produced using methods as disclosed herein.
[0115] In some preferred embodiments, the peptides and functional
equivalents thereof are prepared using chemical synthesis using
standard peptide chemistry synthesis methods, with which the
ordinary artisan will be familiar.
[0116] Modifications made to the nucleic acid molecules which
encode EBPs can include deletions, point mutations, truncations,
potentially resulting in amino acid additions, deletions or
substitutions and can serve to: 1) enhance a property of an EBP,
such as peptide stability in an expression system or the stability
of peptide-protein binding; 2) provide a novel activity or property
to an EBP, such as addition of an antigenic epitope, a detectable
moiety or a localization signal sequence (such as the translocation
sequences discussed herein); or 3) to provide equivalent or better
binding to ErbB2. Alternatively, modifications can be made directly
to the peptide, such as by cleavage, amino acid additions,
deletions or substitutions including substitutions with non-natural
amino acids, formation during peptide or peptidomimetic synthesis
of bonds other than peptide bonds such as pseudo peptide bonds,
addition of a linker molecule, addition of a detectable moiety,
such as biotin, addition of a fatty acid, and the like.
Modifications also embrace fusion proteins comprising all or part
of the EBP amino acid sequence (i.e., SEQ ID NO:1 through to SEQ ID
NO:13, and SEQ ID NO:33 through to SEQ ID NO:39, inclusive).
[0117] Mutations of nucleic acid molecules which encode EBPs
preferably preserve the amino acid reading frame of the coding
sequence, and preferably do not create regions in the nucleic acid
molecule which are likely to hybridize to form secondary
structures, such a hairpins or loops, which can be deleterious to
expression of the variant peptide.
[0118] Mutations can be made by selecting an amino acid
substitution, or by random mutagenesis of a selected site in a
nucleic acid molecule which encodes the peptide. Variant peptides
are then expressed and tested for one or more activities to
determine which mutation provides a variant peptide with the
desired properties. Further mutations can be made to EBPs and
variants thereof that are silent as to the amino acid sequence of
the peptide, but which provide preferred codons for translation in
a particular host. The preferred codons for translation of a
nucleic acid molecule in, e.g., E. coli, are well known to those of
ordinary skill in the art. Still other mutations can be made to the
noncoding sequences of nucleic acid molecules to enhance expression
of the peptide.
[0119] The invention also embraces variants of the EBPs described
above. As used herein, a "variant" of an EBP is a peptide or
peptidomimetic which contains one or more modifications to the
primary amino acid sequence of an EBP. The invention embraces
variants of EBP that possess substituents at various positions.
Modifications to the EBPs that preserve the size, structure, and
charge distribution of the EBP are preferred in some embodiments. A
person of ordinary skill in the art is capable of determining the
size, structure, and charge distribution characteristics of the
EBPs disclosed herein and of designing other putative inhibitory
agents based on this knowledge.
[0120] Variants can include peptides which are modified
specifically to alter a feature of the polypeptide unrelated to its
binding and inhibitory activity. For example, cysteine residues can
be substituted or deleted to prevent disulfide linkages, which may
be desirable if other means of linkage are available in the
peptide. Similarly, certain amino acids can be changed to enhance
expression of the variant by eliminating proteolysis by proteases
in an expression system (e.g., dibasic amino acid residues in yeast
expression systems in which KEX2 protease activity is present).
[0121] It will be apparent to one of ordinary skill in the art that
the invention embraces the synthesis of a wide variety of variants
having any combination of amino acid analogs and/or peptidomimetic
residues as described above and as are known in the art. (See, for
example, Burke, et al., Bio. Med. Chem. Lett., 9:347-352, 1999.)
Non-amino acid substitutions are known in the art and are described
in, inter alia, Burke et al., 1999, Bioorg Med Chem Lett,
9:347-352; Long et al., 1999, Biochem Biophys Res Commun
264:902-908; Yao et al., 1999, J. Med Chem, 42:25-35; Ye et al.,
1995, J Med Chem, 38:4270-4275. Glutamine (Glu) residues may be
replaced with .alpha.-amino-adipate (Adi) molecules and tyrosine
positions may be substituted with 4-carboxymethyl-Phe. Glutamic
acid residues can be modified to possess an additional methylene
group or they may simply be substituted with .alpha.-amino-adipate.
Such modifications have been found to increase binding affinity in
some species of antagonists. (Long et al., 1999, Biochem. Biophys.
Res. Commun. 264:902-908.) Yao et al. reported the synthesis and
use of N-.alpha.-oxalyl groups in certain signaling inhibitory
molecules. (Yao, et al., J. Med. Chem., 42:25-35, 1999) Other
residues which may be incorporated into the ErbB2 specific variants
include the non-naturally occurring amino acid
1-aminocyclohexylcarboxylic acid (Ac.sub.6c) or
1-aminocyclohexanecarboxylic acid as replacements for glutamic acid
residues. (Gay et al., Int. J. Cancer, 83:235-241, 1999). Other
non-naturally occurring amino acids can be used such as
2-azetidinecarboxylic acid or pipecolic acid (which have
6-membered, and 4-membered ring structures respectively) for
proline residues, and S-ethylisothiourea, 2-NH.sub.2-thiazoline and
2-NH.sub.2-thiazole. Also useful in the synthesis of EBP variants
is the use of an asparagine residue substituted with
3-indolyl-propyl at the C terminal carboxyl group. Further
potential modifications of EBPs envisioned by the invention include
modifications of cysteines, histidines, lysines, arginines,
tyrosines, glutamines, asparagines, prolines, and carboxyl groups
as are well known in the art and are described in U.S. Pat. No.
6,037,134. Synthesis of the afore-mentioned variants is described
in the cited references and is well within the realm of one of
ordinary skill in the art.
[0122] The EBP may be modified to introduce or stabilize certain
structural features. As an example, structural features including,
but not limited to, .beta.-turns can be introduced into inhibitory
peptides. .beta.-turns can be introduced into the EBP variants by
synthesizing such variants with a proline residue or a
glycine-proline combination, or a 1-aminocyclohexanecarboxylic acid
as a substitution for glutamic acid residues. (Garcia-Echeverria, J
Med Chem. 41(11):1741-4, 1998) In other embodiments and as
discussed above, it may be preferred that the variants possess a
stable cyclic structure. This may be achieved by generating
thio-ether cyclized peptides such as those reported by Oligino et
al. and Lou et al. (Oligino et al., J. Biol. Chem. 272:29046-29052,
1997; Lou et al., Arch Biochem Biophys, 372:309-314, 1999) This
modification ensures a stable conformation which, in some
instances, may be optimal for ErbB2 binding and potentially
functional inhibition. The cyclic structure can also be formed via
other linkages such as, but not limited to, peptide bonds.
[0123] As used herein with respect to polypeptides, the term
"isolated" means separated from its native environment in
sufficiently pure form so that it can be manipulated or used for
any one of the purposes of the invention. Thus, "isolated" means
sufficiently pure to be used (i) to raise and/or isolate
antibodies, (ii) as a reagent in an assay, (iii) as a therapeutic
agent, or (iv) for sequencing, etc.
[0124] The EBP can be produced in a number of ways. In a preferred
embodiment, the peptides are identified using a phage display
technology as described in the Examples and in PCT patent
application WO98/02176 (PCT/US97/12501). Alternatively, they may be
synthesized using a peptide synthesizer. Alternatively, an
expression vector which incorporates a nucleic acid molecule
encoding the peptide, such as SEQ ID NO:14 through to SEQ ID NO:26,
and SEQ ID NO:40 through to SEQ ID NO:46, inclusive or degenerates
thereof, may be introduced into cells to induce production of the
EBP. In another method, mRNA transcripts encoding the EBP may be
microinjected or otherwise introduced into cells to cause
production of the encoded peptide. Translation of mRNA in cell-free
extracts such as the reticulocyte lysate system also may be used to
produce EBPs. Those skilled in the art also can readily follow
known methods for isolating EBP. These include, but are not limited
to, immunochromatography, HPLC, size-exclusion chromatography, and
ion-exchange chromatography.
[0125] The EBP disclosed herein will be useful as is or as a lead
compound for developing further EBP including ErbB2 antagonists.
Other EBPs may be generated as variants of the EBPs described
herein (as described above) or alternatively they may be produced
in a more random fashion and identified via binding and signaling
assays in comparison with the EBP.
[0126] Known binding peptides, such as the EBP described herein,
may be subjected to directed or random chemical modifications such
as acylation, alkylation, esterification, amidification, etc. to
produce structural analogs which may function as antagonists.
[0127] In an alternative approach, additional EBP can be rationally
designed. One way of doing this involves modeling the binding site
of ErbB2 complexed with or without, for example, a EBP, using X-ray
crystallography or NMR or Raman spectroscopy. In addition, the
successful use of computer-based algorithms to model binding sites
is discussed in U.S. Pat. No. 5,741,713, the entire contents of
which are incorporated by reference herein. The strategy usually
involves computer-based structural modeling of the binding site
including its conformation, reactive groups, and charge groups, and
generally requires knowledge of the three-dimensional structure of
the binding site obtained by X-ray crystallography or NMR or Raman
spectroscopy. With this knowledge, the requirements for a useful
antagonist such as that disclosed herein can be determined, and
rational design of synthetic antagonists can follow.
[0128] Rational design of EBP variants can also be accomplished by
comparing and contrasting the amino acid sequences of the peptides
disclosed herein (i.e., SEQ ID NO:1 through to SEQ ID NO:13, and
SEQ ID NO:33 through to SEQ ID NO:39, inclusive). A study of the
amino acid sequence as well as a structural analysis and subsequent
comparison with peptides which bind to other ErbB family members
(e.g., ErbB4) and not ErB2, can elucidate the amino acid residues
and three-dimensional conformation involved in the binding
specificity. Random or directed mutation of the putative amino acid
residues involved in the recognition and/or binding of the peptide
to ErbB2 can help to identify further binding and inhibitory
peptides, as described herein.
[0129] One of skill in the art will be familiar with methods for
predicting the effect on protein conformation of a change in
protein sequence, and can thus "design" a variant which functions
as an antagonist according to known methods. One example of such a
method is described by Dahiyat and Mayo in Science 278:82-87, 1997,
which describes the design of proteins de novo. The method can be
applied to a known peptide to vary only a portion of the amino acid
sequence. By applying the computational methods of Dahiyat and
Mayo, specific variants of the disclosed peptide can be proposed
and tested to determine whether the variant retains a desired
conformation and the ability to bind and potentially inhibit ErbB2.
Similarly, Blake (U.S. Pat. No. 5,565,325) teaches the use of known
structures to predict and synthesize variants with similar or
modified function.
[0130] Other methods for preparing or identifying peptides which
bind to a particular target are known in the art. Molecular
imprinting, for instance, may be used for the de novo construction
of macromolecular structures such as peptides which bind to a
particular molecule. See, for example, Kenneth J. Shea, Molecular
Imprinting of Synthetic Network Polymers: The De Novo synthesis of
Macromolecular Binding and Catalytic Sites, TRIP Vol. 2, No. 5, May
1994; Klaus Mosbach, Molecular Imprinting, Trends in Biochem. Sci.,
19(9) January 1994; and Wulff, G., in Polymeric Reagents and
Catalysts (Ford, W. T., Ed.) ACS Symposium Series No. 308, pp
186-230, American Chemical Society (1986). One method for preparing
mimics of EBPs involves the steps of: (i) polymerization of
functional monomers around a known substrate (i.e., the template or
in this case, the EBP) that exhibits a desired activity; (ii)
removal of the template molecule; and then (iii) polymerization of
a second class of monomers in the void left by the template, to
provide a new molecule which exhibits one or more desired
properties which are similar to that of the template. This method
can be used to generate both peptide and non-peptide variants.
Non-peptide variants can be comprised of compounds such as
polysaccharides, nucleosides, drugs, nucleoproteins, lipoproteins,
carbohydrates, glycoproteins, steroids, lipids, and other
biologically active materials. This method is useful for designing
a wide variety of biological mimics that are more stable than their
natural counterparts, because they are typically prepared by the
free radical polymerization of functional monomers, resulting in a
compound with a nonbiodegradable backbone. Other methods for
designing such molecules include, for example, drug design based on
structure activity relationships which require the synthesis and
evaluation of a number of compounds and molecular modeling.
[0131] In important embodiments, peptide variants are made and
screened using the phage display technology described herein.
Peptide variants can be synthesized using degenerate
oligonucleotides which are biased for a sequence encoding a known
amino acid sequence. In a preferred embodiment, the phage libraries
are made using the Fuse5 vector. (Scott and Smith, 1990,
249:386-90; Smith and Scott, Methods Enzymol 1993, 217:228-57)
These techniques are well known in the art.
[0132] EBPs can be synthesized from peptides or other biomolecules
including but not limited to saccharides, fatty acids, sterols,
isoprenoids, purines, pyrimidines, derivatives or structural
analogs of the above, or combinations thereof, and the like. Also
envisioned in the invention is the synthesis of ErbB2 specific
binding molecules made from non-natural amino acids (as described
herein), peptoids, random bio-oligomers (U.S. Pat. No. 5,650,489),
benzodiazepines, diversomeres such as dydantoins, dipeptides,
nonpeptidal peptidomimetics with a beta-D-glucose scaffolding,
oligocarbamates or peptidyl phosphonates.
[0133] Many if not all of these compounds can be synthesized using
recombinant or chemical library approaches. One advantage of using
libraries for inhibitor identification is the facile manipulation
of millions of different putative candidates of small size in small
reaction volumes (i.e., in synthesis and screening reactions). A
vast array of candidate agents can be generated from libraries of
synthetic or natural compounds. Libraries of natural compounds in
the form of bacterial, fungal, plant and animal extracts are
available or can readily be produced. Natural and synthetically
produced libraries and compounds can be readily modified through
conventional chemical, physical, and biochemical means. Another
advantage of libraries is the ability to synthesize binding
molecules that might not otherwise be attainable using naturally
occurring sources, particularly in the case of non-peptide
moieties.
[0134] Methods for preparing libraries of molecules are known in
the art and several libraries are commercially available. Libraries
of interest in the invention include peptide libraries (including
synthetic peptide libraries, phage display libraries, and
peptides-on-plasmid libraries), polysome libraries, randomized
oligonucleotide libraries (including aptamer libraries), chemical
libraries, synthetic organic combinatorial libraries (including
small molecule libraries), and the like.
[0135] Degenerate peptide libraries can be readily prepared in
solution. Peptide libraries can also be in immobilized form as
bacterial flagella display libraries or as phage display libraries.
Peptide ligands can be selected from combinatorial libraries of
peptides containing at least one amino acid. Alternatively,
libraries can be synthesized from peptoids and non-peptide
synthetic moieties. Agents that contain non-peptide synthetic
moieties are less subject to enzymatic degradation compared to
their naturally-occurring counterparts. The libraries can also
comprise cyclic carbon or heterocyclic structure and/or aromatic or
polyaromatic structures substitutions.
[0136] Small molecule combinatorial libraries may also be
generated. A combinatorial library of small organic compounds is a
collection of closely related analogs that differ from each other
in one or more points of diversity and are synthesized by organic
techniques using multi-step processes. Combinatorial chemistry
libraries include a large number of small organic compounds. One
type of combinatorial library is prepared by means of parallel
synthesis methods to produce a compound array. A "compound array"
as used herein is a collection of compounds identifiable by their
spatial addresses in Cartesian coordinates and arranged such that
each compound has a common molecular core and one or more variable
structural diversity elements. The compounds in such a compound
array are produced in parallel in separate reaction vessels, with
each compound identified and tracked by its spatial address.
Examples of parallel synthesis mixtures and parallel synthesis
methods are provided in PCT published patent application
WO95/18972, published Jul. 13, 1995 and U.S. Pat. No. 5,712,171
granted Jan. 27, 1998 and its corresponding PCT published patent
application WO96/22529, which are hereby incorporated by
reference.
[0137] As stated herein, other EBP can be generated and identified
by conventional screening methods such as phage display procedures
(e.g., methods described in Hart, et al., J. Biol. Chem. 269:12468
(1994)). Hart et al. report a filamentous phage display library for
identifying novel peptide ligands for mammalian cell receptors. In
general, phage display libraries using, e.g., M13 or fd phage, are
prepared using conventional procedures such as those described in
the foregoing reference. The libraries display inserts containing
from 4 to 80 amino acid residues. The inserts optionally represent
a completely degenerate or a biased array of peptides. Peptides
that bind selectively to the extracellular domain of ErbB2 are
obtained by selecting those phages that express on their surface an
amino acid sequence that recognizes and binds to ErbB2 or the
extracellular domain of ErbB2. These phage then are subjected to
several cycles of re-selection to identify the ErbB2-binding phage
that have the most useful binding characteristics. The minimal
linear portion of the sequence that binds to the extracellular
domain of ErbB2 can be determined. Typically, phage that exhibit
the best binding characteristics (e.g., highest affinity) are
further characterized to identify the particular amino acid
sequences of the peptides expressed on the phage surface and the
optimum length of the expressed peptide to achieve optimum binding.
Phage can also be negatively prescreened for their ability to bind
to other ErbB family members. Preferably, the peptides bind
specifically to ErbB2 extracellular domains and not to the other
ErbB family members. Thus, negative prescreening of phage with
other ErbB family members can enrich for phage of interest.
