U.S. patent application number 13/995086 was filed with the patent office on 2014-10-09 for novel modulators and methods of use.
This patent application is currently assigned to Stem CentRx, Inc.. The applicant listed for this patent is Alex Bankovich, Scott J. Dylla, Orit Foord, Johannes Hampl. Invention is credited to Alex Bankovich, Scott J. Dylla, Orit Foord, Johannes Hampl.
Application Number | 20140302034 13/995086 |
Document ID | / |
Family ID | 46207727 |
Filed Date | 2014-10-09 |
United States Patent
Application |
20140302034 |
Kind Code |
A1 |
Bankovich; Alex ; et
al. |
October 9, 2014 |
NOVEL MODULATORS AND METHODS OF USE
Abstract
Novel modulators, including antibodies and derivatives thereof,
and methods of using such modulators to treat hyperproliferative
disorders are provided.
Inventors: |
Bankovich; Alex; (San
Francisco, CA) ; Foord; Orit; (Foster City, CA)
; Hampl; Johannes; (Santa Clara, CA) ; Dylla;
Scott J.; (Mountain View, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bankovich; Alex
Foord; Orit
Hampl; Johannes
Dylla; Scott J. |
San Francisco
Foster City
Santa Clara
Mountain View |
CA
CA
CA
CA |
US
US
US
US |
|
|
Assignee: |
Stem CentRx, Inc.
San Francisco
CA
|
Family ID: |
46207727 |
Appl. No.: |
13/995086 |
Filed: |
December 7, 2011 |
PCT Filed: |
December 7, 2011 |
PCT NO: |
PCT/US2011/063834 |
371 Date: |
June 23, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61421157 |
Dec 8, 2010 |
|
|
|
Current U.S.
Class: |
424/136.1 ;
424/133.1; 424/178.1; 435/320.1; 435/328; 530/387.3; 530/391.1;
530/391.7; 536/23.53 |
Current CPC
Class: |
A61K 47/6811 20170801;
C07K 2317/24 20130101; C07K 2317/34 20130101; A61P 35/04 20180101;
A61K 47/6855 20170801; C07K 2317/77 20130101; C07K 2317/33
20130101; C07K 2317/565 20130101; C07K 16/3069 20130101; A61K
47/6869 20170801; A61P 43/00 20180101; G01N 33/574 20130101; C07K
16/18 20130101; A61P 35/00 20180101; C07K 2317/56 20130101; C07K
2317/73 20130101; C07K 2317/92 20130101; A61K 47/6815 20170801;
C07K 16/2809 20130101; C07K 2317/76 20130101; A61P 35/02 20180101;
C07K 16/3015 20130101; C07K 16/3061 20130101; C07K 2317/14
20130101; C12Y 302/02022 20130101; A61K 47/6455 20170801; A61K
47/6891 20170801; C07K 16/30 20130101; A61K 47/64 20170801; A61K
47/6867 20170801; C07K 16/2896 20130101; A61K 38/47 20130101; C07K
16/40 20130101; C07K 16/28 20130101 |
Class at
Publication: |
424/136.1 ;
530/387.3; 530/391.1; 530/391.7; 536/23.53; 424/133.1; 424/178.1;
435/320.1; 435/328 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 47/48 20060101 A61K047/48; C07K 16/30 20060101
C07K016/30 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 2, 2011 |
US |
PCT/US2011/050451 |
Claims
1-106. (canceled)
107. An antibody comprising a chimeric, CDR-grafted, humanized,
recombinant human antibody, or multispecific antibody, or fragment
of said antibody, that specifically binds to human EFNA1 and that
competes for binding to human EFNA1 with an antibody comprising:
(a) a heavy chain variable region set forth as SEQ ID NO: 15 and a
light chain variable region set forth as SEQ ID NO: 17; (b) a heavy
chain variable region set forth as SEQ ID NO: 23 and a light chain
variable region set forth as SEQ ID NO: 25; or (c) a heavy chain
variable region set forth as SEQ ID NO: 31 and a light chain
variable region set forth as SEQ ID NO: 33.
108. The antibody of claim 107, which is a neutralizing antibody or
a depleting antibody.
109. The antibody of claim 107, which is an internalizing
antibody.
110. The antibody of claim 107 comprising at least one heavy chain
variable region and at least one light chain variable region,
wherein the at least one heavy chain variable region comprises
three CDRs of a heavy chain variable region set forth as SEQ ID NO:
15, 23, or 31.
111. The antibody of claim 107 comprising at least one heavy chain
variable region and at least one light chain variable region,
wherein the at least one light chain variable region comprises
three CDRs of a light chain variable region set forth as SEQ ID NO:
17, 25, or 33.
112. The antibody of claim 107 comprising: (a) a heavy chain
variable region comprising three CDRs of a heavy chain variable
region set forth as SEQ ID NO: 15 and a light chain variable region
comprising three CDRs of a light chain variable region set forth as
SEQ ID NO: 17; (b) a heavy chain variable region comprising three
CDRs of a heavy chain variable region set forth as SEQ ID NO: 23
and a light chain variable region comprising three CDRs of a light
chain variable region set forth as SEQ ID NO: 25; or (c) a heavy
chain variable region comprising three CDRs of a heavy chain
variable region set forth as SEQ ID NO: 31 and a light chain
variable region comprising three CDRs of a light chain variable
region set forth as SEQ ID NO: 33.
113. The antibody of claim 107 comprising: (a) a heavy chain
variable region having an amino acid sequence that is at least 60%
identical to SEQ ID NO: 15 and a light chain variable having an
amino acid sequence that is at least 60% identical to SEQ ID NO:
17; (b) a heavy chain variable region having an amino acid sequence
that is at least 60% identical to SEQ ID NO: 23 and a light chain
variable having an amino acid sequence that is at least 60%
identical to SEQ ID NO: 25; or (c) a heavy chain variable region
having an amino acid sequence that is at least 60% identical to SEQ
ID NO: 31 and a light chain variable having an amino acid sequence
that is at least 60% identical to SEQ ID NO: 33.
114. The antibody of claim 113 comprising: (a) a heavy chain
variable region set forth as SEQ ID NO: 15 and a light chain
variable region set forth as SEQ ID NO: 17; (b) a heavy chain
variable region set forth as SEQ ID NO: 23 and a light chain
variable region set forth as SEQ ID NO: 25; or (c) a heavy chain
variable region set forth as SEQ ID NO: 31 and a light chain
variable region set forth as SEQ ID NO: 33.
115. The antibody of claim 107 comprising: (a) a heavy chain
variable region set forth as SEQ ID NO: 15, 23, or 31; or (b) a
light chain variable region set forth as SEQ ID NO: 17, 25, or
33.
116. The antibody of claim 107, which is a multispecific antibody
that specifically binds to EFNA1 and to a heterologous epitope.
117. The multispecific antibody of claim 116 that specifically
binds to EFNA1 and to a heterologous epitope on a cancer cell
marker.
118. The multispecific antibody of claim 116 that specifically
binds to EFNA1 and to a heterologous epitope on a cancer stem cell
marker.
119. The multispecific antibody of claim 118 that specifically
binds to EFNA1 and to a heterologous epitope in CD3, CD46, or
CD324.
120. The multispecific antibody of claim 116, which is a
cross-linked or heteroconjugate antibody.
121. The antibody of claim 107, which is conjugated, linked or
otherwise associated with a cytotoxic agent.
122. A pharmaceutical composition comprising the antibody of claim
107.
123. An antibody conjugate comprising a chimeric, CDR-grafted,
humanized, recombinant human antibody, or multispecific antibody,
or a fragment of said antibody, which specifically binds to EFNA1,
and which antibody is conjugated, linked or otherwise associated
with a cytotoxic agent.
124. A pharmaceutical composition comprising the antibody conjugate
of claim 123.
125. A nucleic acid encoding a heavy chain variable region set
forth as SEQ ID NO: 14, 22, or 30, or a light chain variable region
set forth as SEQ ID NO: 16, 24, or 32.
126. A vector comprising the nucleic acid of claim 125.
127. A host cell comprising the nucleic acid of claim 125.
128. A method of treating an EFNA associated disorder comprising
administering to a subject a therapeutically effective amount of a
composition comprising a chimeric, CDR-grafted, humanized,
recombinant human antibody, or multispecific antibody, or a
fragment of said antibody, which specifically binds to EFNA1.
129. The method of claim 128, wherein the composition comprises a
chimeric, CDR-grafted, humanized, recombinant human antibody, or
multispecific antibody, or a fragment of said antibody, which
specifically binds to EFNA1, and which antibody is conjugated,
linked or otherwise associated with a cytotoxic agent.
130. The method of claim 129, wherein the EFNA associated disorder
is a hyperproliferative disorder.
131. The method of claim 130, wherein the hyperproliferative
disorder is a neoplastic disorder.
132. The method of claim 131, wherein the neoplastic disorder
comprises a solid tumor.
133. The method of claim 132, wherein the neoplastic disorder is
lung cancer, ovarian cancer, or colorectal cancer.
Description
CROSS REFERENCED APPLICATIONS
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 61/421,157 filed Dec. 8, 2010 and Patent
Cooperation Treaty (PCT); No. PCT/US2011/050451, filed Sep. 2,
2011, each of which is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] This application generally relates to novel compositions and
methods of their use in preventing, treating or ameliorating
hyperproliferative disorders and any expansion, recurrence, relapse
or metastasis thereof. In a broad aspect, the present invention
relates to the use of ephrin-A ligand (EFNA) modulators, including
anti-EFNA antibodies and fusion constructs, for the treatment or
prophylaxis of neoplastic disorders. Particularly preferred
embodiments of the present invention provide for the use of such
EFNA modulators directed to EFNA1 and EFNA3 for the
immunotherapeutic treatment of malignancies comprising a reduction
in tumor initiating cell frequency.
BACKGROUND OF THE INVENTION
[0003] Stem and progenitor cell differentiation and cell
proliferation are normal ongoing processes that act in concert to
support tissue growth during organogenesis and cell replacement and
repair of most tissues during the lifetime of all living organisms.
Differentiation and proliferation decisions are often controlled by
numerous factors and signals that are balanced to maintain cell
fate decisions and tissue architecture. Normal tissue architecture
is largely maintained by cells responding to microenvironmental
cues that regulate cell division and tissue maturation.
Accordingly, cell proliferation and differentiation normally occurs
only as necessary for the replacement of damaged or dying cells or
for growth. Unfortunately, disruption of cell proliferation and/or
differentiation can result from a myriad of factors including, for
example, the under- or overabundance of various signaling
chemicals, the presence of altered microenvironments, genetic
mutations or some combination thereof. When normal cellular
proliferation and/or differentiation is disturbed or somehow
disrupted it can lead to various diseases or disorders including
hyperproliferative disorders such as cancer.
[0004] Conventional treatments for cancer include chemotherapy,
radiotherapy, surgery, immunotherapy (e.g., biological response
modifiers, vaccines or targeted therapeutics) or combinations
thereof. Sadly, far too many cancers are non-responsive or
minimally responsive to such conventional treatments leaving few
options for patients. For example, in some patients certain cancers
exhibit gene mutations that render them non-responsive despite the
general effectiveness of selected therapies. Moreover, depending on
the type of cancer some available treatments, such as surgery, may
not be viable alternatives. Limitations inherent in current
standard of care therapeutics are particularly evident when
attempting to care for patients who have undergone previous
treatments and have subsequently relapsed. In such cases the failed
therapeutic regimens and resulting patient deterioration may
contribute to refractory tumors which often manifest themselves as
a more aggressive disease that ultimately proves to be incurable.
Although there have been great improvements in the diagnosis and
treatment of cancer over the years, overall survival rates for many
solid tumors have remained largely unchanged due to the failure of
existing therapies to prevent relapse, tumor recurrence and
metastases. Thus, it remains a challenge to develop more targeted
and potent therapies.
[0005] One promising area of research involves the use of targeted
therapeutics to go after the tumorigenic "seed" cells that appear
to underlie many cancers. To that end most solid tissues are now
known to contain adult, tissue-resident stem cell populations
generating the differentiated cell types that comprise the majority
of that tissue. Tumors arising in these tissues similarly consist
of heterogeneous populations of cells that also arise from stem
cells, but differ markedly in their overall proliferation and
organization. While it is increasingly recognized that the majority
of tumor cells have a limited ability to proliferate, a minority
population of cancer cells (commonly known as cancer stem cells or
CSC) have the exclusive ability to extensively self-renew thereby
enabling an inherent tumor reinitiating capacity. More
specifically, the cancer stem cell hypothesis proposes that there
is a distinct subset of cells (i.e. CSC) within each tumor
(approximately 0.1-10%) that is capable of indefinite self-renewal
and of generating tumor cells progressively limited in their
replication capacity as a result of differentiation to tumor
progenitor cells and, subsequently, to terminally differentiated
tumor cells.
[0006] In recent years it has become more evident these CSC (also
known as tumor perpetuating cells or TPC) might be more resistant
to traditional chemotherapeutic agents or radiation and thus
persist after standard of care clinical therapies to later fuel the
growth of refractory tumors, secondary tumors and promote
metastases. Moreover, growing evidence suggests that pathways that
regulate organogenesis and/or the self-renewal of normal
tissue-resident stem cells are deregulated or altered in CSC,
resulting in the continuous expansion of self-renewing cancer cells
and tumor formation. See generally Al-Hajj et al., 2004, PMID:
15378087; and Dalerba et al., 2007, PMID: 17548814; each of which
is incorporated herein in its entirety by reference. Thus, the
effectiveness of traditional, as well as more recent targeted
treatment methods, has apparently been limited by the existence
and/or emergence of resistant cancer cells that are capable of
perpetuating the cancer even in face of these diverse treatment
methods. Huff et al., European Journal of Cancer 42: 1293-1297
(2006) and Zhou et al., Nature Reviews Drug Discovery 8: 806-823
(2009) each of which is incorporated herein in its entirety by
reference. Such observations are confirmed by the consistent
inability of traditional debulking agents to substantially increase
patient survival when suffering from solid tumors, and through the
development of an increasingly sophisticated understanding as to
how tumors grow, recur and metastasize. Accordingly, recent
strategies for treating neoplastic disorders have recognized the
importance of eliminating, depleting, silencing or promoting the
differentiation of tumor perpetuating cells so as to diminish the
possibility of tumor recurrence, metastasis or patient relapse.
[0007] Efforts to develop such strategies have incorporated recent
work involving non-traditional xenograft (NTX) models, wherein
primary human solid tumor specimens are implanted and passaged
exclusively in immunocompromised mice. In numerous cancers such
techniques confirm the existence of a subpopulation of cells with
the unique ability to generate heterogeneous tumors and fuel their
growth indefinitely. As previously hypothesized, work in NTX models
has confirmed that identified CSC subpopulations of tumor cells
appear more resistant to debulking regimens such as chemotherapy
and radiation, potentially explaining the disparity between
clinical response rates and overall survival. Further, employment
of NTX models in CSC research has sparked a fundamental change in
drug discovery and preclinical evaluation of drug candidates that
may lead to CSC-targeted therapies having a major impact on tumor
recurrence and metastasis thereby improving patient survival rates.
While progress has been made, inherent technical difficulties
associated with handling primary and/or xenograft tumor tissue,
along with a lack of experimental platforms to characterize CSC
identity and differentiation potential, pose major challenges. As
such, there remains a substantial need to selectively target cancer
stem cells and develop diagnostic, prophylactic or therapeutic
compounds or methods that may be used in the treatment, prevention
and/or management of hyperproliferative disorders.
SUMMARY OF THE INVENTION
[0008] These and other objectives are provided for by the present
invention which, in a broad sense, is directed to methods,
compounds, compositions and articles of manufacture that may be
used in the treatment of EFNA associated disorders (e.g.,
hyperproliferative disorders or neoplastic disorders). To that end,
the present invention provides novel EFNA (or ephrin-A ligand)
modulators that effectively target tumor cells or cancer stem cells
and may be used to treat patients suffering from a wide variety of
malignancies. As will be discussed in more detail herein, there are
presently six known ephrin-A ligands (i.e., EFNAs 1-6) and the
disclosed modulators preferably comprise or associate with EFNA1
and/or EFNA3. Moreover, in certain embodiments the disclosed EFNA
modulators may comprise any compound that recognizes, competes,
agonizes, antagonizes, interacts, binds or associates with an EFNA1
or EFNA3 polypeptide their receptors or genes and modulates,
adjusts, alters, changes or modifies the impact of the EFNA protein
on one or more physiological pathways. Thus, in a broad sense the
present invention is directed to isolated EFNA modulators selected
from the group consisting of EFNA1 modulators and EFNA3 modulators
(or, as generally used herein, EFNA modulators unless otherwise
dictated by context). In preferred embodiments the invention is
more particularly directed to isolated EFNA1 modulators or isolated
EFNA3 modulators comprising antibodies (i.e., antibodies that
comprise or associate with at least EFNA1 or EFNA3). Moreover, as
discussed extensively below, such modulators may be used to provide
pharmaceutical compositions.
[0009] In selected embodiments of the invention, EFNA modulators
may comprise an EFNA1 or EFNA3 ligand itself or fragments thereof,
either in an isolated form or fused or associated with other
moieties (e.g., Fc-EFNA, PEG-EFNA or EFNA associated with a
targeting moiety). In other selected embodiments EFNA modulators
may comprise EFNA antagonists which, for the purposes of the
instant application, shall be held to mean any construct or
compound that recognizes, competes, interacts, binds or associates
with EFNA1 and/or EFNA3 and neutralizes, eliminates, reduces,
sensitizes, reprograms, inhibits or controls the growth of
neoplastic cells including tumor initiating cells. In preferred
embodiments the EFNA modulators of the instant invention comprise
anti-EFNA1 or anti-EFNA3 antibodies, or fragments or derivatives
thereof, that have unexpectedly been found to silence, neutralize,
reduce, decrease, deplete, moderate, diminish, reprogram,
eliminate, or otherwise inhibit the ability of tumor initiating
cells to propagate, maintain, expand, proliferate or otherwise
facilitate the survival, recurrence, regeneration and/or metastasis
of neoplastic cells. In particularly preferred embodiments the
antibodies or immunoreactive fragments may be associated with or
conjugated to one or more anti-cancer agents.
[0010] In selected embodiments compatible EFNA1 modulators may
comprise an antibody having a light chain variable region and a
heavy chain variable region wherein said light chain variable
region comprises an amino acid sequence having at least 60%
identity to an amino acid sequence selected from the group
consisting of amino acid sequences as set forth in SEQ ID NO: 9,
SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 21, SEQ ID NO: 25, SEQ ID
NO: 29, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 41 and SEQ ID NO:
45 and wherein said heavy chain variable region comprises an amino
acid sequence having at least 60% identity to an amino acid
sequence selected from the group consisting of amino acid sequences
as set forth in SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID
NO: 19, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 31, SEQ ID NO: 35,
SEQ ID NO: 39 and SEQ ID NO: 43.
[0011] In other embodiments compatible EFNA3 modulators may
comprise an antibody having a light chain variable region and a
heavy chain variable region wherein said light chain variable
region comprises an amino acid sequence having at least 60%
identity to an amino acid sequence selected from the group
consisting of amino acid sequences as set forth in SEQ ID NO: 49,
SEQ ID NO: 53, SEQ ID NO: 57, SEQ ID NO: 61, SEQ ID NO: 65, SEQ ID
NO: 69, SEQ ID NO: 73, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85,
SEQ ID NO: 89, SEQ ID NO: 93, SEQ ID NO: 97 and SEQ ID NO: 101 and
wherein said heavy chain variable region comprises an amino acid
sequence having at least 60% identity to an amino acid sequence
selected from the group consisting of amino acid sequences as set
forth in SEQ ID NO: 47, SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO:
59, SEQ ID NO: 63, SEQ ID NO: 67, SEQ ID NO: 71, SEQ ID NO: 75, SEQ
ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87, SEQ ID NO: 91, SEQ ID NO:
95 and SEQ ID NO: 99.
[0012] Of course, in view of the instant disclosure those skilled
in the art could readily identify CDRs associated with each of the
aforementioned heavy and light chain variable regions and use those
CDRs to engineer or fabricate chimeric, humanized or CDR grafted
antibodies without undue experimentation. As such, in selected
embodiments the present invention is directed to anti-EFNA1 or
anti-EFNA3 antibodies comprising one or more CDRs from a variable
region sequence set forth in FIG. 6 or FIG. 7. In preferred
embodiments such antibodies will comprise monoclonal antibodies
and, in even more preferred embodiments will comprise chimeric, CDR
grafted or humanized antibodies. As discussed in more detail below
still other embodiments will comprise such antibodies conjugated or
associated with one or more cytotoxic agents.
[0013] In certain other embodiments the invention will comprise an
EFNA modulator selected from the group consisting of EFNA1
modulators and EFNA3 modulators that reduces the frequency of tumor
initiating cells upon administration to a subject. Preferably the
reduction in frequency will be determined using in vitro or in vivo
limiting dilution analysis. In particularly preferred embodiments
such analysis may be conducted using in vivo limiting dilution
analysis comprising transplant of live human tumor cells into
immunocompromised mice. Alternatively, the limiting dilution
analysis may be conducted using in vitro limiting dilution analysis
comprising limiting dilution deposition of live human tumor cells
into in vitro colony supporting conditions. In either case, the
analysis, calculation or quantification of the reduction in
frequency will preferably comprise the use of Poisson distribution
statistics to provide an accurate accounting. It will be
appreciated that, while such quantification methods are preferred,
other, less labor intensive methodology such as flow cytometry or
immunohistochemistry may also be used to provide the desired values
and, accordingly, are expressly contemplated as being within the
scope of the instant invention. In such cases the reduction in
frequency may be determined using flow cytometric analysis or
immunohistochemical detection of tumor cell surface markers known
to enrich for tumor initiating cells.
[0014] As such, in another preferred embodiment of the instant
invention comprises a method of treating an EFNA associated
disorder comprising administering a therapeutically effective
amount of an EFNA modulator selected from the group consisting of
EFNA1 modulators and EFNA3 modulators to a subject in need thereof
whereby the frequency of tumor initiating cells is reduced. Again,
the reduction in the tumor initiating cell frequency will
preferably be determined using in vitro or in vivo limiting
dilution analysis.
[0015] In this regard it will be appreciated that the present
invention is based, at least in part, upon the discovery that EFNA
polypeptides (i.e., EFNA1 and EFNA3 as discussed below) are
associated with tumor perpetuating cells (i.e., cancer stem cells)
that are involved in the etiology of various neoplasia. More
specifically, the instant application unexpectedly demonstrates
that the administration of various exemplary EFNA modulators can
mediate, reduce, inhibit or eliminate tumorigenic signaling by
tumor initiating cells (i.e., reduce the frequency of tumor
initiating cells). This reduced signaling, whether by reduction,
elimination, reprogramming or silencing of the tumor initiating
cells or by modifying tumor cell morphology (e.g., induced
differentiation, niche disruption), in turn allows for the more
effective treatment of EFNA associated disorders by inhibiting
tumorigenesis, tumor maintenance, expansion and/or metastasis and
recurrence.
[0016] In other embodiments the disclosed modulators of EFNA1 or
EFNA3 may promote, support or otherwise enhance EFNA mediated
signaling that may limit or restrain tumor growth. In other
embodiments the disclosed modulators may interfere, suppress or
otherwise retard EFNA mediated signaling that may fuel tumor
growth. Further, as will be discussed in more detail below, EFNA1
and EFNA3 polypeptides may be involved in generating adhesive and
repulsive forces between cells through integrin and cytoskeleton
rearrangements or cytostructural modifications. Intervention in
such intercellular interactions, using the novel EFNA modulators
described herein, may thereby ameliorate a disorder by more than
one mechanism (i.e., tumor initiating cell reduction and disruption
of cellular adhesion) to provide additive or synergistic effects.
Still other preferred embodiments may take advantage of the
cellular internalization of ephrin-A ligands to deliver a modulator
mediated anti-cancer agent. In this regard it will be appreciated
that the present invention is not limited by any particular
mechanism of action but rather encompasses the broad use of the
disclosed modulators to treat EFNA associated disorders (including
various neoplasia).
[0017] Thus, another preferred embodiment of the invention
comprises a method of treating an EFNA associated disorder in a
subject in need thereof comprising the step of administering an
EFNA modulator selected from the group consisting of EFNA1
modulators and EFNA3 modulators to said subject. In particularly
preferred embodiments the EFNA modulator will be associated (e.g.,
conjugated) with an anti-cancer agent. In yet other embodiments the
EFNA modulator will internalize following association or binding
with the ephrin-A ligand on or near the surface of the cell.
Moreover the beneficial aspects of the instant invention, including
any cellular adhesion disruption and collateral benefits, may be
achieved whether the subject tumor tissue exhibits elevated levels
of EFNA or reduced or depressed levels of EFNA as compared with
normal adjacent tissue.
[0018] As alluded to above and discussed in more detail below there
are currently six known ephrin-A ligands (i.e., EFNAs 1-6). In
accordance with the instant invention it will be appreciated that
the disclosed modulators may be generated, fabricated and/or
selected to react with a single ephrin-A ligand (e.g., EFNA1), a
subset of ephrin-A ligands (e.g., EFNA1 and EFNA3) or all six
ephrin-A ligands. More particularly, as described herein and set
forth in the Examples below, preferred modulators such as
antibodies may be generated and selected so that they react or bind
with domains or epitopes that are expressed on a single ephrin-A
ligand or with epitopes that are conserved (at least to some
extent) and presented across multiple or all EFNA polypeptides
(e.g., EFNAs 1 and 3 or EFNAs 1, 3 and 6). This is significant with
respect to the instant invention in that, as shown in the Examples
below, certain ephrin-A ligands including EFNA1 and EFNA3 have been
found to be preferably expressed on TIC and, in combination, may
serve as particularly effective therapeutic targets that provide
for the selective reduction in tumorigenic cell frequency and/or
depletion of cancer stem cell populations.
[0019] Of course it will be appreciated that the disclosed EFNA
modulators may be generated, fabricated and/or selected to
preferentially react or associate with a single ephrin-A ligand
(e.g., EFNA1) and exhibit minimal or no association with any other
ephrin-A ligand. Accordingly, selected embodiments of the invention
are directed to EFNA modulators that immunospecifically associate
with a selected ephrin-A ligand such as EFNA1 or EFNA3 and exhibit
little or no association with any other ephrin-A ligand. In this
regard preferred embodiments disclosed herein will comprise methods
of treating an EFNA associated disorder in a subject in need
thereof comprising the step of administering an EFNA modulator
wherein the EFNA modulator immunospecifically associates with EFNA1
or EFNA3 and is substantially non-reactive with any other ephrin-A
ligand. Further, methods of generating, fabricating and selecting
such modulators are within the scope of the instant invention.
[0020] Other facets of the instant invention exploit the ability of
the disclosed modulators to potentially disrupt cell interactions
while simultaneously silencing tumor initiating cells. Such
multi-active EFNA modulators (e.g., EFNA antagonists) may prove to
be particularly effective when used in combination with standard of
care anti-cancer agents or debulking agents. In addition, two or
more EFNA antagonists (e.g. antibodies that specifically bind to
two discrete epitopes on an ephrin-A ligand or that associate with
discrete ligands) may be used in combination in accordance with the
present teachings. Moreover, as discussed in some detail below, the
EFNA modulators of the present invention may be used in a
conjugated or unconjugated state and, optionally, as a sensitizing
agent in combination with a variety chemical or biological
anti-cancer agents.
[0021] Thus, another preferred embodiment of the instant invention
comprises a method of sensitizing a tumor in a subject for
treatment with an anti-cancer agent comprising the step of
administering an EFNA modulator selected from the group consisting
of EFNA1 modulators and EFNA3 modulators to said subject. In a
particularly preferred aspect of the invention the EFNA modulator
will specifically result in a reduction of tumor initiating cell
frequency is as determined using in vitro or in vivo limiting
dilution analysis thereby sensitizing the tumor for concomitant or
subsequent debulking.
[0022] Similarly, as the compounds of the instant invention may
exert therapeutic benefits through various physiological
mechanisms, the present invention is also directed to selected
effectors or modulators that are specifically fabricated to exploit
certain cellular processes. For example, in certain embodiments the
preferred modulator may be engineered to associate with EFNA on or
near the surface of the tumor initiating cell and stimulate the
subject's immune response. In other embodiments the modulator may
comprise an antibody directed to an epitope that neutralizes EFNA1
or EFNA3 activity and interactions with ephrin receptors which may
impact adhesive and repulsive forces between cells through integrin
and cytoskeleton rearrangements or cytostructural modifications. In
yet other embodiments the disclosed modulators may act by depleting
or eliminating the EFNA associated cells. As such, it is important
to appreciate that the present invention is not limited to any
particular mode of action but rather encompasses any method or EFNA
modulator that achieves the desired outcome.
[0023] Within such a framework preferred embodiments of the
disclosed embodiments are directed to a method of treating a
subject suffering from neoplastic disorder comprising the step of
administering a therapeutically effective amount of at least one
neutralizing EFNA modulator selected from the group consisting of
EFNA1 modulators and EFNA3 modulators.
[0024] In yet another aspect the present invention will comprise a
method of treating a subject suffering from neoplastic disorder
comprising the step of administering a therapeutically effective
amount of at least one internalizing EFNA modulator selected from
the group consisting of EFNA1 modulators and EFNA3 modulators.
Preferred embodiments will comprise the administration of
internalizing antibody modulators wherein, in other selected
embodiments, the internalizing antibody modulators are conjugated
or associated with a cytotoxic agent.
[0025] Other embodiments are directed to a method of treating a
subject suffering from an EFNA associated disorder comprising the
step of administering a therapeutically effective amount of at
least one depleting EFNA modulator selected from the group
consisting of EFNA1 modulators and EFNA3 modulators. A related
method is directed to depleting EFNA associated cells in a subject
in need thereof comprising the step of administering an EFNA
modulator selected from the group consisting of EFNA1 modulators
and EFNA3 modulators.
[0026] In yet another embodiment the present invention provides
methods of maintenance therapy wherein the disclosed effectors or
modulators are administered over a period of time following an
initial procedure (e.g., chemotherapeutic, radiation or surgery)
designed to remove at least a portion of the tumor mass. Such
therapeutic regimens may be administered over a period of weeks, a
period of months or even a period of years wherein the EFNA
modulators may act prophylactically to inhibit metastasis and/or
tumor recurrence. In yet other embodiments the disclosed modulators
may be administrated in concert with known debulking regimens to
prevent or retard metastasis.
[0027] Beyond the therapeutic uses discussed above it will also be
appreciated that the modulators of the instant invention may be
used to diagnose EFNA related disorders and, in particular,
hyperproliferative disorders. In some embodiments the modulator may
be administered to the subject and detected or monitored in vivo.
Those of skill in the art will appreciate that such modulators may
be labeled or associated with markers or reporters as disclosed
below and detected using any one of a number of standard techniques
(e.g., MRI or CAT scan).
[0028] Thus, in some embodiments the invention will comprise a
method of diagnosing, detecting or monitoring an EFNA associated
disorder in vivo in a subject in need thereof comprising the step
of administering an EFNA modulator selected from the group
consisting of EFNA1 modulators and EFNA3 modulators.
[0029] In other instances the modulators may be used in an in vitro
diagnostic setting using art-recognized procedures. As such, a
preferred embodiment comprises a method of diagnosing a
hyperproliferative disorder in a subject in need thereof comprising
the steps of: [0030] a. obtaining a tissue sample from said
subject; [0031] b. contacting the tissue sample with at least one
EFNA modulator selected from the group consisting of EFNA1
modulators and EFNA3 modulators; and [0032] c. detecting or
quantifying the EFNA modulator associated with the sample.