[0138] In certain embodiments, the libraries may have at least one
constraint imposed upon the displayed peptide sequence. A
constraint includes, e.g., a positive or negative charge,
hydrophobicity, hydrophilicity, a cleavable bond, and the necessary
residues surrounding that bond, one or more cysteines for producing
a cyclic peptide and combinations thereof. In certain embodiments,
more than one constraint is present in each of the peptide
sequences of the library. An example of an imposed constraint is
the length of the peptide. In certain important embodiments,
peptides that are 20 amino acids in length are preferred. An
example of another imposed constraint is the presence of cysteine
residues at or near the ends of the peptide. Generally, the
presence of two cysteine residues in the peptide, provided they are
sufficiently distant from one another, can result in a disulfide
linked cyclic peptide structure.
[0139] The displayed peptide sequence can vary in size. As the size
increases, the potential complexity of the library increases. It is
preferred that the total size of the displayed peptide sequence
(the random amino acids plus any spacer amino acids) should not be
greater than about 100 amino acids long, more preferably not
greater than about 50 amino acids long, and even more preferably
not greater than about 25 amino acids long, and most preferably
less than or equal to 20 amino acids long. The peptides may be as
small as 3 amino acids in length, or 3-6 amino acids in length, or
6-8 amino acids in length.
[0140] ErbB2-binding molecules including peptides can be identified
using a set of screening assays. Compounds such as library members
can be screened for their ability to bind to ErbB2 in vitro using
standard binding assays well known to the ordinary artisan and
described below. ErbB2 may be presented in a number of ways
including, but not limited to, cells expressing ErbB2 (such as
ErbB2 overexpressing cell lines), isolated ErbB2, an isolated
extracellular domain of ErbB2 or a fragment thereof, or a fusion
protein of the extracellular domain and another protein such as an
immunoglobulih or a GST fusion partner. Preferably, the ErbB2
domain is the extracellular domain of ErbB2. For some high
throughput screening assays, the use of purified forms of ErbB2,
its extracellular domain or a fusion of its extracellular domain
with another protein may be preferable. Isolation of binding
partners may be performed in solution or in solid state according
to well-known methods.
[0141] Accordingly, the invention provides a method for screening a
molecular library to identify a compound that inhibits interaction
between ErbB2 and an EBP comprising an amino acid sequence selected
from the group consisting of SEQ ID NO:1 through to SEQ ID NO:13,
and SEQ ID NO:33 through to SEQ ID NO:39, inclusive, or functional
equivalents thereof. The method generally involves performing a
first assay between ErbB2 and the EBP to obtain a first assay
result; performing a second assay between ErbB2 and the EBP in the
presence of a molecular library member to obtain a second assay
result; and comparing the first and second assay results to
determine whether the molecular library member inhibits the
interaction between ErbB2 and the EBP.
[0142] The assay may be a binding assay and it may be performed in
vitro or in vivo. The assay may alternatively be a signaling assay.
The method may involve the initial step of selecting a molecular
library suspected of containing an ErbB2-binding molecule. In still
other embodiments, the assay may be a phosphorylation assay such as
that described in the Examples.
[0143] Such a selection process may involve using libraries which
are made with the preferred constraints mentioned herein. In order
to increase the ErbB2 specificity of a library, the library may be
pre-screened by exposing it to a cell population that does not
express ErbB2. In this way, binding partners which are not specific
for ErbB2 can be eliminated or at least reduced in number from the
library prior to further screening. Procedures for pre-screening
include but are not limited to affinity column purification or
biopanning.
[0144] Standard binding assays are well known in the art, and a
number of these are suitable in the present invention including
ELISA, competition binding assay (particularly suitable in the
present invention since the EBP of the invention may be used),
sandwich assays, radioreceptor assays using radioactively labeled
ErbB2-binding peptides (wherein the binding is blocked in the
presence of the library member), labeled in vitro protein-protein
binding assays, electrophoretic mobility shift assays,
immunoassays, cell-based assays such as two- or three-hybrid
screens, etc. For example, two-hybrid screens are used to rapidly
examine the effect of transfected nucleic acid molecules on the
binding of ErbB2 to EBPs of the invention. The transfected nucleic
acid molecules can derive from, for example, nucleic acid
libraries. Convenient reagents for such assays, e.g., GAL4 fusion
proteins, are known in the art. The nature of the assay is not
essential provided it is sufficiently sensitive to detect binding
of a small number of library members, although this sensitivity may
not be as necessary for phage display based binding assays.
[0145] A variety of other reagents also can be included in the
binding mixture. These include reagents such as salts, buffers,
neutral proteins (e.g., albumin), detergents, etc. which may be
used to facilitate optimal protein-protein and preferably
protein-peptide binding. Such a reagent may also reduce
non-specific or background interactions of the reaction components.
Other reagents that improve the efficiency of the assay may also be
used. The mixture of the foregoing assay materials is incubated
under conditions under which the ErbB2 is normally found. The order
of addition of components, incubation temperature, time of
incubation, and other parameters of the assay may be readily
determined. Such experimentation merely involves optimization of
the assay parameters, not the fundamental composition of the assay.
Incubation temperatures typically are between 4.degree. C. and
40.degree. C. Incubation times preferably are minimized to
facilitate rapid, high throughput screening, and typically are
between 0.1 and 10 hours. After incubation, the presence or absence
of specific binding between ErbB2 and the EBP of the invention can
be detected by any convenient method available to the user.
[0146] Typically, a plurality of assay mixtures are run in parallel
with different compound or library member concentrations to obtain
a different response to the various concentrations. One of these
concentrations serves as a negative control, i.e., at zero
concentration of the compound or at a concentration of compound
below the limits of assay detection.
[0147] For cell-free binding type assays, a separation step is
often used to separate bound from unbound components. The
separation step may be accomplished in a variety of ways.
Conveniently, at least one of the components is immobilized on a
solid substrate, from which the unbound components may be easily
separated. The solid substrate can be made of a wide variety of
materials and in a wide variety of shapes, e.g., colums or gels of
polyacrylamide, agarose or sepharose beads, microtiter plates,
microbeads, resin particles, etc. The separation step preferably
includes multiple rinses or washes. For example, when the solid
substrate is a microtiter plate, the wells may be washed several
times with a washing solution, which typically includes those
components of the incubation mixture that do not participate in
specific bindings such as salts, buffer, detergent, non-specific
protein, etc. Where the solid substrate is a magnetic bead, the
beads may be washed one or more times with a washing solution and
isolated using a magnet.
[0148] One of the components usually comprises, or is coupled to, a
detectable label. A detectable label is a moiety, the presence of
which can be ascertained directly or indirectly. Generally,
detection of the label involves an emission of energy by the label.
The label can be detected directly by its ability to emit and/or
absorb light of a particular wavelength (e.g., radioactivity,
luminescence, optical or electron density, etc.). A label can be
detected indirectly by its ability to bind, recruit and, in some
cases, cleave another moiety which itself may emit or absorb light
of a particular wavelength (e.g., epitope tag such as the FLAG
epitope, enzyme tag such as horseradish peroxidase, etc.). An
example of indirect detection is the use of a first enzyme label
which cleaves a substrate into visible products. The label may be
of a chemical, peptide or nucleic acid molecule nature although it
is not so limited. Other detectable labels include radioactive
isotopes such as P.sup.32 or H.sup.3, luminescent markers such as
fluorochromes, optical or electron density markers, etc., or
epitope tags such as the FLAG epitope or the HA epitope, biotin,
avidin, and enzyme tags such as horseradish peroxidase,
.beta.-galactosidase, etc. Those of ordinary skill in the art will
know of other suitable labels for binding to the binding partners
used in the screening assays, or will be able to ascertain such,
using routine experimentation. Furthermore, the coupling of these
labels to the binding partners used in the screening assays of the
invention can be done using standard techniques common to those of
ordinary skill in the art.
[0149] The label may be bound to a library member, or incorporated
into the structure of the library member. ErbB2 (or the
extracellular domain of ErbB2), the EBP disclosed herein, or the
candidate antagonist may be labeled by a variety of means for use
in screening. In some embodiments, cells expressing ErbB2 may be
used and the detectable label is conjugated to the EBP.
[0150] Another labeling technique which may result in greater
sensitivity consists of coupling the binding partners to low
molecular weight haptens. These haptens can then be specifically
altered by means of a second reaction. For example, it is common to
use haptens such as biotin, which reacts with avidin, or
dinitrophenol, pyridoxal, or fluorescein, which can react with
specific anti-hapten antibodies.
[0151] A variety of methods may be used to detect the label,
depending on the nature of the label and other assay components.
For example, the label may be detected while bound to the solid
substrate or subsequent to separation from the solid substrate.
Labels may be directly detected through optical or electron
density, radioactive emissions, nonradiative energy transfers, etc.
or indirectly detected with antibody conjugates,
streptavidin-biotin conjugates, etc. Methods for detecting the
labels are well known in the art.
[0152] One example of a suitable binding assay involves the use of
ErbB2, an extracellular domain of ErbB2 or an ErbB2 fusion protein
immobilized on resin beads contained within a colum or as a slurry
in test tubes. This can be achieved by using a
glutathione-S-transferase (GST) fusion of ErbB2 or its
extracellular domain and a colum containing anti-GST antibody. The
ErbB2-GST fusion polypeptide is first immobilized on the colum or
resin, followed by the addition of a suspension of candidate
antagonists such as, for example, library members, in a solution
compatible with the binding of EBPs disclosed herein to the
extracellular domain of ErbB2. The colum is then washed to remove
any residual non-bound compounds. The bound compounds are then
eluted by changing the conditions on the colum such that binding to
the ligand binding site is no longer favored, such as pH or ionic
concentration change, or competitive elution with reduced
glutathione. The eluate is collected and the compounds contained
therein are further analyzed. In the case where the compounds are
peptides, the eluted peptides can be sequenced using standard Edman
degradation amino acid sequencing techniques or in the case of
non-peptide moieties, the eluted compounds are analyzed by standard
analytical techniques such as HPLC and mass spectroscopy. Apparati
for performing Edman degradation sequencing, an example of which is
the Applied Biosystems 477A Protein Sequencer, are available
commercially. Analysis of lead candidates from such binding assays
using NMR spectroscopy are described in U.S. Pat. No. 5,877,030,
the contents of which are incorporated herein by reference. In this
way, the sequence or composition of the compounds which bind to the
colum can be deduced.
[0153] In other embodiments, it is preferred that ErbB2 is produced
in eukaryotic expression systems such as insect or COS cells which
can result in more suitable folding and glycosylation of the
peptide.
[0154] A second criteria for testing a putative EBP is its ability
to inhibit signal transduction involving ErbB2. One way in which
EBPs can inhibit ErbB2 mediated signal transduction is by
interfering with the ability of ErbB2 to interact with other
signaling factors. As used herein, signaling factors are proteins
or polypeptides that are involved in transducing a signal into or
within a cell. Signaling factors include endogenous signaling
factors which interact with ErbB2 to transmit a signal to ErbB2 or
to accept a signal from ErbB2. As used herein, an "endogenous"
molecule is one that is known to exist naturally in a cell and
includes intracellular and transmembrane molecules. Examples of
endogenous signaling factors include tyrosine kinases, phosphatases
and adaptor proteins. Tyrosine kinases include receptor tyrosine
kinases and non-receptor tyrosine kinases. Receptor tyrosine
kinases include, but are not limited to, EGRF, ErbB3, ErbB4 (HER4),
PDGFR, CSF-1R (c-fms), c-kit, LET-23R, HGFR/SFR (c-met), FGFR,
IGF1R, flt3/flk2, flk1, c-ret, EphA2, TrkB (BDNFR), tek/tie2, stk,
flt-1 (VEGFR), RON, TrkA (NGFR), MuSK, VEGFR2, ROR, tie1, etc.
Non-receptor tyrosine kinases include, but are not limited to, Fyn,
Lck, Lyn, Syk/ZAP-70, Src, Yes, Hck, Blk, Yrk, Fgr, Rak, Brk, and
Csk. Still other molecules lack tyrosine kinase activity but are
still capable of being phosphorylated by a tyrosine kinase.
Molecules can be phosphorylated by virtue of the fact they contain
a tyrosine, a serine or a threonine residue which can be
phosphorylated. Depending upon the embodiment of the invention,
these molecules may or may not be phosphorylated. Receptor tyrosine
kinases may be ligand-activated or not activated. In some preferred
embodiments, the signaling factors are selected from the group
consisting of Ras, Raf, MAPK, P13K, and adaptor proteins such as
Grb2 and Grb7. In the screening assays described herein, the ErbB2
specific signaling factors may be Grb2 or Grb7, or it may be an
antibody or an antibody fragment specific for ErbB2 such as
Herceptin.RTM. (Trastuzumab) (Genentech, South San Francisco,
Calif.). Other ErbB2 specific antibodies are commercially available
from Oncogene Research, Santa Cruz Biotechnology, and Nova
Castra.
[0155] ErbB2 is known to be phosphorylated during signal
transduction. Phosphorylation status of ErbB2 (such as, for
example, at the Ser 1113 residue) therefore can be used as a
readout for a signaling assay. As shown in the Examples,
phosphorylation of tyrosine residues 877 and 1248 can also be used
as a readout in a signaling assay. Phosphorylation of these
residues is indicative of ErbB2 activation.
[0156] Binding interactions between ErbB2 and factors with which it
interacts can also be carried out through the use of cell-based
assays. As example of this is immunoprecipitation of such ErbB2
interacting factors from cell lysates using purified ErbB2 in the
presence and absence of putative antagonists.
[0157] Binding assays can be followed by screens for biological
antagonist activity. To be useful, the ErbB2 antagonist binds to
ErbB2, precludes or inhibits binding of ErbB2 to one or more of its
endogenous ligands and, in doing so, prevents, inhibits or
interferes with signal transduction from ErbB2 and events
downstream of such signaling. An example of an event downstream of
signaling may be cell proliferation. One way of measuring cell
proliferation involves the use of the MTT and BrdU proliferation
assays. Alternatively, in vitro clonogenic assays can be used.
These assays can be performed using either cell lines known to
express a functional ErbB2 (such as SKBR3, BT474, SKOV3 or MDA361)
or other cells which have been manipulated (i.e., transfected) to
express ErbB2 (such as ErbB2 transfected NIH3T3 cells). Control
cell lines that express no or low levels of ErbB2 include HBL100,
MDA468 and MCF7. The peptides can also be tested for their ability
to inhibit anchorage independent growth in the cancer cells.
[0158] The number and quality of colonies can be determined as a
function of the presence and absence of the library member.
Preferably, the assays are carried out by culturing the cells in a
semi-solid culture under conditions which stimulate maximal colony
growth from the cell population. The library member is then
titrated into the cultures in order to determine the amount
necessary to reduce colony formation. In this manner, in addition
to the amount of antagonist necessary to inhibit colony growth
altogether, one can also determine that amount which inhibits the
growth by a particular percentage. In this way, the amount of
antagonist which impacts upon colony growth from, for example,
aggressive ErbB2-expressing cell lines, but not on the growth of
less aggressive non-ErbB2-expressing cell lines can be determined.
For example, it may be desirable to reduce colony growth of
ErbB2-expressing cell lines by the maximum amount possible while
leaving colony growth by non-ErbB2-expressing cell lines
unaffected. Clonogenic assays such as those described herein are
routinely employed by artisans of ordinary skill. (DeFriend et al.,
1994, Br. J. Cancer, 70(2):204-11; Glinsky et al., 1996, Clin. Exp.
Metastasis, 14(3):253-67; Shen et al., 1998, Oncol. Res.
10(6):325-31; Perez et al., 1998, Cancer Chemother. Pharmacol.
41(6):448-52) Moreover, each of the afore-mentioned in vitro
screening assays is amenable to high-throughput screening.
[0159] Cells useful in these in vitro clonogenic assays are cell
lines or primary cells which preferably are known to express ErbB2
and one or more of its endogenous ligands. Examples include breast
cancer cell lines such as BT474, SKBR3, SKOV3 and MDA361. Cells
which are genetically manipulated to overexpress ErbB2 are also
useful in the invention. Cell lines which can be so manipulated are
preferably breast cancer cell lines, but are not so limited, and
include transfected NIH3T3. A control breast cell line which can be
used is MDA468, MCF7 or HBL100. A control cell line for the
manipulated cell line is NIH3T3.
[0160] Other measures of biological activity include the MTT assay
and signaling assays such as phosphorylation assays. U.S. Pat. No.
6,123,939 describes both assays in the context of ErbB2 expressing
cells.
[0161] Another way of measuring the biological antagonist activity
of the synthetic compound is to perform in vivo assays in which the
putative antagonist is introduced into animals, preferably mice,
which have been made susceptible to, for example, breast cancer
tumors. The mice are then analyzed to determine whether the
putative antagonist ameliorates the symptoms of, for example, the
cancer (e.g., a reduction in tumor growth). As an example of such
an animal model, nude mice can be transplanted with human breast
cancer xenografts, following which the growth of such grafts can be
determined in the presence and absence of peptide administration.
In some instances, breast tissue may also be harvested and plated
into a clonogenic assay. Preferably the size of tumors in vivo
and/or the number and quality of colonies derived from test animals
should be compared to that of animals injected with control carrier
(i.e., saline) lacking the putative antagonist. Adverse side
effects can also be tested in animals injected with putative
antagonists in this manner. Examples of such in vivo mouse models
of breast cancer have been described by Gabri et al., 1999
Pathobiology 67(4):180-5; Liu et al., 1999 Am. J. Pathol.
155(6):1861-7; and Vodovozona et al., 2000 Eur. J. Cancer
36(7):942-9. These assays are also suitable for the testing of
peptide-cytotoxic agent conjugates described herein.
[0162] The antagonists generated as described herein can also be
screened in vivo or in vitro for the ability to prevent metastasis,
using two different animal models of cancer, B16BL6 and LLC. Divino
et al. (2000 Breast Cancer Res. Treat. 650(2):129-34.) specifically
describe a mouse model of breast cancer metastasis. In some
embodiments of the invention, the antagonists can be screened
according to their ability to prevent invasion of tumor cells
across a barrier. The barrier for the tumor cells may be an
artificial barrier in vitro or a natural barrier in vivo. An in
vivo barrier refers to a cellular barrier present in the body of a
subject. In vitro barriers include but are not limited to
extracellular matrix coated membranes, such as Matrigel. Thus the
putative antagonists can be tested for their ability to inhibit
tumor cell invasion in a Matrigel invasion assay system as
described in detail by Parish, C.R., et al., "A Basement-Membrane
Permeability Assay which Correlates with the Metastatic Potential
of Tumour Cells," Int. J. Cancer (1992) 52:378-383, provided the
cells used in the assay have been characterized as having abnormal
interaction of ErbB2 and its ligands. Matrigel is a reconstituted
basement membrane containing type IV collagen, laminin, heparan
sulfate proteoglycans such as perlecan, which bind to and localize
bFGF, vitronectin as well as transforming growth factor-.beta.
(TGF-.beta.), urokinase-type plasminogen activator (uPA), tissue
plasminogen activator (tPA), and the serpin known as plasminogen
activator inhibitor type 1 (PAI-1). Other in vitro and in vivo
assays for metastasis have been described in the prior art, see,
e.g., U.S. Pat. No. 5,935,850, issued on Aug. 10, 1999, which is
incorporated by reference.
[0163] As alluded to earlier, the invention also embraces isolated
nucleic acid molecules that code for EBP of the invention. As will
be appreciated by one of ordinary skill in the art, a number of
nucleic acid molecules code for each EBP, due to the degeneracy of
the genetic code. For example, while the nucleic acid molecule
having the nucleotide sequence SEQ ID NO:14 encodes EBP-1, there
are multiple other nucleic acid molecules that also encode EBP-1.
For example, the threonine residues can each be independently coded
by the following codons: ACU, ACC, ACA and ACG. Each of the four
codons is equivalent for the purposes of encoding an threonine
residue. Thus, it will be apparent to one of ordinary skill in the
art that any of the arginine-encoding nucleotide triplets may be
employed to direct the peptide synthesis apparatus, in vitro or in
vivo, to incorporate an arginine residue into a EBP. Thus, although
SEQ ID NO:14 uses only the first of these codons, it is to be
understood that the invention embraces nucleic acid molecules which
use any of the six codons to code for threonine. Similarly, the
invention embraces nucleic acid molecules that use any of the four
codons coding for valine (GUU, GUC, GUA, and GUG), or either of the
two codons coding for glutamine (CAA, CAG), or either of the two
codons coding for glutamic acid (GAA, GAG), or either of the two
codons coding for cysteine (UGU, UGC), or either of the two codons
coding for lysine (AAA, AAG), or either of the two codons coding
for tyrosine (UAU, UAC), or any of the six codons coding for
leucine (UUA, UUG, CUU, CUC, CUA, and CUG), or either of the two
codons coding for aspartic acid (GAU, GAC), or either of the two
codons coding for asparagine (AAU, AAC), or any of the four codons
coding for glycine (GGU, GGC, GGA, and GGG), or either of the two
codons coding for phenylalanine (UUU, UUC), or either of the two
codons coding for histidine (CAU, CAC), or any of the four codons
coding for proline (CCU, CCC, CCA, CCG), or any of the six codons
coding for arginine (CGU, CGC, CGA, CGG, AGA, and AGG), or any of
the six codons coding for serine (UCU, UCC, UCA, UCG, AGU, and
AGC), or any of the four codons coding for alanine (GCA, GCU, GCC,
and GCG), or any of the three codons coding for isoleucine (AUU,
AUC, and AUA). Methionine and tryptophan are each coded by a single
codon respectively.
[0164] Putative nucleic acids sequences that encode the EBPs of the
invention include TGG ACT GGT TGG TGT TTA AAT CCT GAA GAA TCT ACT
TGG GGT TTT TGT ACT GGT TCT TTT (SEQ ID NO:14); GTT GTT GCA TGT TCT
TGG GAT TGG ACT ATG GGT GCA GTT GTT TGT TAT GAA CGT ATT (SEQ ID
NO:15); GGT TTT TGG ACT TGT GAA TAT GAT TGG TGG TCT GAT GCA ACT GTT
TGT ATG CAT ACT TTA (SEQ ID NO:16); GGT CGT GGT TGG TGT TGG TCT GAA
TGG CAA AAT GAT TGG TTT TGG TGT TGG GAT GTT TGG (SEQ ID NO:17); TGG
ACT GGT TGG TGT TTA AAT CCT GAA GAA TCT ACT TGG GGT TTT TGT ACT GGT
TCT TTT (SEQ ID NO:18); GCA CGT TTA CAA TGT TGG TCT TTA GGT TGG GGT
GGT CCT GTT TAT TGT GGT TTT GGT CAA (SEQ ID NO:19); ATT CAA GAA GTT
TGT TGG TTT GAT TAT AAT TTA TCT CAA TGG CAT TGT ATG ACT GTT ATT
(SEQ ID NO:20); CCT GAT ATT TAT TGT TTA TCT GTT ACT GCA CCT GGT TTT
TTA ATT TGT TAT GAA CGT TAT (SEQ ID NO:21); CAT GAT GAA TTA TGT GTT
TTT TCT TTT GAT TTT AAT GCA TTA TTA TGT TGG CCT GCA GAA (SEQ ID
NO:22); TTA AAT TGG GAA TGT TGG TAT GAT TAT CGT TTA GAA GCA TGG GAT
TGT CGT GGT GAT ATT (SEQ ID NO:23); TGT GAA GTT TGG GGT GAA GTT CCT
TGG ACT TGT (SEQ ID NO:24); TGT GAA GTT TGG GGT TTT GTT CCT TGG GCA
TGT (SEQ ID NO:25); TCT AAT GAA TCT TGT GGT TCT CCT ATT AAT CCT TGG
GGT GAA ATG TGT TTA TTA ATG TTA (SEQ ID NO:26); TGG ACT GGT TGG TGT
TTA AAT CCT GAA GAA TCT ACT TGG GGT TTT TGT CGT TCT GCA GGT (SEQ ID
NO:40); TGG ACT GGT TGG TGT TTA TCT CCT GAA GAA TCT ACT TGG GGT TTT
TGT CGT TCT GCA GGT (SEQ ID NO:41); TGG ACT GGT TGG TGT TTA AAT CCT
GAA GAA TCT ACT TGG GGT TTT TGT TCT GGT TAT ATT (SEQ ID NO:42); TGG
ACT GGT TGG TGT TTT GAT GAT AAT CAT TCT ACT TGG GGT TTT TGT ACT GGT
TCT TTT (SEQ ID NO:43); GAT ACT GAT ATG TGT TGG TGG TGG TCT CGT GAA
TTT GGT TGG GAA TGT GCA GGT GCA GGT (SEQ ID NO:44); TCT TTA GCA TTA
TGT TTA TCT GAA GGT GTT TTA TTA GGT GCA GAT TGT CGT GTT TTA TTT
(SEQ ID NO:45); TGG TCT TCT ATG TGT GGT GAT CCT ACT ATT GCA GAT TGG
TTA TGG TGT TTT TCT GAT GCA (SEQ ID NO:46).
[0165] The foregoing nucleic acid sequences are intended to be
simply illustrative and it is to be understood that depending upon
the subject, the codon usage for particular amino acids will
change, and thus so will the nucleic acid sequence. The invention
intends to embrace all degenerates of the foregoing sequences.
Given the teachings provided herein as the level of skill in the
art, one of ordinary skill can readily ascertain the nucleic acid
sequences of all degenerates.
[0166] It is to be understood that although the codons and
nucleotide sequences listed herein contain thymidine bases, codons
and nucleic acid molecules in which thymidine is replaced with
uracil are equally embraced by the invention. Similarly, modified
nucleotides may also be used in any of the nucleic acid molecules
of the invention provided their function is preserved (e.g.,
hybridization, ability to be cloned or transcribed, etc.). Examples
of modified nucleotides include those with a modified base and/or
sugar, those having backbone sugars which are covalently attached
to low molecular weight organic groups other than a hydroxyl group
at the 3' position and other than a phosphate group at the 5'
position (e.g., a 2'-O-alkylated ribose group). In addition,
modified nucleic acids may include sugars such as arabinose instead
of ribose. The nucleic acids may be heterogeneous in backbone
composition thereby containing any possible combination of polymer
units linked together such as peptide-nucleic acids (which have an
amino acid backbone with nucleic acid bases). In some embodiments,
the nucleic acids are homogeneous in backbone composition. The
purines and pyrimidines of the nucleic acids may also be
substituted e.g., base analogs such as C-5 propyne substituted
bases (Wagner et al., Nature Biotechnology 14:840-844, 1996).
Purines and pyrimidines which can be incorporated into the nucleic
acids of the invention include but are not limited to adenine,
cytosine, guanine, thymine, 5-methylcytosine, 2-aminopurine,
2-amino-6-chloropurine, 2,6-diaminopurine, hypoxanthine, and other
naturally and non-naturally occurring nucleobases, substituted and
unsubstituted aromatic moieties.
[0167] As used herein with respect to nucleic acid molecules, the
term "isolated" means separated from its native environment in
sufficiently pure form so that it can be manipulated or used for
any one of the purposes of the invention. Examples of isolated
nucleic acids include those that are: (i) amplified in vitro by,
for example, polymerase chain reaction (PCR); (ii) recombinantly
produced by cloning; (iii) purified, as by cleavage and gel
separation; or (iv) synthesized by, for example, chemical
synthesis. An isolated nucleic acid molecule is one which is
readily manipulable by recombinant DNA techniques well known in the
art. Thus, a nucleotide sequence contained in a vector in which 5'
and 3' restriction sites are known or for which polymerase chain
reaction (PCR) primer sequences have been disclosed is considered
isolated, but a nucleic acid molecule sequence existing in its
native state in its natural host is not. An isolated nucleic acid
molecule may be substantially purified, but need not be. For
example, a nucleic acid molecule that is isolated within a cloning
or expression vector is not pure in that it may comprise only a
tiny percentage of the material in the cell in which it resides.
Such a nucleic acid molecule is isolated, however, as the term is
used herein because it is readily manipulable by standard
techniques known to those of ordinary skill in the art.
[0168] According to the invention, isolated EBP nucleic acid
molecules include nucleic acid molecules which code for an ErbB2
binding peptide that binds to the extracellular domain of ErbB2
(e.g., EBP-1, EBP-2, EBP-3, EBP-4, EBP-5, EBP-6, EBP-7, EBP-8,
EBP-9, EBP-10, EBP-11, EBP-12, EBP-13, EBP-14, EBP-15, EBP-16,
EBP-17, EBP-18, EBP-19, or EBP-20 (or functionally equivalent
fragments thereof)). These nucleic acid molecule may have a
nucleotide sequence selected from the group consisting of SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ
ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23,
SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:40, SEQ ID
NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, or
SEQ ID NO:46. The nucleic acid molecules also include nucleic acid
molecules that contain deletions, additions and substitutions of
the above sequences that code for an EBP, nucleic acid molecules
that differ from the above nucleic acid molecules in codon sequence
due to the degeneracy of the genetic code (as described above), and
(complements thereof.
[0169] Homologs and alleles of the EBP nucleic acid molecules of
the invention may include naturally occurring peptides or proteins
that bind to ErbB2 (i.e., ErbB2 ligands). These molecules can be
identified by conventional homology search or hybridization
techniques. Example of homologs and alleles are those endogenous
nucleic acid molecules that code for a peptide that binds to ErbB2
(and particularly the extracellular domain of ErbB2), and that
hybridize under stringent conditions to a nucleic acid molecule
having, for example, a nucleotide sequence selected from the group
consisting of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID
NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ
ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26,
SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID
NO:44, SEQ ID NO:45, and SEQ ID NO:46. The term "stringent
conditions" as used herein refers to parameters with which the art
is familiar. Nucleic acid molecule hybridization parameters may be
found in references which compile such methods, e.g. Molecular
Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second
Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., 1989, or Current Protocols in Molecular Biology, F. M.
Ausubel, et al., eds., John Wiley & Sons, Inc., New York. More
specifically, stringent conditions, as used herein, refers, for
example, to hybridization at 65.degree. C. in hybridization buffer
(3.5.times.SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02%
Bovine Serum Albumin, 2.5 mM NaH.sub.2PO.sub.4(pH7), 0.5% SDS, 2 mM
EDTA). SSC is 0.15M sodium chloride/0.15M sodium citrate, pH7; SDS
is sodium dodecyl sulphate; and EDTA is ethylenediaminetetracetic
acid. After hybridization, the membrane upon which the DNA is
transferred is washed at 2.times.SSC at room temperature and then
at 0.1.times.SSC/0.1% SDS at temperatures up to 68.degree. C.
[0170] There are other conditions, reagents, and so forth which can
be used, and would result in a similar degree of stringency. The
skilled artisan will be familiar with such conditions, and thus
they are not given here. It will be understood, however, that the
skilled artisan will be able to manipulate the conditions in a
manner to permit the clear identification of homologs and alleles
of EBP nucleic acid molecules of the invention. The skilled artisan
also is familiar with the methodology for screening phage, cells
and libraries preferably peptide or aptamer libraries for
expression of ErbB2 antagonists which then are isolated, followed
by isolation of the pertinent nucleic acid molecule and
sequencing.
[0171] In general homologs and alleles typically will share at
least 75% nucleotide identity to any EBP nucleic acid molecules
(e.g., SEQ ID NO:14 through to SEQ ID NO:26, and SEQ ID NO:40
through to SEQ ID NO:46, inclusive) and/or at least 90% amino acid
identity to any EBP amino acid sequences (i.e., SEQ ID NO:1 through
to SEQ ID NO:13 and SEQ ID NO:33 through to SEQ ID NO:39,
inclusive). Preferably, homologs and alleles will share at least
85% nucleotide identity and/or at least 95% amino acid identity
and, even more preferably, at least 95% nucleotide identity and/or
at least 99% amino acid identity will be shared. The homology can
be calculated using various, publicly available software tools
developed by NCBI (Bethesda, Maryland) that can be obtained through
the internet at the NIH website. Exemplary tools include the BLAST
system using default settings, available at the NCBI website on the
internet. Pairwise and ClustalW alignments (BLOSUM30 and/or
BLOSUM62 matrix settings) as well as Kyte-Doolittle hydropathic
analysis can be obtained using the MacVetor sequence analysis
software (Oxford Molecular Group). Watson-Crick complements of the
foregoing nucleic acid molecules also are embraced by the
invention.
[0172] The invention also provides isolated unique fragments of EBP
nucleic acid molecules or complements thereof. A unique fragment is
one that is a `signature` for the larger nucleic acid molecule such
as, for example, those encoding EBPs or those encoding endogenous
ErbB2 ligands. For example, the unique fragment is long enough to
assure that its precise sequence is found sparingly in molecules
within the human genome. Those of ordinary skill in the art may
apply no more than routine procedures to determine if a fragment is
unique within the human genome. Unique fragments, however, exclude
fragments completely composed of the nucleotide sequences of a
published GenBank submission or other previously published
sequences as of the date of the invention or the filing date of
this application.
[0173] A fragment which is completely composed of a sequence
described in a GenBank published sequence (as of the date of the
invention or the filing date of this application) is one which does
not include any of the sequences unique to the invention. Thus, a
unique nucleic acid fragment must contain a nucleotide sequence
other than the exact sequence of those in GenBank or fragments
thereof (as of the date of the invention or the filing date of this
application). The difference may be an addition, deletion or
substitution with respect to all or part of the GenBank sequence or
it may be a sequence wholly separate from the GenBank sequence.
[0174] Unique EBP nucleic acid fragments also include nucleic acid
molecules that encode unique EBP peptide fragments, as described
above. As one of ordinary skill will appreciate, these nucleic acid
molecules will be varied given the degeneracy in the nucleic acid
code, however, the sequence of these nucleic acid molecules is
ascertainable with routine reference to a codon table.
[0175] Unique fragments can be used as probes in Southern and
Northern blot assays to identify nucleic acid molecules which
contain these nucleotide sequences, or can be used in amplification
assays such as those employing PCR. Unique fragments also can be
used to produce fusion proteins for generating antibodies or
determining binding of the peptide fragments or for generating
immunoassay components.
[0176] As will be recognized by those skilled in the art, the size
of the unique fragment will depend upon its conservancy in the
genetic code. Thus, some regions of EBP nucleic acid molecules
(e.g., SEQ ID NO:14 through to SEQ ID NO:26, and SEQ ID NO:40
through to SEQ ID NO:46, inclusive (as well as degenerates
thereof)), or complements thereof will require longer segments to
be unique while others will require only short segments, typically
between 12 and 32 nucleotides long (e.g. 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, and 32
bases) or more, up to the entire length of the disclosed sequence.
As mentioned above, this disclosure intends to embrace each and
every fragment of each sequence listed, beginning at the first
nucleotide, the second nucleotide and so on, up to 8 nucleotides
short of the end, and ending anywhere from nucleotide number 8, 9,
10, and so on for each sequence listed, up to the very last
nucleotide, provided the sequence is unique as described above.