[0033] Such methods may be easily discerned in conjunction with the
instant application and may be readily performed using generally
available commercial technology such as automatic plate readers,
dedicated reporter systems, etc. In selected embodiments the EFNA
modulator will be associated with tumor perpetuating cells present
in the sample. In other preferred embodiments the detecting or
quantifying step will comprise a reduction of tumor initiating cell
frequency and detection thereof. Moreover, limiting dilution
analysis may be conducted as previously alluded to above and will
preferably employ the use of Poisson distribution statistics to
provide an accurate accounting as to the reduction of
frequency.
[0034] In a similar vein the present invention also provides kits
that are useful in the diagnosis and monitoring of EFNA associated
disorders such as cancer. To this end the present invention
preferably provides an article of manufacture useful for diagnosing
or treating EFNA associated disorders comprising a receptacle
comprising an EFNA modulator selected from the group consisting of
EFNA1 modulators and EFNA3 modulators and instructional materials
for using said EFNA modulator to treat or diagnose the EFNA
associated disorder.
[0035] Other preferred embodiments of the invention also exploit
the properties of the disclosed modulators as an instrument useful
for identifying, isolating, sectioning or enriching populations or
subpopulations of tumor initiating cells through methods such as
fluorescence activated cell sorting (FACS) or laser mediated
sectioning.
[0036] As such, another preferred embodiment of the instant
invention is directed to a method of identifying, isolating,
sectioning or enriching a population of tumor initiating cells
comprising the step of contacting said tumor initiating cells with
an EFNA modulator.
[0037] The foregoing is a summary and thus contains, by necessity,
simplifications, generalizations, and omissions of detail;
consequently, those skilled in the art will appreciate that the
summary is illustrative only and is not intended to be in any way
limiting. Other aspects, features, and advantages of the methods,
compositions and/or devices and/or other subject matter described
herein will become apparent in the teachings set forth herein. The
summary is provided to introduce a selection of concepts in a
simplified form that are further described below in the Detailed
Description. This summary is not intended to identify key features
or essential features of the claimed subject matter, nor is it
intended to be used as an aid in determining the scope of the
claimed subject matter.
BRIEF DESCRIPTION OF THE FIGURES
[0038] FIGS. 1A-1E depict, respectively, the nucleic acid sequence
encoding human EFNA1 (SEQ ID NO: 1), the corresponding amino acid
sequence of human EFNA1 isoform a (SEQ ID NO: 2), an alignment of
human EFNA1 a, and b isoform sequences showing amino acid
differences (SEQ ID NOS: 2-3), the amino acid sequence encoding
human EFNA3 (SEQ ID NO: 4) and a corresponding nucleic acid
sequence of human EFNA3 (SEQ ID NO: 5);
[0039] FIGS. 2A-2E are graphical representations depicting,
respectively, the gene expression levels of selected human ephrin-A
ligands and ephrin-A receptors in untreated (FIG. 2A) and in
irinotecan treated (FIG. 2B) colorectal tumors and EFNA1 in treated
and untreated colorectal (FIG. 2C), pancreatic (FIG. 2D) and
non-small cell lung cancer (FIG. 2E) tumors as measured using whole
transcriptome sequencing of highly enriched tumor progenitor cell
(TProg) and tumor perpetuating cell (TPC) and non-tumorigenic cell
(NTG) populations obtained from a subset of whole tumor
specimens;
[0040] FIGS. 3A-3C are graphical representations depicting the gene
expression levels of human ephrin-A3 ligand in treated and
untreated colorectal tumor samples (FIG. 3A), pancreatic tumor
samples (FIG. 3B) and non-small cell lung cancer samples (FIG. 3C)
as measured using whole transcriptome sequencing of highly enriched
tumor progenitor cell (TProg) and tumor perpetuating cell (TPC) and
non-tumorigenic cell (NTG) populations or tumorigenic (TG) and
non-tumorigenic cell (NTG) populations;
[0041] FIGS. 4A and 4B are graphical representations showing the
relative gene expression levels of human EFNA1 (FIG. 4A) and EFNA3
(FIG. 4B) as measured using RT-PCR in colorectal and pancreatic
tumor specimens comprising tumor perpetuating cell (TPC) and
non-tumorigenic cell (NTG) populations;
[0042] FIGS. 5A and 5B illustrate the relative gene expression of
EFNA1 (FIG. 5A) and EFNA3 (FIG. 5B) represent gene expression
levels of human EFNA genes as measured by RT-PCR in whole tumor
specimens (grey dot) or matched NAT (white dots) from patients with
one of a number of solid tumor types;
[0043] FIGS. 6A-6J depict the murine heavy and light chain variable
region nucleic acid and amino acid sequences (SEQ ID NOS: 6-45) of
several exemplary EFNA1 modulators isolated and cloned as described
herein;
[0044] FIGS. 7A-7N depict the murine heavy and light chain variable
region nucleic acid and amino acid sequences (SEQ ID NOS: 46-101)
of several exemplary EFNA3 modulators isolated and cloned as
described herein;
[0045] FIG. 8 sets forth biochemical and immunological properties
of exemplary EFNA1 modulators as represented in a tabular
format;
[0046] FIG. 9 sets forth biochemical and immunological properties
of exemplary EFNA3 modulators as represented in a tabular
format;
[0047] FIGS. 10A-10C illustrate, respectively, cell surface binding
properties of an exemplary EFNA1 modulator (open histogram)
compared to isotype control antibody (shaded histogram) with regard
to four selected traditional tumor cell lines (FIG. 10A) and
exemplary EFNA1 (FIG. 10B) and EFNA3 (FIG. 10C) modulator binding
to engineered cells expressing the respective ephrin-A ligand;
[0048] FIGS. 11A-11C are graphical representations illustrating the
ability of ephrin-A ligands to interact selectively with numerous
EPHA receptors wherein HEK293T cells only bind EPHA-ECD-Fc receptor
constructs via endogenously expressed ephrin-A ligands to a limited
degree (FIG. 11A) while HEK293T cells overexpressing EFNA1 (FIG.
11B) and HEK293T cells overexpressing EFNA3 (FIG. 11C) bind tested
EPHA receptor constructs to various degrees;
[0049] FIGS. 12A and 12B illustrate the ability the disclosed
modulators to inhibit the cell surface binding of human EPHA
receptors wherein FIG. 12A demonstrates the ability of 12 exemplary
EFNA1 modulators to reduce EPHA receptor binding to EFNA1
expressing cells and FIG. 12B demonstrates the ability of 16
exemplary EFNA3 modulators to reduce EPHA receptor binding to EFNA3
expressing cells;
[0050] FIGS. 13A and 13B illustrate that exemplary EFNA1 modulators
may effectively be used as targeting moieties to direct cytotoxic
payloads to cells expressing significant levels of ephrin-A1 ligand
(FIG. 13B) wherein the downward sloping curve is indicative of cell
killing through internalized payload, while cells expressing low
levels of ephrin-A1 ligand (FIG. 13A) are not eliminated;
[0051] FIGS. 14A and 14B illustrate that exemplary EFNA3 modulators
may effectively be used as targeting moieties to direct cytotoxic
payloads to cells expressing significant levels of ephrin-A3 ligand
(FIG. 14B) wherein the downward sloping curve is indicative of cell
killing through internalized payload, while cells expressing low
levels of ephrin-A3 ligand (FIG. 13A) are not eliminated; and
[0052] FIGS. 15A and 15B provide evidence that exemplary EFNA1
modulators may effectively be used as targeting moieties to direct
cytotoxic payloads to patient derived NTX lung (FIG. 15A) and
ovarian (FIG. 15B) cancer stem cell populations expressing ephrin-A
ligand.
DETAILED DESCRIPTION OF THE INVENTION
I. Introduction
[0053] While the present invention may be embodied in many
different forms, disclosed herein are specific illustrative
embodiments thereof that exemplify the principles of the invention.
It should be emphasized that the present invention is not limited
to the specific embodiments illustrated. Moreover, any section
headings used herein are for organizational purposes only and are
not to be construed as limiting the subject matter described.
[0054] As previously alluded to, it has surprisingly been found
that the expression of ephrin-A ligands (or EFNA) such as ephrin-A1
and ephrin-A3 are associated with neoplastic growth and
hyperproliferative disorders and that such ligands provide useful
tumor markers which may be exploited in the treatment of related
diseases. More specifically, it has been discovered that EFNA
modulators such as those disclosed herein may advantageously be
used in the diagnosis, theragnosis, treatment or prevention of
neoplastic disorders in subjects in need thereof. Accordingly,
while preferred embodiments of the invention will be discussed
extensively below, particularly in the context of cancer stem cells
and their interactions with the disclosed modulators, those skilled
in the art will appreciate that the scope of the instant invention
is not limited by such exemplary embodiments. Rather, the present
invention and the appended claims are broadly and expressly
directed to EFNA modulators selected from the group consisting of
EFNA1 modulators and EFNA3 modulators and their use in the
diagnosis, theragnosis, treatment or prevention of a variety of
EFNA associated or mediated disorders, including neoplastic or
hyperproliferative disorders, regardless of any particular
mechanism of action or specifically targeted tumor component.
[0055] It will further be appreciated that, in contrast to many
prior art disclosures, the present invention is largely directed to
ephrin ligand modulators (i.e. EFN) rather than ephrin receptor
(i.e. EPH) modulators. That is, while ephrin receptors have been
widely implicated in several types of disorders and generally
targeted for therapeutic intervention, ephrin ligands have
heretofore attracted much less attention. In part this may be as a
result of the promiscuous behavior attributed to the ligands and
the misplaced belief that such varied interactions made them
untenable therapeutic targets as pathway redundancy would likely
compensate for any ligand antagonism. However, as demonstrated
herein the disclosed ephrin-A ligand modulators can effectively be
used to target and eliminate or otherwise incapacitate tumorigenic
cells. Moreover, in selected embodiments the present invention may
comprise modulators that associate or react with more than one
ephrin-A ligand thereby providing an unexpected additive or
synergistic effect that may allow for quiescence of more than one
ephrin ligand mediated pathway.
[0056] Besides the general association discussed immediately above,
the inventors have further discovered a heretofore unknown
phenotypical association between selected "tumor initiating cells"
(TIC) and ephrin-A ligands such as EFNA1 and EFNA3. In this regard,
it has been found that selected TICs express elevated levels of
ephrin-A ligands when compared to normal tissue and non-tumorigenic
cells (NTG), which together comprise much of a solid tumor. Thus,
the ephrin-A ligands comprise tumor associated markers (or
antigens) and have been found to provide effective agents for the
detection and suppression of TIC and associated neoplasia due to
elevated levels of the proteins on cell surfaces or in the tumor
microenvironment. More specifically, it has further been discovered
that EFNA modulators, including immunoreactive antagonists and
antibodies that associate or react with the proteins, effectively
reduce the frequency of tumor initiating cells and are therefore
useful in eliminating, incapacitating, reducing, promoting the
differentiation of, or otherwise precluding or limiting the ability
of these tumor-initiating cells to lie dormant and/or continue to
fuel tumor growth, metastasis or recurrence in a patient. As
discussed in more detail below, the TIC tumor cell subpopulation is
composed of both tumor perpetuating cells (TPC) and highly
proliferative tumor progenitor cells (TProg).
[0057] In view of these discoveries, those skilled in the art will
appreciate that the present invention further provides EFNA
modulators selected from the group consisting of EFNA1 modulators
and EFNA3 modulators and their use in reducing the frequency of
tumor initiating cells. As will be discussed extensively below,
EFNA modulators of the invention broadly comprise any compound that
recognizes, reacts, competes, antagonizes, interacts, binds,
agonizes, or associates with EFNA1 or EFNA3 or their genes. By
these interactions, the EFNA modulators thereby reduce or moderate
the frequency of tumor initiating cells. Exemplary modulators
disclosed herein comprise nucleotides, oligonucleotides,
polynucleotides, peptides or polypeptides. In certain preferred
embodiments the selected modulators will comprise antibodies to
EFNA1 or EFNA3 or immunoreactive fragments or derivatives thereof.
Such antibodies may be antagonistic or agonistic in nature and may
optionally be conjugated or associated with a cytotoxic agent. In
other embodiments, modulators within the instant invention will
comprise an EFNA construct comprising an ephrin-A ligand selected
from the group consisting of EFNA1 modulators and EFNA3 or a
reactive fragment thereof. It will be appreciated that such
constructs may comprise fusion proteins and can include reactive
domains from other polypeptides such as immunoglobulins or
biological response modifiers. In still other aspects, the EFNA
modulator will comprise a nucleic acid assembly that exerts the
desired effects at a genomic level. Still other modulators
compatible with the instant teachings will be discussed in detail
below.
[0058] Whichever form of modulator is ultimately selected it will
preferably be in an isolated and purified state prior to
introduction into a subject. In this regard the term "isolated EFNA
modulator" or "isolated EFNA1 modulator" or "isolated EFNA3
modulator" shall be construed in a broad sense and in accordance
with standard pharmaceutical practice to mean any preparation or
composition comprising the modulator in a state substantially free
of unwanted contaminants (biological or otherwise). As will be
discussed in some detail below these preparations may be purified
and formulated as desired using various art recognized techniques.
Of course, it will be appreciated that such "isolated" preparations
may be intentionally formulated or combined with inert or active
ingredients as desired to improve the commercial, manufacturing or
therapeutic aspects of the finished product and provide
pharmaceutical compositions.
II. EFNA Physiology
[0059] Ephrin receptor tyrosine kinases (EPH), type-I transmembrane
proteins, comprise the largest family of receptor tyrosine kinases
within animal genomes and interact with ephrin ligands (EFN), which
are also cell surface associated. Receptors in the EPH subfamily
typically have a single kinase domain and an extracellular region
containing a Cys-rich domain and 2 fibronectin type III repeats.
Convention holds that ephrin receptors are divided into two groups
based on the similarity of their extracellular domain sequences and
their affinities for binding ephrin-A and ephrin-B ligands.
Previous research has shown that EPH mediated signaling events
control multiple aspects of embryonic development, particularly in
the nervous system and are important mediators of cell-cell
communication regulating cell attachment, shape, and mobility.
Moreover, many members of the ephrin receptor family, as opposed to
ephrin ligands, have been identified as important markers and/or
regulators of the development and progression of cancer. To date
nine ephrin-A receptors and six ephrin-B receptors are known
[0060] For the purposes of the instant application the terms
"ephrin receptor," "ephrin-A receptor," "ephrin-B receptor,"
"EPHA," or "EPHB" (or EphA or EphB) may be used interchangeably and
held to mean the specified family, subfamily or individual receptor
(i.e., EPHA 1, EPHA2, EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8,
EPHA9, EPHB 1, EPHB2, EPHB3, EPHB4, EPHB5, EPHB6) as dictated by
context.
[0061] Based upon sequence analyses, ephrin ligands can be divided
into two groups: six ephrin-A ligands (or EFNA), typically anchored
to the cell surface via glycosylphosphatidylinositol linkages
(although some non-GPI-anchored proteins are produced through
alternative splicing of ephrin mRNAs; e.g. EFNA1) and three
ephrin-B ligands (or EFNB) containing a transmembrane domain and a
short cytoplasmic region with conserved tyrosine residues and a
PDZ-binding motif. EFNA ligands interact preferentially with any of
the nine different EPHA receptors, whereas EFNB ligands interact
preferentially with any of six different EPHB receptors, although
some specific EFNA-EPHB and EFNB-EPHA cross-interactions have been
reported.
[0062] For the purposes of the instant application the terms
"ephrin ligand," "ephrin-A ligand," "ephrin-B ligand," "EFNA," or
"EFNB" may be used interchangeably and held to mean the specified
family, subfamily or individual receptor (i.e., EFNA1, EFNA2,
EFNA3, EFNA4, EFNA5, EFNA6, EFNB 1, EFNB2, EFNB3) as dictated by
context. For example, the terms "ephrin-A1," ephrin-A1 ligand" or
"EFNA 1" shall all be held to designate the same family of protein
isoforms (e.g., as set forth in FIG. 1B) while the terms "ephrin-A
ligand" and "ENFA" shall be held to mean the ephrin subfamily (i.e.
A as opposed to B) comprising all six A type ligands and any
isoforms thereof.
[0063] A more detailed summary of ephrin receptor and ligand
nomenclature may be found in Table 1 immediately below.
TABLE-US-00001 TABLE 1 Receptors Ligands new name previous names
new name previous names EphA1 Eph, Esk ephrin-A1 B61; LERK-1, EFL-1
EphA2 Eck, Myk2, Sek2 ephrin-A2 ELF-1; Cek7-L, LERK-6 EphA3 Cek4,
Mek4, Hek, Tyro4; Hek4 ephrin-A3 Ehk1-L, EFL-2, LERK-3 EphA4 Sek,
Sek1, Cek8, Hek8, Tyro1 ephrin-A4 LERK-4; EFL-4 EphA5 Ehk1, Bsk,
Cek7, Hek7; Rek7 ephrin-A5 AL-1, RAGS; LERK-7, EFL-5 EphA6 Ehk2;
Hek12 ephrin-A6 EphA7 Mdk1, Hek11, Ehk3, Ebk, Cek11 EphA8 Eek; Hek3
EphA9 EphB1 Elk, Cek6, Net; Hek6 ephrin-B1 LERK-2, Elk-L, EFL-3,
Cek5-L; STRA1 EphB2 Cek5, Nuk, Erk, Qek5, Tyro5, Sek3; ephrin-B2
Htk-L, ELF-2; LERK-5, NLERK-1 Hek5, Drt EphB3 Cek10, Hek2, Mdk5,
Tyro6, Sek4 ephrin-B3 NLERK-2, Elk-L3, EFL-6, ELF-3; LERK-8 EphB4
Htk, Myk1, Tyro11; Mdk2 EphB5 Cek9; Hek9 EphB6 Mep
[0064] Eph Nomenclature Committee, Cell. 1997; 90 (3):403-4, which
is incorporated herein in its entirety by reference.
[0065] As with all cell surface receptor-ligand interactions,
engagement of the ephrin receptor by an ephrin ligand ultimately
results in the activation of intracellular signaling cascades.
Although receptor-ligand interactions may take place between
molecules on the surface of the same cell (cis interactions), it is
generally thought that cis interactions do not lead to the
triggering of signaling cascades, or that cis interactions may
actually antagonize signaling cascades initiated by trans
interactions (e.g., between receptors and ligands on separate
cells). One unique aspect of EPH-EFN trans interactions is the
capacity for the triggering of two signaling cascades upon
receptor-ligand engagement--a forward signaling cascade in the cell
expressing the ephrin receptor, and a reverse signaling cascade in
the cell expressing the ephrin ligand. The activation of two
separate signaling cascades may reflect cell sorting and cell
positioning processes that EPH and EFN have evolved to co-ordinate
in animal embryonic development.
[0066] EPH-EFN signaling frequently activates cell-signaling
pathways that regulate cytoskeletal dynamics and lead to modulation
of the adhesive and repulsive interactions between different types
of cells. As a generalization, EPH and EFN proteins are found at
much higher levels during embryogenesis versus those observed in
adult tissues, although continued low-level expression in the adult
may reflect roles for these molecules in the normal function of
tissues such as the adult gut, which has a well defined
architecture arising from the migration of differentiating cells
from their source at the tissue stem cell in the crypt to their
final location at the surface of the villi facing the intestinal
lumen. Since ephrin receptors were first identified in
hepatocellular carcinomas, and EPH and EFN expression is typically
limited in adults, reactivation of the expression of ephrin ligands
and/or ephrin receptors in human cancers may be linked to the
dedifferentiation of the cancer cells and/or the ability of these
cancer cells to invade surrounding normal tissue and to migrate
from the site of the primary tumor to distant locations. Other
studies have suggested that EPH-EFN interactions also have a role
in neoangiogenesis.
[0067] Consistent with findings that EPH-EFN interactions in
non-lymphoid tissues regulate cellular interactions by generating
adhesive or repulsive forces between cells through integrin and
cytoskeleton rearrangements, EPH and EFN molecules found on
lymphoid cells have been shown to mediate cell adhesion to
extracellular matrix components, chemotaxis and cell migration. For
example, EFNA1 engagement on primary CD4 and CD8 T cells has been
found to stimulate cell migration and enhance chemotaxis. Such
properties implicate selected members of the EFNA ligand family as
potential markers for various disorders and, in view of the instant
disclosure and Examples below, as tumor markers. In this regard it
has been found that EFNA1 and EFNA3 are of particular interest as
potential therapeutic and diagnostic targets with respect to
hyperproliferative disorders.
[0068] More specifically, as will be discussed in more detail below
EFNA1 and EFNA3 have been found to display elevated expression in
cancer stem cell populations, while concomitant upregulation of
several EPHA receptors in the bulk tumor raises the possibility
that these ligand receptor interactions may be triggering cell
signaling cascades linked to tumor proliferation, neoangiogenesis
and/or tumor metastasis. While not wishing to be bound by any
particular theory, it is believed that EFNA1 and EFNA3 effectors,
antagonists, and particularly EFNA1 or EFNA3 targeting moieties of
the present invention act, at least in part, by either interfering
with oncogenic survival outside the context of standard of care
therapeutic regimens (e.g. irinotecan), thereby reducing or
eliminating tumor initiating cell frequency or signaling or
delivering an entity able to kill EFNA1 or EFNA3 expressing cells.
For example, elimination of TPC by antagonizing EFNA1 or EFNA3 may
include simply promoting cell proliferation in the face of
chemotherapeutic regimens that eliminate proliferating cells, or
promote differentiation of TPC such that their self-renewal (i.e.
unlimited proliferation and maintenance of multipotency) capacity
is lost. Alternatively, recruitment of cytotoxic T-cells to EFNA1
or EFNA3 expressing cells, or delivery of a potent toxin conjugated
to an anti-EFNA1 or anti-EFNA3 antibody that was able to
internalize, may selectively kill TPC.
[0069] As used herein the term EFNA1 (also known as B61, ligand of
eph-related kinase 1, LERK1; or eph-related receptor tyrosine
kinase ligand 1) to naturally occurring human EFNA1 unless
contextually dictated otherwise. Representative EFNA1 protein
orthologs include, but are not limited to, human (i.e. hEFNA1,
NP.sub.--004419 or NP.sub.--872626), mouse (NP.sub.--034237 or
NP.sub.--001155897) chimpanzee (XP.sub.--001141980 and
XP.sub.--003308473) and rat (NP.sub.--446051). The transcribed
human EFNA1 gene comprises at minimum 7038 bp from chromosome 1 at
q21-22; the transcript may then undergo alternative splicing into a
minimum of two reported forms: (1) a 1590 bp variant
(NM.sub.--004428; EFNA1 transcript variant 1) which encodes a 205
amino acid proprotein (NP.sub.--004419; EFNA1 isoform a); and (2) a
1524 bp variant (NM.sub.--182685; EFNA1 transcript variant 2) which
in encodes a 183 amino acid proprotein (NP.sub.--872626; EFNA
isoform b). An exemplary EFNA1 nucleic acid sequence (variant 1) is
provided in FIG. 1A (SEQ ID NO: 1), an exemplary amino acid
sequence is provided in FIG. 1B (SEQ ID NO: 2) and aligned isoforms
a and b are provided in FIG. 1C (SEQ ID NOS: 2 and 3).
[0070] As used herein the term EFNA3 (also known as ligand of
eph-related kinase 3, LERK3; or eph-related receptor tyrosine
kinase ligand 3) to naturally occurring human EFNA3 unless
contextually dictated otherwise. Representative EFNA3 protein
orthologs include, but are not limited to, human (i.e. hEFNA3,
NP.sub.--004943), mouse (NP.sub.--034238), chimpanzee
(XP.sub.--003308464 and XP.sub.--003308465) and rat
(XP.sub.--574979). The transcribed human EFNA3 gene comprises at
minimum 8667 bp from chromosome 1 at q21-22; the transcript is
subsequently spliced into the mature mRNA (NM.sub.--004952)
encoding a 238 amino acid proprotein (NP.sub.--004943). An
exemplary EFNA3 nucleic acid sequence is provided in FIG. 1D (SEQ
ID NO: 5) while an exemplary amino acid sequence is provided in
FIG. 1C (SEQ ID NO: 4).
[0071] It will be appreciated that both the of the human EFNA1 and
EFNA3 proteins include a predicted signal or leader sequence,
comprising amino acids 1-18 of EFNA1 (NP.sub.--004419) and amino
acids 1-22 of EFNA3 (NP.sub.--004943) according to computer
prediction algorithms, Peterson et al., 2011 PMID: 21959131 which
is incorporated herein by reference. This signal peptide targets
the polypeptide to the cell surface/secretory pathway.
Additionally, the EFNA1 and EFNA3 proteins are post-translationally
processed, like other EFNA family members, into globular proteins
linked to the cell surface via glycosylphosphatidylinositol (GPI)
anchors.
III. Tumor Perpetuating Cells
[0072] In contrast to teachings of the prior art, the present
invention provides EFNA modulators that are particularly useful for
targeting tumor initiating cells, and especially tumor perpetuating
cells, thereby facilitating the treatment, management or prevention
of neoplastic disorders. More specifically, as previously indicated
it has surprisingly been found that specific tumor cell
subpopulations express EFNA and likely modify localized
coordination of morphogen signaling important to cancer stem cell
self-renewal and cell survival. Thus, in preferred embodiments
modulators of EFNA may be used to reduce tumor initiating cell
frequency in accordance with the present teachings and thereby
facilitate the treatment or management of hyperproliferative
diseases.
[0073] As used herein, the term tumor initiating cell (TIC)
encompasses both tumor perpetuating cells (TPC; i.e., cancer stem
cells or CSC) and highly proliferative tumor progenitor cells
(termed TProg), which together generally comprise a unique
subpopulation (i.e. 0.1-40%) of a bulk tumor or mass. For the
purposes of the instant disclosure the terms tumor perpetuating
cells and cancer stem cells are equivalent and may be used
interchangeably herein. Conversely, TPC differ from TProg in that
they can completely recapitulate the composition of tumor cells
existing within a tumor and have unlimited self-renewal capacity as
demonstrated by serial transplantation (two or more passages
through mice) of low numbers of isolated cells. As will be
discussed in more detail below fluorescence-activated cell sorting
(FACS) using appropriate cell surface markers is a reliable method
to isolate highly enriched cell subpopulations (>99.5% purity)
due, at least in part, to its ability to discriminate between
single cells and clumps of cells (i.e. doublets, etc.). Using such
techniques it has been shown that when low cell numbers of highly
purified TProg cells are transplanted into immunocompromised mice
they can fuel tumor growth in a primary transplant. However, unlike
purified TPC subpopulations the TProg generated tumors do not
completely reflect the parental tumor in phenotypic cell
heterogeneity and are demonstrably inefficient at reinitiating
serial tumorigenesis in subsequent transplants. In contrast, TPC
subpopulations completely reconstitute the cellular heterogeneity
of parental tumors and can efficiently initiate tumors when
serially isolated and transplanted. Thus, those skilled in the art
will recognize that a definitive difference between TPC and TProg,
though both may be tumor generating in primary transplants, is the
unique ability of TPC to perpetually fuel heterogeneous tumor
growth upon serial transplantation at low cell numbers. Other
common approaches to characterize TPC involve morphology and
examination of cell surface markers, transcriptional profile, and
drug response although marker expression may change with culture
conditions and with cell line passage in vitro.
[0074] Accordingly, for the purposes of the instant invention tumor
perpetuating cells, like normal stem cells that support cellular
hierarchies in normal tissue, are preferably defined by their
ability to self-renew indefinitely while maintaining the capacity
for multilineage differentiation. Tumor perpetuating cells are thus
capable of generating both tumorigenic progeny (i.e., tumor
initiating cells: TPC and TProg) and non-tumorigenic (NTG) progeny.
As used herein a non-tumorigenic cell (NTG) refers to a tumor cell
that arises from tumor initiating cells, but does not itself have
the capacity to self-renew or generate the heterogeneous lineages
of tumor cells that comprise a tumor. Experimentally, NTG cells are
incapable of reproducibly forming tumors in mice, even when
transplanted in excess cell numbers.
[0075] As indicated, TProg are also categorized as tumor initiating
cells (or TIC) due to their limited ability to generate tumors in
mice. TProg are progeny of TPC and are typically capable of a
finite number of non-self-renewing cell divisions. Moreover, TProg
cells may further be divided into early tumor progenitor cells
(ETP) and late tumor progenitor cells (LTP), each of which may be
distinguished by phenotype (e.g., cell surface markers) and
different capacities to recapitulate tumor cell architecture. In
spite of such technical differences, both ETP and LTP differ
functionally from TPC in that they are generally less capable of
serially reconstituting tumors when transplanted at low cell
numbers and typically do not reflect the heterogeneity of the
parental tumor. Notwithstanding the foregoing distinctions, it has
also been shown that various TProg populations can, on rare
occasion, gain self-renewal capabilities normally attributed to
stem cells and themselves become TPC (or CSC). In any event both
types of tumor-initiating cells are likely represented in the
typical tumor mass of a single patient and are subject to treatment
with the modulators as disclosed herein. That is, the disclosed
compositions are generally effective in reducing the frequency or
altering the chemosensitivity of such EFNA positive tumor
initiating cells regardless of the particular embodiment or mix
represented in a tumor.
[0076] In the context of the instant invention, TPC are more
tumorigenic, relatively more quiescent and often more
chemoresistant than the TProg (both ETP and LTP), NTG cells and the
tumor-infiltrating non-TPC derived cells (e.g., fibroblasts/stroma,
endothelial & hematopoietic cells) that comprise the bulk of a
tumor. Given that conventional therapies and regimens have, in
large part, been designed to both debulk tumors and attack rapidly
proliferating cells, TPC are likely to be more resistant to
conventional therapies and regimens than the faster proliferating
TProg and other bulk tumor cell populations. Further, TPC often
express other characteristics that make them relatively
chemoresistant to conventional therapies, such as increased
expression of multi-drug resistance transporters, enhanced DNA
repair mechanisms and anti-apoptotic proteins. These properties,
each of which contribute to drug tolerance by TPC, constitute a key
reason for the failure of standard oncology treatment regimens to
ensure long-term benefit for most patients with advanced stage
neoplasia; i.e. the failure to adequately target and eradicate
those cells that fuel continued tumor growth and recurrence (i.e.
TPC or CSC).
[0077] Unlike many of the aforementioned prior art treatments, the
novel compositions of the present invention preferably reduce the
frequency of tumor initiating cells upon administration to a
subject regardless of the form or specific target (e.g., genetic
material, EFNA antibody or ligand fusion construct) of the selected
modulator. As noted above, the reduction in tumor initiating cell
frequency may occur as a result of a) elimination, depletion,
sensitization, silencing or inhibition of tumor initiating cells;
b) controlling the growth, expansion or recurrence of tumor
initiating cells; c) interrupting the initiation, propagation,
maintenance, or proliferation of tumor initiating cells; or d) by
otherwise hindering the survival, regeneration and/or metastasis of
the tumorigenic cells. In some embodiments, the reduction in the
frequency of tumor initiating cells occurs as a result of a change
in one or more physiological pathways. The change in the pathway,
whether by reduction or elimination of the tumor initiating cells
or by modifying their potential (e.g., induced differentiation,
niche disruption) or otherwise interfering with their ability to
exert affects on the tumor environment or other cells, in turn
allows for the more effective treatment of EFNA-associated
disorders by inhibiting tumorigenesis, tumor maintenance and/or
metastasis and recurrence.
[0078] Among the methods that can be used to assess such a
reduction in the frequency of tumor initiating cells is limiting
dilution analysis either in vitro or in vivo, preferably followed
by enumeration using Poisson distribution statistics or assessing
the frequency of predefined definitive events such as the ability
to generate tumors in vivo or not. While such limiting dilution
analysis are the preferred methods of calculating reduction of
tumor initiating cell frequency, other, less demanding methods, may
also be used to effectively determine the desired values, albeit
slightly less accurately, and are entirely compatible with the
teachings herein. Thus, as will be appreciated by those skilled in
the art, it is also possible to determine reduction of frequency
values through well-known flow cytometric or immunohistochemical
means. As to all the aforementioned methods see, for example, Dylla
et al. 2008, PMCID: PMC2413402 & Hoey et al. 2009, PMID:
19664991; each of which is incorporated herein by reference in its
entirety.
[0079] With respect to limiting dilution analysis, in vitro
enumeration of tumor initiating cell frequency may be accomplished
by depositing either fractionated or unfractionated human tumor
cells (e.g. from treated and untreated tumors, respectively) into
in vitro growth conditions that foster colony formation. In this
manner, colony forming cells might be enumerated by simple counting
and characterization of colonies, or by analysis consisting of, for
example, the deposition of human tumor cells into plates in serial
dilutions and scoring each well as either positive or negative for
colony formation at least 10 days after plating. In vivo limiting
dilution experiments or analyses, which are generally more accurate
in their ability to determine tumor initiating cell frequency
encompass the transplantation of human tumor cells, from either
untreated control or treated conditions, for example, into
immunocompromised mice in serial dilutions and subsequently scoring
each mouse as either positive or negative for tumor formation at
least 60 days after transplant. The derivation of cell frequency
values by limiting dilution analysis in vitro or in vivo is
preferably done by applying Poisson distribution statistics to the
known frequency of positive and negative events, thereby providing
a frequency for events fulfilling the definition of a positive
event; in this case, colony or tumor formation, respectively.
[0080] As to other methods compatible with the instant invention
that may be used to calculate tumor initiating cell frequency, the
most common comprise quantifiable flow cytometric techniques and
immunohistochemical staining procedures. Though not as precise as
the limiting dilution analysis techniques described immediately
above, these procedures are much less labor intensive and provide
reasonable values in a relatively short time frame. Thus, it will
be appreciated that a skilled artisan may use flow cytometric cell
surface marker profile determination employing one or more
antibodies or reagents that bind art recognized cell surface
proteins known to enrich for tumor initiating cells (e.g.,
potentially compatible markers as are set forth in Example 1 below)
and thereby measure TIC levels from various samples. In still
another compatible method one skilled in the art might enumerate
TIC frequency in situ (e.g., in a tissue section) by
immunohistochemistry using one or more antibodies or reagents that
are able to bind cell surface proteins thought to demarcate these
cells.
[0081] Using any of the above-referenced methods it is then
possible to quantify the reduction in frequency of TIC (or the TPC
therein) provided by the disclosed EFNA modulators (including those
conjugated to cytotoxic agents) in accordance with the teachings
herein. In some instances, the compounds of the instant invention
may reduce the frequency of TIC (by a variety of mechanisms noted
above, including elimination, induced differentiation, niche
disruption, silencing, etc.) by 10%, 15%, 20%, 25%, 30% or even by
35%. In other embodiments, the reduction in frequency of TIC may be
on the order of 40%, 45%, 50%, 55%, 60% or 65%. In certain
embodiments, the disclosed compounds my reduce the frequency of TIC
by 70%, 75%, 80%, 85%, 90% or even 95%. Of course it will be
appreciated that any reduction of the frequency of the TIC likely
results in a corresponding reduction in the tumorigenicity,
persistence, recurrence and aggressiveness of the neoplasia.
IV. EFNA Modulators
[0082] In any event, the present invention is directed to the use
of EFNA modulators selected from the group consisting of EFNA1
modulators and EFNA3 modulators, including EFNA antagonists, for
the diagnosis, treatment and/or prophylaxis of any one of a number
of EFNA associated malignancies. The disclosed modulators may be
used alone or in conjunction with a wide variety of anti-cancer
compounds such as chemotherapeutic or immunotherapeutic agents
(e.g., therapeutic antibodies) or biological response modifiers. In
other selected embodiments, two or more discrete EFNA modulators
may be used in combination to provide enhanced anti-neoplastic
effects or may be used to fabricate multispecific constructs.
[0083] In certain embodiments, the EFNA modulators of the present
invention will comprise nucleotides, oligonucleotides,
polynucleotides, peptides or polypeptides. Even more preferably the
modulators will comprise soluble EFNA (sEFNA) or a form, variant,
derivative or fragment thereof including, for example, EFNA fusion
constructs (e.g., EFNA-Fc, EFNA-targeting moiety, etc.) or
EFNA-conjugates (e.g., EFNA-PEG, EFNA-cytotoxic agent, EFNA-brm,
etc.). It will also be appreciated that, in other embodiments, the
EFNA modulators comprise antibodies (e.g., anti-EFNA1 or anti-EFNA3
mAbs) or immunoreactive fragments or derivatives thereof. In
particularly preferred embodiments the modulators of the instant
invention will comprise neutralizing antibodies or derivatives or
fragments thereof. In other embodiments the EFNA modulators may
comprise internalizing antibodies or fragments thereof. In still
other embodiments the EFNA modulators may comprise depleting
antibodies or fragments thereof. Moreover, as with the
aforementioned fusion constructs, these antibody modulators may be
conjugated, linked or otherwise associated with selected cytotoxic
agents, polymers, biological response modifiers (BRMs) or the like
to provide directed immunotherapies with various (and optionally
multiple) mechanisms of action. As alluded to above such antibodies
may be pan-EFNA antibodies and associate with two or more ephrin-A
ligands or immunospecific antibodies that selectively react with
one of the six ephrin-A ligands. In yet other embodiments the
modulators may operate on the genetic level and may comprise
compounds as antisense constructs, siRNA, micro RNA and the
like.
[0084] Based on the teachings herein, those skilled in the art will
appreciate that particularly preferred embodiments of the invention
may comprise soluble Fc-constructs (e.g., sEFNA1 or sEFNA3) or
antibody modulators that associate with either, or both, of EFNA1
or EFNA3.
[0085] It will further be appreciated that the disclosed EFNA
modulators may deplete, silence, neutralize, eliminate or inhibit
growth, propagation or survival of tumor cells, particularly TPC,
and/or associated neoplasia through a variety of mechanisms,
including agonizing or antagonizing selected pathways or
eliminating specific cells depending, for example, on the form of
EFNA modulator, any associated payload or dosing and method of
delivery. Accordingly, while preferred embodiments disclosed herein
are directed to the depletion, inhibition or silencing of specific
tumor cell subpopulations such as tumor perpetuating cells, it must
be emphasized that such embodiments are merely illustrative and not
limiting in any sense. Rather, as set forth in the appended claims,
the present invention is broadly directed to EFNA modulators and
their use in the treatment, management or prophylaxis of various
EFNA associated hyperproliferative disorders irrespective of any
particular mechanism or target tumor cell population.
[0086] In the same sense disclosed embodiments of the instant
invention may comprise one or more EFNA antagonists that associate
with EFNA1 or EFNA3. To that end it will be appreciated that EFNA
antagonists of the instant invention may comprise any ligand,
polypeptide, peptide, fusion protein, antibody or immunologically
active fragment or derivative thereof that recognizes, reacts,
binds, combines, competes, associates or otherwise interacts with
the EFNA1 or EFNA3 protein or fragment thereof and eliminates,
silences, reduces, inhibits, hinders, restrains or controls the
growth of tumor initiating cells or other neoplastic cells
including bulk tumor or NTG cells. In selected embodiments the EFNA
modulators comprise EFNA antagonists.
[0087] As used herein an antagonist refers to a molecule capable of
neutralizing, blocking, inhibiting, abrogating, reducing or
interfering with the activities of a particular or specified
protein, including the binding of receptors to ligands or the
interactions of enzymes with substrates. More generally antagonists
of the invention may comprise antibodies and antigen-binding
fragments or derivatives thereof, proteins, peptides,
glycoproteins, glycopeptides, glycolipids, polysaccharides,
oligosaccharides, nucleic acids, antisense constructs, siRNA,
miRNA, bioorganic molecules, peptidomimetics, pharmacological
agents and their metabolites, transcriptional and translation
control sequences, and the like. Antagonists may also include small
molecule inhibitors, fusion proteins, receptor molecules and
derivatives which bind specifically to the protein thereby
sequestering its binding to its substrate target, antagonist
variants of the protein, antisense molecules directed to the
protein, RNA aptamers, and ribozymes against the protein.
[0088] As used herein and applied to two or more molecules or
compounds, the terms recognizes or associates shall be held to mean
the reaction, binding, specific binding, combination, interaction,
connection, linkage, uniting, coalescence, merger or joining,
covalently or non-covalently, of the molecules whereby one molecule
exerts an effect on the other molecule.
[0089] Moreover, as demonstrated in the examples herein, some
modulators of human EFNA may, in certain cases, cross-react with
EFNA from a species other than human (e.g., murine). In other cases
exemplary modulators may be specific for one or more isoforms of
human EFNA and will not exhibit cross-reactivity with EFNA
orthologs from other species. Of course, in conjunction with the
teachings herein such embodiments may comprise pan-EFNA antibodies
that associate with two or more ephrin-A ligands from a single
species or antibodies that exclusively associate with a single
ephrin-A ligand.
[0090] In any event, and as will be discussed in more detail below,
those skilled in the art will appreciate that the disclosed
modulators may be used in a conjugated or unconjugated form. That
is, the modulator may be associated with or conjugated to (e.g.
covalently or non-covalently) pharmaceutically active compounds,
biological response modifiers, anti-cancer agents, cytotoxic or
cytostatic agents, diagnostic moieties or biocompatible modifiers.
In this respect it will be understood that such conjugates may
comprise peptides, polypeptides, proteins, fusion proteins, nucleic
acid molecules, small molecules, mimetic agents, synthetic drugs,
inorganic molecules, organic molecules and radioisotopes. Moreover,
as indicated herein the selected conjugate may be covalently or
non-covalently linked to the EFNA modulator in various molar ratios
depending, at least in part, on the method used to effect the
conjugation.
V. Antibodies
[0091] a. Overview
[0092] As previously alluded to particularly preferred embodiments
of the instant invention comprise EFNA modulators in the form of
antibodies that preferentially associate with EFNA1 or EFNA3. The
term antibody is used in the broadest sense and specifically covers
synthetic antibodies, monoclonal antibodies, oligoclonal or
polyclonal antibodies, multiclonal antibodies, recombinantly
produced antibodies, intrabodies, multispecific antibodies,
bispecific antibodies, monovalent antibodies, multivalent
antibodies, human antibodies, humanized antibodies, chimeric
antibodies, CDR-grafted antibodies, primatized antibodies, Fab
fragments, F(ab') fragments, single-chain FvFcs (scFvFc),
single-chain Fvs (scFv), anti-idiotypic (anti-Id) antibodies and
any other immunologically active antibody fragments so long as they
exhibit the desired biological activity (i.e., immunospecific or
immunopreferential EFNA1 or EFNA3 association or binding). In a
broader sense, the antibodies of the present invention include
immunoglobulin molecules and immunologically active fragments of
immunoglobulin molecules, i.e., molecules that contain an antigen
binding site, where these fragments may or may not be fused to
another immunoglobulin domain including, but not limited to, an Fc
region or fragment thereof. Further, as outlined in more detail
herein, the terms antibody and antibodies specifically include Fc
variants as described below, including full length antibodies and
variant Fc-Fusions comprising Fc regions, or fragments thereof,
optionally comprising at least one amino acid residue modification
and fused to an immunologically active fragment of an
immunoglobulin.
[0093] As discussed in more detail below, the generic terms
antibody or immunoglobulin comprises five distinct classes of
antibody that can be distinguished biochemically and, depending on
the amino acid sequence of the constant domain of their heavy
chains, can readily be assigned to the appropriate class. For
historical reasons, the major classes of intact antibodies are
termed IgA, IgD, IgE, IgG, and IgM. In humans, the IgG and IgA
classes may be further divided into recognized subclasses
(isotypes), i.e., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 depending
on structure and certain biochemical properties. It will be
appreciated that the IgG isotypes in humans are named in order of
their abundance in serum with IgG1 being the most abundant.
[0094] While all five classes of antibodies (i.e. IgA, IgD, IgE,
IgG, and IgM) and all isotypes (i.e., IgG1, IgG2, IgG3, IgG4, IgA1,
and IgA2), as well as variations thereof, are within the scope of
the present invention, preferred embodiments comprising the IgG
class of immunoglobulin will be discussed in some detail solely for
the purposes of illustration. It will be understood that such
disclosure is, however, merely demonstrative of exemplary
compositions and methods of practicing the present invention and
not in any way limiting of the scope of the invention or the claims
appended hereto.
[0095] In this respect, human IgG immunoglobulins comprise two
identical light polypeptide chains of molecular weight
approximately 23,000 Daltons, and two identical heavy chains of
molecular weight 53,000-70,000 depending on the isotype.
Heavy-chain constant domains that correspond to the different
classes of antibodies are denoted by the corresponding lower case
Greek letter .alpha., .delta., .epsilon., .gamma., and .mu.,
respectively. The light chains of the antibodies from any
vertebrate species can be assigned to one of two clearly distinct
types, called kappa (.eta.) and lambda (.lamda.), based on the
amino acid sequences of their constant domains. Those skilled in
the art will appreciate that the subunit structures and
three-dimensional configurations of different classes of
immunoglobulins are well known.
[0096] The four chains are joined by disulfide bonds in a Y
configuration wherein the light chains bracket the heavy chains
starting at the mouth of the Y and continuing through the variable
region to the dual ends of the Y. Each light chain is linked to a
heavy chain by one covalent disulfide bond while two disulfide
linkages in the hinge region join the heavy chains. The respective
heavy and light chains also have regularly spaced intrachain
disulfide bridges the number of which may vary based on the isotype
of IgG.
[0097] Each heavy chain has at one end a variable domain (V.sub.H)
followed by a number of constant domains. Each light chain has a
variable domain at one end (V.sub.L) and a constant domain at its
other end; the constant domain of the light chain is aligned with
the first constant domain of the heavy chain, and the light chain
variable domain is aligned with the variable domain of the heavy
chain. In this regard, it will be appreciated that the variable
domains of both the light (V.sub.L) and heavy (V.sub.H) chain
portions determine antigen recognition and specificity. Conversely,
the constant domains of the light chain (C.sub.L) and the heavy
chain (C.sub.H1, C.sub.H2 or C.sub.H3) confer and regulate
important biological properties such as secretion, transplacental
mobility, circulation half-life, complement binding, and the like.
By convention the numbering of the constant region domains
increases as they become more distal from the antigen binding site
or amino-terminus of the antibody. Thus, the amino or N-terminus of
the antibody comprises the variable region and the carboxy or
C-terminus comprises the constant region. Thus, the C.sub.H3 and
C.sub.L domains actually comprise the carboxy-terminus of the heavy
and light chain, respectively.
[0098] The term variable refers to the fact that certain portions
of the variable domains differ extensively in sequence among
immunoglobulins and these hot spots largely define the binding and
specificity characteristics of a particular antibody. These
hypervariable sites manifest themselves in three segments, known as
complementarity determining regions (CDRs), in both the light-chain
and the heavy-chain variable domains respectively. The more highly
conserved portions of variable domains flanking the CDRs are termed
framework regions (FRs). More specifically, in naturally occurring
monomeric IgG antibodies, the six CDRs present on each arm of the
antibody are short, non-contiguous sequences of amino acids that
are specifically positioned to form the antigen binding site as the
antibody assumes its three dimensional configuration in an aqueous
environment.
[0099] The framework regions comprising the remainder of the heavy
and light variable domains show less inter-molecular variability in
amino acid sequence. Rather, the framework regions largely adopt a
.beta.-sheet conformation and the CDRs form loops which connect,
and in some cases form part of, the .beta.-sheet structure. Thus,
these framework regions act to form a scaffold that provides for
positioning the six CDRs in correct orientation by inter-chain,
non-covalent interactions. The antigen-binding site formed by the
positioned CDRs defines a surface complementary to the epitope on
the immunoreactive antigen (i.e. EFNA1 or EFNA3). This
complementary surface promotes the non-covalent binding of the
antibody to the immunoreactive antigen epitope. It will be
appreciated that the position and composition of CDRs can be
readily identified by one of ordinary skill in the art using the
definitions provided herein.
[0100] As discussed in more detail below all or part of the heavy
and light chain variable regions may be recombined or engineered
using standard recombinant and expression techniques to provide
effective antibodies. That is, the heavy or light chain variable
region from a first antibody (or any portion thereof) may be mixed
and matched with any selected portion of the heavy or light chain
variable region from a second antibody. For example, in one
embodiment, the entire light chain variable region comprising the
three light chain CDRs of a first antibody may be paired with the
entire heavy chain variable region comprising the three heavy chain
CDRs of a second antibody to provide an operative antibody.
Moreover, in other embodiments, individual heavy and light chain
CDRs derived from various antibodies may be mixed and matched to
provide the desired antibody having optimized characteristics.
Thus, an exemplary antibody may comprise three light chain CDRs
from a first antibody, two heavy chain CDRs derived from a second
antibody and a third heavy chain CDR from a third antibody.
[0101] More specifically, in the context of the instant invention
it will be appreciated that any of the disclosed heavy and light
chain CDRs derived from the sequences set forth in FIG. 6 or FIG. 7
may be rearranged in this manner to provide optimized anti-EFNA
(e.g. anti-hEFNA1 or anti-hEFNA3) antibodies in accordance with the
instant teachings. That is, one or more of the CDRs derived from
the sequences set forth in FIG. 6 (SEQ ID NOS: 6-45) comprising
anti-EFNA1 antibodies or those set forth in FIG. 7 (SEQ ID NOS:
46-101) comprising anti-EFNA3 antibodies may be incorporated in an
EFNA modulator and, in particularly preferred embodiments, in a CDR
grafted or humanized antibody that immunospecifically associates
with one or more ephrin-A ligands.
[0102] In any event, the complementarity determining regions
residue numbers may be defined as those of Kabat et al. (1991, NIH
Publication 91-3242, National Technical Information Service,
Springfield, Va.), specifically, residues 24-34 (CDR1), 50-56
(CDR2) and 89-97 (CDR3) in the light chain variable domain and
31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) in the heavy chain
variable domain. Note that CDRs vary considerably from antibody to
antibody (and by definition will not exhibit homology with the
Kabat consensus sequences). Maximal alignment of framework residues
frequently requires the insertion of spacer residues in the
numbering system, to be used for the Fv region. In addition, the
identity of certain individual residues at any given Kabat site
number may vary from antibody chain to antibody chain due to
interspecies or allelic divergence. See also Chothia et al., J.
Mol. Biol. 196:901-917 (1987); Chothia et al., Nature 342, pp.
877-883 (1989) and by MacCallum et al., J. Mol. Biol. 262:732-745
(1996) where the definitions include overlapping or subsets of
amino acid residues when compared against each other. Each of the
aforementioned references is incorporated herein by reference in
its entirety and the amino acid residues which encompass CDRs as
defined by each of the above cited references are set forth for
comparison.
TABLE-US-00002 CDR Definitions Kabat.sup.1 Chothia.sup.2
MacCallum.sup.3 V.sub.H CDR1 31-35 26-32 30-35 V.sub.H CDR2 50-65
53-55 47-58 V.sub.H CDR3 95-102 96-101 93-101 V.sub.L CDR1 24-34
26-32 30-36 V.sub.L CDR2 50-56 50-52 46-55 V.sub.L CDR3 89-97 91-96
89-96 .sup.1Residue numbering follows the nomenclature of Kabat et
al., supra .sup.2Residue numbering follows the nomenclature of
Chothia et al., supra .sup.3Residue numbering follows the
nomenclature of MacCallum et al., supra
[0103] As discussed one skilled in the art could readily define,
identify derive and/or enumerate the CDRs as defined by Kabat et
al., Chothia et al. or MacCallum et al. for each respective heavy
and light chain sequence set forth in FIG. 6 or FIG. 7.
Accordingly, each of the subject CDRs and antibodies comprising
CDRs defined by all such nomenclature are expressly included within
the scope of the instant invention. More broadly the term variable
region CDR amino acid residue includes amino acids in a CDR as
identified using any sequence or structure based method as set
forth above.
[0104] As used herein the term variable region framework (FR) amino
acid residues refers to those amino acids in the framework region
of an Ig chain. The term framework region or FR region as used
herein, includes the amino acid residues that are part of the
variable region, but are not part of the CDRs (e.g., using the
Kabat definition of CDRs). Therefore, a variable region framework
is a non-contiguous sequence between about 100-120 amino acids in
length but includes only those amino acids outside of the CDRs.
[0105] For the specific example of a heavy chain variable region
and for the CDRs as defined by Kabat et al., framework region 1
corresponds to the domain of the variable region encompassing amino
acids 1-30; framework region 2 corresponds to the domain of the
variable region encompassing amino acids 36-49; framework region 3
corresponds to the domain of the variable region encompassing amino
acids 66-94, and framework region 4 corresponds to the domain of
the variable region from amino acids 103 to the end of the variable
region. The framework regions for the light chain are similarly
separated by each of the light claim variable region CDRs.
Similarly, using the definition of CDRs by Chothia et al. or
McCallum et al. the framework region boundaries are separated by
the respective CDR termini as described above.
[0106] With the aforementioned structural considerations in mind,
those skilled in the art will appreciate that the antibodies of the
present invention may comprise any one of a number of functional
embodiments. In this respect, compatible antibodies may comprise
any immunoreactive antibody (as the term is defined herein) that
provides the desired physiological response in a subject. While any
of the disclosed antibodies may be used in conjunction with the
present teachings, certain embodiments of the invention will
comprise chimeric, humanized or human monoclonal antibodies or
immunoreactive fragments thereof. Yet other embodiments may, for
example, comprise homogeneous or heterogeneous multimeric
constructs, Fc variants and conjugated or glycosylationally altered
antibodies. Moreover, it will be understood that such
configurations are not mutually exclusive and that compatible
individual antibodies may comprise one or more of the functional
aspects disclosed herein. For example, a compatible antibody may
comprise a single chain diabody with humanized variable regions or
a fully human full length IgG3 antibody with Fc modifications that
alter the glycosylation pattern to modulate serum half-life. Other
exemplary embodiments are readily apparent to those skilled in the
art and may easily be discernable as being within the scope of the
invention.
[0107] b. Antibody Generation
[0108] As is well known, and shown in the Examples herein, various
host animals, including rabbits, mice, rats, etc. may be inoculated
and used to provide antibodies in accordance with the teachings
herein. Art known adjuvants that may be used to increase the
immunological response, depending on the inoculated species
include, but are not limited to, Freund's (complete and
incomplete), mineral gels such as aluminum hydroxide, surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins,
dinitrophenol, and potentially useful human adjuvants such as BCG
(bacille Calmette-Guerin) and corynebacterium parvum. Such
adjuvants may protect the antigen from rapid dispersal by
sequestering it in a local deposit, or they may contain substances
that stimulate the host to secrete factors that are chemotactic for
macrophages and other components of the immune system. Preferably,
if a polypeptide is being administered, the immunization schedule
will involve two or more administrations of the polypeptide, spread
out over several weeks.
[0109] After immunization of an animal with an EFNA immunogen
(e.g., soluble EFNA1 or EFNA3) which may comprise selected isoforms
and/or peptides, or live cells or cell preparations expressing the
desired protein, antibodies and/or antibody-producing cells can be
obtained from the animal using art recognized techniques. In some
embodiments, polyclonal anti-EFNA antibody-containing serum is
obtained by bleeding or sacrificing the animal. The serum may be
used for research purposes in the form obtained from the animal or,
in the alternative, the anti-EFNA antibodies may be partially or
fully purified to provide immunoglobulin fractions or homogeneous
antibody preparations.
[0110] c. Monoclonal Antibodies
[0111] While polyclonal antibodies may be used in conjunction with
certain aspects of the present invention, preferred embodiments
comprise the use of EFNA reactive monoclonal antibodies. As used
herein, the term monoclonal antibody or mAb refers to an antibody
obtained from a population of substantially homogeneous antibodies,
i.e., the individual antibodies comprising the population are
identical except for possible mutations, e.g., naturally occurring
mutations, that may be present in minor amounts. Thus, the modifier
monoclonal indicates the character of the antibody as not being a
mixture of discrete antibodies and may be used in conjunction with
any type of antibody. In certain embodiments, such a monoclonal
antibody includes an antibody comprising a polypeptide sequence
that binds or associates with EFNA, wherein the EFNA-binding
polypeptide sequence was obtained by a process that includes the
selection of a single target binding polypeptide sequence from a
plurality of polypeptide sequences.
[0112] In preferred embodiments, antibody-producing cell lines are
prepared from cells isolated from the immunized animal. After
immunization, the animal is sacrificed and lymph node and/or
splenic B cells are immortalized by means well known in the art as
shown in the appended Examples). Methods of immortalizing cells
include, but are not limited to, transfecting them with oncogenes,
infecting them with an oncogenic virus and cultivating them under
conditions that select for immortalized cells, subjecting them to
carcinogenic or mutating compounds, fusing them with an
immortalized cell, e.g., a myeloma cell, and inactivating a tumor
suppressor gene. If fusion with myeloma cells is used, the myeloma
cells preferably do not secrete immunoglobulin polypeptides (a
non-secretory cell line). Immortalized cells are screened using an
ephrin-A ligand (including selected isoforms), or an immunoreactive
portion thereof. In a preferred embodiment, the initial screening
is performed using an enzyme-linked immunoassay (ELISA) or a
radioimmunoassay.
[0113] More generally, discrete monoclonal antibodies consistent
with the present invention can be prepared using a wide variety of
techniques known in the art including hybridoma, recombinant
techniques, phage display technologies, yeast libraries, transgenic
animals (e.g. a XenoMouse.RTM. or HuMAb Mouse.RTM.) or some
combination thereof. For example, monoclonal antibodies can be
produced using hybridoma techniques such as broadly described above
and taught in more detail in Harlow et al., Antibodies: A
Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell
Hybridomas 563-681 (Elsevier, N.Y., 1981) each of which is
incorporated herein. Using the disclosed protocols, antibodies are
preferably raised in mammals by multiple subcutaneous or
intraperitoneal injections of the relevant antigen and an adjuvant.
As previously discussed, this immunization generally elicits an
immune response that comprises production of antigen-reactive
antibodies (that may be fully human if the immunized animal is
transgenic) from activated splenocytes or lymphocytes. While the
resulting antibodies may be harvested from the serum of the animal
to provide polyclonal preparations, it is generally more desirable
to isolate individual lymphocytes from the spleen, lymph nodes or
peripheral blood to provide homogenous preparations of monoclonal
antibodies. Most typically, the lymphocytes are obtained from the
spleen and immortalized to provide hybridomas.
[0114] For example, as described above, the selection process can
be the selection of a unique clone from a plurality of clones, such
as a pool of hybridoma clones, phage clones, or recombinant DNA
clones. It should be understood that a selected EFNA binding
sequence can be further altered, for example, to improve affinity
for the target, to humanize the target binding sequence, to improve
its production in cell culture, to reduce its immunogenicity in
vivo, to create a multispecific antibody, etc., and that an
antibody comprising the altered target binding sequence is also a
monoclonal antibody of this invention. In contrast to polyclonal
antibody preparations, which typically include discrete antibodies
directed against different determinants (epitopes), each monoclonal
antibody of a monoclonal antibody preparation is directed against a
single determinant on an antigen. In addition to their specificity,
monoclonal antibody preparations are advantageous in that they are
typically uncontaminated by other immunoglobulins that may be
cross-reactive.
[0115] d. Chimeric Antibodies
[0116] In another embodiment, the antibody of the invention may
comprise chimeric antibodies derived from covalently joined protein
segments from at least two different species or types of
antibodies. It will be appreciated that, as used herein, the term
chimeric antibodies is directed to constructs in which a portion of
the heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567; Morrison et
al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one
exemplary embodiment, a chimeric antibody in accordance with the
teachings herein may comprise murine V.sub.H and V.sub.L amino acid
sequences and constant regions derived from human sources. In other
compatible embodiments a chimeric antibody of the present invention
may comprise a CDR grafted or humanized antibody as described
herein.
[0117] Generally, a goal of making a chimeric antibody is to create
a chimera in which the number of amino acids from the intended
subject species is maximized. One example is the CDR-grafted
antibody, in which the antibody comprises one or more
complementarity determining regions (CDRs) from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the antibody chain(s) is/are identical with
or homologous to a corresponding sequence in antibodies derived
from another species or belonging to another antibody class or
subclass. For use in humans, the variable region or selected CDRs
from a rodent antibody often are grafted into a human antibody,
replacing the naturally occurring variable regions or CDRs of the
human antibody. These constructs generally have the advantages of
providing full strength modulator functions (e.g., CDC, ADCC, etc.)
while reducing unwanted immune responses to the antibody by the
subject.
[0118] e. Humanized Antibodies
[0119] Similar to the CDR grafted antibody is a humanized antibody.
Generally, a humanized antibody is produced from a monoclonal
antibody raised initially in a non-human animal. As used herein
humanized forms of non-human (e.g., murine) antibodies are chimeric
antibodies that contain a minimal sequence derived from a non-human
immunoglobulin. In one embodiment, a humanized antibody is a human
immunoglobulin (recipient or acceptor antibody) in which residues
from a CDR of the recipient antibody are replaced by residues from
a CDR of a non-human species (donor antibody) such as mouse, rat,
rabbit, or nonhuman primate having the desired specificity,
affinity, and/or capacity.
[0120] Generally humanization of an antibody comprises an analysis
of the sequence homology and canonical structures of both the donor
and recipient antibodies. In selected embodiments, the recipient
antibody may comprise consensus sequences. To create consensus
human frameworks, frameworks from several human heavy chain or
light chain amino acid sequences may be aligned to identify a
consensus amino acid sequence. Moreover, in many instances, one or
more framework residues in the variable domain of the human
immunoglobulin are replaced by corresponding non-human residues
from the donor antibody. These framework substitutions are
identified by methods well known in the art, e.g., by modeling of
the interactions of the CDR and framework residues to identify
framework residues important for antigen binding and sequence
comparison to identify unusual framework residues at particular
positions. Such substitutions help maintain the appropriate
three-dimensional configuration of the grafted CDR(s) and often
improve infinity over similar constructs with no framework
substitutions. Furthermore, humanized antibodies may comprise
residues that are not found in the recipient antibody or in the
donor antibody. These modifications may be made to further refine
antibody performance using well-known techniques.
[0121] CDR grafting and humanized antibodies are described, for
example, in U.S. Pat. Nos. 6,180,370, 5,693,762, 5,693,761,
5,585,089, and 5,530,101. In general, a humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the CDRs
correspond to those of a non-human immunoglobulin, and all or
substantially all of the framework regions are those of a human
immunoglobulin sequence. The humanized antibody optionally will
also comprise at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. For further
details, see, e.g., Jones et al., Nature 321:522-525 (1986);
Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op.
Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and
Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105-115 (1998);
Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and
Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos.
6,982,321 and 7,087,409. Still another method is termed humaneering
and is described, for example, in U.S. 2005/0008625. For the
purposes of the present application the term humanized antibodies
will be held to expressly include CDR grafted antibodies (i.e.
human antibodies comprising one or more grafted non-human CDRs)
with no or minimal framework substitutions.
[0122] Additionally, a non-human anti-EFNA antibody may also be
modified by specific deletion of human T cell epitopes or
deimmunization by the methods disclosed in WO 98/52976 and WO
00/34317. Briefly, the heavy and light chain variable regions of an
antibody can be analyzed for peptides that bind to MHC Class II;
these peptides represent potential T-cell epitopes (as defined in
WO 98/52976 and WO 00/34317). For detection of potential T-cell
epitopes, a computer modeling approach termed peptide threading can
be applied, and in addition a database of human MHC class II
binding peptides can be searched for motifs present in the V.sub.H
and V.sub.L sequences, as described in WO 98/52976 and WO 00/34317.