Taking into account the exclusion described above, virtually any
segment of the region of, for example, SEQ ID NO14, beginning at
nucleotide 1 and ending at nucleotide 30, or complements thereof,
that is 20 or more nucleotides in length will be unique. Those
skilled in the art are well versed in methods for selecting such
sequences, typically on the basis of the ability of the unique
fragment to selectively distinguish the sequence of interest from
other sequences in the human genome of the fragment to those on
known databases typically is all that is necessary, although in
vitro confirmatory hybridization and sequencing analysis may be
performed.
[0177] Most if not all of the characteristics of nucleic acid
molecule unique fragments are shared with peptide unique fragments
disclosed herein.
[0178] The invention also involves expression vectors coding for
EBPs and fragments and variants thereof and host cells containing
those expression vectors. As used herein, a "vector" may be any of
a number of nucleic acid molecules into which a desired sequence
may be inserted by restriction and ligation for transport between
different genetic environments or for expression in a host cell.
Vectors are typically composed of DNA although RNA vectors are also
available. Vectors include, but are not limited to, plasmids,
phagemids and virus genomes. In some preferred embodiments, the
expression system is a phage.
[0179] A cloning vector is one which is able to replicate in a host
cell, and which is further characterized by one or more
endonuclease restriction sites at which the vector may be cut in a
determinable fashion and into which a desired DNA sequence may be
ligated such that the new recombinant vector retains its ability to
replicate in the host cell. In the case of plasmids, replication of
the desired sequence may occur many times as the plasmid increases
in copy number within the host bacterium or just a single time per
host before the host reproduces by mitosis. In the case of phage,
replication may occur actively during a lytic phase or passively
during a lysogenic phase.
[0180] An expression vector is one into which a desired DNA
sequence may be inserted by restriction and ligation such that it
is operably joined to regulatory sequences and may be expressed as
an RNA transcript. Vectors may further contain one or more marker
sequences suitable for use in the identification of cells which
have or have not been transformed or transfected with the vector.
Markers include, for example, genes encoding proteins which
increase or decrease either resistance or sensitivity to
antibiotics or other compounds, genes which encode enzymes whose
activities are detectable by standard assays known in the art
(e.g., .beta.-galactosidase or alkaline phosphatase), and genes
which visibly affect the phenotype of transformed or transfected
cells, hosts, colonies or plaques (e.g., green fluorescent
protein). Preferred vectors are those capable of autonomous
replication and expression of the structural gene products present
in the DNA segments to which they are operably joined.
[0181] As used herein, a marker or coding sequence and regulatory
sequences are said to be "operably" joined when they are covalently
linked in such a way as to place the expression or transcription of
the coding sequence under the influence or control of the
regulatory sequences. If it is desired that the coding sequences be
translated into a functional peptide or polypeptide, two DNA
sequences are said to be operably joined if induction of a promoter
in the 5' regulatory sequences results in the transcription of the
coding sequence and if the nature of the linkage between the two
DNA sequences does not (1) result in the introduction of a
frame-shift mutation, (2) interfere with the ability of the
promoter region to direct the transcription of the coding
sequences, or (3) interfere with the ability of the corresponding
RNA transcript to be translated into a protein. Thus, a promoter
region would be operably joined to a coding sequence if the
promoter region were capable of effecting transcription of that DNA
sequence such that the resulting transcript might be translated
into the desired protein or polypeptide.
[0182] The precise nature of the regulatory sequences needed for
gene expression may vary between species or cell types, but shall
in general include, as necessary, 5' non-transcribed and 5'
non-translated sequences involved with the initiation of
transcription and translation respectively, such as a TATA box,
capping sequence, CCAAT sequence, and the like. Especially, such 5'
non-transcribed regulatory sequences will include a promoter region
which includes a promoter sequence for transcriptional control of
the operably joined coding sequence. Regulatory sequences may also
include enhancer sequences or upstream activator sequences as
desired. The vectors of the invention may optionally include 5'
leader or signal sequences. The choice and design of an appropriate
vector is within the ability and discretion of one of ordinary
skill in the art.
[0183] Expression vectors containing all the necessary elements for
expression are commercially available and known to those skilled in
the art. See, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, 1989. Cells are genetically engineered for EBP expression by
the introduction of a heterologous nucleic acid molecule, usually
DNA, encoding a EBP or fragment or a variant thereof into the
cells. The heterologous nucleic acid molecules are placed under
operable control of transcriptional elements to permit the
expression of the heterologous nucleic acid molecules in the host
cell.
[0184] Preferred systems for mRNA expression in mammalian cells are
those such as pcDNA3.1 (available from Invitrogen, Carlsbad,
Calif.) that contain a selectable marker such as a gene that
confers G418 resistance (which facilitates the selection of stably
transfected cell lines) and the human cytomegalovirus (CMV)
enhancer-promoter sequences. Additionally suitable for expression
in primate or canine cell lines is the pCEP4 vector (Invitrogen,
Carlsbad, Calif.), which contains an Epstein Barr virus (EBV)
origin of replication, facilitating the maintenance of plasmid as a
multicopy extrachromosomal element. Another expression vector is
the pEF-BOS plasmid containing the promoter of polypeptide
Elongation Factor 1.alpha., which stimulates efficiently
transcription in vitro. The plasmid is described by Mishizuma and
Nagata (Nuc. Acids Res. 18:5322, 1990), and its use in transfection
experiments is disclosed by, for example, Demoulin (Mol. Cell.
Biol. 16:4710-4716, 1996). Still another preferred expression
vector is an adenovirus, described by Stratford-Perricaudet, which
is defective for E1 and E3 proteins (J. Clin. Invest. 90:626-630,
1992). The use of the adenovirus as an Adeno. P1A recombinant is
disclosed by Warnier et al., in intradermal injection in mice for
immunization against P1A (Int. J. Cancer, 67:303-310, 1996).
[0185] It will also be recognized that the invention embraces the
use of the above described EBP nucleotide sequence-containing
expression vectors to transfect host cells. Virtually any cells, be
these prokaryotic (e.g., E. coli), or eukaryotic (e.g., CHO cells,
COS cells, yeast expression systems, and recombinant baculovirus
expression in insect cells) which can be transformed with
heterologous DNA or RNA, and which can be grown or maintained in
culture, may be used in the practice of the invention. Especially
useful are mammalian cells such as human, mouse, hamster, pig,
goat, primate, etc., from a wide variety of tissue types, including
primary cells and established cell lines. Specific examples include
mammalian epithelial cells, fibroblast cells, and kidney epithelial
cells, either as primary cells or cell lines. Cell-free
transcription systems also may be used in lieu of cells.
[0186] As eluded to earlier, the EBP of the invention, and
preferably the nucleic acid molecules that encode these peptides
can be used to screen cells or peptide or nucleic acid libraries
for naturally occurring EBP (i.e., ErbB2 ligands) with homology to
the EBPs of the invention. Naturally occurring EBP may comprise or
share homology with the amino acid sequences of SEQ ID NO:1 through
to SEQ ID NO:13 or SEQ ID NO:33 through to SEQ ID NO:39, inclusive
or functionally equivalent fragments thereof. Naturally occurring
peptides or polypeptides with such homology may be identified
through homology searches in protein and peptide databases (such as
GenBank) or binding to antibodies specific for EBP. In this way,
native, naturally occurring binding partners of ErbB2 may be
identified. Polypeptides identified in the manner may be useful,
for example, as ErbB2 antagonists or agonists as well as in
elucidating the natural mechanism through which ErbB2 and its
interactions are inhibited or stimulated respectively. Polypeptides
which are identified in this manner can be isolated from biological
samples including tissue or cell homogenates, or alternatively can
also be expressed recombinantly in a variety of prokaryotic and
eukaryotic expression systems by constructing an expression vector
appropriate to the expression system, introducing the expression
vector into the expression system, and isolating the recombinantly
expressed protein. The invention also embraces the identification
of ErbB2 agonists which can also be useful in the treatment of
certain diseases, particularly those affecting tissues in which
ErbB2 is believed to play a physiological role such as CNS
tissues.
[0187] In addition, the nucleic acid molecules which code for
naturally occurring EBP may have homology to the nucleic acid
molecules which encode the EBPs of the invention (e.g., SEQ ID
NO:14 through to SEQ ID NO:26, and SEQ ID NO:40 through to SEQ ID
NO:46, inclusive). Naturally occurring nucleic acid molecules with
such homology may be identified through stringent hybridization of
the EBP nucleic acid molecules to cells (e.g., whole cell filter
hybridization) or nucleic acid libraries (e.g., cDNA libraries), or
by homology searching in nucleic acid sequence databases such as
GenBank.
[0188] The peptides, and unique fragments thereof, may be used in
the diagnostic or therapeutic methods of the invention either in a
free form (i.e., unconjugated or not complexed with another
compound), or conjugated form. In one aspect, the EBP can be used
as homing molecules to allow specific delivery of particular agents
to diseased tissue. These agents include detectable labels (e.g.,
imaging agents) and therapeutic agents. (e.g., cytotoxic agents
such as anti-cancer agents).
[0189] The conjugations or modifications described herein employ
routine chemistry, which chemistry does not form a part of the
invention and which chemistry is well known to those skilled in the
art of chemistry. The use of protecting groups and known linkers
such as mono- and hetero-bifunctional linkers are well documented
in the literature and will not be repeated here.
[0190] As used herein, "conjugated" means two entities stably bound
to one another by any physiochemical means. It is important that
the nature of the attachment be of such a nature that it does not
impair substantially the effectiveness of either entity. Keeping
these parameters in mind, any linkage known to those of ordinary
skill in the art may be employed, covalent or noncovalent. Covalent
is preferred. Noncovalent methods of conjugation may also be used.
Noncovalent conjugation includes hydrophobic interactions, ionic
interactions, high affinity interactions such as biotin-avidin and
biotin-streptavidin complexation and other affinity interactions.
Such means and methods of attachment are well known to those of
ordinary skill in the art.
[0191] Conjugation of the EBP to a detectable label facilitates,
among other things, detection of cells and tissues that overexpress
ErbB2, and subsequent diagnosis. These agents can be imaging agents
such as contrast agents and radioactive agents that can be detected
using medical imaging techniques such as nuclear medicine scans and
magnetic resonance imaging (MRI). Detectable labels for magnetic
resonance imaging (MRI) include Gd(DOTA); for nuclear medicine
include .sup.201TI, gamma-emitting radionuclide 99mTc; for
positron-emission tomography (PET) include positron-emitting
isotopes, (18)F-fluorodeoxyglucose ((18)FDG), (18)F-fluoride,
copper-64, gadodiamide, and radioisotopes of Pb(II) such as 203Pb,
and 111In. In other embodiments, the peptides can also be
conjugated or coupled to the surface of gas filled particles which
can be detected using ultrasound.
[0192] In one particularly important aspect of the invention, the
peptides are used to identify, isolate and remove cells that
overexpress ErbB2 from a tissue or a cell population. As an
example, the peptides can be used to remove breast cancer cells
from a bone marrow population. Breast cancer patients often undergo
autologous bone marrow transplantation following aggressive
marrow-ablative chemotherapy and/or radiation therapy in order to
reconstitute their marrow. Due to the autologous nature of these
transplants there exists a possibility of re-administering cancer
cells into the patient by way of the transplant. "Purging" of the
marrow (i.e., removal of the cancer cells from the marrow cell
population) prior to its re-infusion into the patient is generally
recommended. The peptides of the invention can be used to identify
and remove ErbB2 overexpressing cells. Separation or removal of
ErbB2 expressing cells from the marrow population can be achieved
using for example flow cytometry or panning. For flow cytometry
purposes, the peptides should be labeled with a detectable label,
preferably a fluorescent label, as described herein. The ability to
purge marrow populations of, for example, breast cancer cells can
reduce the recurrence of breast cancer metastases following
transplant.
[0193] The EBP can also be conjugated to therapeutic agents. As
used herein, a therapeutic agent is a compound that has been shown
to have therapeutic benefit in a subject having a particular
disorder. Therapeutic agents include cytotoxic agents (such as
anti-cancer agents), immunomodulatory agents, anti-angiogenic
agents, and translocation agents.
[0194] The EBP can be co-administered with therapeutic agents in an
unconjugated form. In some embodiments, the EBP may be administered
substantially simultaneously with another agent (e.g., an
anti-cancer agent). By substantially simultaneously, it is meant
that the EBP is administered to a subject close enough in time with
the administration of the other therapeutic agent, so that the two
compounds exert an additive or even synergistic effect.
[0195] One important category of therapeutic agents is cytotoxic
agents. As used herein, a cytotoxic agent is an agent that can
induce the death of a cell with which it is in contact. When
conjugated to the EBP, the conjugate serves to deliver a cytotoxic
agent directly and specifically to diseased cells (e.g., cancer
cells). Prior art polypeptides including Herceptin have been
co-administered with cytotoxic agents in clinical trials. Although
the results of such trials have been promising, a smaller agent
that binds to ErbB2 (such as the peptides of the invention) will be
a significantly more effective therapeutic. In some instances,
direct covalent linkage of the homing molecule and the cytotoxic
agent will enhance the therapeutic effectiveness of both the
peptide and the cytotoxic agent. Arap et al. (Science 279:377) have
reported the ability of a small peptide generated using random
peptide phage display technology and conjugated to doxorubicin to
bind specifically to endothelial cells of tumor blood vessels.
Administration of the novel peptide-doxorubicin conjugate to
animals bearing breast cancer xenographs resulted in a dramatic
increase in survival, with less associated toxicity than
doxorubicin alone.
[0196] Chemotherapeutic agents that are useful in the invention in
combination with the EBP include alkylating agents,
antimetabolites, natural products, hormones and antagonists, and
miscellaneous chemotherapeutic agents.
[0197] Alkylating agents include nitrogen mustards such as
mechlorethamine (HN.sub.2), cyclophosphamide, melphalan
(L-sarcolysin), uracil mustard, chlorambucil; alkyl sulfonates such
as busulfan; nitrosoureas such as carmustine (BCNU), lomustine
(CCNU), semustine (methyl-CCNU), streptozocin (streptozotocin);
triazenes such as dacarbazine (DTIC;
dimethyltriazen-oimidazolecarbox-amide); platinum co-ordination
complexes such as cisplatin (cis-DDP), carboplatin, tetraplatin,
ipraplatin; and ethylenimine.
[0198] Antimetabolites include folic acid analogs such as
methotrexate (amethopterin); pyrimidine analogs such as
fluorouracil (5-fluorouracil; 5-FU) and cytarabine (cytosine
arabinoside); purine analogs such as mercaptopurine
(6-mercaptopurine; 6-MP) and thioguanine (6-thioguanine; TG).
[0199] Natural products include vinca alkaloids such as vinblastine
(VLB), vincristine and vindesine; Epipodophyllo-toxins such as
etoposide and teniposide; antibiotics such as dactinomycin
(actinomycin D), daunorubicin (daunomycin; rubidomycin),
doxorubicin, bleomycin, plicamycin (mithramycin), mitomycin
(mitomycin C); and enzymes such as L-asparaginase.
[0200] Miscellaneous chemotherapeutic agents include substituted
urea such as hydroxyurea; methyl hydrazine derivatives such as
procarbazine (N-methylhydrazine, MIH); adrenocortical suppressants
such as mitotane (o,p'-DDD) and aminoglutethimide.
[0201] Hormones and antagonists include adrenocorticosteroids such
as prednisone; progestins such as hydroxyprogesterone caproate,
medroxyprogesterone acetate, megestrol acetate; estrogens such as
diethylstilbestrol and ethinyl estradiol; antiestrogens such as
tamoxifen; and androgens such as testosterone propionate and
fluoxymesterone.
[0202] Other cytotoxic agents are toxins such as diptheria toxin A
chain, P. aeruginosa exotoxin A chain, ricin A chain, abrin A
chain, modeccin A chain, .alpha.-sarcin, Aleurites fordii proteins,
dianthin protein, Phytolacca americana proteins (PAPI, PAPII,
PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, pokeweed anti-viral protein, cholera toxin,
pertussis toxin.
[0203] One particularly important class of cytotoxic agents is the
anti-cancer agents. Anti-cancer agents are agents which possess a
preferential cytotoxicity towards malignant cells. In some
preferred embodiments, the anti-cancer agents are specific for
cancers such as breast cancer, ovarian cancer, thyroid cancer,
gastric adenocarcinoma, cervical cancer, prostate cancer, lung
cancer, colorectal cancer, and salivary gland cancer. Some of these
preferred anti-cancer agents include epirubicin, doxorubicin,
taxol, taxanes (paclitaxel and docetaxel), vinorelbine,
gemcitabine, capecitabine, cyclophosphamide, methotrexate, and
fluorouracil.