These motifs bind to any of the 18 major MHC class II DR allotypes,
and thus constitute potential T cell epitopes. Potential T-cell
epitopes detected can be eliminated by substituting small numbers
of amino acid residues in the variable regions, or by single amino
acid substitutions. As far as possible, conservative substitutions
are made. Often, but not exclusively, an amino acid common to a
position in human germline antibody sequences may be used. After
the deimmunizing changes are identified, nucleic acids encoding
V.sub.H and V.sub.L can be constructed by mutagenesis or other
synthetic methods (e.g., de novo synthesis, cassette replacement,
and so forth). A mutagenized variable sequence can, optionally, be
fused to a human constant region.
[0123] In selected embodiments, at least 60%, 65%, 70%, 75%, or 80%
of the humanized antibody variable region residues will correspond
to those of the parental framework region (FR) and CDR sequences.
In other embodiments at least 85% or 90% of the humanized antibody
residues will correspond to those of the parental framework region
(FR) and CDR sequences. In a further preferred embodiment, greater
than 95% of the humanized antibody residues will correspond to
those of the parental framework region (FR) and CDR sequences.
[0124] Humanized antibodies may be fabricated using common
molecular biology and biomolecular engineering techniques as
described herein. These methods include isolating, manipulating,
and expressing nucleic acid sequences that encode all or part of
immunoglobulin Fv variable regions from at least one of a heavy or
light chain. Sources of such nucleic acid are well known to those
skilled in the art and, for example, may be obtained from a
hybridoma, eukaryotic cell or phage producing an antibody or
immunoreactive fragment against a predetermined target, as
described above, from germline immunoglobulin genes, or from
synthetic constructs. The recombinant DNA encoding the humanized
antibody can then be cloned into an appropriate expression
vector.
[0125] Human germline sequences, for example, are disclosed in
Tomlinson, I. A. et al. (1992) J. Mol. Biol. 227:776-798; Cook, G.
P. et al. (1995) Immunol. Today 16: 237-242; Chothia, D. et al.
(1992) J. Mol. Bio. 227:799-817; and Tomlinson et al. (1995) EMBO J
14:4628-4638. The V BASE directory provides a comprehensive
directory of human immunoglobulin variable region sequences (See
Retter et al., (2005) Nuc Acid Res 33: 671-674). These sequences
can be used as a source of human sequence, e.g., for framework
regions and CDRs. As set forth herein consensus human framework
regions can also be used, e.g., as described in U.S. Pat. No.
6,300,064.
[0126] f. Human Antibodies
[0127] In addition to the aforementioned antibodies, those skilled
in the art will appreciate that the antibodies of the present
invention may comprise fully human antibodies. For the purposes of
the instant application the term human antibody comprises an
antibody which possesses an amino acid sequence that corresponds to
that of an antibody produced by a human and/or has been made using
any of the techniques for making human antibodies as disclosed
herein. This definition of a human antibody specifically excludes a
humanized antibody comprising non-human antigen-binding
residues.
[0128] Human antibodies can be produced using various techniques
known in the art. As alluded to above, phage display techniques may
be used to provide immunoactive binding regions in accordance with
the present teachings. Thus, certain embodiments of the invention
provide methods for producing anti-EFNA antibodies or
antigen-binding portions thereof comprising the steps of
synthesizing a library of (preferably human) antibodies on phage,
screening the library with a selected EFNA or an antibody-binding
portion thereof, isolating phage that binds EFNA, and obtaining the
immunoreactive fragments from the phage. By way of example, one
method for preparing the library of antibodies for use in phage
display techniques comprises the steps of immunizing a non-human
animal comprising human or non-human immunoglobulin loci with the
selected EFNA or an antigenic portion thereof to create an immune
response, extracting antibody-producing cells from the immunized
animal; isolating RNA encoding heavy and light chains of antibodies
of the invention from the extracted cells, reverse transcribing the
RNA to produce cDNA, amplifying the cDNA using primers, and
inserting the cDNA into a phage display vector such that antibodies
are expressed on the phage. More particularly, DNA encoding the
V.sub.H and V.sub.L domains are recombined together with an scFv
linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6
or pComb 3 HSS). The vector may then be electroporated in E. coli
and then the E. coli is infected with helper phage. Phage used in
these methods are typically filamentous phage including fd and M13
and the V.sub.H and V.sub.L domains are usually recombinantly fused
to either the phage gene III or gene VIII.
[0129] Recombinant human anti-EFNA antibodies of the invention may
be isolated by screening a recombinant combinatorial antibody
library prepared as above. In a preferred embodiment, the library
is a scFv phage display library, generated using human V.sub.L and
V.sub.H cDNAs prepared from mRNA isolated from B cells. Methods for
preparing and screening such libraries are well known in the art
and kits for generating phage display libraries are commercially
available (e.g., the Pharmacia Recombinant Phage Antibody System,
catalog no. 27-9400-01; and the Stratagene SurfZAP.TM. phage
display kit, catalog no. 240612). There also are other methods and
reagents that can be used in generating and screening antibody
display libraries (see, e.g., U.S. Pat. No. 5,223,409; PCT
Publication Nos. WO 92/18619, WO 91/17271, WO 92/20791, WO
92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al.,
Bio/Technology 9:1370-1372 (1991); Hay et al., Hum. Antibod.
Hybridomas 3:81-85 (1992); Huse et al., Science 246:1275-1281
(1989); McCafferty et al., Nature 348:552-554 (1990); Griffiths et
al., EMBO J. 12:725-734 (1993); Hawkins et al., J. Mol. Biol.
226:889-896 (1992); Clackson et al., Nature 352:624-628 (1991);
Gram et al., Proc. Natl. Acad. Sci. USA 89:3576-3580 (1992); Garrad
et al., Bio/Technology 9:1373-1377 (1991); Hoogenboom et al., Nuc.
Acid Res. 19:4133-4137 (1991); and Barbas et al., Proc. Natl. Acad.
Sci. USA 88:7978-7982 (1991).
[0130] The antibodies produced by naive libraries (either natural
or synthetic) can be of moderate affinity (K.sub.a of about
10.sup.6 to 10.sup.7 M.sup.-1), but affinity maturation can also be
mimicked in vitro by constructing and reselecting from secondary
libraries as described in the art. For example, mutation can be
introduced at random in vitro by using error-prone polymerase
(reported in Leung et al., Technique, 1: 11-15 (1989)) in the
method of Hawkins et al., J. Mol. Biol., 226: 889-896 (1992) or in
the method of Gram et al., Proc. Natl. Acad. Sci. USA, 89:
3576-3580 (1992). Additionally, affinity maturation can be
performed by randomly mutating one or more CDRs, e.g. using PCR
with primers carrying random sequence spanning the CDR of interest,
in selected individual Fv clones and screening for higher affinity
clones. WO 9607754 described a method for inducing mutagenesis in a
complementarity determining region of an immunoglobulin light chain
to create a library of light chain genes. Another effective
approach is to recombine the V.sub.H or V.sub.L domains selected by
phage display with repertoires of naturally occurring V domain
variants obtained from unimmunized donors and screen for higher
affinity in several rounds of chain reshuffling as described in
Marks et al., Biotechnol., 10: 779-783 (1992). This technique
allows the production of antibodies and antibody fragments with a
dissociation constant K.sub.d (k.sub.off/k.sub.on) of about
10.sup.-9 M or less.
[0131] It will further be appreciated that similar procedures may
be employed using libraries comprising eukaryotic cells (e.g.,
yeast) that express binding pairs on their surface. As with phage
display technology, the eukaryotic libraries are screened against
the antigen of interest (i.e., EFNA) and cells expressing
candidate-binding pairs are isolated and cloned. Steps may be taken
to optimize library content and for affinity maturation of the
reactive binding pairs. See, for example, U.S. Pat. No. 7,700,302
and U.S. Ser. No. 12/404,059. In one embodiment, the human antibody
is selected from a phage library, where that phage library
expresses human antibodies (Vaughan et al. Nature Biotechnology
14:309-314 (1996): Sheets et al. Proc. Natl. Acad. Sci.
95:6157-6162 (1998)); Hoogenboom and Winter, J. Mol. Biol, 227:381
(1991); Marks et al., J. Mol. Biol, 222:581 (1991)). In other
embodiments human binding pairs may be isolated from combinatorial
antibody libraries generated in eukaryotic cells such as yeast. See
e.g., U.S. Pat. No. 7,700,302. Such techniques advantageously allow
for the screening of large numbers of candidate modulators and
provide for relatively easy manipulation of candidate sequences
(e.g., by affinity maturation or recombinant shuffling).
[0132] Human antibodies can also be made by introducing human
immunoglobulin loci into transgenic animals, e.g., mice in which
the endogenous immunoglobulin genes have been partially or
completely inactivated. Upon challenge, human antibody production
is observed, which closely resembles that seen in humans in all
respects, including gene rearrangement, assembly, and antibody
repertoire. This approach is described, for example, in U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;
5,661,016, and U.S. Pat. Nos. 6,075,181 and 6,150,584 regarding
Xenomouse.RTM. technology along with the following scientific
publications: Marks et al., Bio/Technology 10: 779-783 (1992);
Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature
368:812-13 (1994); Fishwild et al., Nature Biotechnology 14: 845-51
(1996); Neuberger, Nature Biotechnology 14: 826 (1996); Lonberg and
Huszar, Intern. Rev. Immunol. 13:65-93 (1995). Alternatively, the
human antibody may be prepared via immortalization of human
B-lymphocytes producing an antibody directed against a target
antigen (such B lymphocytes may be recovered from an individual
suffering from a neoplastic disorder or may have been immunized in
vitro). See, e.g., Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol,
147 (1):86-95 (1991); and U.S. Pat. No. 5,750,373.
VI. Antibody Characteristics
[0133] No matter how obtained or which of the aforementioned forms
the antibody modulator takes (e.g., humanized, human, etc.) the
preferred embodiments of the disclosed modulators may exhibit
various characteristics. In this regard anti-EFNA
antibody-producing cells (e.g., hybridomas or yeast colonies) may
be selected, cloned and further screened for desirable
characteristics including, for example, robust growth, high
antibody production and, as discussed in more detail below,
desirable antibody characteristics. Hybridomas can be expanded in
vivo in syngeneic animals, in animals that lack an immune system,
e.g., nude mice, or in cell culture in vitro. Methods of selecting,
cloning and expanding hybridomas and/or colonies, each of which
produces a discrete antibody species, are well known to those of
ordinary skill in the art.
[0134] a. Neutralizing Antibodies
[0135] In particularly preferred embodiments the modulators of the
instant invention will comprise neutralizing antibodies or
derivative or fragment thereof. The term neutralizing antibody or
neutralizing antagonist refers to an antibody or antagonist that
binds to or interacts with an ephrin-A ligand and prevents binding
or association of the ligand to its binding partner (e.g., EphA
receptor) thereby interrupting the biological response that
otherwise would result from the interaction of the molecules. In
assessing the binding and specificity of an antibody or
immunologically functional fragment or derivative thereof, an
antibody or fragment will substantially inhibit binding of the
ligand to its binding partner or substrate when an excess of
antibody reduces the quantity of binding partner bound to the
target molecule by at least about 20%, 30%, 40%, 50%, 60%, 70%,
80%, 85%, 90%, 95%, 97%, 99% or more as measured, for example, in
an in vitro competitive binding assay (see e.g., Examples 10 and 11
herein). In the case of antibodies to EFNA1 for example, a
neutralizing antibody or antagonist will preferably diminish the
ability of EFNA1 to bind to a selected EphA receptor by at least
about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or
more. It will be appreciated that this diminished activity may be
measured directly using art recognized techniques or may be
measured by the impact such reduction will have on EphA receptor
activity.
[0136] b. Internalizing Antibodies
[0137] While evidence indicates that selected ephrin-A ligands or
their isoforms may be present in a soluble form, at least some EFNA
(e.g., EFNA1 and EFNA3) likely remains associated with the cell
surface thereby allowing for internalization of the disclosed
modulators. Accordingly, the anti-EFNA antibodies of the instant
invention may be internalized, at least to some extent, by cells
that express an ephrin-A ligand. For example, an anti-EFNA1
antibody that binds to EFNA1 on the surface of a tumor-initiating
cell may be internalized by the tumor-initiating cell. In
particularly preferred embodiments such anti-EFNA antibodies may be
associated with or conjugated to anti-cancer agents such as
cytotoxic moieties that kill the cell upon internalization.
[0138] As used herein, an anti-EFNA antibody that internalizes is
one that is taken up by the cell upon binding to an EFNA associated
with a mammalian cell. The internalizing antibody includes antibody
fragments, human or humanized antibody and antibody conjugates.
Internalization may occur in vitro or in vivo. For therapeutic
applications, internalization may occur in vivo. The number of
antibody molecules internalized may be sufficient or adequate to
kill an EFNA-expressing cell, especially an EFNA-expressing tumor
initiating cell. Depending on the potency of the antibody or
antibody conjugate, in some instances, the uptake of a single
antibody molecule into the cell is sufficient to kill the target
cell to which the antibody binds. For example, certain toxins are
highly potent in killing such that internalization of one molecule
of the toxin conjugated to the antibody is sufficient to kill the
tumor cell. Whether an anti-EFNA antibody internalizes upon binding
EFNA on a mammalian cell can be determined by various assays
including those described in the Examples below (e.g., Examples
12-14). Methods of detecting whether an antibody internalizes into
a cell are also described in U.S. Pat. No. 7,619,068 which is
incorporated herein by reference in its entirety.
[0139] c. Depleting Antibodies
[0140] In other preferred embodiments the modulators of the instant
invention will comprise depleting antibodies or derivatives or
fragments thereof. The term depleting antibody refers to an
antibody or fragment that binds to or associates with an EFNA on or
near the cell surface and induces, promotes or causes the death,
incapacitation or elimination of the cell (e.g., by
complement-dependent cytotoxicity or antibody-dependent cellular
cytotoxicity). In some embodiments discussed more fully below the
selected depleting antibodies will be associated or conjugated to a
cytotoxic agent. Preferably a depleting antibody will be able to
remove, incapacitate, eliminate or kill at least 20%, 30%, 40%,
50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% of tumor
perpetuating cells in a defined cell population. In some
embodiments the cell population may comprise enriched, sectioned,
purified or isolated tumor perpetuating cells. In other embodiments
the cell population may comprise whole tumor samples or
heterogeneous tumor extracts that comprise tumor perpetuating
cells. Those skilled in the art will appreciate that standard
biochemical techniques as described in the Examples below (e.g.,
Example 14) may be used to monitor and quantify the depletion of
tumorigenic cells or tumor perpetuating cells in accordance with
the teachings herein.
[0141] d. Epitope Binding
[0142] It will further be appreciated the disclosed anti-EFNA
antibodies will associate with, or bind to, discrete epitopes or
determinants presented by the selected target(s). As used herein
the term epitope refers to that portion of the target antigen
capable of being recognized and specifically bound by a particular
antibody. When the antigen is a polypeptide such as EFNA, epitopes
can be formed both from contiguous amino acids and noncontiguous
amino acids juxtaposed by tertiary folding of a protein. Epitopes
formed from contiguous amino acids are typically retained upon
protein denaturing, whereas epitopes formed by tertiary folding are
typically lost upon protein denaturing. An epitope typically
includes at least 3, and more usually, at least 5 or 8-10 amino
acids in a unique spatial conformation. More specifically, the
skilled artisan will appreciate the term epitope includes any
protein determinant capable of specific binding to an
immunoglobulin or T-cell receptor or otherwise interacting with a
molecule. Epitopic determinants generally consist of chemically
active surface groupings of molecules such as amino acids or
carbohydrate or sugar side chains and generally have specific three
dimensional structural characteristics, as well as specific charge
characteristics. Additionally an epitope may be linear or
conformational. In a linear epitope, all of the points of
interaction between the protein and the interacting molecule (such
as an antibody) occur linearly along the primary amino acid
sequence of the protein. In a conformational epitope, the points of
interaction occur across amino acid residues on the protein that
are linearly separated from one another.
[0143] Once a desired epitope on an antigen is determined, it is
possible to generate antibodies to that epitope, e.g., by
immunizing with a peptide comprising the epitope using techniques
described in the present invention. Alternatively, during the
discovery process, the generation and characterization of
antibodies may elucidate information about desirable epitopes. From
this information, it is then possible to competitively screen
antibodies for binding to the same epitope. An approach to achieve
this is to conduct competition studies to find antibodies that
competitively bind with one another, i.e. the antibodies compete
for binding to the antigen. A high throughput process for binning
antibodies based upon their cross-competition is described in WO
03/48731.
[0144] As used herein, the term binning refers to a method to group
antibodies based on their antigen binding characteristics. The
assignment of bins is somewhat arbitrary, depending on how
different the observed binding patterns of the antibodies tested.
Thus, while the technique is a useful tool for categorizing
antibodies of the instant invention, the bins do not always
directly correlate with epitopes and such initial determinations
should be further confirmed by other art recognized
methodology.
[0145] With this caveat one can determine whether a selected
primary antibody (or fragment thereof) binds to the same epitope or
cross competes for binding with a second antibody by using methods
known in the art and set forth in the Examples herein. In one
embodiment, one allows the primary antibody of the invention to
bind to EFNA under saturating conditions and then measures the
ability of the secondary antibody to bind to EFNA. If the test
antibody is able to bind to EFNA at the same time as the primary
anti-EFNA antibody, then the secondary antibody binds to a
different epitope than the primary antibody. However, if the
secondary antibody is not able to bind to EFNA at the same time,
then the secondary antibody binds to the same epitope, an
overlapping epitope, or an epitope that is in close proximity to
the epitope bound by the primary antibody. As known in the art and
detailed in the Examples below, the desired data can be obtained
using solid phase direct or indirect radioimmunoassay (RIA), solid
phase direct or indirect enzyme immunoassay (EIA), sandwich
competition assay, a Biacore.TM. system (i.e., surface plasmon
resonance--GE Healthcare), a ForteBio.RTM. Analyzer (i.e.,
bio-layer interferometry--ForteBio, Inc.) or flow cytometric
methodology. The term surface plasmon resonance, as used herein,
refers to an optical phenomenon that allows for the analysis of
real-time specific interactions by detection of alterations in
protein concentrations within a biosensor matrix. In a particularly
preferred embodiment, the analysis is performed using a Biacore or
ForteBio instrument as demonstrated in the Examples below.
[0146] The term compete when used in the context of antibodies that
compete for the same epitope means competition between antibodies
is determined by an assay in which the antibody or immunologically
functional fragment under test prevents or inhibits specific
binding of a reference antibody to a common antigen. Typically,
such an assay involves the use of purified antigen bound to a solid
surface or cells bearing either of these, an unlabeled test
immunoglobulin and a labeled reference immunoglobulin. Competitive
inhibition is measured by determining the amount of label bound to
the solid surface or cells in the presence of the test
immunoglobulin. Usually the test immunoglobulin is present in
excess. Antibodies identified by competition assay (competing
antibodies) include antibodies binding to the same epitope as the
reference antibody and antibodies binding to an adjacent epitope
sufficiently proximal to the epitope bound by the reference
antibody for steric hindrance to occur. Additional details
regarding methods for determining competitive binding are provided
in the Examples herein. Usually, when a competing antibody is
present in excess, it will inhibit specific binding of a reference
antibody to a common antigen by at least 40%, 45%, 50%, 55%, 60%,
65%, 70% or 75%. In some instance, binding is inhibited by at least
80%, 85%, 90%, 95%, or 97% or more.
[0147] Besides epitope specificity the disclosed antibodies may be
characterized using a number of different physical characteristics
including, for example, binding affinities, melting temperature
(Tm), and isoelectric points.
[0148] e. Binding Affinity
[0149] In this respect, the present invention further encompasses
the use of antibodies that have a high binding affinity for a
selected EFNA or, in the case of pan-antibodies, more than one type
of ephrin-A ligand. An antibody of the invention is said to
specifically bind its target antigen when the dissociation constant
K.sub.d(k.sub.off/k.sub.on) is .ltoreq.10.sup.-8M. The antibody
specifically binds antigen with high affinity when the K.sub.d is
.ltoreq.5.times.10.sup.-9M, and with very high affinity when the
K.sub.d is .ltoreq.5.times.10.sup.-10M. In one embodiment of the
invention, the antibody has a K.sub.d of .ltoreq.10.sup.-9M and an
off-rate of about 1.times.10.sup.-4/sec. In one embodiment of the
invention, the off-rate is <1.times.10.sup.-5/sec. In other
embodiments of the invention, the antibodies will bind to EFNA with
a IQ of between about 10.sup.-8M and 10.sup.-10M, and in yet
another embodiment it will bind with a
K.sub.d.ltoreq.2.times.10.sup.-10M. Still other selected
embodiments of the present invention comprise antibodies that have
a disassociation constant or K.sub.d (k.sub.off/k.sub.on) of less
than 10.sup.-2M, less than 5.times.10.sup.-2M, less than
10.sup.-3M, less than 5.times.10.sup.-3M, less than 10.sup.-4M,
less than 5.times.10.sup.-4M, less than 10.sup.-5M, less than
5.times.10.sup.-5M, less than 10.sup.-6M, less than
5.times.10.sup.-6M, less than 10.sup.-7M, less than
5.times.10.sup.-7M, less than 10.sup.-8M, less than
5.times.10.sup.-8M, less than 10.sup.-9M, less than
5.times.10.sup.-9M, less than 10.sup.-10M, less than
5.times.10.sup.-10M, less than 10.sup.-11M, less than
5.times.10.sup.-11M, less than 10.sup.-12M, less than
5.times.10.sup.-12M, less than 10.sup.-13M, less than
5.times.10.sup.-13M, less than 10.sup.-14M, less than
5.times.10.sup.-14M, less than 10.sup.-15M or less than
5.times.10.sup.-15M.
[0150] In specific embodiments, an antibody of the invention that
immunospecifically binds to EFNA has an association rate constant
or k.sub.on rate (EFNA (Ab)+antigen (Ag).sup.k.sub.on.rarw.Ab-Ag)
of at least 10.sup.5M.sup.-1s.sup.-1, at least
2.times.10.sup.5M.sup.-1s.sup.-1, at least
5.times.10.sup.5M.sup.-1s.sup.-1, at least
10.sup.6M.sup.-1s.sup.-1, at least
5.times.10.sup.6M.sup.-1s.sup.-1, at least
10.sup.7M.sup.-1s.sup.-1, at least
5.times.10.sup.7M.sup.-1s.sup.-1, or at least
10.sup.8M.sup.-1s.sup.-1.
[0151] In another embodiment, an antibody of the invention that
immunospecifically binds to EFNA has a k.sub.off rate (EFNA
(Ab)+antigen (Ag).sup.k.sub.off.rarw.Ab-Ag) of less than
10.sup.-1s.sup.-1, less than 5.times.10.sup.-1s.sup.-1, less than
10.sup.-2s.sup.-1, less than 5.times.10.sup.-2s.sup.-1, less than
10.sup.-3s.sup.-1, less than 5.times.10.sup.-3s.sup.-1, less than
10.sup.-4s.sup.-1, less than 5.times.10.sup.-4s.sup.-1, less than
10.sup.-5s.sup.-1, less than 5.times.10.sup.-5s.sup.-1, less than
10.sup.-6s.sup.-1, less than 5.times.10.sup.-6s.sup.-1 less than
10.sup.-7s.sup.-1, less than 5.times.10.sup.-7s.sup.-1, less than
10.sup.-8s.sup.-1, less than 5.times.10.sup.-8s.sup.-1, less than
10.sup.-9s.sup.-1, less than 5.times.10.sup.-9s.sup.-1 or less than
10.sup.-10s.sup.-1.
[0152] In other selected embodiments of the present invention
anti-EFNA antibodies will have an affinity constant or K.sub.a
(k.sub.on/k.sub.off) of at least 10.sup.2M.sup.-1, at least
5.times.10.sup.2M.sup.-1, at least 10.sup.3M.sup.-1, at least
5.times.10.sup.3M.sup.-1, at least 10.sup.4M.sup.-1, at least
5.times.10.sup.4M.sup.-1, at least 10.sup.5M.sup.-1, at least
5.times.10.sup.5M.sup.-1, at least 10.sup.6M.sup.-1, at least
5.times.10.sup.6M.sup.-1, at least 10.sup.7M.sup.-1, at least
5.times.10.sup.7M.sup.-1, at least 10.sup.8M.sup.-1, at least
5.times.10.sup.8M.sup.-1, at least 10.sup.9M.sup.-1, at least
5.times.10.sup.9M.sup.-1, at least 10.sup.10 M.sup.-1, at least
5.times.10.sup.10 M.sup.-1, at least 10.sup.11M.sup.-1, at least
5.times.10.sup.11M.sup.-1, at least 10.sup.12M.sup.-1, at least
5.times.10.sup.12M.sup.-1, at least 10.sup.13M.sup.-1, at least
5.times.10.sup.13M.sup.-1, at least 10.sup.14M.sup.-1, at least
5.times.10.sup.14M.sup.-1, at least 10.sup.15M.sup.-1 or at least
5.times.10.sup.15M.sup.-1.
[0153] f. Isoelectric Points
[0154] In addition to the aforementioned binding properties,
anti-EFNA antibodies and fragments thereof, like all polypeptides,
have an Isoelectric Point (pI), which is generally defined as the
pH at which a polypeptide carries no net charge. It is known in the
art that protein solubility is typically lowest when the pH of the
solution is equal to the isoelectric point (pI) of the protein.
Therefore it is possible to optimize solubility by altering the
number and location of ionizable residues in the antibody to adjust
the pI. For example the pI of a polypeptide can be manipulated by
making the appropriate amino acid substitutions (e.g., by
substituting a charged amino acid such as a lysine, for an
uncharged residue such as alanine). Without wishing to be bound by
any particular theory, amino acid substitutions of an antibody that
result in changes of the pI of said antibody may improve solubility
and/or the stability of the antibody. One skilled in the art would
understand which amino acid substitutions would be most appropriate
for a particular antibody to achieve a desired pI.
[0155] The pI of a protein may be determined by a variety of
methods including but not limited to, isoelectric focusing and
various computer algorithms (see for example Bjellqvist et al.,
1993, Electrophoresis 14:1023). In one embodiment, the pI of the
anti-EFNA antibodies of the invention is between is higher than
about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or about
9.0. In another embodiment, the pI of the anti-EFNA antibodies of
the invention is between is higher than 6.5, 7.0, 7.5, 8.0, 8.5, or
9.0. In yet another embodiment, substitutions resulting in
alterations in the pI of antibodies of the invention will not
significantly diminish their binding affinity for EFNA. As
discussed in more detail below, it is specifically contemplated
that the substitution(s) of the Fc region that result in altered
binding to Fc.gamma.R may also result in a change in the pI. In a
preferred embodiment, substitution(s) of the Fc region are
specifically chosen to effect both the desired alteration in
Fc.gamma.R binding and any desired change in pI. As used herein,
the pI value is defined as the pI of the predominant charge
form.
[0156] g. Thermal Stability
[0157] It will further be appreciated that the Tm of the Fab domain
of an antibody can be a good indicator of the thermal stability of
an antibody and may further provide an indication of the
shelf-life. Tm is merely the temperature of 50% unfolding for a
given domain or sequence. A lower Tm indicates more
aggregation/less stability, whereas a higher Tm indicates less
aggregation/more stability. Thus, antibodies or fragments or
derivatives having higher Tm are preferable. Moreover, using
art-recognized techniques it is possible to alter the composition
of the anti-EFNA antibodies or domains thereof to increase or
optimize molecular stability. See, for example, U.S. Pat. No.
7,960,142. Thus, in one embodiment, the Fab domain of a selected
antibody has a Tm value higher than at least 50.degree. C.,
55.degree. C., 60.degree. C., 65.degree. C., 70.degree. C.,
75.degree. C., 80.degree. C., 85.degree. C., 90.degree. C.,
95.degree. C., 100.degree. C., 105.degree. C., 110.degree. C.,
115.degree. C. or 120.degree. C. In another embodiment, the Fab
domain of an antibody has a Tm value higher than at least about
50.degree. C., about 55.degree. C., about 60.degree. C., about
65.degree. C., about 70.degree. C., about 75.degree. C., about
80.degree. C., about 85.degree. C., about 90.degree. C., about
95.degree. C., about 100.degree. C., about 105.degree. C., about
110.degree. C., about 115.degree. C. or about 120.degree. C.
Thermal melting temperatures (Tm) of a protein domain (e.g., a Fab
domain) can be measured using any standard method known in the art,
for example, by differential scanning calorimetry (see, e.g.,
Vermeer et al., 2000, Biophys. J. 78:394-404; Vermeer et al., 2000,
Biophys. J. 79: 2150-2154 both incorporated herein by
reference).
VII. EFNA Modulator Fragments and Derivatives
[0158] Whether the agents of the present invention comprise intact
fusion constructs, antibodies, fragments or derivatives, the
selected modulators will react, bind, combine, complex, connect,
attach, join, interact or otherwise associate with EFNA and thereby
provide the desired anti-neoplastic effects. Those of skill in the
art will appreciate that modulators comprising anti-EFNA antibodies
interact or associate with EFNA through one or more binding sites
expressed on the antibody. More specifically, as used herein the
term binding site comprises a region of a polypeptide that is
responsible for selectively binding to a target molecule of
interest (e.g., enzyme, antigen, ligand, receptor, substrate or
inhibitor). Binding domains comprise at least one binding site
(e.g. an intact IgG antibody will have two binding domains and two
binding sites). Exemplary binding domains include an antibody
variable domain, a receptor-binding domain of a ligand, a
ligand-binding domain of a receptor or an enzymatic domain. For the
purpose of the instant invention the typical active region of EFNA
(e.g., as part of an Fc-EFNA fusion construct) may comprise a
binding site for a substrate (e.g., an Eph receptor).
[0159] a. Fragments
[0160] Regardless of which form of the modulator (e.g. chimeric,
humanized, etc.) is selected to practice the invention, it will be
appreciated that immunoreactive fragments of the same may be used
in accordance with the teachings herein. In the broadest sense, the
term antibody fragment comprises at least a portion of an intact
antibody (e.g. a naturally occurring immunoglobulin). More
particularly the term fragment refers to a part or portion of an
antibody or antibody chain (or EFNA molecule in the case of Fc
fusions) comprising fewer amino acid residues than an intact or
complete antibody or antibody chain. The term antigen-binding
fragment refers to a polypeptide fragment of an immunoglobulin or
antibody that binds antigen or competes with intact antibody (i.e.,
with the intact antibody from which they were derived) for antigen
binding (i.e., specific binding). As used herein, the term fragment
of an antibody molecule includes antigen-binding fragments of
antibodies, for example, an antibody light chain (V.sub.L), an
antibody heavy chain (V.sub.H), a single chain antibody (scFv), a
F(ab')2 fragment, a Fab fragment, an Fd fragment, an Fv fragment,
single domain antibody fragments, diabodies, linear antibodies,
single-chain antibody molecules and multispecific antibodies formed
from antibody fragments. Similarly, an active fragment of EFNA
comprises a portion of the EFNA molecule that retains its ability
to interact with EFNA substrates or receptors and modify them in a
manner similar to that of an intact EFNA (though maybe with
somewhat less efficiency).
[0161] Those skilled in the art will appreciate fragments can be
obtained via chemical or enzymatic treatment of an intact or
complete modulator (e.g., antibody or antibody chain) or by
recombinant means. In this regard, while various antibody fragments
are defined in terms of the digestion of an intact antibody, one of
skill will appreciate that such fragments may be synthesized de
novo either chemically or by using recombinant DNA methodology.
Thus, the term antibody, as used herein, explicitly includes
antibodies or fragments or derivatives thereof either produced by
the modification of whole antibodies or synthesized de novo using
recombinant DNA methodologies.
[0162] More specifically, papain digestion of antibodies produces
two identical antigen-binding fragments, called Fab fragments, each
with a single antigen-binding site, and a residual Fc fragment,
whose name reflects its ability to crystallize readily. Pepsin
treatment yields an F(ab'), fragment that has two antigen-binding
sites and is still capable of cross-linking antigen. The Fab
fragment also contains the constant domain of the light chain and
the first constant domain (C.sub.H1) of the heavy chain. Fab'
fragments differ from Fab fragments by the addition of a few
residues at the carboxy terminus of the heavy-chain C.sub.H1 domain
including one or more cysteines from the antibody hinge region.
Fab'-SH is the designation herein for Fab' in which the cysteine
residue(s) of the constant domains bear at least one free thiol
group. F(ab')2 antibody fragments originally were produced as pairs
of Fab' fragments that have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known. See, e.g.,
Fundamental Immunology, W. E. Paul, ed., Raven Press, N.Y. (1999),
for a more detailed description of other antibody fragments.
[0163] It will further be appreciated that an Fv fragment is an
antibody fragment that contains a complete antigen recognition and
binding site. This region is made up of a dimer of one heavy and
one light chain variable domain in tight association, which can be
covalent in nature, for example in scFv. It is in this
configuration that the three CDRs of each variable domain interact
to define an antigen binding site on the surface of the
V.sub.H-V.sub.L dimer. Collectively, the six CDRs or a subset
thereof confer antigen binding specificity to the antibody.
However, even a single variable domain (or half of an Fv comprising
only three CDRs specific for an antigen) has the ability to
recognize and bind antigen, although usually at a lower affinity
than the entire binding site.
[0164] In other embodiments an antibody fragment, for example, is
one that comprises the Fc region, retains at least one of the
biological functions normally associated with the Fc region when
present in an intact antibody, such as FcRn binding, antibody half
life modulation, ADCC function and complement binding. In one
embodiment, an antibody fragment is a monovalent antibody that has
an in vivo half life substantially similar to an intact antibody.
For example, such an antibody fragment may comprise on antigen
binding arm linked to an Fc sequence capable of conferring in vivo
stability to the fragment.
[0165] b. Derivatives
[0166] In another embodiment, it will further be appreciated that
the modulators of the invention may be monovalent or multivalent
(e.g., bivalent, trivalent, etc.). As used herein the term valency
refers to the number of potential target (i.e., EFNA) binding sites
associated with an antibody. Each target binding site specifically
binds one target molecule or specific position or locus on a target
molecule. When an antibody of the instant invention comprises more
than one target binding site (multivalent), each target binding
site may specifically bind the same or different molecules (e.g.,
may bind to different ligands or different antigens, or different
epitopes or positions on the same antigen). For the purposes of the
instant invention, the subject antibodies will preferably have at
least one binding site specific for human EFNA. In one embodiment
the antibodies of the instant invention will be monovalent in that
each binding site of the molecule will specifically bind to a
single EFNA position or epitope. In other embodiments, the
antibodies will be multivalent in that they comprise more than one
binding site and the different binding sites specifically associate
with more than a single position or epitope. In such cases the
multiple epitopes may be present on the selected EFNA polypeptide
or spice variant or a single epitope may be present on EFNA while a
second, different epitope may be present on another molecule or
surface. See, for example, U.S.P.N. 2009/0130105.
[0167] As alluded to above, multivalent antibodies may
immunospecifically bind to different epitopes of the desired target
molecule or may immunospecifically bind to both the target molecule
as well as a heterologous epitope, such as a heterologous
polypeptide or solid support material. While preferred embodiments
of the anti-EFNA antibodies only bind two antigens (i.e. bispecific
antibodies), antibodies with additional specificities such as
trispecific antibodies are also encompassed by the instant
invention. Examples of bispecific antibodies include, without
limitation, those with one arm directed against EFNA and the other
arm directed against any other antigen (e.g., a modulator cell
marker). Methods for making bispecific antibodies are known in the
art. Traditional production of full-length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., 1983, Nature, 305:537-539). Other more
sophisticated compatible multispecific constructs and methods of
their fabrication are set forth in U.S.P.N. 2009/0155255.
[0168] In yet other embodiments, antibody variable domains with the
desired binding specificities (antibody-antigen combining sites)
are fused to immunoglobulin constant domain sequences. The fusion
preferably is with an immunoglobulin heavy chain constant domain,
comprising at least part of the hinge, C.sub.H2, and/or C.sub.H3
regions. In one example, the first heavy-chain constant region
(C.sub.H1) containing the site necessary for light chain binding is
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when, the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
[0169] In one embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm (e.g., EFNA1), and a hybrid
immunoglobulin heavy chain-light chain pair (providing a second
binding specificity) in the other arm. It was found that this
asymmetric structure facilitates the separation of the desired
bispecific compound from unwanted immunoglobulin chain
combinations, as the presence of an immunoglobulin light chain in
only one half of the bispecific molecule provides for a facile way
of separation. This approach is disclosed in WO 94/04690. For
further details of generating bispecific antibodies see, for
example, Suresh et al., 1986, Methods in Enzymology, 121:210.
According to another approach described in WO96/27011, a pair of
antibody molecules can be engineered to maximize the percentage of
heterodimers that are recovered from recombinant cell culture. The
preferred interface comprises at least a part of the C.sub.H3
domain of an antibody constant domain. In this method, one or more
small amino acid side chains from the interface of the first
antibody molecule are replaced with larger side chains (e.g.
tyrosine or tryptophan). Compensatory cavities of identical or
similar size to the large side chain(s) are created on the
interface of the second antibody molecule by replacing large amino
acid side chains with smaller ones (e.g. alanine or threonine).
This provides a mechanism for increasing the yield of the
heterodimer over other unwanted end-products such as
homodimers.
[0170] Bispecific antibodies also include cross-linked or
heteroconjugate antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
VIII. EFNA Modulators
Constant Region Modifications
[0171] a. Fc Region and Fc Receptors
[0172] In addition to the various modifications, substitutions,
additions or deletions to the variable or binding region of the
disclosed modulators (e.g., Fc-EFNA or anti-EFNA antibodies) set
forth above, those skilled in the art will appreciate that selected
embodiments of the present invention may also comprise
substitutions or modifications of the constant region (i.e. the Fc
region). More particularly, it is contemplated that the EFNA
modulators of the invention may contain inter alia one or more
additional amino acid residue substitutions, mutations and/or
modifications which result in a compound with preferred
characteristics including, but not limited to: altered
pharmacokinetics, increased serum half life, increase binding
affinity, reduced immunogenicity, increased production, altered Fc
ligand binding, enhanced or reduced ADCC or CDC activity, altered
glycosylation and/or disulfide bonds and modified binding
specificity. In this regard it will be appreciated that these Fc
variants may advantageously be used to enhance the effective
anti-neoplastic properties of the disclosed modulators.
[0173] The term Fc region herein is used to define a C-terminal
region of an immunoglobulin heavy chain, including native sequence
Fc regions and variant Fc regions. Although the boundaries of the
Fc region of an immunoglobulin heavy chain might vary, the human
IgG heavy chain Fc region is usually defined to stretch from an
amino acid residue at position Cys226, or from Pro230, to the
carboxyl-terminus thereof. The C-terminal lysine (residue 447
according to the EU numbering system) of the Fc region may be
removed, for example, during production or purification of the
antibody, or by recombinantly engineering the nucleic acid encoding
a heavy chain of the antibody. Accordingly, a composition of intact
antibodies may comprise antibody populations with all K447 residues
removed, antibody populations with no K447 residues removed, and
antibody populations having a mixture of antibodies with and
without the K447 residue. A functional Fc region possesses an
effector function of a native sequence Fc region. Exemplary
effector functions include C1q binding; CDC; Fc receptor binding;
ADCC; phagocytosis; down regulation of cell surface receptors (e.g.
B cell receptor; BCR), etc. Such effector functions generally
require the Fc region to be combined with a binding domain (e.g.,
an antibody variable domain) and can be assessed using various
assays as disclosed, for example, in definitions herein.
[0174] Fc receptor or FcR describes a receptor that binds to the Fc
region of an antibody. In some embodiments, an FcR is a native
human FcR. In some embodiments, an FcR is one that binds an IgG
antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of those
receptors. Fc.gamma.II receptors include Fc.gamma.RIIA (an
activating receptor) and Fc.gamma.RIIB (an inhibiting receptor),
which have similar amino acid sequences that differ primarily in
the cytoplasmic domains thereof. Activating receptor Fc.gamma. RIIA
contains an immunoreceptor tyrosine-based activation motif (ITAM)
in its cytoplasmic domain. Inhibiting receptor F.gamma.RIIB
contains an immunoreceptor tyrosine-based inhibition motif (ITIM)
in its cytoplasmic domain. (see, e.g., Daeron, Annu. Rev. Immunol.
15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and
Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al.,
Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin.
Med. 126:330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term FcR herein.
The term Fc receptor or FcR also includes the neonatal receptor,
FcRn, which, in certain instances, is responsible for the transfer
of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587
(1976) and Kim et al., J. Immunol. 24:249 (1994)) and regulation of
homeostasis of immunoglobulins. Methods of measuring binding to
FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today
18(12):592-598 (1997); Ghetie et al., Nature Biotechnology,
15(7):637-640 (1997); Hinton et al., J. Biol. Chem.
279(8):6213-6216 (2004); WO 2004/92219 (Hinton et al.).
[0175] b. Fc Functions
[0176] As used herein complement dependent cytotoxicity and CDC
refer to the lysing of a target cell in the presence of complement.
The complement activation pathway is initiated by the binding of
the first component of the complement system (C1q) to a molecule,
an antibody for example, complexed with a cognate antigen. To
assess complement activation, a CDC assay, e.g. as described in
Gazzano-Santoro et al., 1996, J. Immunol. Methods, 202:163, may be
performed.
[0177] Further, antibody-dependent cell-mediated cytotoxicity or
ADCC refers to a form of cytotoxicity in which secreted Ig bound
onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g.,
Natural Killer (NK) cells, neutrophils, and macrophages) enables
these cytotoxic effector cells to bind specifically to an
antigen-bearing target cell and subsequently kill the target cell
with cytotoxins. Specific high-affinity IgG antibodies directed to
the target arm cytotoxic cells and are absolutely required for such
killing. Lysis of the target cell is extracellular, requires direct
cell-to-cell contact, and does not involve complement.
[0178] EFNA modulator variants with altered FcR binding affinity or
ADCC activity is one which has either enhanced or diminished FcR
binding activity and/or ADCC activity compared to a parent or
unmodified antibody or to a modulator comprising a native sequence
Fc region. The modulator variant which displays increased binding
to an FcR binds at least one FcR with better affinity than the
parent or unmodified antibody or to a modulator comprising a native
sequence Fc region. A variant which displays decreased binding to
an FcR, binds at least one FcR with worse affinity than the parent
or unmodified antibody or to a modulator comprising a native
sequence Fc region. Such variants which display decreased binding
to an FcR may possess little or no appreciable binding to an FcR,
e.g., 0-20% binding to the FcR compared to a native sequence IgG Fc
region, e.g. as determined techniques well known in the art.
[0179] As to FcRn, the antibodies of the instant invention also
comprise or encompass Fc variants with modifications to the
constant region that provide half-lives (e.g., serum half-lives) in
a mammal, preferably a human, of greater than 5 days, greater than
10 days, greater than 15 days, preferably greater than 20 days,
greater than 25 days, greater than 30 days, greater than 35 days,
greater than 40 days, greater than 45 days, greater than 2 months,
greater than 3 months, greater than 4 months, or greater than 5
months. The increased half-lives of the antibodies (or Fc
containing molecules) of the present invention in a mammal,
preferably a human, results in a higher serum titer of said
antibodies or antibody fragments in the mammal, and thus, reduces
the frequency of the administration of said antibodies or antibody
fragments and/or reduces the concentration of said antibodies or
antibody fragments to be administered. Antibodies having increased
in vivo half-lives can be generated by techniques known to those of
skill in the art. For example, antibodies with increased in vivo
half-lives can be generated by modifying (e.g., substituting,
deleting or adding) amino acid residues identified as involved in
the interaction between the Fc domain and the FcRn receptor (see,
e.g., International Publication Nos. WO 97/34631; WO 04/029207;
U.S. Pat. No. 6,737,056 and U.S.P.N. 2003/0190311. Binding to human
FcRn in vivo and serum half life of human FcRn high affinity
binding polypeptides can be assayed, e.g., in transgenic mice or
transfected human cell lines expressing human FcRn, or in primates
to which the polypeptides with a variant Fc region are
administered. WO 2000/42072 describes antibody variants with
improved or diminished binding to FcRns. See also, e.g., Shields et
al. J. Biol. Chem. 9(2):6591-6604 (2001).
[0180] c. Glycosylation Modifications
[0181] In still other embodiments, glycosylation patterns or
compositions of the antibodies of the invention are modified. More
particularly, preferred embodiments of the present invention may
comprise one or more engineered glycoforms, i.e., an altered
glycosylation pattern or altered carbohydrate composition that is
covalently attached to a molecule comprising an Fc region.
Engineered glycoforms may be useful for a variety of purposes,
including but not limited to enhancing or reducing effector
function, increasing the affinity of the antibody for a target
antigen or facilitating production of the antibody. In cases where
reduced effector function is desired, it will be appreciated that
the molecule may be engineered to express in an aglycosylated form.
Such carbohydrate modifications can be accomplished by, for
example, altering one or more sites of glycosylation within the
antibody sequence. That is, one or more amino acid substitutions
can be made that result in elimination of one or more variable
region framework glycosylation sites to thereby eliminate
glycosylation at that site (see e.g. U.S. Pat. Nos. 5,714,350 and
6,350,861. Conversely, enhanced effector functions or improved
binding may be imparted to the Fc containing molecule by
engineering in one or more additional glycosylation sites.
[0182] Additionally or alternatively, an Fc variant can be made
that has an altered glycosylation composition, such as a
hypofucosylated antibody having reduced amounts of fucosyl residues
or an antibody having increased bisecting GlcNAc structures. These
and similar altered glycosylation patterns have been demonstrated
to increase the ADCC ability of antibodies. Engineered glycoforms
may be generated by any method known to one skilled in the art, for
example by using engineered or variant expression strains, by
coexpression with one or more enzymes (for example
N-acetylglucosaminyltransferase III (GnTI11)), by expressing a
molecule comprising an Fc region in various organisms or cell lines
from various organisms or by modifying carbohydrate(s) after the
molecule comprising Fc region has been expressed. See, for example,
Shields, R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana
et al. (1999) Nat. Biotech. 17:176-1, as well as, European Patent
No: EP 1,176,195; PCT Publications WO 03/035835; WO 99/54342, Umana
et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 20017
Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem
277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473)
U.S. Pat. No. 6,602,684; U.S. Ser. Nos. 10/277,370; 10/113,929; PCT
WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1; PCT WO
02/30954A1; Potillegent.TM. technology (Biowa, Inc.); GlycoMAb.TM.
glycosylation engineering technology (GLYCART biotechnology AG); WO
00061739; EA01229125; U.S.P.N. 2003/0115614; Okazaki et al., 2004,
JMB, 336: 1239-49.
IX. Modulator Expression
[0183] a. Overview
[0184] DNA encoding the desired EFNA modulators may be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding antibody heavy and light chains).
Isolated and subcloned hybridoma cells (or phage or yeast derived
colonies) may serve as a preferred source of such DNA if the
modulator is an antibody. If desired, the nucleic acid can further
be manipulated as described herein to create agents including
fusion proteins, or chimeric, humanized or fully human antibodies.
More particularly, the isolated DNA (which may be modified) can be
used to clone constant and variable region sequences for the
manufacture antibodies as described in U.S. Pat. No. 7,709,611.
[0185] This exemplary method entails extraction of RNA from the
selected cells, conversion to cDNA, and amplification by PCR using
antibody specific primers. Suitable primers are well known in the
art and, as exemplified herein, are readily available from numerous
commercial sources. It will be appreciated that, to express a
recombinant human or non-human antibody isolated by screening of a
combinatorial library, the DNA encoding the antibody is cloned into
a recombinant expression vector and introduced into host cells
including mammalian cells, insect cells, plant cells, yeast, and
bacteria. In yet other embodiments, the modulators are introduced
into and expressed by simian COS cells, NS0 cells, Chinese Hamster
Ovary (CHO) cells or myeloma cells that do not otherwise produce
the desired construct. As will be discussed in more detail below,
transformed cells expressing the desired modulator may be grown up
in relatively large quantities to provide clinical and commercial
supplies of the fusion construct or immunoglobulin.
[0186] Whether the nucleic acid encoding the desired portion of the
EFNA modulator is obtained or derived from phage display
technology, yeast libraries, hybridoma based technology,
synthetically or from commercial sources, it is to be understood
that the present invention explicitly encompasses nucleic acid
molecules and sequences encoding EFNA modulators including fusion
proteins and anti-EFNA antibodies or antigen-binding fragments or
derivatives thereof. The invention further encompasses nucleic
acids or nucleic acid molecules (e.g., polynucleotides) that
hybridize under high stringency, or alternatively, under
intermediate or lower stringency hybridization conditions (e.g., as
defined below), to polynucleotides complementary to nucleic acids
having a polynucleotide sequence that encodes a modulator of the
invention or a fragment or variant thereof. The term nucleic acid
molecule or isolated nucleic acid molecule, as used herein, is
intended to include at least DNA molecules and RNA molecules. A
nucleic acid molecule may be single-stranded or double-stranded,
but preferably is double-stranded DNA. Moreover, the present
invention comprises any vehicle or construct, incorporating such
modulator encoding polynucleotide including, without limitation,
vectors, plasmids, host cells, cosmids or viral constructs.
[0187] The term isolated nucleic acid means a that the nucleic acid
was (i) amplified in vitro, for example by polymerase chain
reaction (PCR), (ii) recombinantly produced by cloning, (iii)
purified, for example by cleavage and gel-electrophoretic
fractionation, or (iv) synthesized, for example by chemical
synthesis. An isolated nucleic acid is a nucleic acid that is
available for manipulation by recombinant DNA techniques.
[0188] More specifically, nucleic acids that encode a modulator,
including one or both chains of an antibody of the invention, or a
fragment, derivative, mutein, or variant thereof, polynucleotides
sufficient for use as hybridization probes, PCR primers or
sequencing primers for identifying, analyzing, mutating or
amplifying a polynucleotide encoding a polypeptide, anti-sense
nucleic acids for inhibiting expression of a polynucleotide, and
complementary sequences of the foregoing are also provided. The
nucleic acids can be any length. They can be, for example, 5, 10,
15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250,
300, 350, 400, 450, 500, 750, 1,000, 1,500, 3,000, 5,000 or more
nucleotides in length, and/or can comprise one or more additional
sequences, for example, regulatory sequences, and/or be part of a
larger nucleic acid, for example, a vector. These nucleic acids can
be single-stranded or double-stranded and can comprise RNA and/or
DNA nucleotides, and artificial variants thereof (e.g., peptide
nucleic acids). Nucleic acids encoding modulators of the invention,
including antibodies or immunoreactive fragments or derivatives
thereof, have preferably been isolated as described above.
[0189] b. Hybridization and Identity
[0190] As indicated, the invention further provides nucleic acids
that hybridize to other nucleic acids under particular
hybridization conditions. Methods for hybridizing nucleic acids are
well known in the art. See, e.g., Current Protocols in Molecular
Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For the
purposes of the instant application, a moderately stringent
hybridization condition uses a prewashing solution containing
5.times. sodium chloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM
EDTA (pH 8.0), hybridization buffer of about 50% formamide,
6.times.SSC, and a hybridization temperature of 55.degree. C. (or
other similar hybridization solutions, such as one containing about
50% formamide, with a hybridization temperature of 42.degree. C.),
and washing conditions of 60.degree. C., in 0.5.times.SSC, 0.1%
SDS. A stringent hybridization condition hybridizes in 6.times.SSC
at 45.degree. C., followed by one or more washes in 0.1.times.SSC,
0.2% SDS at 68.degree. C. Furthermore, one of skill in the art can
manipulate the hybridization and/or washing conditions to increase
or decrease the stringency of hybridization such that nucleic acids
comprising nucleotide sequences that are at least 65, 70, 75, 80,
85, 90, 95, 98 or 99% identical to each other typically remain
hybridized to each other. More generally, for the purposes of the
instant disclosure the term substantially identical with regard to
a nucleic acid sequence may be construed as a sequence of
nucleotides exhibiting at least about 85%, or 90%, or 95%, or 97%
sequence identity to the reference nucleic acid sequence.
[0191] The basic parameters affecting the choice of hybridization
conditions and guidance for devising suitable conditions are set
forth by, for example, Sambrook, Fritsch, and Maniatis (1989,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11; and
Current Protocols in Molecular Biology, 1995, Ausubel et al., eds.,
John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4), and can be
readily determined by those having ordinary skill in the art based
on, for example, the length and/or base composition of the nucleic
acid.
[0192] It will further be appreciated that nucleic acids may,
according to the invention, be present alone or in combination with
other nucleic acids, which may be homologous or heterologous. In
preferred embodiments, a nucleic acid is functionally linked to
expression control sequences that may be homologous or heterologous
with respect to said nucleic acid. In this context the term
homologous means that a nucleic acid is also functionally linked to
the expression control sequence naturally and the term heterologous
means that a nucleic acid is not functionally linked to the
expression control sequence naturally.
[0193] c. Expression
[0194] A nucleic acid, such as a nucleic acid expressing RNA and/or
protein or peptide, and an expression control sequence are
functionally linked to one another, if they are covalently linked
to one another in such a way that expression or transcription of
said nucleic acid is under the control or under the influence of
said expression control sequence. If the nucleic acid is to be
translated into a functional protein, then, with an expression
control sequence functionally linked to a coding sequence,
induction of said expression control sequence results in
transcription of said nucleic acid, without causing a frame shift
in the coding sequence or said coding sequence not being capable of
being translated into the desired protein or peptide.
[0195] The term expression control sequence comprises according to
the invention promoters, ribosome binding sites, enhancers and
other control elements that regulate transcription of a gene or
translation of mRNA. In particular embodiments of the invention,
the expression control sequences can be regulated. The exact
structure of expression control sequences may vary as a function of
the species or cell type, but generally comprises 5'-untranscribed
and 5'- and 3'-untranslated sequences which are involved in
initiation of transcription and translation, respectively, such as
TATA box, capping sequence, CAAT sequence, and the like. More
specifically, 5'-untranscribed expression control sequences
comprise a promoter region that includes a promoter sequence for
transcriptional control of the functionally linked nucleic acid.
Expression control sequences may also comprise enhancer sequences
or upstream activator sequences.
[0196] According to the invention the term promoter or promoter
region relates to a nucleic acid sequence which is located upstream
(5') to the nucleic acid sequence being expressed and controls
expression of the sequence by providing a recognition and binding
site for RNA-polymerase. The promoter region may include further
recognition and binding sites for further factors that are involved
in the regulation of transcription of a gene. A promoter may
control the transcription of a prokaryotic or eukaryotic gene.
Furthermore, a promoter may be inducible and may initiate
transcription in response to an inducing agent or may be
constitutive if transcription is not controlled by an inducing
agent. A gene that is under the control of an inducible promoter is
not expressed or only expressed to a small extent if an inducing
agent is absent. In the presence of the inducing agent the gene is
switched on or the level of transcription is increased. This is
mediated, in general, by binding of a specific transcription
factor.
[0197] Promoters which are preferred according to the invention
include promoters for SP6, T3 and T7 polymerase, human U6 RNA
promoter, CMV promoter, and artificial hybrid promoters thereof
(e.g. CMV) where a part or parts are fused to a part or parts of
promoters of genes of other cellular proteins such as e.g. human
GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and including or
not including (an) additional intron(s).
[0198] According to the invention, the term expression is used in
its most general meaning and comprises the production of RNA or of
RNA and protein/peptide. It also comprises partial expression of
nucleic acids. Furthermore, expression may be carried out
transiently or stably.
[0199] In a preferred embodiment, a nucleic acid molecule is
according to the invention present in a vector, where appropriate
with a promoter, which controls expression of the nucleic acid. The
term vector is used here in its most general meaning and comprises
any intermediary vehicle for a nucleic acid which enables said
nucleic acid, for example, to be introduced into prokaryotic and/or
eukaryotic cells and, where appropriate, to be integrated into a
genome. Vectors of this kind are preferably replicated and/or
expressed in the cells. Vectors may comprise plasmids, phagemids,
bacteriophages or viral genomes. The term plasmid as used herein
generally relates to a construct of extrachromosomal genetic
material, usually a circular DNA duplex, which can replicate
independently of chromosomal DNA.
[0200] In practicing the present invention it will be appreciated
that many conventional techniques in molecular biology,
microbiology, and recombinant DNA technology are optionally used.
Such conventional techniques relate to vectors, host cells and
recombinant methods as defined herein. These techniques are well
known and are explained in, for example, Berger and Kimmel, Guide
to Molecular Cloning Techniques, Methods in Enzymology volume 152
Academic Press, Inc., San Diego, Calif.; Sambrook et al., Molecular
Cloning-A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 2000 and Current Protocols in
Molecular Biology, F. M. Ausubel et al., eds., supra Other useful
references, e.g. for cell isolation and culture (e.g., for
subsequent nucleic acid or protein isolation) include Freshney
(1994) Culture of Animal Cells, a Manual of Basic Technique, third
edition, Wiley-Liss, New York and the references cited therein;
Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems
John Wiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips
(Eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental
Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New
York) and Atlas and Parks (Eds.) The Handbook of Microbiological
Media (1993) CRC Press, Boca Raton, Fla. Methods of making nucleic
acids (e.g., by in vitro amplification, purification from cells, or
chemical synthesis), methods for manipulating nucleic acids (e.g.,
site-directed mutagenesis, by restriction enzyme digestion,
ligation, etc.), and various vectors, cell lines and the like
useful in manipulating and making nucleic acids are described in
the above references. In addition, essentially any polynucleotide
(including, e.g., labeled or biotinylated polynucleotides) can be
custom or standard ordered from any of a variety of commercial
sources.
[0201] Thus, in one aspect, the present invention provides
recombinant host cells allowing recombinant expression of
antibodies of the invention or portions thereof. Antibodies
produced by expression in such recombinant host cells are referred
to herein as recombinant antibodies. The present invention also
provides progeny cells of such host cells, and antibodies produced
by the same.
[0202] The term recombinant host cell (or simply host cell), as
used herein, means a cell into which a recombinant expression
vector has been introduced. It should be understood that
recombinant host cell and host cell mean not only the particular
subject cell but also the progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but are still included
within the scope of the term host cell as used herein. Such cells
may comprise a vector according to the invention as described
above.
[0203] In another aspect, the present invention provides a method
for making an antibody or portion thereof as described herein.
According to one embodiment, said method comprises culturing a cell
transfected or transformed with a vector as described above, and
retrieving the antibody or portion thereof.
[0204] As indicated above, expression of an antibody of the
invention (or fragment or variants thereof) preferably comprises
expression vector(s) containing a polynucleotide that encodes the
desired anti-EFNA antibody. Methods that are well known to those
skilled in the art can be used to construct expression vectors
comprising antibody coding sequences and appropriate
transcriptional and translational control signals. These methods
include, for example, in vitro recombinant DNA techniques,
synthetic techniques, and in vivo genetic recombination.
Embodiments of the invention, thus, provide replicable vectors
comprising a nucleotide sequence encoding an anti-EFNA antibody of
the invention (e.g., a whole antibody, a heavy or light chain of an
antibody, a heavy or light chain variable domain of an antibody, or
a portion thereof, or a heavy or light chain CDR, a single chain
Fv, or fragments or variants thereof), operably linked to a
promoter. In preferred embodiments such vectors may include a
nucleotide sequence encoding the heavy chain of an antibody
molecule (or fragment thereof), a nucleotide sequence encoding the
light chain of an antibody (or fragment thereof) or both the heavy
and light chain.
[0205] Once the nucleotides of the present invention have been
isolated and modified according to the teachings herein, they may
be used to produce selected modulators including anti-EFNA
antibodies or fragments thereof.
X. Modulator Production and Purification
[0206] Using art recognized molecular biology techniques and
current protein expression methodology, substantial quantities of
the desired modulators may be produced. More specifically, nucleic
acid molecules encoding modulators, such as antibodies obtained and
engineered as described above, may be integrated into well known
and commercially available protein production systems comprising
various types of host cells to provide preclinical, clinical or
commercial quantities of the desired pharmaceutical product. It
will be appreciated that in preferred embodiments the nucleic acid
molecules encoding the modulators are engineered into vectors or
expression vectors that provide for efficient integration into the
selected host cell and subsequent high expression levels of the
desired EFNA modulator.
[0207] Preferably nucleic acid molecules encoding EFNA modulators
and vectors comprising these nucleic acid molecules can be used for
transfection of a suitable mammalian, plant, bacterial or yeast
host cell though it will be appreciated that prokaryotic systems
may be used for modulator production. Transfection can be by any
known method for introducing polynucleotides into a host cell.
Methods for the introduction of heterologous polynucleotides into
mammalian cells are well known in the art and include
dextran-mediated transfection, calcium phosphate precipitation,
polybrene-mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, and direct microinjection of the DNA into nuclei. In
addition, nucleic acid molecules may be introduced into mammalian
cells by viral vectors. Methods of transforming mammalian cells are
well known in the art. See, e.g., U.S. Pat. Nos. 4,399,216,
4,912,040, 4,740,461, and 4,959,455. Further, methods of
transforming plant cells are well known in the art, including,
e.g., Agrobacterium-mediated transformation, biolistic
transformation, direct injection, electroporation and viral
transformation. Methods of transforming bacterial and yeast cells
are also well known in the art.
[0208] Moreover, the host cell may be co-transfected with two
expression vectors of the invention, for example, the first vector
encoding a heavy chain derived polypeptide and the second vector
encoding a light chain derived polypeptide. The two vectors may
contain identical selectable markers that enable substantially
equal expression of heavy and light chain polypeptides.
Alternatively, a single vector may be used which encodes, and is
capable of expressing, both heavy and light chain polypeptides. In
such situations, the light chain is preferably placed before the
heavy chain to avoid an excess of toxic free heavy chain. The
coding sequences for the heavy and light chains may comprise cDNA
or genomic DNA.
[0209] a. Host-Expression Systems
[0210] A variety of host-expression vector systems, many
commercially available, are compatible with the teachings herein
and may be used to express the modulators of the invention. Such
host-expression systems represent vehicles by which the coding
sequences of interest may be expressed and subsequently purified,
but also represent cells which may, when transformed or transfected
with the appropriate nucleotide coding sequences, express a
molecule of the invention in situ. Such systems include, but are
not limited to, microorganisms such as bacteria (e.g., E. coli, B.
subtilis, streptomyces) transformed with recombinant bacteriophage
DNA, plasmid DNA or cosmid DNA expression vectors containing
modulator coding sequences; yeast (e.g., Saccharomyces, Pichia)
transfected with recombinant yeast expression vectors containing
modulator coding sequences; insect cell systems infected with
recombinant virus expression vectors (e.g., baculovirus) containing
modulator coding sequences; plant cell systems (e.g., Nicotiana,
Arabidopsis, duckweed, corn, wheat, potato, etc.) infected with
recombinant virus expression vectors (e.g., cauliflower mosaic
virus, CaMV; tobacco mosaic virus, TMV) or transfected with
recombinant plasmid expression vectors (e.g., Ti plasmid)
containing modulator coding sequences; or mammalian cell systems
(e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant
expression constructs containing promoters derived from the genome
of mammalian cells (e.g., metallothionein promoter) or from
mammalian viruses (e.g., the adenovirus late promoter; the vaccinia
virus 7.5K promoter).