[0204] Other examples of anti-cancer agents include but are not
limited to: Acivicin; Aclarubicin; Acodazole Hydrochloride;
Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin;
Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole;
Anthramycin; Asparaginase; Asperlin; Azacitidine; Azetepa;
Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene
Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate;
Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone;
Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin
Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin;
Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide;
Cytarabine; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride;
Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate;
Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride;
Droloxifene; Droloxifene Citrate; Dromostanolone Propionate;
Duazomycin; Edatrexate; Eflornithine Hydrochloride; Elsamitrucin;
Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride;
Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine
Phosphate Sodium; Etanidazole; Etoposide; Etoposide Phosphate;
Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide;
Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine;
Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine
Hydrochloride; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide;
Ilmofosine; Interferon Alfa-2a; Interferon Alfa-2b; Interferon
Alfa-n1; Interferon Alfa-n3; Interferon Beta-I a; Interferon
Gamma-I b; Iproplatin; Irinotecan Hydrochloride; Lanreotide
Acetate; Letrozole; Leuprolide Acetate; Liarozole Hydrochloride;
Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride;
Masoprocol; Maytansine; Mechlorethamine Hydrochloride; Megestrol
Acetate; Melengestrol Acetate; Melphalan; Menogaril;
Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine;
Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin;
Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone
Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin;
Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin;
Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman;
Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane;
Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine
Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin;
Riboprine; Rogletimide; Safingol; Safingol Hydrochloride;
Semustine; Simtrazene; Sparfosate Sodium; Sparsomycin;
Spirogermanium Hydrochloride; Spiromustine; Spiroplatin;
Streptonigrin; Streptozocin; Sulofenur; Talisomycin; Tecogalan
Sodium; Tegafur; Teloxantrone Hydrochloride; Taxol; Taxotere;
Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine;
Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan
Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine
Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin;
Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide;
Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine;
Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate;
Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate;
Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin
Hydrochloride.
[0205] Other anti-cancer agents include: 20-epi-1,25
dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin;
acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK
antagonists; altretamine; ambamustine; amidox; amifostine;
aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole;
andrographolide; angiogenesis inhibitors; antagonist D; antagonist
G; antarelix; anti-dorsalizing morphogenetic protein-1;
antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston;
antisense oligonucleotides; aphidicolin glycinate; apoptosis gene
modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA;
arginine deaminase; asulacrine; atamestane; atrimustine;
axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin;
azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL
antagonists; benzochlorins; benzoylstaurosporine; beta lactam
derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF
inhibitor; bicalutamide; bisantrene; bisaziridinylspermine;
bisnafide; bistratene A; bizelesin; breflate; bropirimine;
budotitane; buthionine sulfoximine; calcipotriol; calphostin C;
camptothecin derivatives; canarypox IL-2; capecitabine;
carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN
700; cartilage derived inhibitor; carzelesin; casein kinase
inhibitors (ICOS); castanospermine; cecropin B; cetrorelix;
chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;
dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B;
didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-;
dioxamycin; diphenyl spiromustine; docosanol; dolasetron;
doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen;
ecomustine; edelfosine; edrecolomab; eflomithine; elemene;
emitefur; epirubicin; epristeride; estramustine analogue; estrogen
agonists; estrogen antagonists; etanidazole; etoposide phosphate;
exemestane; fadrozole; fazarabine; fenretinide; filgrastim;
finasteride; flavopiridol; flezelastine; fluasterone; fludarabine;
fluorodaunorunicin hydrochloride; forfenimex; formestane;
fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;
galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;
glutathione inhibitors; hepsulfam; heregulin; hexamethylene
bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;
idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;
immunostimulant peptides; insulin-like growth factor-1 receptor
inhibitor; interferon agonists; interferons; interleukins;
iobenguane; iododoxorubicin; ipomeanol, 4-; irinotecan; iroplact;
irsogladine; isobengazole; isohomohalicondrin B; itasetron;
jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide;
leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole;
leukemia inhibiting factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole;
liarozole; linear polyamine analogue; lipophilic disaccharide
peptide; lipophilic platinum compounds; lissoclinamide 7;
lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;
lovastatin; loxoribine; lurtotecan; lutetium texaphyrin;
lysofylline; lytic peptides; maitansine; mannostatin A; marimastat;
masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase
inhibitors; menogaril; merbarone; meterelin; methioninase;
metoclopramide; MIF inhibitor; mifepristone; miltefosine;
mirimostim; mismatched double stranded RNA; mitoguazone;
mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast
growth factor-saporin; mitoxantrone; mofarotene; molgramostim;
monoclonal antibody, human chorionic gonadotrophin; monophosphoryl
lipid A+myobacterium cell wall sk; mopidamol; multiple drug
resistance gene inhibitor; multiple tumor suppressor 1-based
therapy; mustard anti cancer compound; mycaperoxide B;
mycobacterial cell wall extract; myriaporone; N-acetyldinaline;
N-substituted benzamides; nafarelin; nagrestip;
naloxone+pentazocine; napavin; naphterpin; nartograstim;
nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase;
nilutamide; nisamycin; nitric oxide modulators; nitroxide
antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone;
oligonucleotides; onapristone; ondansetron; ondansetron; oracin;
oral cytokine inducer; ormaplatin; osaterone; oxaliplatin;
oxaunomycin; paclitaxel analogues; paclitaxel derivatives;
palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol;
panomifene; parabactin; pazelliptine; pegaspargase; peldesine;
pentosan polysulfate sodium; pentostatin; pentrozole; perflubron;
perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate;
phosphatase inhibitors; picibanil; pilocarpine hydrochloride;
pirarubicin; piritrexim; placetin A; placetin B; plasminogen
activator inhibitor; platinum complex; platinum compounds;
platinum-triamine complex; porfimer sodium; porfiromycin; propyl
bis-acridone; prostaglandin J2; proteasome inhibitors; protein
A-based immune modulator; protein kinase C inhibitor; protein
kinase C inhibitors, microalgal; protein tyrosine phosphatase
inhibitors; purine nucleoside phosphorylase inhibitors; purpurins;
pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene
conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl
protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;
roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU;
sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence
derived inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; signal transduction modulators; single chain antigen
binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium
phenylacetate; solverol; somatomedin binding protein; sonermin;
sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1; squalamine; stem cell inhibitor; stem-cell division
inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;
superactive vasoactive intestinal peptide antagonist; suradista;
suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;
tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;
tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;
temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;
thaliblastine; thalidomide; thiocoraline; thrombopoietin;
thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist;
thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin;
tirapazamine; titanocene dichloride; topotecan; topsentin;
toremifene; totipotent stem cell factor; translation inhibitors;
tretinoin; triacetyluridine; triciribine; trimetrexate;
triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors;
tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived
growth inhibitory factor; urokinase receptor antagonists;
vapreotide; variolin B; vector system, erythrocyte gene therapy;
velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine;
vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatin
stimalamer.
[0206] Anti-cancer supplementary potentiating compounds include:
Tricyclic anti-depressant drugs (e.g., imipramine, desipramine,
amitryptyline, clomipramine, trimipramine, doxepin, nortriptyline,
protriptyline, amoxapine and maprotiline); non-tricyclic
anti-depressant drugs (e.g., sertraline, trazodone and citalopram);
Ca.sup.++ antagonists (e.g., verapamil, nifedipine, nitrendipine
and caroverine); Calmodulin inhibitors (e.g., prenylamine,
trifluoroperazine and clomipramine); Amphotericin B; Triparanol
analogues (e.g., tamoxifen); antiarrhythmic drugs (e.g.,
quinidine); antihypertensive drugs (e.g., reserpine); Thiol
depleters (e.g., buthionine and sulfoximine) and multiple drug
resistance reducing compounds such as Cremaphor EL.
[0207] Other compounds which are useful in combination therapy for
the purpose of the invention include the antiproliferation
compound, Piritrexim Isethionate; the antiprostatic hypertrophy
compound, Sitogluside; the benign prostatic hyperplasia therapy
compound, Tamsulosin Hydrochloride; the prostate growth inhibitor,
Pentomone; radioactive compounds such as Fibrinogen 1 125,
Fludeoxyglucose F 18, Fluorodopa F 18, Insulin I 125, Insulin I
131, lobenguane I 123, lodipamide Sodium I 131, lodoantipyrine I
131, Iodocholesterol I 131, lodohippurate Sodium I 123,
lodohippurate Sodium I 125, lodohippurate Sodium I 131, lodopyracet
I 125, lodopyracet I 131, lofetamine Hydrochloride I 123, Iomethin
I 125, lomethin I 131, lothalamate Sodium I 125, lothalamate Sodium
I 131, lotyrosine 1 131, Liothyronine I 125, Liothyronine I 131,
Merisoprol Acetate Hg 197, Merisoprol Acetate Hg 203, Merisoprol Hg
197, Selenomethionine Se 75, Technetium Tc 99m Antimony Trisulfide
Colloid, Technetium Tc 99m Bicisate, Technetium Tc 99m Disofenin,
Technetium Tc 99m Etidronate, Technetium Tc 99m Exametazime,
Technetium Tc 99m Furifosmin, Technetium Tc 99m Gluceptate,
Technetium Tc 99m Lidofenin, Technetium Tc 99m Mebrofenin,
Technetium Tc 99m Medronate, Technetium Tc 99m Medronate Disodium,
Technetium Tc 99m Mertiatide, Technetium Tc 99m Oxidronate,
Technetium Tc 99m Pentetate, Technetium Tc 99m Pentetate Calcium
Trisodium, Technetium Tc 99m Sestamibi, Technetium Tc 99m
Siboroxime, Technetium Tc 99m Succimer, Technetium Tc 99m Sulfur
Colloid, Technetium Tc 99m Teboroxime, Technetium Tc 99m
Tetrofosmin, Technetium Tc 99m Tiatide, Thyroxine I 125, Thyroxine
I 131, Tolpovidone I 131, Triolein I 125 and Triolein I 131.
[0208] Other agents that are suitable for use with the EBP
described herein (whether in conjugated or unconjugated form)
include, but are not limited to, small molecule quinazoline and
pyrimidine-based inhibitors, and radioisotopes such as 212Pb.
Cytotoxic agents can also be high energy-emitting radionuclides
such as cobalt-60.
[0209] Other therapeutic agents that can be administered with the
EBP (either unconjugated or unconjugated form) include
immunomodulatory agents, apoptosis-inducing agents, and
anti-angiogenic agents. An immunomodulatory agent is an agent which
is capable of modulating an immune response, preferably at the
tissue having the disorder. Immunomodulatory agents include
adjuvants such as BCG, BCG cell walls, alum, DETOX, SYNTEX,
Comeybacterium parvum, and immunostimulatory nucleic acids; and
cytokines such as IFN-.gamma., IL-1, IL-2, and IL-6. Preferably the
immunomodulatory agent is an immune response-inducing compound
which induces an immune response at the tissue. In one embodiment,
the immune response-inducing compound is a peptide. In another
embodiment the immune response-inducing compound is a carbohydrate.
As used herein an immune response-inducing or an immune
response-inhibiting compound can be a peptide, a carbohydrate or
some combination thereof. In some embodiments, the immunomodulatory
agent is an antigen or an antigenic moiety capable of inducing an
immune response.
[0210] An apoptosis-inducing agent is an agent that induces
apoptosis of the cell with which it comes in contact, or in the
cell within which it enters. An example of an apoptosis-inducing
agent is described by Jodo et al. (J Biol Chem 2001 Aug. 23; epub).
Other examples include curcumin, the chemotherapeutic agent
suberoylanilide hydroxamic acid (SAHA), 7-hydroxystaurosporine,
Indole-3-carbinol (I3C), bis(4,7-dimethyl-1,10 phenanthroline)
sulfatooxovanadium(iv), and the like.
[0211] An anti-angiogenic agent is an agent which can inhibit
angiogenesis, including inhibiting new blood vessel budding or
growth of pre-existing vessels. Such agents are useful in
restricting the blood supply to a diseased tissue or tumor.
Anti-angiogenic agents include sulfated beta-cyclodextrins,
sulfated malto-oligosaccharides, suramin, angiostatin, endostatin,
fumagillin, non-glucocorticoid steroids, and heparin or heparin
fragments, and antibodies to one or more angiogenic peptides such
as .alpha.FGF, .beta.FGF, VEGF, IL-8, and GM-CSF. Angiogenic
inhibitory peptides such as the VEGF-R binding peptide reported by
Binetruy-Tournaire et al. is also embraced by the invention as an
anti-angiogenic agent. (Binetruy-Tournaire et al. EMBO J. 19:1525,
2000.)
[0212] The invention additionally provides methods which use the
EBP disclosed herein. These methods include methods of diagnosis,
including medical imaging, and methods of prevention and treatment
of disorders characterized by ErbB2 overexpression. The invention
seeks, in one aspect, to prophylactically or therapeutically treat
subjects having or at risk of having a disorder characterized by
ErbB2 overexpression. The method involves administering to a
subject in need of such treatment (i.e., a subject who has been
diagnosed as having or at risk of having the disorder) an EBP (such
as EBP-1, EBP-2, EBP-3, etc.) or a functional equivalent thereof.
The EBP or its functional equivalent is administered in an amount
effective to inhibit the disorder. In preferred embodiments, the
EBP is a peptide having an amino acid sequence selected from the
group consisting of SEQ ID NO:1 through to SEQ ID NO:13 and SEQ ID
NO:33 through to SEQ ID NO:39, inclusive. The peptides may be
linear, but in some preferred embodiments, they are cyclic.
[0213] As used herein, a subject is a human, non-human primate,
cow, horse, pig, sheep, goat, dog, cat or rodent. In all
embodiments, human subjects are preferred.
[0214] ErbB2 overexpression is defined as a level higher than that
observed in a control normal population as described herein. A
"normal" level, as used herein in reference to the level of ErbB2
mRNA or polypeptide, may be a level in a control population, which
preferably includes subjects having similar characteristics as the
treated individual, such as age and sex. The "normal" level can
also be a range, for example, where a population is used to obtain
a baseline range for a particular group into which the subject
falls. Thus, the "normal" value can depend upon a particular
population selected. Preferably, the normal levels are those of
apparently healthy subjects who have no prior history of ErbB2
overexpression related disorders. As an example, if the subject to
be treated has been diagnosed as having breast cancer or is at risk
of having breast cancer, then the control population is one that
does not have breast cancer and is not at risk of having breast
cancer (e.g., that does not have a family history of breast
cancer). Such normal levels can then be established as preselected
values, taking into account the category into which an individual
falls. Appropriate ranges and categories can be selected with no
more than routine experimentation by those of ordinary skill in the
art. Either the mean or another preselected number within the range
can be established as the normal preselected value.
[0215] More preferably, the normal level is that level in a tissue
of a normal subject corresponding to the tissue sampled for the
test subject. In other instances, the normal levels can also be
determined by measuring mRNA and/or peptide or polypeptide levels
in a sample of normal tissue adjacent to the suspected diseased
tissue in the subject to be treated. As an example, breast tumors
are, in some cases, sufficiently delineated to the extent that such
tissue can be distinguished from the surrounding normal breast
tissue. This delineation facilitates selective removal of diseased
breast tissue, such as occurs in non-radical mastectomies (e.g.,
lumpectomy). Similarly, such delineation can be used in the present
invention to harvest both suspected diseased tissue and normal
tissue from a given subject.
[0216] The disorders to be prevented or treated according to the
invention may occur in tissues in which ErbB2 is known to be
expressed normally. ErbB2 is expressed during fetal development,
particularly in neural tissue, however it is minimally expressed in
normal adult tissue. Disorders to be prevented or treated may also
occur in tissues in which ErbB2 expression has not been detected
normally (e.g., most normal adult tissues). Tissues at risk of
developing a disorder similarly include tissues in which the
disorders listed herein have been found previously (e.g., breast
and esophageal tissue).
[0217] Preferably, the disorder being diagnosed or treated is a
proliferative disorder such as cancer. As used herein, a cancer is
defined as an uncontrolled (e.g., factor independent) growth of
abnormal cells, which can either remain localized, or may
disseminate throughout the body via the bloodstream or the
lymphatic system, and thereby seed a secondary site (i.e., a
metastasis). The diagnostic, prophylactic, and treatment methods of
the invention are intended to be used to in the prevention and
treatment of primary tumors and secondary tumors (i.e.,
metastases). ErbB2 overexpression has been reportedly associated
with particular forms of cancer including most notably breast
cancer and ovarian cancer. Examples of cancers to be diagnosed,
prevented, and/or treated include: biliary tract cancer; brain
cancer, including glioblastomas and medulloblastomas; breast
cancer; cervical cancer; choriocarcinoma; colon cancer; endometrial
cancer; esophageal cancer; gastric cancer; hematological neoplasms,
including acute lymphocytic and myelogenous leukemia; chronic
lymphocytic and myelogenous leukemia; multiple myeloma;
AIDS-associated leukemias and adult T-cell leukemia lymphoma;
intraepithelial neoplasms, including Bowen's disease and Paget's
disease; liver cancer; lung cancer; lymphomas, including Hodgkin's
disease and lymphocytic lymphomas; neuroblastomas; oral cancer,
including squamous cell carcinoma; ovarian cancer, including those
arising from epithelial cells, stromal cells, germ cells, and
mesenchymal cells; pancreas cancer; prostate cancer; colorectal
cancer; sarcomas, including leiomyosarcoma, rhabdomyosarcoma,
liposarcoma, fibrosarcoma, and osteosarcoma; skin cancer, including
melanoma, Kaposi's sarcoma, basocellular cancer, and squamous cell
cancer; testicular cancer, including germinal tumors (seminoma,
non-seminoma teratomas, and choriocarcinomas), stromal tumors, and
germ cell tumors; thyroid cancer, including thyroid adenocarcinoma
and medullar carcinoma; and renal cancer including adenocarcinoma
and Wilms' tumor. In some important embodiments, the cancer is
defined as an ErbB2 expressing cancer such as breast cancer, lung
cancer, ovarian cancer, gastric cancer and bladder cancer.
Preferably, the invention is directed at breast cancer and
esophageal cancer.
[0218] The EBP of the invention can also be used to prevent or
inhibit metastasis. Tumor metastasis involves the spread of tumor
cells primarily via the vasculature following the disassembly of
tumor cell-extracellular matrix (ECM) interactions through the
degradation of the ECM, and tumor cell extravasation through the
capillary bed. The invasion and metastasis of cancer is a complex
process which involves changes in cell adhesion properties which
allow a transformed cell to invade and migrate through the ECM and
acquire anchorage-independent growth properties. Liotta, L. A., et
al., Cell 64:327-336 (1991). Some of these changes occur at focal
adhesions, which are cell/ECM contact points containing
membrane-associated, cytoskeletal, and intracellular signaling
molecules. Metastatic disease occurs when the disseminated foci of
tumor cells seed a tissue which supports their growth and
propagation, and this secondary spread of tumor cells is
responsible for the morbidity and mortality associated with the
majority of cancers. Thus the term "metastasis" as used herein
refers to the invasion and migration of tumor cells away from the
primary tumor site.
[0219] Subjects having the disorder characterized by ErbB2
overexpression include subjects who have a disorder such as cancer.
These subjects may be identified using the diagnostic methods
described herein, and/or the methods used to diagnose the cell
proliferative disorders listed above, including physical exam and
diagnostic imaging tests. The diagnosis of such disorders,
including cell proliferative disorders such as cancer and
metastasis, are well known in the art and are routinely practiced
by medical professionals. The treatment method may further comprise
the selection of a subject having the disorder prior to the
administration of the ErbB2 antagonist, according to the teaching
provided herein.
[0220] The prophylactic methods of the invention are directed to
subjects who are at risk of developing the disorder. Such a subject
may also be identified using the diagnostic methods provided
herein. Namely, a subject at risk may be one who exhibits ErbB2
overexpression in a particular tissue yet who does not manifest
other symptoms of the disorder (e.g., no discernible breast lump in
the case of a subject at risk of breast cancer). Other subjects at
risk of developing such a disorder may be those with a family
history of such disorders. As an example, subjects with a family
history of breast cancer may be considered subjects for
prophylactic treatment. Subjects at risk of certain disorders
characterized by ErbB2 overexpression may also be those who have
previously been diagnosed and treated for such a disorder. An
example of this is a subject who has previously been diagnosed and
treated for breast cancer. This subject is at risk of re-developing
breast cancer either as a primary tumor or as a metastasis at a
secondary site. In certain embodiments, the prophylactic methods
further comprise first selecting a subject who is at risk of
developing the disorder prior to the administration of the EBP.
[0221] In still other aspects, the invention embraces the use of
ErbB2 agonists as therapeutic agents, particularly in the treatment
of disorder such as osteoporosis, paralysis, or degenerative nerve
disease (e.g., Parkinson's disease). Such agonists can be
identified via homology to the EBPs identified herein.
[0222] The EBP may have varying binding affinity for ErbB2. This
variation can be exploited in the treatment of subjects where it is
necessary to control the extent of ErbB2 inhibition desired either
as a function of development or of time in a treatment regimen.
Thus, early on in a subject's treatment it may be desirable to
administer a higher affinity EBP while later in the treatment (for
example, during a remission) it may be more suitable to administer
a lesser affinity EBP. In addition, lower affinity EBP may also be
desired in some instances in order to effect better solid tumor
perfusion.
[0223] The EBP are administered to a subject in an effective
amount. The effective amount will depend upon the mode of
administration, the particular condition being treated, and the
desired outcome. It will also depend upon, as discussed above, the
stage and severity of the condition, the subject to be treated
including the age and physical condition of the subject, the nature
of concurrent therapy, if any, and like factors well-known to the
medical practitioner. For prophylactic applications, it is
generally that amount sufficient to delay the onset of, inhibit the
progression of, or halt altogether the particular condition sought
to be prevented. For therapeutic applications, it is generally that
amount sufficient to achieve a medically desirable result.
[0224] When used therapeutically, an effective amount is that
amount which inhibits the disorder. Such inhibition may be measured
by an inhibition or a decrease in cell proliferation or, in some
instances, tumor growth. Inhibition of tumor growth may be manifest
as a reduction in the size of a tumor mass, or as a failure of the
tumor to increase in size. Inhibition of the disorder may also be
measured in terms of the occurrence and diminution of metastatic
lesions in the subject. When used prophylactically, an effective
amount may be that amount which prevents a disorder from arising.
Such inhibition may be measured by an absence of a tumor, perhaps
manifest as a failure of the suspect tissue to increase in size or
mass, or to develop a discernible tumor. If the subject to be
treated already has a tumor, and is at risk of having a metastasis,
the effective amount may also be that amount which prevents the
spread of a primary tumor to secondary sites (i.e., an inhibition
in metastasis). Thus, in one embodiment, the agent may be
administered in an effective amount to inhibit metastasis,
independent of its ability to inhibit primary tumor growth.
[0225] Generally, doses of active compounds of the present
invention would be from about 0.01 mg/kg per day to 1000 mg/kg per
day. It is expected that doses ranging from 1-500 mg/kg, and
preferably doses ranging from 1-100 mg/kg, and even more preferably
doses ranging from 1-50 mg/kg, will be suitable.
[0226] The methods of the invention, generally speaking, may be
practiced using any mode of administration that is medically
acceptable, meaning any mode that produces effective levels of the
active compounds without causing clinically unacceptable adverse
effects. Such modes of administration include oral, rectal,
topical, nasal, interdermal, or parenteral routes. The term
"parenteral" includes subcutaneous, intravenous, intramuscular, or
infusion.
[0227] Intravenous or intramuscular routes are not particularly
suitable for long-term therapy and prophylaxis. They could,
however, be preferred in emergency situations. Oral administration
may be preferred for prophylactic treatment because of the
convenience to the patient as well as the dosing schedule.
[0228] When the compounds described herein (including peptide and
non-peptide varieties) are used therapeutically, in certain
embodiments a desirable route of administration may be by pulmonary
aerosol. Techniques for preparing aerosol delivery systems
containing compounds are well known to those of skill in the art.
Generally, such systems should utilize components which will not
significantly impair the biological properties of the compounds,
for example the ErbB2 binding capacity of the EBPs (see, for
example, Sciarra and Cutie, "Aerosols," in Remington's
Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712;
incorporated by reference). Those of skill in the art can readily
determine the various parameters and conditions for producing
aerosols without resort to undue experimentation.
[0229] The EBPs, functional equivalents thereof and/or nucleic acid
molecules that encode EBPs, may be administered directly to a
tissue. Preferably, the tissue is itself a tumor or it is a tissue
in which the disorder exists. Alternatively, the tissue is one in
which a tumor or disorder is likely to exist. For example, a
subject at risk of developing breast cancer may be prophylactically
treated by administering an EBP into the breast tissue of the
subject. Direct tissue administration may be achieved by direct
injection. The EBPs may be administered once, or alternatively they
may be administered in a plurality of administrations. If
administered multiple times, the EBPs may be administered via
different routes. For example, the first (or the first few)
administrations may be made directly into the affected tissue while
later administrations may be systemic. These later administrations
may also comprise lower doses of EBP, particularly if their purpose
is remission maintenance rather than remission induction.
[0230] Although the EBPs of the invention can act as targeting
agents, it may also be desirable in some instances to conjugate an
EBP to a particular targeting agent or compound such as a ligand
specific for a particular tissue or tumor type. In this way, a
subset of ErbB2 expressing cells can be targeted, and in some
instances cells that express low levels of ErbB2 (i.e., levels
insufficient to bind large amounts of EBP in an unconjugated form)
can also be targeted. The agents of the invention may be targeted
to primary or, in some instances, secondary (i.e., metastatic)
lesions through the use of targeting compounds which preferentially
recognize a cell surface marker. The targeting compound may be
directly conjugated to the agents of the invention via a covalent
linkage. The agent may be indirectly conjugated to a targeting
compound via a linker. Methods of conjugation suitable in the
invention have been described elsewhere herein.
[0231] Alternatively, the targeting compound (including the EBP)
may be conjugated or associated with an intermediary compound such
as, for example, a liposome within which the agent is encapsulated.
Liposomes are artificial membrane vessels which are useful as a
delivery vector in vivo or in vitro. It has been shown that large
unilamellar vessels (LUV), which range in size from 0.2-4.0 .mu.m
can encapsulate large macromolecules. Liposomes may be targeted to
a particular tissue, such as the vascular endothelium, by coupling
the liposome to a specific ligand such as a monoclonal antibody,
sugar, glycolipid, or protein. Liposomes are commercially available
from Gibco BRL, for example, as LIPOFECTIN.TM. and LIPOFECTACE.TM.,
which are formed of cationic lipids such as N-[1-(2,3
dioleyloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA) and
dimethyl dioctadecylammonium bromide (DDAB). Methods for making
liposomes are well known in the art and have been described in many
publications. Liposomes also have been reviewed by Gregoriadis, G.
in Trends in Biotechnology, V. 3, p. 235-241 (1985). In still other
embodiments, the targeting compound may be loosely associated with
the EBP, such as within a microparticle comprising a polymer, the
EBP and the targeting compound.
[0232] In some instances, the EBP of the invention can exist on the
surface of a liposome that contains a therapeutic agent. In this
way, the EBP acts as a targeting agent that delivers the
therapeutic agent to a cell that expresses ErbB2. The liposome may
alternatively carry a nucleic acid sequence intended for use in
gene therapy.
[0233] Targeting compounds useful according to the methods of the
invention are those which direct the antagonist to a site of a
disorder characterized by ErbB2 overexpression (e.g., a tumor). The
targeting compound of choice will depend upon the nature of, for
example, the tumor or the tissue origin of the metastasis. In some
instances it may be desirable to target the agent to the tissue in
which the tumor is located. As an example, agents can be delivered
to breast epithelium by using a targeting compound specific for
breast tissue. In important embodiments, the target is specific for
malignant breast epithelium. Examples of compounds which may
localize to malignant breast epithelium include, but are not
limited to, estrogen and progesterone, among others. Ovarian
cancers are also known to express EGFR and c-fms, and thus could be
targeted through the use of ligands for either receptor. In the
case of c-fms which is also expressed by macrophages and monocytes,
targeted delivery to an ovarian cancer may require a combination of
local administration such as a vaginal suppository as well as a
targeting compound. Prostate cancers can be targeted using
compounds such as peptides (e.g., antibodies or antibody fragments)
which bind to prostate specific antigen (PSA) or prostate specific
membrane antigen (PSMA). Other markers which may be used for
targeting of the agent to specific tissues include, for example, in
liver: HGF, insulin-like growth factor I, II, insulin, OV-6,
HEA-125, hyaluronic acid, collagen, N-terminal propeptide of
collagen type III, mannose/N-acetylglucosamine, asialoglycoprotein,
tissue plasminogen activator, low density lipoprotein,
carcinoembryonic antigen; in kidney cells: angiotensin II, 20
vasopressin, antibodies to CD44v6; in keratinocytes and skin
fibroblasts: KGF, very low density lipoprotein, RGD-containing
peptides, collagen, laminin; in melanocytes: kit ligand;
[0234] in gut: cobalamin-intrinsic factor, heat stable enterotoxin
of E. coli; in breast epithelium:
[0235] heregulin, prolactin, transferrin, cadherin-11. Other
markers specific to particular tissues are available and would be
known to one of ordinary skill in the art. In still other
embodiments, the agent of the invention may be targeted to
fibroblasts via ligands or binding partners for fibroblast specific
markers. Examples of these markers include, but are not limited to
fibroblast growth factors (FGF) and platelet derived growth factor
(PDGF).
[0236] In still other embodiments, the invention provides
bifunctional peptides that are capable of binding to ErbB2 as well
as another protein such as for example EGFR, ErbB3 or ErbB4.
[0237] The invention further provides a composition of the EBPs or
their functional equivalents for use as a medicament, methods for
preparing the medicament, and methods for the sustained release of
the medicament in vivo.
[0238] Pharmaceutical preparations of EBP (e.g., EBPs), functional
equivalents thereof, and/or nucleic acid molecules encoding such
EBP are provided by the invention. The pharmaceutical preparation
also contain a pharmaceutically acceptable carrier. An EBP is
present in the pharmaceutical preparation in a prophylactically or
therapeutically effective amount.
[0239] The term "pharmaceutically-acceptable carrier" as used
herein means one or more compatible solid or liquid filler,
diluents or encapsulating substances which are suitable for
administration into a human. The term "carrier" denotes an organic
or inorganic ingredient, natural or synthetic, with which the
active ingredient is combined to facilitate the application. The
components of the pharmaceutical compositions also are capable of
being commingled with the agents of the present invention, and with
each other, in a manner such that there is no interaction which
would substantially impair the desired pharmaceutical efficacy.
[0240] The pharmaceutical preparations may routinely contain salt,
buffering agents, preservatives, compatible carriers, and
optionally other therapeutic agents. When used in medicine, the
salts should be pharmaceutically acceptable, but
non-pharmaceutically acceptable salts may conveniently be used to
prepare pharmaceutically-acceptable salts thereof and are not
excluded from the scope of the invention. Such pharmacologically
and pharmaceutically-acceptable salts include, but are not limited
to, those prepared from the following acids: hydrochloric,
hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic,
salicylic, citric, formic, malonic, succinic, and the like. Also,
pharmaceutically-acceptable salts can be prepared as alkaline metal
or alkaline earth salts, such as sodium, potassium or calcium
salts.
[0241] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives may also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like. Lower doses will result from other forms of
administration, such as intravenous administration. In the event
that a response in a subject is insufficient at the initial doses
applied, higher doses (or effectively higher doses by a different,
more localized delivery route) may be employed to the extent that
patient tolerance permits. Multiple doses per day are contemplated
to achieve appropriate systemic levels of compounds.
[0242] Compositions suitable for oral administration may be
presented as discrete units, such as capsules, tablets, lozenges,
each containing a predetermined amount of the active agent. Other
compositions include suspensions in aqueous liquids or non-aqueous
liquids such as a syrup, elixir or an emulsion.
[0243] In yet other embodiments, the EBP is administered via a
biocompatible microparticle or implant that is suitable for
implantation into the mammalian recipient. Exemplary bioerodible
implants that are useful in accordance with this method are
described in PCT International Application No. PCT/US/03307
(Publication No. WO 95/24929, entitled "Polymeric Gene Delivery
System", claiming priority to U.S. patent application Ser. No.
213,668, filed Mar. 15, 1994). PCT/US/0307 describes a
biocompatible, preferably biodegradable polymeric matrix for
containing a biological macromolecule. The polymeric matrix may be
used to achieve sustained release of the agent in a subject. In
accordance with one aspect of the instant invention, the agent
described herein may be encapsulated or dispersed within the
biocompatible, preferably biodegradable polymeric matrix disclosed
in PCT/US/03307. The polymeric matrix preferably is in the form of
a microparticle such as a microsphere (wherein the agent is
dispersed throughout a solid polymeric matrix) or a microcapsule
(wherein the agent is stored in the core of a polymeric shell).
Other forms of the polymeric matrix for containing the agent
include films, coatings, gels, implants, and stents. The size and
composition of the polymeric matrix device is selected to result in
favorable release kinetics in the tissue into which the matrix
device is implanted. The size of the polymeric matrix device
further is selected according to the method of delivery which is to
be used, typically injection into a tissue or administration of a
suspension by aerosol into the nasal and/or pulmonary areas. The
polymeric matrix composition can be selected to have both favorable
degradation rates and also to be formed of a material which is
bioadhesive, to further increase the effectiveness of transfer when
the device is administered to a vascular, pulmonary, or other
surface. The matrix composition also can be selected not to
degrade, but rather, to release by diffusion over an extended
period of time.
[0244] Both non-biodegradable and biodegradable polymeric matrices
can be used to deliver the agents of the invention to the subject.
Biodegradable matrices are preferred. Such polymers may be natural
or synthetic polymers. Synthetic polymers are preferred. The
polymer is selected based on the period of time over which release
is desired, generally in the order of a few hours to a year or
longer. Typically, release over a period ranging from between a few
hours and three to twelve months is most desirable. The polymer
optionally is in the form of a hydrogel that can absorb up to about
90% of its weight in water and further, optionally is cross-linked
with multivalent ions or other polymers.
[0245] In general, the agents of the invention may be delivered
using the bioerodible implant by way of diffusion, or more
preferably, by degradation of the polymeric matrix. Exemplary
synthetic polymers which can be used to form the biodegradable
delivery system include: polyamides, polycarbonates, polyalkylenes,
polyalkylene glycols, polyalkylene oxides, polyalkylene
terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl
esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides,
polysiloxanes, polyurethanes and co-polymers thereof, alkyl
cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose
esters, nitro celluloses, polymers of acrylic and methacrylic
esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose,
hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose,
cellulose acetate, cellulose propionate, cellulose acetate
butyrate, cellulose acetate phthalate, carboxylethyl cellulose,
cellulose triacetate, cellulose sulphate sodium salt, poly(methyl
methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),
poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl
methacrylate), poly(lauryl methacrylate), poly(phenyl
methacrylate), poly(methyl acrylate), poly(isopropyl acrylate),
poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene,
polypropylene, poly(ethylene glycol), poly(ethylene oxide),
poly(ethylene terephthalate), poly(vinyl alcohols), polyvinyl
acetate, poly vinyl chloride, polystyrene and
polyvinylpyrrolidone.
[0246] Examples of non-biodegradable polymers include ethylene
vinyl acetate, poly(meth)acrylic acid, polyamides, copolymers and
mixtures thereof.