[0211] In bacterial systems, a number of expression vectors may be
advantageously selected depending upon the use intended for the
molecule being expressed. For example, when a large quantity of
such a protein is to be produced, for the generation of
pharmaceutical compositions of a modulator, vectors which direct
the expression of high levels of fusion protein products that are
readily purified may be desirable. Such vectors include, but are
not limited to, the E. coli expression vector pUR278 (Ruther et
al., EMBO 1. 2:1791 (1983)), in which the coding sequence may be
ligated individually into the vector in frame with the lac Z coding
region so that a fusion protein is produced; pIN vectors (Inouye
& Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke
& Schuster, J. Biol. Chem. 24:5503-5509 (1989)); and the like.
pGEX vectors may also be used to express foreign polypeptides as
fusion proteins with glutathione 5-transferase (GST). In general,
such fusion proteins are soluble and can easily be purified from
lysed cells by adsorption and binding to matrix glutathione agarose
beads followed by elution in the presence of free glutathione. The
pGEX vectors are designed to include thrombin or factor Xa protease
cleavage sites so that the cloned target gene product can be
released from the GST moiety.
[0212] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) may be used as a vector to express
foreign genes. The virus grows in Spodoptera frugiperda cells. The
coding sequences may be cloned individually into non-essential
regions (for example, the polyhedrin gene) of the virus and placed
under control of an AcNPV promoter (for example, the polyhedrin
promoter).
[0213] In mammalian host cells, a number of viral-based expression
systems may be used to introduce the desired nucleotide sequence.
In cases where an adenovirus is used as an expression vector, the
coding sequence of interest may be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter
and tripartite leader sequence. This chimeric gene may then be
inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non-essential region of the viral
genome (e.g., region E1 or E3) will result in a recombinant virus
that is viable and capable of expressing the molecule in infected
hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA 8
1:355-359 (1984)). Specific initiation signals may also be required
for efficient translation of inserted coding sequences. These
signals include the ATG initiation codon and adjacent sequences.
Furthermore, the initiation codon must be in phase with the reading
frame of the desired coding sequence to ensure translation of the
entire insert. These exogenous translational control signals and
initiation codons can be of a variety of origins, both natural and
synthetic. The efficiency of expression may be enhanced by the
inclusion of appropriate transcription enhancer elements,
transcription terminators, etc. (see, e.g., Bittner et al., Methods
in Enzymol. 153:51-544 (1987)). Thus, compatible mammalian cell
lines available as hosts for expression are well known in the art
and include many immortalized cell lines available from the
American Type Culture Collection (ATCC). These include, inter alia,
Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T
cells, 293 Freestyle cells (Life Technologies), NIH-3T3 cells, HeLa
cells, baby hamster kidney (BHK) cells, African green monkey kidney
cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2),
A549 cells, and a number of other cell lines.
[0214] For long-term, high-yield production of recombinant proteins
stable expression is preferred. Accordingly, cell lines that stably
express the selected modulator may be engineered using standard art
recognized techniques. Rather than using expression vectors that
contain viral origins of replication, host cells can be transformed
with DNA controlled by appropriate expression control elements
(e.g., promoter, enhancer, sequences, transcription terminators,
polyadenylation sites, etc.), and a selectable marker. Following
the introduction of the foreign DNA, engineered cells may be
allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines which express the molecule. Such engineered cell lines may be
particularly useful in screening and evaluation of compositions
that interact directly or indirectly with the molecule.
[0215] A number of selection systems are well known in the art and
may be used including, but not limited to, the herpes simplex virus
thymidine kinase (Wigler et al., Cell 11:223 (1977)),
hypoxanthineguanine phosphoribosyltransferase (Szybalska &
Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine
phosphoribosyltransferase (Lowy et al., Cell 22:8 17 (1980)) genes
can be employed in tk-, hgprt- or aprt-cells, respectively. Also,
antimetabolite resistance can be used as the basis of selection for
the following genes: dhfr, which confers resistance to methotrexate
(Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al.,
Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers
resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl.
Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to
the aminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu and Wu,
Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol.
Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993);
and Morgan and Anderson, Ann. Rev. Biochem. 62: 191-217 (1993); TIB
TECH 11(5):155-2 15 (May, 1993)); and hygro, which confers
resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).
Methods commonly known in the art of recombinant DNA technology may
be routinely applied to select the desired recombinant clone, and
such methods are described, for example, in Ausubel et al. (eds.),
Current Protocols in Molecular Biology, John Wiley & Sons, NY
(1993); Kriegler, Gene Transfer and Expression, A Laboratory
Manual, Stockton Press, NY (1990); and in Chapters 12 and 13,
Dracopoli et al. (eds), Current Protocols in Human Genetics, John
Wiley & Sons, NY (1994); Colberre-Garapin et al., J. Mol. Biol.
150:1 (1981). It will be appreciated that one particularly
preferred method of establishing a stable, high yield cell line
comprises the glutamine synthetase gene expression system (the GS
system) which provides an efficient approach for enhancing
expression under certain conditions. The GS system is discussed in
whole or part in connection with EP patents 0 216 846, 0 256 055, 0
323 997 and 0 338 841 each of which is incorporated herein by
reference.
[0216] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products may be important for the function and/or
purification of the protein. Different host cells have
characteristic and specific mechanisms for the post-translational
processing and modification of proteins and gene products. As known
in the art appropriate cell lines or host systems can be chosen to
ensure the desired modification and processing of the expressed
polypeptide. To this end, eukaryotic host cells that possess the
cellular machinery for proper processing of the primary transcript,
glycosylation, and phosphorylation of the gene product are
particularly effective for use in the instant invention.
Accordingly, particularly preferred mammalian host cells include,
but are not limited to, CHO, VERY, BHK, HeLa, COS, NS0, MDCK, 293,
3T3, W138, as well as breast cancer cell lines such as, for
example, BT483, Hs578T, HTB2, BT2O and T47D, and normal mammary
gland cell line such as, for example, CRL7O3O and HsS78Bst.
Depending on the modulator and the selected production system,
those of skill in the art may easily select and optimize
appropriate host cells for efficient expression of the
modulator.
[0217] b. Chemical Synthesis
[0218] Besides the aforementioned host cell systems, it will be
appreciated that the modulators of the invention may be chemically
synthesized using techniques known in the art (e.g., see Creighton,
1983, Proteins: Structures and Molecular Principles, W.H. Freeman
& Co., N.Y., and Hunkapiller, M., et al., 1984, Nature
310:105-111). For example, a peptide corresponding to a polypeptide
fragment of the invention can be synthesized by use of a peptide
synthesizer. Furthermore, if desired, nonclassical amino acids or
chemical amino acid analogs can be introduced as a substitution or
addition into a polypeptide sequence. Non-classical amino acids
include, but are not limited to, to the D-isomers of the common
amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid,
4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx,
6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino
propionic acid, ornithine, norleucine, norvaline, hydroxyproline,
sarcosine, citrulline, homocitrulline, cysteic acid,
t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine,
b-alanine, fluoro-amino acids, designer amino acids such as
b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids,
and amino acid analogs in general. Furthermore, the amino acid can
be D (dextrorotary) or L (levorotary).
[0219] c. Transgenic Systems
[0220] The EFNA modulators of the invention also can be produced
transgenically through the generation of a mammal or plant that is
transgenic for the immunoglobulin heavy and light chain sequences
(or fragments or derivatives or variants thereof) of interest and
production of the desired compounds in a recoverable form. In
connection with the transgenic production in mammals, anti-EFNA
antibodies, for example, can be produced in, and recovered from,
the milk of goats, cows, or other mammals. See, e.g., U.S. Pat.
Nos. 5,827,690, 5,756,687, 5,750,172, and 5,741,957. In some
embodiments, non-human transgenic animals that comprise human
immunoglobulin loci are immunized with EFNA or an immunogenic
portion thereof, as described above. Methods for making antibodies
in plants are described, e.g., in U.S. Pat. Nos. 6,046,037 and
5,959,177.
[0221] In accordance with the teachings herein non-human transgenic
animals or plants may be produced by introducing one or more
nucleic acid molecules encoding an EFNA modulator of the invention
into the animal or plant by standard transgenic techniques. See
Hogan and U.S. Pat. No. 6,417,429. The transgenic cells used for
making the transgenic animal can be embryonic stem cells or somatic
cells or a fertilized egg. The transgenic non-human organisms can
be chimeric, nonchimeric heterozygotes, and nonchimeric
homozygotes. See, e.g., Hogan et al., Manipulating the Mouse
Embryo: A Laboratory Manual 2nd ed., Cold Spring Harbor Press
(1999); Jackson et al., Mouse Genetics and Transgenics: A Practical
Approach, Oxford University Press (2000); and Pinkert, Transgenic
Animal Technology: A Laboratory Handbook, Academic Press (1999). In
some embodiments, the transgenic non-human animals have a targeted
disruption and replacement by a targeting construct that encodes,
for example, a heavy chain and/or a light chain of interest. In one
embodiment, the transgenic animals comprise and express nucleic
acid molecules encoding heavy and light chains that specifically
bind to EFNA. While anti-EFNA antibodies may be made in any
transgenic animal, in particularly preferred embodiments the
non-human animals are mice, rats, sheep, pigs, goats, cattle or
horses. In further embodiments the non-human transgenic animal
expresses the desired pharmaceutical product in blood, milk, urine,
saliva, tears, mucus and other bodily fluids from which it is
readily obtainable using art recognized purification
techniques.
[0222] It is likely that modulators, including antibodies,
expressed by different cell lines or in transgenic animals will
have different glycosylation patterns from each other. However, all
modulators encoded by the nucleic acid molecules provided herein,
or comprising the amino acid sequences provided herein are part of
the instant invention, regardless of the glycosylation state of the
molecule, and more generally, regardless of the presence or absence
of post-translational modification(s). In addition the invention
encompasses modulators that are differentially modified during or
after translation, e.g., by glycosylation, acetylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to an
antibody molecule or other cellular ligand, etc. Any of numerous
chemical modifications may be carried out by known techniques,
including but not limited, to specific chemical cleavage by
cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease,
NaBH4, acetylation, formylation, oxidation, reduction, metabolic
synthesis in the presence of tunicamycin, etc. Various
post-translational modifications are also encompassed by the
invention include, for example, e.g., N-linked or O-linked
carbohydrate chains, processing of N-terminal or C-terminal ends),
attachment of chemical moieties to the amino acid backbone,
chemical modifications of N-linked or O-linked carbohydrate chains,
and addition or deletion of an N-terminal methionine residue as a
result of prokaryotic host cell expression. Moreover, as set forth
in the text and Examples below the polypeptides may also be
modified with a detectable label, such as an enzymatic,
fluorescent, radioisotopic or affinity label to allow for detection
and isolation of the modulator.
[0223] d. Purification
[0224] Once a modulator of the invention has been produced by
recombinant expression or any one of the other techniques disclosed
herein, it may be purified by any method known in the art for
purification of immunoglobulins, or more generally by any other
standard technique for the purification of proteins. In this
respect the modulator may be isolated. As used herein, an isolated
EFNA modulator is one that has been identified and separated and/or
recovered from a component of its natural environment. Contaminant
components of its natural environment are materials that would
interfere with diagnostic or therapeutic uses for the polypeptide
and may include enzymes, hormones, and other proteinaceous or
nonproteinaceous solutes. Isolated modulators include a modulator
in situ within recombinant cells because at least one component of
the polypeptide's natural environment will not be present.
[0225] When using recombinant techniques, the EFNA modulator (e.g.
an anti-EFNA antibody or derivative or fragment thereof) can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the desired molecule is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, may be removed, for example, by
centrifugation or ultrafiltration. For example, Carter, et al.,
Bio/Technology 10:163 (1992) describe a procedure for isolating
antibodies that are secreted to the periplasmic space of E. coli.
Briefly, cell paste is thawed in the presence of sodium acetate (pH
3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30
minutes. Cell debris can be removed by centrifugation. Where the
antibody is secreted into the medium, supernatants from such
expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. A protease
inhibitor such as PMSF may be included in any of the foregoing
steps to inhibit proteolysis and antibiotics may be included to
prevent the growth of adventitious contaminants.
[0226] The modulator (e.g., fc-EFNA or anti-EFNA antibody)
composition prepared from the cells can be purified using, for
example, hydroxylapatite chromatography, gel electrophoresis,
dialysis, and affinity chromatography, with affinity chromatography
being the preferred purification technique. The suitability of
protein A as an affinity ligand depends on the species and isotype
of any immunoglobulin Fc domain that is present in the selected
construct. Protein A can be used to purify antibodies that are
based on human IgG1, IgG2 or IgG4 heavy chains (Lindmark, et al., J
Immunol Meth 62:1 (1983)). Protein G is recommended for all mouse
isotypes and for human IgG3 (Guss, et al., EMBO J 5:1567 (1986)).
The matrix to which the affinity ligand is attached is most often
agarose, but other matrices are available. Mechanically stable
matrices such as controlled pore glass or
poly(styrenedivinyl)benzene allow for faster flow rates and shorter
processing times than can be achieved with agarose. Where the
antibody comprises a C.sub.H3 domain, the Bakerbond ABX.TM.' resin
(J. T. Baker; Phillipsburg, N.J.) is useful for purification. Other
techniques for protein purification such as fractionation on an
ion-exchange column, ethanol precipitation, reverse phase HPLC,
chromatography on silica, chromatography on heparin, sepharose
chromatography on an anion or cation exchange resin (such as a
polyaspartic acid column), chromatofocusing, SDS-PAGE and ammonium
sulfate precipitation are also available depending on the antibody
to be recovered. In particularly preferred embodiments the
modulators of the instant invention will be purified, at least in
part, using Protein A or Protein G affinity chromatography.
XI. Conjugated EFNA Modulators
[0227] Once the modulators of the invention have been purified
according to the teachings herein they may be linked with, fused
to, conjugated to (e.g., covalently or non-covalently) or otherwise
associated with pharmaceutically active or diagnostic moieties or
biocompatible modifiers. As used herein the term conjugate will be
used broadly and held to mean any molecule associated with the
disclosed modulators regardless of the method of association. In
this respect it will be understood that such conjugates may
comprise peptides, polypeptides, proteins, polymers, nucleic acid
molecules, small molecules, mimetic agents, synthetic drugs,
inorganic molecules, organic molecules and radioisotopes. Moreover,
as indicated above the selected conjugate may be covalently or
non-covalently linked to the modulator and exhibit various molar
ratios depending, at least in part, on the method used to effect
the conjugation.
[0228] In preferred embodiments it will be apparent that the
modulators of the invention may be conjugated or associated with
proteins, polypeptides or peptides that impart selected
characteristics (e.g., biotoxins, biomarkers, purification tags,
etc.). More generally, in selected embodiments the present
invention encompasses the use of modulators or fragments thereof
recombinantly fused or chemically conjugated (including both
covalent and non-covalent conjugations) to a heterologous protein
or polypeptide wherein the polypeptide comprises at least 10, at
least 20, at least 30, at least 40, at least 50, at least 60, at
least 70, at least 80, at least 90 or at least 100 amino acids. The
construct does not necessarily need to be directly linked, but may
occur through linker sequences. For example, antibodies may be used
to target heterologous polypeptides to particular cell types
expressing EFNA, either in vitro or in vivo, by fusing or
conjugating the modulators of the present invention to antibodies
specific for particular cell surface receptors. Moreover,
modulators fused or conjugated to heterologous polypeptides may
also be used in in vitro immunoassays and may be compatible with
purification methodology known in the art. See e.g., International
publication No. WO 93/21232; European Patent No. EP 439,095;
Naramura et al., 1994, Immunol. Lett. 39:91-99; U.S. Pat. No.
5,474,981; Gillies et al., 1992, PNAS 89:1428-1432; and Fell et
al., 1991, J. Immunol. 146:2446-2452.
[0229] a. Biocompatible Modifiers
[0230] In a preferred embodiment, the modulators of the invention
may be conjugated or otherwise associated with biocompatible
modifiers that may be used to adjust, alter, improve or moderate
modulator characteristics as desired. For example, antibodies or
fusion constructs with increased in vivo half-lives can be
generated by attaching relatively high molecular weight polymer
molecules such as commercially available polyethylene glycol (PEG)
or similar biocompatible polymers. Those skilled in the art will
appreciate that PEG may be obtained in many different molecular
weight and molecular configurations that can be selected to impart
specific properties to the antibody (e.g. the half-life may be
tailored). PEG can be attached to modulators or antibody fragments
or derivatives with or without a multifunctional linker either
through site-specific conjugation of the PEG to the N- or
C-terminus of said antibodies or antibody fragments or via
epsilon-amino groups present on lysine residues. Linear or branched
polymer derivatization that results in minimal loss of biological
activity may be used. The degree of conjugation can be closely
monitored by SDS-PAGE and mass spectrometry to ensure optimal
conjugation of PEG molecules to antibody molecules. Unreacted PEG
can be separated from antibody-PEG conjugates by, e.g., size
exclusion or ion-exchange chromatography. In a similar manner, the
disclosed modulators can be conjugated to albumin in order to make
the antibody or antibody fragment more stable in vivo or have a
longer half life in vivo. The techniques are well known in the art,
see e.g., International Publication Nos. WO 93/15199, WO 93/15200,
and WO 01/77137; and European Patent No. 0 413, 622. Other
biocompatible conjugates are evident to those of ordinary skill and
may readily be identified in accordance with the teachings
herein.
[0231] b. Diagnostic or Detection Agents
[0232] In other preferred embodiments, modulators of the present
invention, or fragments or derivatives thereof, are conjugated to a
diagnostic or detectable agent, marker or reporter which may be a
biological molecule (e.g., a peptide or nucleotide), a small
molecule, flourophore, or radioisotope. Labeled modulators can be
useful for monitoring the development or progression of a
hyperproliferative disorder or as part of a clinical testing
procedure to determine the efficacy of a particular therapy
including the disclosed modulators (i.e. theragnostics). Such
markers or reporters may also be useful in purifying the selected
modulator, separating or isolating TIC or in preclinical procedures
or toxicology studies.
[0233] Such diagnosis and detection can be accomplished by coupling
the modulator to detectable substances including, but not limited
to, various enzymes comprising for example horseradish peroxidase,
alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
prosthetic groups, such as but not limited to streptavidinlbiotin
and avidin/biotin; fluorescent materials, such as but not limited
to, umbelliferone, fluorescein, fluorescein isothiocynate,
rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; luminescent materials, such as but not limited to,
luminol; bioluminescent materials, such as but not limited to,
luciferase, luciferin, and aequorin; radioactive materials, such as
but not limited to iodine (.sup.131I, .sup.125I, .sup.123I,
.sup.121I,), carbon (.sup.14C), sulfur (.sup.35S), tritium
(.sup.3H), indium (.sup.115In, .sup.113In, .sup.112In,
.sup.111In,), and technetium (.sup.99Tc), thallium (.sup.201Ti),
gallium (.sup.68Ga, .sup.67Ga), palladium (.sup.103Pd), molybdenum
(.sup.99Mo), xenon (.sup.133Xe), fluorine (.sup.18F), .sup.153Sm,
.sup.177Lu, .sup.159Gd, .sup.149Pm, .sup.140La, .sup.175Yb,
.sup.166Ho, .sup.90Y, .sup.47Sc, .sup.186Re, .sup.188Re,
.sup.142Pr, .sup.105Rh, .sup.97Ru, .sup.68Ge, .sup.57Co, .sup.65Zn,
.sup.85Sr, .sup.32P, .sup.153Gd, .sup.169Yb, .sup.51Cr, .sup.54Mn,
.sup.75Se, .sup.113Sn, and .sup.117Tin; positron emitting metals
using various positron emission tomographies, noradioactive
paramagnetic metal ions, and molecules that are radiolabeled or
conjugated to specific radioisotopes. In such embodiments
appropriate detection methodology is well known in the art and
readily available from numerous commercial sources.
[0234] As indicated above, in other embodiments the modulators or
fragments thereof can be fused to marker sequences, such as a
peptide or fluorophore to facilitate purification or diagnostic
procedures such as immunohistochemistry or FACs. In preferred
embodiments, the marker amino acid sequence is a hexa-histidine
peptide, such as the tag provided in a pQE vector (Qiagen), among
others, many of which are commercially available. As described in
Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for
instance, hexa-histidine provides for convenient purification of
the fusion protein. Other peptide tags useful for purification
include, but are not limited to, the hemagglutinin "HA" tag, which
corresponds to an epitope derived from the influenza hemagglutinin
protein (Wilson et al., 1984, Cell 37:767) and the "flag" tag (U.S.
Pat. No. 4,703,004).
[0235] c. Therapeutic Moieties
[0236] As previously alluded to the modulators or fragments or
derivatives thereof may also be conjugated, linked or fused to or
otherwise associated with a therapeutic moiety such as anti-cancer
agents, a cytotoxin or cytotoxic agent, e.g., a cytostatic or
cytocidal agent, a therapeutic agent or a radioactive metal ion,
e.g., alpha or beta-emitters. As used herein a cytotoxin or
cytotoxic agent includes any agent or therapeutic moiety that is
detrimental to cells and may inhibit cell growth or survival.
Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium
bromide, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy
anthracin, maytansinoids such as DM-1 and DM-4 (Immunogen, Inc.),
dione, mitoxantrone, mithramycin, actinomycin D,
1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide
and analogs or homologs thereof. Additional cytotoxins comprise
auristatins, including monomethyl auristatin E (MMAE) and
monomethyl auristatin F (MMAF) (Seattle Genetics, Inc.), amanitins
such as alpha-amanitin, beta-amanitin, gamma-amanitin or
epsilon-amanitin (Heidelberg Pharma AG), DNA minor groove binding
agents such as duocarmycin derivatives (Syntarga, B.V.) and
modified pyrrolobenzodiazepine dimers (PBDs, Spirogen, Ltd).
Furthermore, in one embodiment the EFNA modulators of the instant
invention may be associated with anti-CD3 binding molecules to
recruit cytotoxic T-cells and have them target the tumor initiating
cells (BiTE technology; see e.g., Fuhrmann, S. et. al. Annual
Meeting of AACR Abstract No. 5625 (2010) which is incorporated
herein by reference).
[0237] Additional compatible therapeutic moieties comprise
cytotoxic agents including, but are not limited to, antimetabolites
(e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-fluorouracil decarbazine), alkylating agents (e.g.,
mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU)
and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,
streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II)
(DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly
daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin
(formerly actinomycin), bleomycin, mithramycin, and anthramycin
(AMC)), and anti-mitotic agents (e.g., vincristine and
vinblastine). A more extensive list of therapeutic moieties can be
found in PCT publication WO 03/075957 and U.S.P.N. 2009/0155255
each of which is incorporated herein by reference.
[0238] The selected modulators can also be conjugated to
therapeutic moieties such as radioactive materials or macrocyclic
chelators useful for conjugating radiometal ions (see above for
examples of radioactive materials). In certain embodiments, the
macrocyclic chelator is
1,4,7,10-tetraazacyclododecane-N,N',N'',N''-tetraacetic acid (DOTA)
which can be attached to the antibody via a linker molecule. Such
linker molecules are commonly known in the art and described in
Denardo et al., 1998, Clin Cancer Res. 4:2483; Peterson et al.,
1999, Bioconjug. Chem. 10:553; and Zimmerman et al., 1999, Nucl.
Med. Biol. 26:943.
[0239] Exemplary radioisotopes that may be compatible with this
aspect of the invention include, but are not limited to, iodine
(.sup.131I, .sup.125I, .sup.123I, .sup.121I) carbon (.sup.14C),
copper (.sup.62Cu, .sup.64Cu, .sup.67Cu), sulfur (.sup.35S),
tritium (.sup.3H), indium (.sup.115In, .sup.113In, .sup.112In,
.sup.111In,), bismuth (.sup.212Bi, .sup.213Bi), technetium
(.sup.99Tc), thallium (.sup.201Ti), gallium (.sup.68Ga, .sup.67Ga),
palladium (.sup.103Pd), molybdenum (.sup.99Mo), xenon (.sup.133Xe),
fluorine (.sup.18F), .sup.153Sm, .sup.177Lu, .sup.159Gd,
.sup.149Pm, .sup.140La, .sup.175Yb, .sup.166Ho, .sup.90Y,
.sup.47Sc, .sup.186Re, .sup.188Re, .sup.142Pr, .sup.105Rh,
.sup.97Ru, .sup.68Ge, .sup.57Co, .sup.65Zn, .sup.85Sr, .sup.32P,
.sup.153Gd, .sup.169Yb, .sup.51Cr, .sup.54Mn, .sup.75Se,
.sup.113Sn, .sup.117Tin, .sup.225Ac, .sup.76Br, and .sup.211At.
Other radionuclides are also available as diagnostic and
therapeutic agents, especially those in the energy range of 60 to
4,000 keV. Depending on the condition to be treated and the desired
therapeutic profile, those skilled in the art may readily select
the appropriate radioisotope for use with the disclosed
modulators.
[0240] EFNA modulators of the present invention may also be
conjugated to a therapeutic moiety or drug that modifies a given
biological response (e.g., biological response modifiers or BRMs).
That is, therapeutic agents or moieties compatible with the instant
invention are not to be construed as limited to classical chemical
therapeutic agents. For example, in particularly preferred
embodiments the drug moiety may be a protein or polypeptide or
fragment thereof possessing a desired biological activity. Such
proteins may include, for example, a toxin such as abrin, ricin A,
Onconase (or another cytotoxic RNase), pseudomonas exotoxin,
cholera toxin, or diphtheria toxin; a protein such as tumor
necrosis factor, .alpha.-interferon, .beta.-interferon, nerve
growth factor, platelet derived growth factor, tissue plasminogen
activator, an apoptotic agent, e.g., TNF-.alpha., TNF-.beta., AIM I
(see, International Publication No. WO 97/33899), AIM II (see,
International Publication No. WO 97/34911), Fas Ligand (Takahashi
et al., 1994, J. Immunol., 6:1567), and VEGI (see, International
Publication No. WO 99/23105), a thrombotic agent or an
anti-angiogenic agent, e.g., angiostatin or endostatin; or, a
biological response modifier such as, for example, a lymphokine
(e.g., interleukin-1 interleukin-2 ("IL-2"), interleukin-6
("IL-6"), granulocyte macrophage colony stimulating factor
("GM-CSF"), and granulocyte colony stimulating factor ("G-CSF")),
or a growth factor (e.g., growth hormone ("GH")). As set forth
above, methods for fusing or conjugating modulators to polypeptide
moieties are known in the art. In addition to the previously
disclosed subject references see, e.g., U.S. Pat. Nos. 5,336,603;
5,622,929; 5,359,046; 5,349,053; 5,447,851, and 5,112,946; EP
307,434; EP 367,166; PCT Publications WO 96/04388 and WO 91/06570;
Ashkenazi et al., 1991, PNAS USA 88:10535; Zheng et al., 1995, J
Immunol 154:5590; and Vil et al., 1992, PNAS USA 89:11337 each of
which is incorporated herein by reference. The association of a
modulator with a moiety does not necessarily need to be direct, but
may occur through linker sequences. Such linker molecules are
commonly known in the art and described in Denardo et al., 1998,
Clin Cancer Res 4:2483; Peterson et al., 1999, Bioconjug Chem
10:553; Zimmerman et al., 1999, Nucl Med Biol 26:943; Garnett,
2002, Adv Drug Deliv Rev 53:171 each of which is incorporated
herein.
[0241] More generally, techniques for conjugating therapeutic
moieties or cytotoxic agents to modulators are well known. Moieties
can be conjugated to modulators by any art-recognized method,
including, but not limited to aldehyde/Schiff linkage, sulphydryl
linkage, acid-labile linkage, cis-aconityl linkage, hydrazone
linkage, enzymatically degradable linkage (see generally Garnett,
2002, Adv Drug Deliv Rev 53:171). Also see, e.g., Amon et al.,
"Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer
Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et
al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al.,
"Antibodies For Drug Delivery", in Controlled Drug Delivery (2nd
Ed.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc.
1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer
Therapy: A Review", in Monoclonal Antibodies '84: Biological And
Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982, Immunol.
Rev. 62:119. In preferred embodiments an EFNA modulator that is
conjugated to a therapeutic moiety or cytotoxic agent may be
internalized by a cell upon binding to an EFNA molecule associated
with the cell surface thereby delivering the therapeutic
payload.
XII. Diagnostics and Screening
[0242] a. Diagnostics
[0243] As indicated, the present invention provides in vitro or in
vivo methods for detecting, diagnosing or monitoring
hyperproliferative disorders and methods of screening cells from a
patient to identify tumorigenic cells including TPCs. Such methods
include identifying an individual having cancer for treatment or
monitoring progression of a cancer comprising contacting the
patient or a sample obtained from a patient with a selected EFNA
modulator as described herein and detecting presence or absence, or
level of association of the modulator to bound or free ephrin-A
ligand in the sample. When the modulator comprises an antibody or
immunologically active fragment thereof the association with
particular EFNA in the sample likely denotes that the sample may
contain tumor perpetuating cells (e.g., a cancer stem cells)
indicating that the individual having cancer may be effectively
treated with an EFNA modulator as described herein. The methods may
further comprise a step of comparing the level of binding to a
control. Conversely, when the selected modulator is Fc-EFNA the
binding properties of the selected ephrin-A ligand may be exploited
and monitored (directly or indirectly, in vivo or in vitro) when in
contact with the sample to provide the desired information. Other
diagnostic or theragnostic methods compatible with the teachings
herein are well known in the art and can be practiced using
commercial materials such as dedicated reporting systems.
[0244] In a particularly preferred embodiment the modulators of the
instant invention may be used to detect and quantify EFNA levels in
a patient sample (e.g., plasma or blood) which may, in turn, be
used to detect, diagnose or monitor EFNA associated disorders
including hyperproliferative disorders.
[0245] Exemplary compatible assay methods include
radioimmunoassays, enzyme immunoassays, competitive-binding assays,
fluorescent immunoassay, immunoblot assays, Western Blot analysis,
flow cytometry assays, and ELISA assays. More generally detection
of EFNA in a biological sample or the measurement of EFNA enzymatic
activity (or inhibition thereof) may be accomplished using any
art-known assay. Compatible in vivo theragnostics or diagnostics
may comprise art recognized imaging or monitoring techniques such
as magnetic resonance imaging (MRI), computerized tomography (e.g.
CAT scan), positron tomography (e.g., PET scan) radiography,
ultrasound, etc. Those skilled in the art will readily be able to
recognize and implement appropriate detection, monitoring or
imaging techniques (often comprising commercially available
sources) based on the etiology, pathological manifestation or
clinical progression of the disorder.
[0246] In another embodiment, the invention provides a method of
analyzing cancer progression and/or pathogenesis in vivo. In
another embodiment, analysis of cancer progression and/or
pathogenesis in vivo comprises determining the extent of tumor
progression. In another embodiment, analysis comprises the
identification of the tumor. In another embodiment, analysis of
tumor progression is performed on the primary tumor. In another
embodiment, analysis is performed over time depending on the type
of cancer as known to one skilled in the art. In another
embodiment, further analysis of secondary tumors originating from
metastasizing cells of the primary tumor is analyzed in-vivo. In
another embodiment, the size and shape of secondary tumors are
analyzed. In some embodiments, further ex vivo analysis is
performed.