[0247] Examples of biodegradable polymers include synthetic
polymers such as polymers of lactic acid and glycolic acid,
polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid),
poly(valeric acid), and poly(lactide-cocaprolactone), and natural
polymers such as alginate and other polysaccharides including
dextran and cellulose, collagen, chemical derivatives thereof
(substitutions, additions of chemical groups, for example, alkyl,
alkylene, hydroxylations, oxidations, and other modifications
routinely made by those skilled in the art), albumin and other
hydrophilic proteins, zein and other prolamines and hydrophobic
proteins, copolymers and mixtures thereof. In general, these
materials degrade either by enzymatic hydrolysis or exposure to
water in vivo, by surface or bulk erosion.
[0248] Bioadhesive polymers of particular interest include
bioerodible hydrogels described by H. S. Sawhney, C. P. Pathak and
J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of
which are incorporated herein, polyhyaluronic acids, casein,
gelatin, glutin, polyanhydrides, polyacrylic acid, alginate,
chitosan, poly(methyl methacrylates), poly(ethyl methacrylates),
poly(butylmethacrylate), poly(isobutyl methacrylate),
poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl
methacrylate), poly(phenyl methacrylate), poly(methyl acrylate),
poly(isopropyl acrylate), poly(isobutyl acrylate), and
poly(octadecyl acrylate).
[0249] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the EBP or function equivalent,
increasing convenience to the subject and the physician. Many types
of release delivery systems are available and known to those of
ordinary skill in the art. They include polymer base systems such
as poly(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides. Microcapsules of the foregoing polymers containing
drugs are described in, for example, U.S. Pat. No. 5,075,109.
Delivery systems also include non-polymer systems that are: lipids
including sterols such as cholesterol, cholesterol esters and fatty
acids or neutral fats such as mono- di- and tri-glycerides;
hydrogel release systems; silastic systems; peptide based systems;
wax coatings; compressed tablets using conventional binders and
excipients; partially fused implants; and the like. Specific
examples include, but are not limited to: (a) erosional systems in
which the platelet reducing agent is contained in a form within a
matrix such as those described in U.S. Pat. Nos. 4,452,775,
4,675,189, and 5,736,152 and (b) diffusional systems in which an
active component permeates at a controlled rate from a polymer such
as described in U.S. Pat. Nos. 3,854,480, 5,133,974 and 5,407,686.
In addition, pump-based hardware delivery systems can be used, some
of which are adapted for implantation.
[0250] Use of a long-term sustained release implant may be
particularly suitable for prophylactic treatment of subjects at
risk of having a disorder characterized by ErbB2 overexpression.
Long-term release, as used herein, means that the implant is
constructed and arranged to delivery therapeutic levels of the
active ingredient for at least 30 days, and preferably 60 days.
Long-term sustained release implants are well-known to those of
ordinary skill in the art and include some of the release systems
described above.
[0251] The following examples are included for purposes of
illustration and are not intended to limit the scope of the
invention.
EXAMPLES
Example 1
Identification of Peptide Phage from Random and Biased Peptide
Phage Libraries
[0252] Clones were identified by screening random as well as biased
peptide phage library. Peptides identified by screening random
libraries include those having the following sequences:
TABLE-US-00001 CB022701-20 DTDMCWWWSREFGWECAGAG (SEQ ID NO: 37)
(E-20) CB051701-19 SLALCLSEGVLLGADCRVLF (SREQ ID NO: 38) (C-19)
CB051701-25 WSSMCGDPTIADWLWCFSDA. (SEQ ID NO: 39) (C-25)
[0253] The following example describes the methodology used to
identify four clones from four biased peptide phage libraries. The
libraries were enriched for the sequence of the EC-1 phage clone
(SEQ ID NO:1). The four libraries had the following design:
TABLE-US-00002 X.sub.4CLNPEESTWGFCRSAG, (SEQ ID NO: 29)
WTGWCX.sub.5STWGFCRSAG, (SEQ ID NO: 30) WTGWCLNPEEX.sub.5CRSAG,
(SEQ ID NO: 31) and WTGWCLNPEESTWGFCX.sub.4, (SEQ ID NO: 32)
where X=any amino acid.
[0254] The peptides so identified had the following sequences:
TABLE-US-00003 02-124 WTGWCLNPEESTWGFCRSAG (SEQ ID NO: 33) 02-137
WTGWCLSPEESTWGFCRSAG (SEQ ID NO: 34) 02-140 WTGWCLNPEESTWGFCSGYI
(SEQ ID NO: 35) 02-135 WTGWCFDDNHSTWGFCTGSF. (SEQ ID NO: 36)
Example 2
Peptide Phage Bind Specifically to Purified ErbB2 Extracellular
Domain
[0255] The peptide libraries were screened for the ability to bind
to the purified extracellular domain of ErbB2 with a His tag
(denoted CBECD). The following example demonstrates the methodology
for such screening. Binding results are shown in FIGS. 1A and
1B.
[0256] SfmECDAP labeled wells were plated with 250 ng of a purified
ErbB2 ECD-alkaline phosphatase fusion protein; CBECD labeled wells
were plated with 250 ng of a purified ErbB2
ECD-polyhistidine-tagged protein; bovine serum albumin (denoted
BSA) was used as a negative target control. E12 and CB1 are peptide
phage clones identified independently that display an identical
(ErbB2-binding) peptide. E12CB1-NA peptide phage clones were
constructed to present, with and without a glycine-glycine-alanine
linker, a shortened form of the peptide. Results indicate that the
deletion mutants were inactive (FIG. 1A). G78 is a peptide phage
clone that binds strongly to the Grb7 SH2 domain, used here as a
negative peptide phage control. Library phage also serve as a
negative peptide phage control. 9G6 is an antibody to the ECD of
ErbB2.
[0257] FIG. 1B demonstrates the binding to the extracellular domain
of ErbB2 of the peptides identified from the biased libraries.
Example 2
Binding of Peptide Phage to Membrane Lysates from ErbB2 Expressing
Cells
[0258] The above identified peptide phage specifically bind to
native, intact ErbB2 extracted from the membranes of human breast
cancer cells that overexpress ErbB2. Plates were coated with 1
.mu.g of an antibody that binds to the intracellular domain of
ErbB2, followed by 100 .mu.l of solubilized membrane preparations
from two different breast cancer cell lines that overexpress ErbB2
(BT474 and SKBR3), and probed with CB1 peptide-phage, control
library phage, and 9G6 anti-ErbB2. MCF7 cells serve as a (relative)
negative target control, as MCF7 breast cancer cells express only a
low level of ErbB2. (See FIG. 2A.) The ELISA data indicates that
the EBP gives a signal higher than the anti-ErbB2 antibody used as
a positive control in the assay.
[0259] Peptide phage derived from the EC-1 biases library similarly
bind specifically to membrane lysates from ErbB2 expressing SKBR3
cells but not to lysates from minimally expressing MCF7 cells. (See
FIG. 2B.)
[0260] Immunofluorescence assay results indicate that the peptide
is a highly effective ErbB2-binding agent as demonstrated by the
ability of phage bearing the peptide to label brightly the surface
of ErbB2-overexpressing cells, but to not bind to cells that do not
overexpress ErbB2 (FIGS. 3, 4, 5).
Example 3
Effect of Phagepeptide on ErbB2 Phosphorylation
[0261] ErbB2 overexpressing SKBR3 cells were treated with the EC-1
free peptide to determine its effect on ErbB2 activation. When
ErbB2 is activated it becomes phosphorylated on specific tyrosine
residues (i.e., pY1248 and pY877). This is turn triggers downstream
signal transduction events, culminating in increased cellular
proliferation. Viable SKBR3 cells were treated with 25 .mu.M EC-1
peptide for 15 minutes. At 0.5, 2, 4, 8, 22 and 48 hours
thereafter, cell lysates were prepared and run on a Western blot
using phospho-specific ErbB2 antibodies. (See FIG. 6A.)
Densitometric analysis of the Western blots showed that EC-1
peptide inhibits 40% of the phosphorylation of residues pY1248 and
pY877 after 0.5 hrs. The results demonstrate that 25 .mu.M EC-1
inhibits the phosphorylation of ErbB2 for at least 8 hours after
treatment. (See FIG. 6B.) The peptide had no effect on total ErbB2
expression.
Example 4
Localization of Agents to and Within ErbB2 Expressing Cells
[0262] EBP either unconjugated or conjugated to a therapeutic agent
such as a cytotoxic agent are produced. These peptides are tested
in the following assays with the SKBR3 and BT474 cell lines that
are known to overexpress ErbB2, cell lines that express low levels
of ErbB2 (MCF-7) and breast cells that do not overexpress ErbB2 (Hs
578Bst) as controls. These cell lines are tested using the
following assays: a) viability by trypan blue and hemocytometer
counts (standard); b) cell proliferation by MTT assay (Hansen et
al. J. Immunol. Meth. 119:203-210, 1989); c) cell proliferation by
BrdU assay (Roche); d) clonogenic assays (as described above); e)
human breast cancer xenographs in nude mice (as described
above).
[0263] The peptides are modified, depending on the results of the
above assays, using mutagenesis phage display technology and
medicinal chemistry techniques. For example, peptides that require
a cyclic structure may have their disulfide bond changed to a
thioether bond, to increase stability in vivo (Oligino et al., JBC
272:29046).
[0264] The cytotoxicity and specificity of the therapeutic for
ErbB2 expressing cells, in vitro, in animal models, and in clinical
studies, can be significantly improved by covalent attachment to an
EBP. EBP in an unconjugated form can effectively inhibit the
function of ErbB2 (including the susceptibility of ErbB2 to
phosphorylation) and, therefore, the proliferation of cells
overexpressing ErbB2. Inhibition of proliferation can ameliorate
disease caused by the overexpression of ErbB2 directly and/or can
lead to induction of apoptosis of cells overexpressing ErbB2.
[0265] The EBP can also be used in some embodiments to modulate
trafficking of agents within a cell. Some EBP are capable of
localizing therapeutic agents preferentially in the cytoplasm,
while others allow movement into the nucleus. Agents that exert
their effects in the cytoplasm can be conjugated to the EBP
described herein in order to increase their concentration in the
cytoplasm.
Equivalents
[0266] It should be understood that the preceding is merely a
detailed description of certain preferred embodiments. It therefore
should be apparent to those of ordinary skill in the art that
various modifications and equivalents can be made without departing
from the spirit and scope of the invention. It is intended that the
invention encompass all such modifications within the scope of the
appended claims.
[0267] All references, patents and patent applications and
publications that are cited or referred to in this application are
incorporated in their entirety herein by reference.
Sequence CWU 1
1
48 1 20 PRT Artificial sequence Synthetic peptide 1 Trp Thr Gly Trp
Cys Leu Asn Pro Glu Glu Ser Thr Trp Gly Phe Cys 1 5 10 15 Thr Gly
Ser Phe 20 2 19 PRT Artificial sequence Synthetic peptide 2 Val Val
Ala Cys Ser Trp Asp Trp Thr Met Gly Ala Val Val Cys Tyr 1 5 10 15
Glu Arg Ile 3 20 PRT Artificial sequence Synthetic peptide 3 Gly
Phe Trp Thr Cys Glu Tyr Asp Trp Trp Ser Asp Ala Thr Val Cys 1 5 10
15 Met His Thr Leu 20 4 20 PRT Artificial sequence Synthetic
peptide 4 Gly Arg Gly Trp Cys Trp Ser Glu Trp Gln Asn Asp Trp Phe
Trp Cys 1 5 10 15 Trp Asp Val Trp 20 5 20 PRT Artificial sequence
Synthetic peptide 5 Trp Thr Gly Trp Cys Leu Asn Pro Glu Glu Ser Thr
Trp Gly Phe Cys 1 5 10 15 Thr Gly Ser Phe 20 6 20 PRT Artificial
sequence Synthetic peptide 6 Ala Arg Leu Gln Cys Trp Ser Leu Gly
Trp Gly Gly Pro Val Tyr Cys 1 5 10 15 Gly Phe Gly Gln 20 7 20 PRT
Artificial sequence Synthetic peptide 7 Ile Gln Glu Val Cys Trp Phe
Asp Tyr Asn Leu Ser Gln Trp His Cys 1 5 10 15 Met Thr Val Ile 20 8
20 PRT Artificial sequence Synthetic peptide 8 Pro Asp Ile Tyr Cys
Leu Ser Val Thr Ala Pro Gly Phe Leu Ile Cys 1 5 10 15 Tyr Glu Arg
Tyr 20 9 20 PRT Artificial sequence Synthetic peptide 9 His Asp Glu
Leu Cys Val Phe Ser Phe Asp Phe Asn Ala Leu Leu Cys 1 5 10 15 Trp
Pro Ala Glu 20 10 20 PRT Artificial sequence Synthetic peptide 10
Leu Asn Trp Glu Cys Trp Tyr Asp Tyr Arg Leu Glu Ala Trp Asp Cys 1 5
10 15 Arg Gly Asp Ile 20 11 11 PRT Artificial sequence Synthetic
peptide 11 Cys Glu Val Trp Gly Glu Val Pro Trp Thr Cys 1 5 10 12 11
PRT Artificial sequence Synthetic peptide 12 Cys Glu Val Trp Gly
Phe Val Pro Trp Ala Cys 1 5 10 13 20 PRT Artificial sequence
Synthetic peptide 13 Ser Asn Glu Ser Cys Gly Ser Pro Ile Asn Pro
Trp Gly Glu Met Cys 1 5 10 15 Leu Leu Met Leu 20 14 57 DNA
Artificial sequence Synthetic oligonucleotide 14 actggttggt
gtttaaatcc tgaagaatct acttggggtt tttgtactgg ttctttt 57 15 57 DNA
Artificial sequence Synthetic oligonucleotide 15 gttgttgcat
gttcttggga ttggactatg ggtgcagttg tttgttatga acgtatt 57 16 60 DNA
Artificial sequence Synthetic oligonucleotide 16 ggtttttgga
cttgtgaata tgattggtgg tctgatgcaa ctgtttgtat gcatacttta 60 17 60 DNA
Artificial sequence Synthetic oligonucleotide 17 ggtcgtggtt
ggtgttggtc tgaatggcaa aatgattggt tttggtgttg ggatgtttgg 60 18 60 DNA
Artificial sequence Synthetic oligonucleotide 18 tggactggtt
ggtgtttaaa tcctgaagaa tctacttggg gtttttgtac tggttctttt 60 19 60 DNA
Artificial sequence Synthetic oligonucleotide 19 gcacgtttac
aatgttggtc tttaggttgg ggtggtcctg tttattgtgg ttttggtcaa 60 20 60 DNA
Artificial sequence Synthetic oligonucleotide 20 attcaagaag
tttgttggtt tgattataat ttatctcaat ggcattgtat gactgttatt 60 21 60 DNA
Artificial sequence Synthetic oligonucleotide 21 cctgatattt
attgtttatc tgttactgca cctggttttt taatttgtta tgaacgttat 60 22 60 DNA
Artificial sequence Synthetic oligonucleotide 22 catgatgaat
tatgtgtttt ttcttttgat tttaatgcat tattatgttg gcctgcagaa 60 23 60 DNA
Artificial sequence Synthetic oligonucleotide 23 ttaaattggg
aatgttggta tgattatcgt ttagaagcat gggattgtcg tggtgatatt 60 24 33 DNA
Artificial sequence Synthetic oligonucleotide 24 tgtgaagttt
ggggtgaagt tccttggact tgt 33 25 33 DNA Artificial sequence
Synthetic oligonucleotide 25 tgtgaagttt ggggttttgt tccttgggca tgt
33 26 60 DNA Artificial sequence Synthetic oligonucleotide 26
tctaatgaat cttgtggttc tcctattaat ccttggggtg aaatgtgttt attaatgtta