[0247] In another embodiment, the invention provides a method of
analyzing cancer progression and/or pathogenesis in vivo including
determining cell metastasis. In yet another embodiment, analysis of
cell metastasis comprises determination of progressive growth of
cells at a site that is discontinuous from the primary tumor. In
another embodiment, the site of cell metastasis analysis comprises
the route of neoplastic spread. In some embodiment, cells can
disperse via blood vasculature, lymphatics, within body cavities or
combinations thereof. In another embodiment, cell metastasis
analysis is performed in view of cell migration, dissemination,
extravasation, proliferation or combinations thereof.
[0248] In certain examples, the tumorigenic cells in a subject or a
sample from a subject may be assessed or characterized using the
disclosed modulators prior to therapy or regimen to establish a
baseline. In other examples the sample is derived from a subject
that was treated. In some examples the sample is taken from the
subject at least about 1, 2, 4, 6, 7, 8, 10, 12, 14, 15, 16, 18,
20, 30, 60, 90 days, 6 months, 9 months, 12 months, or >12
months after the subject begins or terminates treatment. In certain
examples, the tumorigenic cells are assessed or characterized after
a certain number of doses (e.g., after 2, 5, 10, 20, 30 or more
doses of a therapy). In other examples, the tumorigenic cells are
characterized or assessed after 1 week, 2 weeks, 1 month, 2 months,
1 year, 2 years, 3 years, 4 years or more after receiving one or
more therapies.
[0249] In another aspect, and as discussed in more detail below,
the present invention provides kits for detecting, monitoring or
diagnosing a hyperproliferative disorder, identifying individual
having such a disorder for possible treatment or monitoring
progression (or regression) of the disorder in a patient, wherein
the kit comprises a modulator as described herein, and reagents for
detecting the impact of the modulator on a sample.
[0250] b. Screening
[0251] The EFNA modulators and cells, cultures, populations and
compositions comprising the same, including progeny thereof, can
also be used to screen for or identify compounds or agents (e.g.,
drugs) that affect a function or activity of tumor initiating cells
or progeny thereof by interacting with an ephrin-A ligand (e.g.,
the polypeptide or genetic components thereof). The invention
therefore further provides systems and methods for evaluation or
identification of a compound or agent that can affect a function or
activity tumor initiating cells or progeny thereof by associating
with EFNA or its substrates. Such compounds and agents can be drug
candidates that are screened for the treatment of a
hyperproliferative disorder, for example. In one embodiment, a
system or method includes tumor initiating cells exhibiting EFNA
and a compound or agent (e.g., drug), wherein the cells and
compound or agent (e.g., drug) are in contact with each other.
[0252] The invention further provides methods of screening and
identifying EFNA modulators or agents and compounds for altering an
activity or function of tumor initiating cells or progeny cells. In
one embodiment, a method includes contacting tumor initiating cells
or progeny thereof with a test agent or compound; and determining
if the test agent or compound modulates an activity or function of
the ephrin-A ligand associated tumor initiating cells.
[0253] A test agent or compound modulating an EFNA related activity
or function of such tumor initiating cells or progeny thereof
within the population identifies the test agent or compound as an
active agent. Exemplary activity or function that can be modulated
include changes in cell morphology, expression of a marker,
differentiation or dedifferentiation, maturation, proliferation,
viability, apoptosis or cell death neuronal progenitor cells or
progeny thereof.
[0254] Contacting, when used in reference to cells or a cell
culture or method step or treatment, means a direct or indirect
interaction between the composition (e.g., an ephrin-A ligand
associated cell or cell culture) and another referenced entity. A
particular example of a direct interaction is physical interaction.
A particular example of an indirect interaction is where a
composition acts upon an intermediary molecule which in turn acts
upon the referenced entity (e.g., cell or cell culture).
[0255] In this aspect of the invention modulates indicates
influencing an activity or function of tumor initiating cells or
progeny cells in a manner compatible with detecting the effects on
cell activity or function that has been determined to be relevant
to a particular aspect (e.g., metastasis or proliferation) of the
tumor initiating cells or progeny cells of the invention. Exemplary
activities and functions include, but are not limited to, measuring
morphology, developmental markers, differentiation, proliferation,
viability, cell respiration, mitochondrial activity, membrane
integrity, or expression of markers associated with certain
conditions. Accordingly, a compound or agent (e.g., a drug
candidate) can be evaluated for its effect on tumor initiating
cells or progeny cells, by contacting such cells or progeny cells
with the compound or agent and measuring any modulation of an
activity or function of tumor initiating cells or progeny cells as
disclosed herein or would be known to the skilled artisan.
[0256] Methods of screening and identifying agents and compounds
include those suitable for high throughput screening, which include
arrays of cells (e.g., microarrays) positioned or placed,
optionally at pre-determined locations or addresses.
High-throughput robotic or manual handling methods can probe
chemical interactions and determine levels of expression of many
genes in a short period of time. Techniques have been developed
that utilize molecular signals (e.g., fluorophores) and automated
analyses that process information at a very rapid rate (see, e.g.,
Pinhasov et al., Comb. Chem. High Throughput Screen. 7:133 (2004)).
For example, microarray technology has been extensively utilized to
probe the interactions of thousands of genes at once, while
providing information for specific genes (see, e.g., Mocellin and
Rossi, Adv. Exp. Med. Biol. 593:19 (2007)).
[0257] Such screening methods (e.g., high-throughput) can identify
active agents and compounds rapidly and efficiently. For example,
cells can be positioned or placed (pre-seeded) on a culture dish,
tube, flask, roller bottle or plate (e.g., a single multi-well
plate or dish such as an 8, 16, 32, 64, 96, 384 and 1536 multi-well
plate or dish), optionally at defined locations, for identification
of potentially therapeutic molecules. Libraries that can be
screened include, for example, small molecule libraries, phage
display libraries, fully human antibody yeast display libraries
(Adimab, LLC), siRNA libraries, and adenoviral transfection
vectors.
XIII. Pharmaceutical Preparations and Therapeutic Uses
[0258] a. Formulations and Routes of Administration
[0259] Depending on the form of the modulator along with any
optional conjugate, the mode of intended delivery, the disease
being treated or monitored and numerous other variables,
compositions of the instant invention may be formulated as desired
using art recognized techniques. That is, in various embodiments of
the instant invention compositions comprising EFNA modulators are
formulated with a wide variety of pharmaceutically acceptable
carriers (see, e.g., Gennaro, Remington: The Science and Practice
of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed.
(2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery
Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et
al., Handbook of Pharmaceutical Excipients, 3.sup.rd ed.,
Pharmaceutical Press (2000)). Various pharmaceutically acceptable
carriers, which include vehicles, adjuvants, and diluents, are
readily available from numerous commercial sources. Moreover, an
assortment of pharmaceutically acceptable auxiliary substances,
such as pH adjusting and buffering agents, tonicity adjusting
agents, stabilizers, wetting agents and the like, are also
available. Certain non-limiting exemplary carriers include saline,
buffered saline, dextrose, water, glycerol, ethanol, and
combinations thereof.
[0260] More particularly it will be appreciated that, in some
embodiments, the therapeutic compositions of the invention may be
administered neat or with a minimum of additional components.
Conversely the EFNA modulators of the present invention may
optionally be formulated to contain suitable pharmaceutically
acceptable carriers comprising excipients and auxiliaries that are
well known in the art and are relatively inert substances that
facilitate administration of the modulator or which aid processing
of the active compounds into preparations that are pharmaceutically
optimized for delivery to the site of action. For example, an
excipient can give form or consistency or act as a diluent to
improve the pharmacokinetics of the modulator. Suitable excipients
include but are not limited to stabilizing agents, wetting and
emulsifying agents, salts for varying osmolarity, encapsulating
agents, buffers, and skin penetration enhancers.
[0261] Disclosed modulators for systemic administration may be
formulated for enteral, parenteral or topical administration.
Indeed, all three types of formulation may be used simultaneously
to achieve systemic administration of the active ingredient.
Excipients as well as formulations for parenteral and nonparenteral
drug delivery are set forth in Remington, The Science and Practice
of Pharmacy 20th Ed. Mack Publishing (2000). Suitable formulations
for parenteral administration include aqueous solutions of the
active compounds in water-soluble form, for example, water-soluble
salts. In addition, suspensions of the active compounds as
appropriate for oily injection suspensions may be administered.
Suitable lipophilic solvents or vehicles include fatty oils, for
example, sesame oil, or synthetic fatty acid esters, for example,
ethyl oleate or triglycerides. Aqueous injection suspensions may
contain substances that increase the viscosity of the suspension
and include, for example, sodium carboxymethyl cellulose, sorbitol,
and/or dextran. Optionally, the suspension may also contain
stabilizers. Liposomes can also be used to encapsulate the agent
for delivery into the cell.
[0262] Suitable formulations for enteral administration include
hard or soft gelatin capsules, pills, tablets, including coated
tablets, elixirs, suspensions, syrups or inhalations and controlled
release forms thereof.
[0263] In general the compounds and compositions of the invention,
comprising EFNA modulators may be administered in vivo, to a
subject in need thereof, by various routes, including, but not
limited to, oral, intravenous, intra-arterial, subcutaneous,
parenteral, intranasal, intramuscular, intracardiac,
intraventricular, intratracheal, buccal, rectal, intraperitoneal,
intradermal, topical, transdermal, and intrathecal, or otherwise by
implantation or inhalation. The subject compositions may be
formulated into preparations in solid, semi-solid, liquid, or
gaseous forms; including, but not limited to, tablets, capsules,
powders, granules, ointments, solutions, suppositories, enemas,
injections, inhalants, and aerosols. The appropriate formulation
and route of administration may be selected according to the
intended application and therapeutic regimen.
[0264] b. Dosages
[0265] Similarly, the particular dosage regimen, i.e., dose, timing
and repetition, will depend on the particular individual and that
individual's medical history. Empirical considerations such as
pharmacokinetics (e.g., half-life, clearance rate, etc.) will
contribute to the determination of the dosage. Frequency of
administration may be determined and adjusted over the course of
therapy, and is based on reducing the number of hyperproliferative
or neoplastic cells, including tumor initiating cells, maintaining
the reduction of such neoplastic cells, reducing the proliferation
of neoplastic cells, or delaying the development of metastasis.
Alternatively, sustained continuous release formulations of a
subject therapeutic composition may be appropriate. As alluded to
above various formulations and devices for achieving sustained
release are known in the art.
[0266] From a therapeutic standpoint the pharmaceutical
compositions are administered in an amount effective for treatment
or prophylaxis of the specific indication. The therapeutically
effective amount is typically dependent on the weight of the
subject being treated, his or her physical or health condition, the
extensiveness of the condition to be treated, or the age of the
subject being treated. In general, the EFNA modulators of the
invention may be administered in an amount in the range of about 10
.mu.g/kg body weight to about 100 mg/kg body weight per dose. In
certain embodiments, the EFNA modulators of the invention may be
administered in an amount in the range of about 50 .mu.g/kg body
weight to about 5 mg/kg body weight per dose. In certain other
embodiments, the EFNA modulators of the invention may be
administered in an amount in the range of about 100 .mu.g/kg body
weight to about 10 mg/kg body weight per dose. Optionally, the EFNA
modulators of the invention may be administered in an amount in the
range of about 100 .mu.g/kg body weight to about 20 mg/kg body
weight per dose. Further optionally, the EFNA modulators of the
invention may be administered in an amount in the range of about
0.5 mg/kg body weight to about 20 mg/kg body weight per dose. In
certain embodiments the compounds of present invention are provided
a dose of at least about 100 .mu.g/kg body weight, at least about
250 .mu.g/kg body weight, at least about 750 .mu.g/kg body weight,
at least about 3 mg/kg body weight, at least about 5 mg/kg body
weight, at least about 10 mg/kg body weight is administered.
[0267] Other dosing regimens may be predicated on Body Surface Area
(BSA) calculations as disclosed in U.S. Pat. No. 7,744,877 which is
incorporated herein by reference in its entirety. As is well known
in the art the BSA is calculated using the patient's height and
weight and provides a measure of a subject's size as represented by
the surface area of his or her body. In selected embodiments of the
invention using the BSA the modulators may be administered in
dosages from 10 mg/m.sup.2 to 800 mg/m.sup.2. In other preferred
embodiments the modulators will be administered in dosages from 50
m g/m.sup.2 to 500 mg/m.sup.2 and even more preferably at dosages
of 100 mg/m.sup.2, 150 mg/m.sup.2, 200 mg/m.sup.2, 250 mg/m.sup.2,
300 mg/m.sup.2, 350 mg/m.sup.2, 400 mg/m.sup.2 or 450 mg/m.sup.2.
Of course it will be appreciated that, regardless of how the
dosages are calculated, multiple dosages may be administered over a
selected time period to provide an absolute dosage that is
substantially higher than the individual administrations.
[0268] In any event, the EFNA modulators are preferably
administered as needed to subjects in need thereof. Determination
of the frequency of administration may be made by persons skilled
in the art, such as an attending physician based on considerations
of the condition being treated, age of the subject being treated,
severity of the condition being treated, general state of health of
the subject being treated and the like. Generally, an effective
dose of the EFNA modulator is administered to a subject one or more
times. More particularly, an effective dose of the modulator is
administered to the subject once a month, more than once a month,
or less than once a month. In certain embodiments, the effective
dose of the EFNA modulator may be administered multiple times,
including for periods of at least a month, at least six months, or
at least a year.
[0269] Dosages and regimens may also be determined empirically for
the disclosed therapeutic compositions in individuals who have been
given one or more administration(s). For example, individuals may
be given incremental dosages of a therapeutic composition produced
as described herein. To assess efficacy of the selected
composition, a marker of the specific disease, disorder or
condition can be followed as described previously. In embodiments
where the individual has cancer, these include direct measurements
of tumor size via palpation or visual observation, indirect
measurement of tumor size by x-ray or other imaging techniques; an
improvement as assessed by direct tumor biopsy and microscopic
examination of the tumor sample; the measurement of an indirect
tumor marker (e.g., PSA for prostate cancer) or an antigen
identified according to the methods described herein, a decrease in
pain or paralysis; improved speech, vision, breathing or other
disability associated with the tumor; increased appetite; or an
increase in quality of life as measured by accepted tests or
prolongation of survival. It will be apparent to one of skill in
the art that the dosage will vary depending on the individual, the
type of neoplastic condition, the stage of neoplastic condition,
whether the neoplastic condition has begun to metastasize to other
location in the individual, and the past and concurrent treatments
being used.
[0270] c. Combination Therapies
[0271] Combination therapies contemplated by the invention may be
particularly useful in decreasing or inhibiting unwanted neoplastic
cell proliferation (e.g. endothelial cells), decreasing the
occurrence of cancer, decreasing or preventing the recurrence of
cancer, or decreasing or preventing the spread or metastasis of
cancer. In such cases the compounds of the instant invention may
function as sensitizing or chemosensitizing agent by removing the
TPC propping up and perpetuating the tumor mass (e.g. NTG cells)
and allow for more effective use of current standard of care
debulking or anti-cancer agents. That is, a combination therapy
comprising an EFNA modulator and one or more anti-cancer agents may
be used to diminish established cancer e.g., decrease the number of
cancer cells present and/or decrease tumor burden, or ameliorate at
least one manifestation or side effect of cancer. As such,
combination therapy refers to the administration of an EFNA
modulator and one or more anti-cancer agent that includes, but is
not limited to, cytotoxic agents, cytostatic agents,
chemotherapeutic agents, targeted anti-cancer agents, biological
response modifiers, immunotherapeutic agents, cancer vaccines,
anti-angiogenic agents, cytokines, hormone therapies, radiation
therapy and anti-metastatic agents.
[0272] According to the methods of the present invention, there is
no requirement for the combined results to be additive of the
effects observed when each treatment (e.g., anti-EFNA antibody and
anti-cancer agent) is conducted separately. Although at least
additive effects are generally desirable, any increased anti-tumor
effect above one of the single therapies is beneficial.
Furthermore, the invention does not require the combined treatment
to exhibit synergistic effects. However, those skilled in the art
will appreciate that with certain selected combinations that
comprise preferred embodiments, synergism may be observed.
[0273] To practice combination therapy according to the invention,
an EFNA modulator (e.g., anti-EFNA antibody) in combination with
one or more anti-cancer agent may be administered to a subject in
need thereof in a manner effective to result in anti-cancer
activity within the subject. The EFNA modulator and anti-cancer
agent are provided in amounts effective and for periods of time
effective to result in their combined presence and their combined
actions in the tumor environment as desired. To achieve this goal,
the EFNA modulator and anti-cancer agent may be administered to the
subject simultaneously, either in a single composition, or as two
or more distinct compositions using the same or different
administration routes.
[0274] Alternatively, the modulator may precede, or follow, the
anti-cancer agent treatment by, e.g., intervals ranging from
minutes to weeks. In certain embodiments wherein the anti-cancer
agent and the antibody are applied separately to the subject, the
time period between the time of each delivery is such that the
anti-cancer agent and modulator are able to exert a combined effect
on the tumor. In a particular embodiment, it is contemplated that
both the anti-cancer agent and the EFNA modulator are administered
within about 5 minutes to about two weeks of each other.
[0275] In yet other embodiments, several days (2, 3, 4, 5, 6 or 7),
several weeks (1, 2, 3, 4, 5, 6, 7 or 8) or several months (1, 2,
3, 4, 5, 6, 7 or 8) may lapse between administration of the
modulator and the anti-cancer agent. The EFNA modulator and one or
more anti-cancer agent (combination therapy) may be administered
once, twice or at least the period of time until the condition is
treated, palliated or cured. Preferably, the combination therapy is
administered multiple times. The combination therapy may be
administered from three times daily to once every six months. The
administering may be on a schedule such as three times daily, twice
daily, once daily, once every two days, once every three days, once
weekly, once every two weeks, once every month, once every two
months, once every three months, once every six months or may be
administered continuously via a minipump. As previously indicated
the combination therapy may be administered via an oral, mucosal,
buccal, intranasal, inhalable, intravenous, subcutaneous,
intramuscular, parenteral, intratumor or topical route. The
combination therapy may be administered at a site distant from the
site of the tumor. The combination therapy generally will be
administered for as long as the tumor is present provided that the
combination therapy causes the tumor or cancer to stop growing or
to decrease in weight or volume.
[0276] In one embodiment an EFNA modulator is administered in
combination with one or more anti-cancer agents for a short
treatment cycle to a subject in need thereof. The duration of
treatment with the antibody may vary according to the particular
anti-cancer agent used. The invention also contemplates
discontinuous administration or daily doses divided into several
partial administrations. An appropriate treatment time for a
particular anti-cancer agent will be appreciated by the skilled
artisan, and the invention contemplates the continued assessment of
optimal treatment schedules for each anti-cancer agent.
[0277] The present invention contemplates at least one cycle,
preferably more than one cycle during which the combination therapy
is administered. An appropriate period of time for one cycle will
be appreciated by the skilled artisan, as will the total number of
cycles, and the interval between cycles. The invention contemplates
the continued assessment of optimal treatment schedules for each
modulator and anti-cancer agent. Moreover, the invention also
provides for more than one administration of either the anti-EFNA
antibody or the anti-cancer agent. The modulator and anti-cancer
agent may be administered interchangeably, on alternate days or
weeks; or a sequence of antibody treatment may be given, followed
by one or more treatments of anti-cancer agent therapy. In any
event, as will be understood by those of ordinary skill in the art,
the appropriate doses of chemotherapeutic agents will be generally
around those already employed in clinical therapies wherein the
chemotherapeutics are administered alone or in combination with
other chemotherapeutics.
[0278] In another preferred embodiment the EFNA modulators of the
instant invention may be used in maintenance therapy to reduce or
eliminate the chance of tumor recurrence following the initial
presentation of the disease. Preferably the disorder will have been
treated and the initial tumor mass eliminated, reduced or otherwise
ameliorated so the patient is asymptomatic or in remission. As such
time the subject may be administered pharmaceutically effective
amounts of the disclosed modulators one or more times even though
there is little or no indication of disease using standard
diagnostic procedures. In some embodiments the effectors will be
administered on a regular schedule over a period of time. For
example the EFNA modulators could be administered weekly, every two
weeks, monthly, every six weeks, every two months, every three
months every six months or annually. Given the teachings herein,
one skilled in the art could readily determine favorable dosages
and dosing regimens to reduce the potential of disease recurrence.
Moreover such treatments could be continued for a period of weeks,
months, years or even indefinitely depending on the patient
response and clinical and diagnostic parameters.
[0279] In yet another preferred embodiment the effectors of the
present invention may be used to prophylactically to prevent or
reduce the possibility of tumor metastasis following a debulking
procedure. As used in the instant disclosure a debulking procedure
is defined broadly and shall mean any procedure, technique or
method that eliminates, reduces, treats or ameliorates a tumor or
tumor proliferation. Exemplary debulking procedures include, but
are not limited to, surgery, radiation treatments (i.e., beam
radiation), chemotherapy or ablation. At appropriate times readily
determined by one skilled in the art in view of the instant
disclosure the EFNA modulators may be administered as suggested by
clinical and diagnostic or theragnostic procedures to reduce tumor
metastasis. The modulators may be administered one or more times at
pharmaceutically effective dosages as determined using standard
techniques. Preferably the dosing regimen will be accompanied by
appropriate diagnostic or monitoring techniques that allow it to be
modified as necessary.
[0280] d. Anti-Cancer Agents
[0281] As used herein the term anti-cancer agent means any agent
that can be used to treat a cell proliferative disorder such as
cancer, including cytotoxic agents, cytostatic agents,
anti-angiogenic agents, debulking agents, chemotherapeutic agents,
radiotherapy and radiotherapeutic agents, targeted anti-cancer
agents, biological response modifiers, antibodies, and
immunotherapeutic agents. It will be appreciated that, in selected
embodiments as discussed above, anti-cancer agents may comprise
conjugates and may be associated with modulators prior to
administration.
[0282] The term cytotoxic agent means a substance that decreases or
inhibits the function of cells and/or causes destruction of cells,
i.e., the substance is toxic to the cells. Typically, the substance
is a naturally occurring molecule derived from a living organism.
Examples of cytotoxic agents include, but are not limited to, small
molecule toxins or enzymatically active toxins of bacteria (e.g.,
Diptheria toxin, Pseudomonas endotoxin and exotoxin, Staphylococcal
enterotoxin A), fungal (e.g., .alpha.-sarcin, restrictocin), plants
(e.g., abrin, ricin, modeccin, viscumin, pokeweed anti-viral
protein, saporin, gelonin, momoridin, trichosanthin, barley toxin,
Aleurites fordii proteins, dianthin proteins, Phytolacca mericana
proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor,
curcin, crotin, saponaria officinalis inhibitor, gelonin,
mitegellin, restrictocin, phenomycin, neomycin, and the
tricothecenes) or animals, e.g., cytotoxic RNases, such as
extracellular pancreatic RNases; DNase I, including fragments
and/or variants thereof.
[0283] A chemotherapeutic agent means a chemical compound that
non-specifically decreases or inhibits the growth, proliferation,
and/or survival of cancer cells (e.g., cytotoxic or cytostatic
agents). Such chemical agents are often directed to intracellular
processes necessary for cell growth or division, and are thus
particularly effective against cancerous cells, which generally
grow and divide rapidly. For example, vincristine depolymerizes
microtubules, and thus inhibits cells from entering mitosis. In
general, chemotherapeutic agents can include any chemical agent
that inhibits, or is designed to inhibit, a cancerous cell or a
cell likely to become cancerous or generate tumorigenic progeny
(e.g., TIC). Such agents are often administered, and are often most
effective, in combination, e.g., in the formulation CHOP.
[0284] Examples of anti-cancer agents that may be used in
combination with (or conjugated to) the modulators of the present
invention include, but are not limited to, alkylating agents, alkyl
sulfonates, aziridines, ethylenimines and methylamelamines,
acetogenins, a camptothecin, bryostatin, callystatin, CC-1065,
cryptophycins, dolastatin, duocarmycin, eleutherobin,
pancratistatin, a sarcodictyin, spongistatin, nitrogen mustards,
antibiotics, enediyne antibiotics, dynemicin, bisphosphonates, an
esperamicin, chromoprotein enediyne antiobiotic chromophores,
aclacinomysins, actinomycin, authramycin, azaserine, bleomycins,
cactinomycin, carabicin, carminomycin, carzinophilin,
chromomycinis, dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM. doxorubicin,
epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins,
mycophenolic acid, nogalamycin, olivomycins, peplomycin,
potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubercidin, ubenimex, zinostatin, zorubicin;
anti-metabolites, folic acid analogues, purine analogs, androgens,
anti-adrenals, folic acid replenisher such as frolinic acid,
aceglatone, aldophosphamide glycoside, aminolevulinic acid,
eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate,
defofamine, demecolcine, diaziquone, elfornithine, elliptinium
acetate, an epothilone, etoglucid, gallium nitrate, hydroxyurea,
lentinan, lonidainine, maytansinoids, mitoguazone, mitoxantrone,
mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin,
losoxantrone, podophyllinic acid, 2-ethylhydrazide, procarbazine,
PSK.RTM. polysaccharide complex (JHS Natural Products, Eugene,
Oreg.), razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic
acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxoids, chloranbucil; GEMZAR.RTM.
gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum
analogs, vinblastine; platinum; etoposide (VP-16); ifosfamide;
mitoxantrone; vincristine; NAVELBINE.RTM. vinorelbine; novantrone;
teniposide; edatrexate; daunomycin; aminopterin; xeloda;
ibandronate; irinotecan (Camptosar, CPT-11), topoisomerase
inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids;
capecitabine; combretastatin; leucovorin (LV); oxaliplatin;
inhibitors of PKC-alpha, Raf, H-Ras, EGFR and VEGF-A that reduce
cell proliferation and pharmaceutically acceptable salts, acids or
derivatives of any of the above. Also included in this definition
are anti-hormonal agents that act to regulate or inhibit hormone
action on tumors such as anti-estrogens and selective estrogen
receptor modulators (SERMs), aromatase inhibitors that inhibit the
enzyme aromatase, which regulates estrogen production in the
adrenal glands, and anti-androgens; as well as troxacitabine (a
1,3-dioxolane nucleoside cytosine analog); antisense
oligonucleotides; ribozymes such as a VEGF expression inhibitor and
a HER2 expression inhibitor; vaccines, PROLEUKIN.RTM. rIL-2;
LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM. rmRH;
Vinorelbine and Esperamicins and pharmaceutically acceptable salts,
acids or derivatives of any of the above. Other embodiments
comprise the use of immunotherapeutic agents, such as antibodies,
approved for cancer therapy including, but not limited to,
rituximab, trastuzumab, gemtuzumab ozogamcin, alemtuzumab,
ibritumomab tiuxetan, tositumomab, bevacizumab, cetuximab,
patitumumab, ofatumumab, ipilimumab and brentuximab vedotin. Those
skilled in the art will be able to readily identify additional
anti-cancer agents that are compatible with the teachings
herein.
[0285] e. Radiotherapy
[0286] The present invention also provides for the combination of
EFNA modulators with radiotherapy (i.e., any mechanism for inducing
DNA damage locally within tumor cells such as gamma.-irradiation,
X-rays, UV-irradiation, microwaves, electronic emissions and the
like). Combination therapy using the directed delivery of
radioisotopes to tumor cells is also contemplated, and may be used
in connection with a targeted anti-cancer agent or other targeting
means. Typically, radiation therapy is administered in pulses over
a period of time from about 1 to about 2 weeks. The radiation
therapy may be administered to subjects having head and neck cancer
for about 6 to 7 weeks. Optionally, the radiation therapy may be
administered as a single dose or as multiple, sequential doses.
[0287] f. Neoplastic Conditions
[0288] Whether administered alone or in combination with an
anti-cancer agent or radiotherapy, the EFNA modulators of the
instant invention are particularly useful for generally treating
neoplastic conditions in patients or subjects which may include
benign or malignant tumors (e.g., renal, liver, kidney, bladder,
breast, gastric, ovarian, colorectal, prostate, pancreatic, lung,
thyroid, hepatic carcinomas; sarcomas; glioblastomas; and various
head and neck tumors); leukemias and lymphoid malignancies; other
disorders such as neuronal, glial, astrocytal, hypothalamic and
other glandular, macrophagal, epithelial, stromal and blastocoelic
disorders; and inflammatory, angiogenic, immunologic disorders and
disorders caused by pathogens. Particularly preferred targets for
treatment with therapeutic compositions and methods of the present
invention are neoplastic conditions comprising solid tumors. In
other preferred embodiments the modulators of the present invention
may be used for the diagnosis, prevention or treatment of
hematologic malignancies. Preferably the subject or patient to be
treated will be human although, as used herein, the terms are
expressly held to comprise any mammalian species.
[0289] More specifically, neoplastic conditions subject to
treatment in accordance with the instant invention may be selected
from the group including, but not limited to, adrenal gland tumors,
AIDS-associated cancers, alveolar soft part sarcoma, astrocytic
tumors, bladder cancer (squamous cell carcinoma and transitional
cell carcinoma), bone cancer (adamantinoma, aneurismal bone cysts,
osteochondroma, osteosarcoma), brain and spinal cord cancers,
metastatic brain tumors, breast cancer, carotid body tumors,
cervical cancer, chondrosarcoma, chordoma, chromophobe renal cell
carcinoma, clear cell carcinoma, colon cancer, colorectal cancer,
cutaneous benign fibrous histiocytomas, desmoplastic small round
cell tumors, ependymomas, Ewing's tumors, extraskeletal myxoid
chondrosarcoma, fibrogenesis imperfecta ossium, fibrous dysplasia
of the bone, gallbladder and bile duct cancers, gestational
trophoblastic disease, germ cell tumors, head and neck cancers,
islet cell tumors, Kaposi's Sarcoma, kidney cancer (nephroblastoma,
papillary renal cell carcinoma), leukemias, lipoma/benign
lipomatous tumors, liposarcoma/malignant lipomatous tumors, liver
cancer (hepatoblastoma, hepatocellular carcinoma), lymphomas, lung
cancers (small cell carcinoma, adenocarcinoma, squamous cell
carcinoma, large cell carcinoma etc.), medulloblastoma, melanoma,
meningiomas, multiple endocrine neoplasia, multiple myeloma,
myelodysplastic syndrome, neuroblastoma, neuroendocrine tumors,
ovarian cancer, pancreatic cancers, papillary thyroid carcinomas,
parathyroid tumors, pediatric cancers, peripheral nerve sheath
tumors, phaeochromocytoma, pituitary tumors, prostate cancer,
posterious unveal melanoma, rare hematologic disorders, renal
metastatic cancer, rhabdoid tumor, rhabdomysarcoma, sarcomas, skin
cancer, soft-tissue sarcomas, squamous cell cancer, stomach cancer,
synovial sarcoma, testicular cancer, thymic carcinoma, thymoma,
thyroid metastatic cancer, and uterine cancers (carcinoma of the
cervix, endometrial carcinoma, and leiomyoma). In certain preferred
embodiments, the cancerous cells are selected from the group of
solid tumors including but not limited to breast cancer, non-small
cell lung cancer (NSCLC), small cell lung cancer, pancreatic
cancer, colon cancer, prostate cancer, sarcomas, renal metastatic
cancer, thyroid metastatic cancer, and clear cell carcinoma.