60 27 4530 DNA Homo sapiens 27 aattctcgag ctcgtcgacc ggtcgacgag
ctcgagggtc gacgagctcg agggcgcgcg 60 cccggccccc acccctcgca
gcaccccgcg ccccgcgccc tcccagccgg gtccagccgg 120 agccatgggg
ccggagccgc agtgagcacc atggagctgg cggccttgtg ccgctggggg 180
ctcctcctcg ccctcttgcc ccccggagcc gcgagcaccc aagtgtgcac cggcacagac
240 atgaagctgc ggctccctgc cagtcccgag acccacctgg acatgctccg
ccacctctac 300 cagggctgcc aggtggtgca gggaaacctg gaactcacct
acctgcccac caatgccagc 360 ctgtccttcc tgcaggatat ccaggaggtg
cagggctacg tgctcatcgc tcacaaccaa 420 gtgaggcagg tcccactgca
gaggctgcgg attgtgcgag gcacccagct ctttgaggac 480 aactatgccc
tggccgtgct agacaatgga gacccgctga acaataccac ccctgtcaca 540
ggggcctccc caggaggcct gcgggagctg cagcttcgaa gcctcacaga gatcttgaaa
600 ggaggggtct tgatccagcg gaacccccag ctctgctacc aggacacgat
tttgtggaag 660 gacatcttcc acaagaacaa ccagctggct ctcacactga
tagacaccaa ccgctctcgg 720 gcctgccacc cctgttctcc gatgtgtaag
ggctcccgct gctggggaga gagttctgag 780 gattgtcaga gcctgacgcg
cactgtctgt gccggtggct gtgcccgctg caaggggcca 840 ctgcccactg
actgctgcca tgagcagtgt gctgccggct gcacgggccc caagcactct 900
gactgcctgg cctgcctcca cttcaaccac agtggcatct gtgagctgca ctgcccagcc
960 ctggtcacct acaacacaga cacgtttgag tccatgccca atcccgaggg
ccggtataca 1020 ttcggcgcca gctgtgtgac tgcctgtccc tacaactacc
tttctacgga cgtgggatcc 1080 tgcaccctcg tctgccccct gcacaaccaa
gaggtgacag cagaggatgg aacacagcgg 1140 tgtgagaagt gcagcaagcc
ctgtgcccga gtgtgctatg gtctgggcat ggagcacttg 1200 cgagaggtga
gggcagttac cagtgccaat atccaggagt ttgctggctg caagaagatc 1260
tttgggagcc tggcatttct gccggagagc tttgatgggg acccagcctc caacactgcc
1320 ccgctccagc cagagcagct ccaagtgttt gagactctgg aagagatcac
aggttaccta 1380 tacatctcag catggccgga cagcctgcct gacctcagcg
tcttccagaa cctgcaagta 1440 atccggggac gaattctgca caatggcgcc
tactcgctga ccctgcaagg gctgggcatc 1500 agctggctgg ggctgcgctc
actgagggaa ctgggcagtg gactggccct catccaccat 1560 aacacccacc
tctgcttcgt gcacacggtg ccctgggacc agctctttcg gaacccgcac 1620
caagctctgc tccacactgc caaccggcca gaggacgagt gtgtgggcga gggcctggcc
1680 tgccaccagc tgtgcgcccg agggcactgc tggggtccag ggcccaccca
gtgtgtcaac 1740 tgcagccagt tccttcgggg ccaggagtgc gtggaggaat
gccgagtact gcaggggctc 1800 cccagggagt atgtgaatgc caggcactgt
ttgccgtgcc accctgagtg tcagccccag 1860 aatggctcag tgacctgttt
tggaccggag gctgaccagt gtgtggcctg tgcccactat 1920 aaggaccctc
ccttctgcgt ggcccgctgc cccagcggtg tgaaacctga cctctcctac 1980
atgcccatct ggaagtttcc agatgaggag ggcgcatgcc agccttgccc catcaactgc
2040 acccactcct gtgtggacct ggatgacaag ggctgccccg ccgagcagag
agccagccct 2100 ctgacgtcca tcgtctctgc ggtggttggc attctgctgg
tcgtggtctt gggggtggtc 2160 tttgggatcc tcatcaagcg acggcagcag
aagatccgga agtacacgat gcggagactg 2220 ctgcaggaaa cggagctggt
ggagccgctg acacctagcg gagcgatgcc caaccaggcg 2280 cagatgcgga
tcctgaaaga gacggagctg aggaaggtga aggtgcttgg atctggcgct 2340
tttggcacag tctacaaggg catctggatc cctgatgggg agaatgtgaa aattccagtg
2400 gccatcaaag tgttgaggga aaacacatcc cccaaagcca acaaagaaat
cttagacgaa 2460 gcatacgtga tggctggtgt gggctcccca tatgtctccc
gccttctggg catctgcctg 2520 acatccacgg tgcagctggt gacacagctt
atgccctatg gctgcctctt agaccatgtc 2580 cgggaaaacc gcggacgcct
gggctcccag gacctgctga actggtgtat gcagattgcc 2640 aaggggatga
gctacctgga ggatgtgcgg ctcgtacaca gggacttggc cgctcggaac 2700
gtgctggtca agagtcccaa ccatgtcaaa attacagact tcgggctggc tcggctgctg
2760 gacattgacg agacagagta ccatgcagat gggggcaagg tgcccatcaa
gtggatggcg 2820 ctggagtcca ttctccgccg gcggttcacc caccagagtg
atgtgtggag ttatggtgtg 2880 actgtgtggg agctgatgac ttttggggcc
aaaccttacg atgggatccc agcccgggag 2940 atccctgacc tgctggaaaa
gggggagcgg ctgccccagc cccccatctg caccattgat 3000 gtctacatga
tcatggtcaa atgttggatg attgactctg aatgtcggcc aagattccgg 3060
gagttggtgt ctgaattctc ccgcatggcc agggaccccc agcgctttgt ggtcatccag
3120 aatgaggact tgggcccagc cagtcccttg gacagcacct tctaccgctc
actgctggag 3180 gacgatgaca tgggggacct ggtggatgct gaggagtatc
tggtacccca gcagggcttc 3240 ttctgtccag accctgcccc gggcgctggg
ggcatggtcc accacaggca ccgcagctca 3300 tctaccagga gtggcggtgg
ggacctgaca ctagggctgg agccctctga agaggaggcc 3360 cccaggtctc
cactggcacc ctccgaaggg gctggctccg atgtatttga tggtgacctg 3420
ggaatggggg cagccaaggg gctgcaaagc ctccccacac atgaccccag ccctctacag
3480 cggtacagtg aggaccccac agtacccctg ccctctgaga ctgatggcta
cgttgccccc 3540 ctgacctgca gcccccagcc tgaatatgtg aaccagccag
atgttcggcc ccagccccct 3600 tcgccccgag agggccctct gcctgctgcc
cgacctgctg gtgccactct ggaaagggcc 3660 aagactctct ccccagggaa
gaatggggtc gtcaaagacg tttttgcctt tgggggtgcc 3720 gtggagaacc
ccgagtactt gacaccccag ggaggagctg cccctcagcc ccaccctcct 3780
cctgccttca gcccagcctt cgacaacctc tattactggg accaggaccc accagagcgg
3840 ggggctccac ccagcacctt caaagggaca cctacggcag agaacccaga
gtacctgggt 3900 ctggacgtgc cagtgtgaac cagaaggcca agtccgcaga
agccctgatg tgtcctcagg 3960 gagcagggaa ggcctgactt ctgctggcat
caagaggtgg gagggccctc cgaccacttc 4020 caggggaacc tgccatgcca
ggaacctgtc ctaaggaacc ttccttcctg cttgagttcc 4080 cagatggctg
gaaggggtcc agcctcgttg gaagaggaac agcactgggg agtctttgtg 4140
gattctgagg ccctgcccaa tgagactcta gggtccagtg gatgccacag cccagcttgg
4200 ccctttcctt ccagatcctg ggtactgaaa gccttaggga agctggcctg
agaggggaag 4260 cggccctaag ggagtgtcta agaacaaaag cgacccattc
agagactgtc cctgaaacct 4320 agtactgccc cccatgagga aggaacagca
atggtgtcag tatccaggct ttgtacagag 4380 tgcttttctg tttagttttt
actttttttg ttttgttttt ttaaagacga aataaagacc 4440 caggggagaa
tgggtgttgt atggggaggc aagtgtgggg ggtccttctc cacacccact 4500
ttgtccattt gcaaatatat tttggaaaac 4530 28 1255 PRT Homo sapiens 28
Met Glu Leu Ala Ala Leu Cys Arg Trp Gly Leu Leu Leu Ala Leu Leu 1 5
10 15 Pro Pro Gly Ala Ala Ser Thr Gln Val Cys Thr Gly Thr Asp Met
Lys 20 25 30 Leu Arg Leu Pro Ala Ser Pro Glu Thr His Leu Asp Met
Leu Arg His 35 40 45 Leu Tyr Gln Gly Cys Gln Val Val Gln Gly Asn
Leu Glu Leu Thr Tyr 50 55 60 Leu Pro Thr Asn Ala Ser Leu Ser Phe
Leu Gln Asp Ile Gln Glu Val 65 70 75 80 Gln Gly Tyr Val Leu Ile Ala
His Asn Gln Val Arg Gln Val Pro Leu 85 90 95 Gln Arg Leu Arg Ile
Val Arg Gly Thr Gln Leu Phe Glu Asp Asn Tyr 100 105 110 Ala Leu Ala
Val Leu Asp Asn Gly Asp Pro Leu Asn Asn Thr Thr Pro 115 120 125 Val
Thr Gly Ala Ser Pro Gly Gly Leu Arg Glu Leu Gln Leu Arg Ser 130 135
140 Leu Thr Glu Ile Leu Lys Gly Gly Val Leu Ile Gln Arg Asn Pro Gln
145 150 155 160 Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe
His Lys Asn 165 170 175 Asn Gln Leu Ala Leu Thr Leu Ile Asp Thr Asn
Arg Ser Arg Ala Cys 180 185 190 His Pro Cys Ser Pro Met Cys Lys Gly
Ser Arg Cys Trp Gly Glu Ser 195 200 205 Ser Glu Asp Cys Gln Ser Leu
Thr Arg Thr Val Cys Ala Gly Gly Cys 210 215 220 Ala Arg Cys Lys Gly
Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys 225 230 235 240 Ala Ala
Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu 245 250 255
His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val 260
265 270 Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly
Arg 275 280 285 Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr
Asn Tyr Leu 290 295 300 Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys
Pro Leu His Asn Gln 305 310 315 320 Glu Val Thr Ala Glu Asp Gly Thr
Gln Arg Cys Glu Lys Cys Ser Lys 325 330 335 Pro Cys Ala Arg Val Cys
Tyr Gly Leu Gly Met Glu His Leu Arg Glu 340 345 350 Val Arg Ala Val
Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys 355 360 365 Lys Ile
Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp 370 375 380
Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe 385
390 395 400 Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala
Trp Pro 405 410 415 Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu
Gln Val Ile Arg 420 425 430 Gly Arg Ile Leu His Asn Gly Ala Tyr Ser
Leu Thr Leu Gln Gly Leu 435 440 445 Gly Ile Ser Trp Leu Gly Leu Arg
Ser Leu Arg Glu Leu Gly Ser Gly 450 455 460 Leu Ala Leu Ile His His
Asn Thr His Leu Cys Phe Val His Thr Val 465 470 475 480 Pro Trp Asp
Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr 485 490 495 Ala
Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His 500 505
510 Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys
515 520 525 Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu
Glu Cys 530 535 540 Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn
Ala Arg His Cys 545 550 555 560 Leu Pro Cys His Pro Glu Cys Gln Pro
Gln Asn Gly Ser Val Thr Cys 565 570 575 Phe Gly Pro Glu Ala Asp Gln
Cys Val Ala Cys Ala His Tyr Lys Asp 580 585 590 Pro Pro Phe Cys Val
Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu 595 600 605 Ser Tyr Met
Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln 610 615 620 Pro
Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys 625 630
635 640 Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Val
Ser 645 650 655 Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val
Val Phe Gly 660 665 670 Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg
Lys Tyr Thr Met Arg 675 680 685 Arg Leu Leu Gln Glu Thr Glu Leu Val
Glu Pro Leu Thr Pro Ser Gly 690 695 700 Ala Met Pro Asn Gln Ala Gln
Met Arg Ile Leu Lys Glu Thr Glu Leu 705 710 715 720 Arg Lys Val Lys
Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys 725 730 735 Gly Ile
Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile 740 745 750
Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu 755
760 765 Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser
Arg 770 775 780 Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val
Thr Gln Leu 785 790 795 800 Met Pro Tyr Gly Cys Leu Leu Asp His Val
Arg Glu Asn Arg Gly Arg 805 810 815 Leu Gly Ser Gln Asp Leu Leu Asn
Trp Cys Met Gln Ile Ala Lys Gly 820 825 830 Met Ser Tyr Leu Glu Asp
Val Arg Leu Val His Arg Asp Leu Ala Ala 835 840 845 Arg Asn Val Leu
Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe 850 855 860 Gly Leu
Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp 865 870 875
880 Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg
885 890 895 Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val
Thr Val 900 905 910 Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp
Gly Ile Pro Ala 915 920 925 Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly
Glu Arg Leu Pro Gln Pro 930 935 940 Pro Ile Cys Thr Ile Asp Val Tyr
Met Ile Met Val Lys Cys Trp Met 945 950 955 960 Ile Asp Ser Glu Cys
Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe 965 970 975 Ser Arg Met
Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu 980 985 990 Asp
Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu 995
1000 1005 Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu
Tyr 1010 1015 1020 Leu Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro
Ala Pro Gly 1025 1030 1035 Ala Gly Gly Met Val His His Arg His Arg
Ser Ser Ser Thr Arg 1040 1045 1050 Ser Gly Gly Gly Asp Leu Thr Leu
Gly Leu Glu Pro Ser Glu Glu 1055 1060 1065 Glu Ala Pro Arg Ser Pro
Leu Ala Pro Ser Glu Gly Ala Gly Ser 1070 1075 1080 Asp Val Phe Asp
Gly Asp Leu Gly Met Gly Ala Ala Lys Gly Leu 1085 1090 1095 Gln Ser
Leu Pro Thr His Asp Pro Ser Pro Leu Gln Arg Tyr Ser
1100 1105 1110 Glu Asp Pro Thr Val Pro Leu Pro Ser Glu Thr Asp Gly
Tyr Val 1115 1120 1125 Ala Pro Leu Thr Cys Ser Pro Gln Pro Glu Tyr
Val Asn Gln Pro 1130 1135 1140 Asp Val Arg Pro Gln Pro Pro Ser Pro
Arg Glu Gly Pro Leu Pro 1145 1150 1155 Ala Ala Arg Pro Ala Gly Ala
Thr Leu Glu Arg Ala Lys Thr Leu 1160 1165 1170 Ser Pro Gly Lys Asn
Gly Val Val Lys Asp Val Phe Ala Phe Gly 1175 1180 1185 Gly Ala Val
Glu Asn Pro Glu Tyr Leu Thr Pro Gln Gly Gly Ala 1190 1195 1200 Ala
Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala Phe Asp 1205 1210
1215 Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala Pro
1220 1225 1230 Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro
Glu Tyr 1235 1240 1245 Leu Gly Leu Asp Val Pro Val 1250 1255 29 20
PRT Artificial sequence Synthetic peptide 29 Xaa Xaa Xaa Xaa Cys
Leu Asn Pro Glu Glu Ser Thr Trp Gly Phe Cys 1 5 10 15 Arg Ser Ala
Gly 20 30 20 PRT Artificial sequence Synthetic peptide 30 Trp Thr
Gly Trp Cys Xaa Xaa Xaa Xaa Xaa Ser Thr Trp Gly Phe Cys 1 5 10 15
Arg Ser Ala Gly 20 31 20 PRT Artificial sequence Synthetic peptide
31 Trp Thr Gly Trp Cys Leu Asn Pro Glu Glu Xaa Xaa Xaa Xaa Xaa Cys
1 5 10 15 Arg Ser Ala Gly 20 32 21 PRT Artificial sequence
Synthetic peptide 32 Trp Thr Gly Trp Cys Leu Asn Pro Glu Glu Ser
Thr Trp Gly Phe Cys 1 5 10 15 Xaa Xaa Xaa Xaa Xaa 20 33 20 PRT
Artificial sequence Synthetic peptide 33 Trp Thr Gly Trp Cys Leu
Asn Pro Glu Glu Ser Thr Trp Gly Phe Cys 1 5 10 15 Arg Ser Ala Gly
20 34 20 PRT Artificial sequence Synthetic peptide 34 Trp Thr Gly
Trp Cys Leu Ser Pro Glu Glu Ser Thr Trp Gly Phe Cys 1 5 10 15 Arg
Ser Ala Gly 20 35 20 PRT Artificial sequence Synthetic peptide 35
Trp Thr Gly Trp Cys Leu Asn Pro Glu Glu Ser Thr Trp Gly Phe Cys 1 5
10 15 Ser Gly Tyr Ile 20 36 20 PRT Artificial sequence Synthetic
peptide 36 Trp Thr Gly Trp Cys Phe Asp Asp Asn His Ser Thr Trp Gly
Phe Cys 1 5 10 15 Thr Gly Ser Phe 20 37 20 PRT Artificial sequence
Synthetic peptide 37 Asp Thr Asp Met Cys Trp Trp Trp Ser Arg Glu
Phe Gly Trp Glu Cys 1 5 10 15 Ala Gly Ala Gly 20 38 20 PRT
Artificial sequence Synthetic peptide 38 Ser Leu Ala Leu Cys Leu
Ser Glu Gly Val Leu Leu Gly Ala Asp Cys 1 5 10 15 Arg Val Leu Phe
20 39 20 PRT Artificial sequence Synthetic peptide 39 Trp Ser Ser
Met Cys Gly Asp Pro Thr Ile Ala Asp Trp Leu Trp Cys 1 5 10 15 Phe
Ser Asp Ala 20 40 60 DNA Artificial sequence Synthetic
oligonucleotide 40 tggactggtt ggtgtttaaa tcctgaagaa tctacttggg
gtttttgtcg ttctgcaggt 60 41 60 DNA Artificial sequence Synthetic
oligonucleotide 41 tggactggtt ggtgtttatc tcctgaagaa tctacttggg
gtttttgtcg ttctgcaggt 60 42 60 DNA Artificial sequence Synthetic
oligonucleotide 42 tggactggtt ggtgtttaaa tcctgaagaa tctacttggg
gtttttgttc tggttatatt 60 43 60 DNA Artificial sequence Synthetic
oligonucleotide 43 tggactggtt ggtgttttga tgataatcat tctacttggg
gtttttgtac tggttctttt 60 44 60 DNA Artificial sequence Synthetic
oligonucleotide 44 gatactgata tgtgttggtg gtggtctcgt gaatttggtt
gggaatgtgc aggtgcaggt 60 45 60 DNA Artificial sequence Synthetic
oligonucleotide 45 tctttagcat tatgtttatc tgaaggtgtt ttattaggtg
cagattgtcg tgttttattt 60 46 60 DNA Artificial sequence Synthetic
oligonucleotide 46 tggtcttcta tgtgtggtga tcctactatt gcagattggt
tatggtgttt ttctgatgca 60 47 4 PRT Artificial sequence Synthetic
peptide 47 Trp Thr Gly Trp 1 48 5 PRT Artificial sequence Synthetic
peptide 48 Ser Thr Trp Gly Phe 1 5
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