[0290] With regard to hematologic malignancies it will be further
be appreciated that the compounds and methods of the present
invention may be particularly effective in treating a variety of
B-cell lymphomas, including low grade/NHL follicular cell lymphoma
(FCC), mantle cell lymphoma (MCL), diffuse large cell lymphoma
(DLCL), small lymphocytic (SL) NHL, intermediate grade/follicular
NHL, intermediate grade diffuse NHL, high grade immunoblastic NHL,
high grade lymphoblastic NHL, high grade small non-cleaved cell
NHL, bulky disease NHL, Waldenstrom's Macroglobulinemia,
lymphoplasmacytoid lymphoma (LPL), mantle cell lymphoma (MCL),
follicular lymphoma (FL), diffuse large cell lymphoma (DLCL),
Burkitt's lymphoma (BL), AIDS-related lymphomas, monocytic B cell
lymphoma, angioimmunoblastic lymphoadenopathy, small lymphocytic,
follicular, diffuse large cell, diffuse small cleaved cell, large
cell immunoblastic lymphoblastoma, small, non-cleaved, Burkitt's
and non-Burkitt's, follicular, predominantly large cell;
follicular, predominantly small cleaved cell; and follicular, mixed
small cleaved and large cell lymphomas. See, Gaidono et al.,
"Lymphomas", IN CANCER: PRINCIPLES & PRACTICE OF ONCOLOGY, Vol.
2: 2131-2145 (DeVita et al., eds., 5.sup.th ed. 1997). It should be
clear to those of skill in the art that these lymphomas will often
have different names due to changing systems of classification, and
that patients having lymphomas classified under different names may
also benefit from the combined therapeutic regimens of the present
invention.
[0291] In yet other preferred embodiments the EFNA modulators may
be used to effectively treat certain myeloid and hematologic
malignancies including leukemias such as chronic lymphocytic
leukemia (CLL or B-CLL). CLL is predominantly a disease of the
elderly that starts to increase in incidence after fifty years of
age and reaches a peak by late sixties. It generally involves the
proliferation of neoplastic peripheral blood lymphocytes. Clinical
finding of CLL involves lymphocytosis, lymphadenopatliy,
splenomegaly, anemia and thrombocytopenia. A characteristic feature
of CLL is monoclonal B cell proliferation and accumulation of
B-lymphocytes arrested at an intermediate state of differentiation
where such B cells express surface IgM (sIgM) or both sIgM and
sIgD, and a single light chain at densities lower than that on the
normal B cells.
[0292] The present invention also provides for a preventative or
prophylactic treatment of subjects who present with benign or
precancerous tumors. It is not believed that any particular type of
tumor or neoplastic disorder should be excluded from treatment
using the present invention. However, the type of tumor cells may
be relevant to the use of the invention in combination with
secondary therapeutic agents, particularly chemotherapeutic agents
and targeted anti-cancer agents.
[0293] Still other preferred embodiments of the instant invention
comprise the use of EFNA modulators to treat subjects suffering
from solid tumors. In such subjects many of these solid tumors
comprise tissue exhibiting various genetic mutations that may
render them particularly susceptible to treatment with the
disclosed effectors. For example, KRAS, APC and CTNNB1 and CDH1
mutations are relatively common in patients with colorectal cancer.
Moreover, patients suffering from tumors with these mutations are
usually the most refractory to current therapies; especially those
patients with KRAS mutations. KRAS activating mutations, which
typically result in single amino acid substitutions, are also
implicated in other difficult to treat malignancies, including lung
adenocarcinoma, mucinous adenoma, and ductal carcinoma of the
pancreas.
[0294] Currently, the most reliable prediction of whether
colorectal cancer patients will respond to EGFR- or VEGF-inhibiting
drugs, for example, is to test for certain KRAS "activating"
mutations. KRAS is mutated in 35-45% of colorectal cancers, and
patients whose tumors express mutated KRAS do not respond well to
these drugs. For example, KRAS mutations are predictive of a lack
of response to panitumumab and cetuximab therapy in colorectal
cancer (Lievre et al. Cancer Res 66:3992-5; Karapetis et al. NEJM
359:1757-1765). Approximately 85% of patients with colorectal
cancer have mutations in the APC gene (Markowitz & Bertagnolli.
NEJM 361:2449-60), and more than 800 APC mutations have been
characterized in patients with familial adenomatous polyposis and
colorectal cancer. A majority of these mutations result in a
truncated APC protein with reduced functional ability to mediate
the destruction of beta-catenin. Mutations in the beta-catenin
gene, CTNNB1, can also result in increased stabilization of the
protein, resulting in nuclear import and subsequent activation of
several oncogenic transcriptional programs, which is also the
mechanism of oncogenesis resulting from failure of mutated APC to
appropriately mediate beta-catenin destruction, which is required
to keep normal cell proliferation and differentiation programs in
check.
[0295] Loss of CDH1 (E-cadherin) expression is yet another common
occurrence in colorectal cancer, often observed in more advanced
stages of the disease. E-cadherin is the central member of adherin
junctions that connect and organize cells in epithelial layers.
Normally E-cadherin physically sequesters beta-catenin (CTNNB1) at
the plasma membrane; loss of E-cadherin expression in colorectal
cancer results in localization of beta-catenin to the nucleus and
transcriptional activation of the beta-catenin/WNT pathway.
Aberrant beta-catenin/WNT signaling is central to oncogenesis and
nuclear beta-catenin has been implicated in cancer stemness
(Schmalhofer et al., 2009 PMID 19153669). E-cadherin is required
for the expression and function of EphA2 a known binding partner
for EFNA ligands in epithelia cells (Dodge Zantek et al., 1999 PMID
10511313; Orsulic S and Kemler R, 2000 PMID 10769210). Using
modulators that bind to EFNA ligands and agonize with or antagonize
Eph receptor binding may modify, interrupt or reverse the
pro-oncogenic processes. Alternatively, EFNA modulators may
preferentially bind to tumor cells with aberrant EphA/EFNA
interactions based on the binding preferences of the EFNA
modulators. Hence patients with cancers carrying the above
mentioned genetic traits may benefits from treatment with
aforementioned EFNA modulators.
XIV. Articles of Manufacture
[0296] Pharmaceutical packs and kits comprising one or more
containers, comprising one or more doses of an EFNA modulator are
also provided. In certain embodiments, a unit dosage is provided
wherein the unit dosage contains a predetermined amount of a
composition comprising, for example, an anti-EFNA antibody, with or
without one or more additional agents. For other embodiments, such
a unit dosage is supplied in single-use prefilled syringe for
injection. In still other embodiments, the composition contained in
the unit dosage may comprise saline, sucrose, or the like; a
buffer, such as phosphate, or the like; and/or be formulated within
a stable and effective pH range. Alternatively, in certain
embodiments, the composition may be provided as a lyophilized
powder that may be reconstituted upon addition of an appropriate
liquid, for example, sterile water. In certain preferred
embodiments, the composition comprises one or more substances that
inhibit protein aggregation, including, but not limited to, sucrose
and arginine. Any label on, or associated with, the container(s)
indicates that the enclosed composition is used for diagnosing or
treating the disease condition of choice.
[0297] The present invention also provides kits for producing
single-dose or multi-dose administration units of an EFNA modulator
and, optionally, one or more anti-cancer agents. The kit comprises
a container and a label or package insert on or associated with the
container. Suitable containers include, for example, bottles,
vials, syringes, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition that is effective for treating the condition and may
have a sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). Such kits will generally contain in a
suitable container a pharmaceutically acceptable formulation of the
EFNA modulator and, optionally, one or more anti-cancer agents in
the same or different containers. The kits may also contain other
pharmaceutically acceptable formulations, either for diagnosis or
combined therapy. For example, in addition to the EFNA modulator of
the invention such kits may contain any one or more of a range of
anti-cancer agents such as chemotherapeutic or radiotherapeutic
drugs; anti-angiogenic agents; anti-metastatic agents; targeted
anti-cancer agents; cytotoxic agents; and/or other anti-cancer
agents. Such kits may also provide appropriate reagents to
conjugate the EFNA modulator with an anti-cancer agent or
diagnostic agent (e.g., see U.S. Pat. No. 7,422,739 which is
incorporated herein by reference in its entirety).
[0298] More specifically the kits may have a single container that
contains the EFNA modulator, with or without additional components,
or they may have distinct containers for each desired agent. Where
combined therapeutics are provided for conjugation, a single
solution may be pre-mixed, either in a molar equivalent
combination, or with one component in excess of the other.
Alternatively, the EFNA modulator and any optional anti-cancer
agent of the kit may be maintained separately within distinct
containers prior to administration to a patient. The kits may also
comprise a second/third container means for containing a sterile,
pharmaceutically acceptable buffer or other diluent such as
bacteriostatic water for injection (BWFI), phosphate-buffered
saline (PBS), Ringer's solution and dextrose solution.
[0299] When the components of the kit are provided in one or more
liquid solutions, the liquid solution is preferably an aqueous
solution, with a sterile aqueous solution being particularly
preferred. However, the components of the kit may be provided as
dried powder(s). When reagents or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container.
[0300] As indicated briefly above the kits may also contain a means
by which to administer the antibody and any optional components to
an animal or patient, e.g., one or more needles or syringes, or
even an eye dropper, pipette, or other such like apparatus, from
which the formulation may be injected or introduced into the animal
or applied to a diseased area of the body. The kits of the present
invention will also typically include a means for containing the
vials, or such like, and other component in close confinement for
commercial sale, such as, e.g., injection or blow-molded plastic
containers into which the desired vials and other apparatus are
placed and retained. Any label or package insert indicates that the
EFNA modulator composition is used for treating cancer, for example
colorectal cancer.
XV. Research Reagents
[0301] Other preferred embodiments of the invention also exploit
the properties of the disclosed modulators as an instrument useful
for identifying, isolating, sectioning or enriching populations or
subpopulations of tumor initiating cells through methods such as
fluorescent activated cell sorting (FACS), magnetic activated cell
sorting (MACS) or laser mediated sectioning. Those skilled in the
art will appreciate that the modulators may be used in several
compatible techniques for the characterization and manipulation of
TIC including cancer stem cells (e.g., see U.S. Ser. Nos.
12/686,359, 12/669,136 and 12/757,649 each of which is incorporated
herein by reference in its entirety).
XVI. Miscellaneous
[0302] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. More specifically, as used in this specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "a protein" includes a plurality of
proteins; reference to "a cell" includes mixtures of cells, and the
like. In addition, ranges provided in the specification and
appended claims include both end points and all points between the
end points. Therefore, a range of 2.0 to 3.0 includes 2.0, 3.0, and
all points between 2.0 and 3.0.
[0303] Generally, nomenclature used in connection with, and
techniques of, cell and tissue culture, molecular biology,
immunology, microbiology, genetics and protein and nucleic acid
chemistry and hybridization described herein are those well known
and commonly used in the art. The methods and techniques of the
present invention are generally performed according to conventional
methods well known in the art and as described in various general
and more specific references that are cited and discussed
throughout the present specification unless otherwise indicated.
See, e.g., Sambrook J. & Russell D. Molecular Cloning: A
Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (2000); Ausubel et al., Short Protocols in
Molecular Biology: A Compendium of Methods from Current Protocols
in Molecular Biology, Wiley, John & Sons, Inc. (2002); Harlow
and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et
al., Short Protocols in Protein Science, Wiley, John & Sons,
Inc. (2003). Enzymatic reactions and purification techniques are
performed according to manufacturer's specifications, as commonly
accomplished in the art or as described herein. The nomenclature
used in connection with, and the laboratory procedures and
techniques of, analytical chemistry, synthetic organic chemistry,
and medicinal and pharmaceutical chemistry described herein are
those well known and commonly used in the art.
[0304] All references or documents disclosed or cited within this
specification are, without limitation, incorporated herein by
reference in their entirety. Moreover, any section headings used
herein are for organizational purposes only and are not to be
construed as limiting the subject matter described.
EXAMPLES
[0305] The present invention, thus generally described above, will
be understood more readily by reference to the following examples,
which are provided by way of illustration and are not intended to
be limiting of the instant invention. The examples are not intended
to represent that the experiments below are all or the only
experiments performed. Unless indicated otherwise, parts are parts
by weight, molecular weight is weight average molecular weight,
temperature is in degrees Centigrade, and pressure is at or near
atmospheric.
Example 1
Enrichment of Tumor Initiating Cell Populations
[0306] To characterize the cellular heterogeneity of solid tumors
as they exist in cancer patients, elucidate the identity of tumor
perpetuating cells (TPC; i.e. cancer stem cells: CSC) using
particular phenotypic markers and identify clinically relevant
therapeutic targets, a large non-traditional xenograft (NTX) tumor
bank was developed and maintained using art recognized techniques.
The NTX tumor bank, comprising a large number of discrete tumor
cell lines, was propagated in immunocompromised mice through
multiple passages of heterogeneous tumor cells originally obtained
from numerous cancer patients afflicted by a variety of solid tumor
malignancies. The continued availability of a large number of
discrete early passage NTX tumor cell lines having well defined
lineages greatly facilitate the identification and isolation of TPC
as they allow for the reproducible and repeated characterization of
cells purified from the cell lines. More particularly, isolated or
purified TPC are most accurately defined retrospectively according
to their ability to generate phenotypically and morphologically
heterogeneous tumors in mice that recapitulate the patient tumor
sample from which the cells originated. Thus, the ability to use
small populations of isolated cells to generate fully heterogeneous
tumors in mice is strongly indicative of the fact that the isolated
cells comprise TPC. In such work the use of minimally passaged NTX
cell lines greatly simplifies in vivo experimentation and provides
readily verifiable results. Moreover, early passage NTX tumors also
respond to therapeutic agents such as irinotecan (i.e.
Camptosar.RTM.), which provides clinically relevant insights into
underlying mechanisms driving tumor growth, resistance to current
therapies and tumor recurrence.
[0307] As the NTX tumor cell lines were established the constituent
tumor cell phenotypes were analyzed using flow cytometry to
identify discrete markers that might be used to characterize,
isolate, purify or enrich tumor initiating cells (TIC) and separate
or analyze TPC and TProg cells within such populations. In this
regard the inventors employed a proprietary proteomic based
platform (i.e. PhenoPrint.TM. Array) that provided for the rapid
characterization of cells based on protein expression and the
concomitant identification of potentially useful markers. The
PhenoPrint Array is a proprietary proteomic platform comprising
hundreds of discrete binding molecules, many obtained from
commercial sources, arrayed in 96 well plates wherein each well
contains a distinct antibody in the phycoerythrin fluorescent
channel and multiple additional antibodies in different
fluorochromes arrayed in every well across the plate. This allows
for the determination of expression levels of the antigen of
interest in a subpopulation of selected tumor cells through rapid
inclusion of relevant cells or elimination of non-relevant cells
via non-phycoerythrin channels. When the PhenoPrint Array was used
in combination with tissue dissociation, transplantation and stem
cell techniques well known in the art (Al-Hajj et al., 2004,
Dalerba et al., 2007 and Dylla et al., 2008, all supra, each of
which is incorporated herein by reference in its entirety), it was
possible to effectively identify relevant markers and subsequently
isolate and transplant specific human tumor cell subpopulations
with great efficiency.
[0308] Accordingly, upon establishing various NTX tumor cell lines
as is commonly done for human tumors in severely immune compromised
mice, the tumors were resected from mice upon reaching 800-2,000
mm.sup.3 and the cells were dissociated into single cell
suspensions using art-recognized enzymatic digestion techniques
(See for example U.S.P.N. 2007/0292414 which is incorporated
herein). Data obtained from these suspensions using the PhenoPrint
Array provided both absolute (per cell) and relative (vs. other
cells in the population) surface protein expression on a
cell-by-cell basis, leading to more complex characterization and
stratification of cell populations. More specifically, use of the
PhenoPrint Array allowed for the rapid identification of proteins
or markers that prospectively distinguished TIC or TPC from NTG
bulk tumor cells and tumor stroma and, when isolated from NTX tumor
models, provided for the relatively rapid characterization of tumor
cell subpopulations expressing differing levels of specific cell
surface proteins. In particular, proteins with heterogeneous
expression across the tumor cell population allow for the isolation
and transplantation of distinct, and highly purified, tumor cell
subpopulations expressing either high and low levels of a
particular protein or marker into immune-compromised mice, thereby
facilitating the assessment of whether TPC were enriched in one
subpopulation or another.
[0309] The term enriching is used synonymously with isolating cells
and means that the yield (fraction) of cells of one type is
increased over the fraction of other types of cells as compared to
the starting or initial cell population. Preferably, enriching
refers to increasing the percentage by about 10%, by about 20%, by
about 30%, by about 40%, by about 50% or greater than 50% of one
type of cell in a population of cells as compared to the starting
population of cells.
[0310] As used herein a marker, in the context of a cell or tissue,
means any characteristic in the form of a chemical or biological
entity that is identifiably associated with, or specifically found
in or on a particular cell, cell population or tissue including
those identified in or on a tissue or cell population affected by a
disease or disorder. As manifested, markers may be morphological,
functional or biochemical in nature. In preferred embodiments the
marker is a cell surface antigen that is differentially or
preferentially expressed by specific cell types (e.g., TPC) or by
cells under certain conditions (e.g., during specific points of the
cell life cycle or cells in a particular niche). Preferably, such
markers are proteins, and more preferably, possess an epitope for
antibodies, aptamers or other binding molecules as known in the
art. However, a marker may consist of any molecule found on the
surface or within a cell including, but not limited to, proteins
(peptides and polypeptides), lipids, polysaccharides, nucleic acids
and steroids. Examples of morphological marker characteristics or
traits include, but are not limited to, shape, size, and nuclear to
cytoplasmic ratio. Examples of functional marker characteristics or
traits include, but are not limited to, the ability to adhere to
particular substrates, ability to incorporate or exclude particular
dyes, for example but not limited to exclusions of lipophilic dyes,
ability to migrate under particular conditions and the ability to
differentiate along particular lineages. Markers can also be a
protein expressed from a reporter gene, for example a reporter gene
expressed by the cell as a result of introduction of the nucleic
acid sequence encoding the reporter gene into the cell and its
transcription resulting in the production of the reporter protein
that can be used as a marker. Such reporter genes that can be used
as markers are, for example but not limited to fluorescent proteins
enzymes, chromomeric proteins, resistance genes and the like.
[0311] In a related sense the term marker phenotype in the context
of a tissue, cell or cell population (e.g., a stable TPC phenotype)
means any marker or combination of markers that may be used to
characterize, identify, separate, isolate or enrich a particular
cell or cell population (e.g., by FACS). In specific embodiments,
the marker phenotype is a cell surface phenotype that may be
determined by detecting or identifying the expression of a
combination of cell surface markers.
[0312] Those skilled in the art will recognize that numerous
markers (or their absence) have been associated with various
populations of cancer stem cells and used to isolate or
characterize tumor cell subpopulations. In this respect exemplary
cancer stem cell markers comprise OCT4, Nanog, STAT3, EPCAM, CD24,
CD34, NB84, TrkA, GD2, CD133, CD20, CD56, CD29, B7H3, CD46,
transferrin receptor, JAM3, carboxypeptidase M, ADAM9, oncostatin
M, Lgr5, Lgr6, CD324, CD325, nestin, Sox1, Bmi-1, eed, easyh1,
easyh2, mf2, yy1, smarcA3, smarckA5, smarcD3, smarcE1, mllt3, FZD1,
FZD2, FZD3, FZD4, FZD6, FZD7, FZD8, FZD9, FZD10, WNT2, WNT2B, WNT3,
WNT5A, WNT10B, WNT16, AXIN1, BCL9, MYC, (TCF4) SLC7A8, IL1RAP,
TEM8, TMPRSS4, MUC16, GPRC5B, SLC6A14, SLC4A11, PPAP2C, CAV1, CAV2,
PTPN3, EPHA1, EPHA2, SLC1A1, CX3CL1, ADORA2A, MPZL1, FLJ10052,
C4.4A, EDG3, RARRES1, TMEPAI, PTS, CEACAM6, NID2, STEAP, ABCA3,
CRIM1, IL1R1, OPN3, DAF, MUC1, MCP, CPD, NMA, ADAM9, GJA1, SLC19A2,
ABCA1, PCDH7, ADCY9, SLC39A1, NPC1, ENPP1, N33, GPNMB, LY6E,
CELSR1, LRP3, C20orf52, TMEPAI, FLVCR, PCDHA10, GPR54, TGFBR3,
SEMA4B, PCDHB2, ABCG2, CD166, AFP, BMP-4, .beta.-catenin, CD2, CD3,
CD9, CD14, CD31, CD38, CD44, CD45, CD74, CD90, CXCR4, decorin,
EGFR, CD105, CD64, CD16, CD16a, CD16b, GLI1, GLI2, CD49b, and
CD49f. See, for example, Schulenburg et al., 2010, PMID: 20185329,
U.S. Pat. No. 7,632,678 and U.S.P.Ns. 2007/0292414, 2008/0175870,
2010/0275280, 2010/0162416 and 2011/0020221 each of which is
incorporated herein by reference. It will be appreciated that a
number of these markers were included in the PhenoPrint Array
described above.
[0313] Similarly, non-limiting examples of cell surface phenotypes
associated with cancer stem cells of certain tumor types include
CD44.sup.hiCD24.sup.low, ALDH.sup.+, CD133.sup.+, CD123.sup.+,
CD34.sup.+CD38.sup.-, CD44.sup.+CD24.sup.-,
CD46.sup.hiCD324.sup.+CD66c.sup.-,
CD133.sup.+CD34.sup.+CD10.sup.-CD19.sup.-,
CD138.sup.-CD34.sup.-CD19.sup.+, CD133.sup.+RC2.sup.+,
CD44.sup.+.alpha..sub.2.beta..sub.1.sup.hiCD133.sup.+,
CD44.sup.+CD24.sup.+ESA.sup.+, CD271.sup.+, ABCB5.sup.+ as well as
other cancer stem cell surface phenotypes that are known in the
art. See, for example, Schulenburg et al., 2010, supra, Visvader et
al., 2008, PMID: 18784658 and U.S.P.N. 2008/0138313, each of which
is incorporated herein in its entirety by reference. Those skilled
in the art will appreciate that marker phenotypes such as those
exemplified immediately above may be used in conjunction with
standard flow cytometric analysis and cell sorting techniques to
characterize, isolate, purify or enrich TIC and/or TPC cells or
cell populations for further analysis. Of interest with regard to
the instant invention CD46, CD324 and, optionally, CD66c are either
highly or heterogeneously expressed on the surface of many human
colorectal ("CR"), breast ("BR"), non-small cell lung (NSCLC),
small cell lung (SCLC), pancreatic ("PA"), melanoma ("Mel"),
ovarian ("OV"), and head and neck cancer ("HN") tumor cells,
regardless of whether the tumor specimens being analyzed were
primary patient tumor specimens or patient-derived NTX tumors.
[0314] Cells with negative expression (i.e."-") are herein defined
as those cells expressing less than, or equal to, the 95.sup.th
percentile of expression observed with an isotype control antibody
in the channel of fluorescence in the presence of the complete
antibody staining cocktail labeling for other proteins of interest
in additional channels of fluorescence emission. Those skilled in
the art will appreciate that this procedure for defining negative
events is referred to as "fluorescence minus one", or "FMO",
staining. Cells with expression greater than the 95.sup.th
percentile of expression observed with an isotype control antibody
using the FMO staining procedure described above are herein defined
as "positive" (i.e."+"). As defined herein there are various
populations of cells broadly defined as "positive." First, cells
with low expression (i.e. "lo") are generally defined as those
cells with observed expression above the 95.sup.th percentile
determined using FMO staining with an isotype control antibody and
within one standard deviation of the 95.sup.th percentile of
expression observed with an isotype control antibody using the FMO
staining procedure described above. Cells with "high" expression
(i.e. "hi") may be defined as those cells with observed expression
above the 95.sup.th percentile determined using FMO staining with
an isotype control antibody and greater than one standard deviation
above the 95.sup.th percentile of expression observed with an
isotype control antibody using the FMO staining procedure described
above. In other embodiments the 99.sup.th percentile may preferably
be used as a demarcation point between negative and positive FMO
staining and in particularly preferred embodiments the percentile
may be greater than 99%.
[0315] Using techniques such as those described above to quickly
identify and rank colorectal tumor antigens based on expression
intensity and heterogeneity across several NTX tumors from
colorectal cancer patients, candidate TPC antigens were further
assessed by comparison of tumor versus normal adjacent tissue and
then selected based, at least in part, on the up- or
down-regulation of the particular antigen in malignant cells.
Moreover, systematic analysis of a variety of cell surface markers
for their ability to enrich for the ability to transplant fully
heterogeneous tumors into mice (i.e. tumorigenic ability), and
subsequent combination of these markers substantially improved the
resolution of the method and improved the ability to tailor
fluorescence activated cell sorting (FACS) techniques to identify
and characterize distinct, highly enriched tumor cell
subpopulations that exclusively contained all tumor generating
ability upon transplantation (i.e. tumor initiating cells). To
reiterate, the term tumor initiating cell (TIC) or tumorigenic (TG)
cell encompasses both Tumor Perpetuating Cells (TPC; i.e. cancer
stem cells) and highly proliferative Tumor Progenitor cells
(TProg), which together generally comprise a unique subpopulation
(i.e. 0.1-25%) of a bulk tumor or mass; the characteristics of
which are defined above. The majority of tumor cells characterized
in this fashion are devoid of this tumor forming ability, and can
thus be characterized as non-tumorigenic (NTG). Surprisingly, it
was observed that most distinct markers identified using the
proprietary PhenoPrint Array did not demonstrate an ability to
enrich tumor initiating cell populations in colorectal tumors using
standard FACS protocols, but that distinct marker combinations
could be used to identify two subpopulations of tumor initiating
cells: TPC and TProg. Those skilled in the art will recognize that
the defining difference between TPC and TProg, though both are
tumor initiating in primary transplants, is the ability of TPC to
perpetually fuel tumor growth upon serial transplantation at low
cell numbers. Furthermore, the marker/proteins used in combination
to enrich for both TPC and TProg were unknown to be associated with
cells containing such activity in any tissue or neoplasm prior to
discovery by current inventors though others have defined cell
surface markers or enzymatic activity that can similarly be used to
enrich for tumorigenic cells (Dylla et al 2008, supra). As set
forth below, specific tumor cell subpopulations isolated using cell
surface marker combinations alluded to above were then analyzed
using whole transcriptome next generation sequencing to identify
and characterize differentially expressed genes.
Example 2
Isolation and Analysis of RNA Samples from Enriched Tumor
Initiating Cell Populations
[0316] Several established colorectal NTX cell lines (SCRX-CR4,
CR11, CR33, PA3, PA6 & PA14) generated and passaged as
described in Example 1 were used to initiate tumors in immune
compromised mice. For mice bearing SCRX-CR4, PA3 or PA6 tumors,
once the mean tumor burden reached .about.300 mm.sup.3 the mice
were randomized and treated with 15 mg/kg irinotecan, 25 mg/kg
Gemcitabine, or vehicle control (PBS) twice weekly for a period of
at least twenty days prior to euthanization. Tumors arising from
all six NTX lines, including those from mice undergoing
chemotherapeutic treatment were removed and TPC, TProg and NTG
cells, respectively, were isolated from freshly resected colorectal
NTX tumors and, similarly, TG and NTG cells were isolated from
pancreatic NTX tumors, generally using the technique set out in
Example 1. More particularly, cell populations were isolated by
FACS and immediately pelleted and lysed in Qiagen RLTplus RNA lysis
buffer (Qiagen, Inc.). The lysates were then stored at -80.degree.
C. until used. Upon thawing, total RNA was extracted using the
Qiagen RNeasy isolation kit (Qiagen, Inc.) following vendor's
instructions and quantified on the Nanodrop (Thermo Scientific) and
a Bioanalyzer 2100 (Agilent Technologies) again using the vendor's
protocols and recommended instrument settings. The resulting total
RNA preparation was suitable for genetic sequencing and
analysis.
[0317] Total RNA samples obtained from the respective cell
populations isolated as described above from vehicle or
chemotherapeutic agent-treated mice were prepared for whole
transcriptome sequencing using an Applied Biosystems SOLiD 3.0
(Sequencing by Oligo Ligation/Detection) next generation sequencing
platform (Life Technologies), starting with 5 ng of total RNA per
sample. The data generated by the SOLiD platform mapped to 34,609
genes from the human genome and was able to detect ephrin-A
ligands, including EFNA1 and EFNA3, and provided verifiable
measurements of ENFA levels in most samples.
[0318] Generally the SOLiD3 next generation sequencing platform
enables parallel sequencing of clonally-amplified RNA/DNA fragments
linked to beads. Sequencing by ligation with dye-labeled
oligonucleotides is then used to generate 50 base reads of each
fragment that exists in the sample with a total of greater than 50
million reads generating a much more accurate representation of the
mRNA transcript level expression of proteins in the genome. The
SOLiD3 platform is able to capture not only expression, but SNPs,
known and unknown alternative splicing events, and potentially new
exon discoveries based solely on the read coverage (reads mapped
uniquely to genomic locations). Thus, use of this next generation
platform allowed the determination of differences in transcript
level expression as well as differences or preferences for specific
splice variants of those expressed mRNA transcripts. Moreover,
analysis with the SOLiD3 platform using a modified whole
transcriptome protocol from Applied Biosystems only required
approximately 5 ng of starting material pre-amplification. This is
significant as extraction of total RNA from sorted cell populations
where the TPC subset of cells is, for example, vastly smaller in
number than the NTG or bulk tumors and thus results in very small
quantities of usable starting material.
[0319] Duplicate runs of sequencing data from the SOLiD3 platform
were normalized and transformed and fold ratios calculated as is
standard industry practice. As seen in FIG. 2, levels of EFNA1,
EFNA3 and EFNA4 from a tumor were measured as well as levels of Eph
receptors EPHA1, EPHA2 and EPHA10. An analysis of the data showed
that EFNA1 is the most highly expressed ephrin-A ligand in SCRx-CR4
NTX tumors, with slightly elevated expression in the TPC population
(FIG. 2A). In mice being treated with 15 mg/kg irinotecan twice
weekly, EFNA1 expression was maintained in TPC whereas NTG cells
saw reduced levels (FIG. 2B). It will further be appreciated that
EFNA1 was also elevated in TPC versus TProg and NTG cells,
respectively, in NTX tumors derived from additional patients
(SCRx-CR7 and SCRx-CR11; FIG. 2C). This expression was also
independent of whether tumors had been exposed to standard of care
chemotherapeutic regimens such as FOLFIRI (i.e. 5-FU, oxaliplatin
and irinotecan). Furthermore, when pancreatic (SCRx-PA3, PA4, PA6
& PA14; FIG. 2D) and non-small cell lung tumor samples (FIG.
2E) were analyzed by SOLiD3 whole-transcriptome sequencing, EFNA1
gene expression was similarly elevated in TPC versus NTG cells, and
in non-small cell lung tumor subpopulations versus normal lung
(FIG. 2E), in most patients, as defined using CD46.sup.+CD324.sup.+
cell populations as illustrated previously. Furthermore, EFNA1 gene
expression was elevated in pancreatic tumor cell subpopulations
exposed to the standard of care chemotherapeutic agent, gemcitibine
(FIG. 2D).
[0320] Close examination of whole transcriptome gene expression
data revealed that EPHA2 receptor (with which both EFNA1 and EFNA3
ligands interact) expression inversely reflects that of both EFNA1
and EFNA3 during the progression of differentiation from TPC to NTG
cells in colorectal tumors (FIGS. 2A and 2B). This inverse
expression pattern of the EFNA1/EFNA3 ligands and EPHA2 receptor
suggests that crosstalk between these ligand/receptor pairs might
play a role in cell fate decisions during colorectal cancer stem
cell differentiation and that neutralizing these interactions might
negatively impact tumor growth. Specifically, by blocking EphA2
interactions with EFNA1 and/or EFNA3 using neutralizing modulators
as disclosed herein, TPC might be sensitized to chemotherapeutic
agents, for example, or forced to differentiate. Moreover, by
targeting TPC using EFNA1 and/or EFNA3-internalizing antibodies,
TPC might be killed directly by the naked modulator or through the
use of a toxin or antibody drug conjugate.
[0321] Analysis of whole transcriptome data, as discussed above,
also showed elevated EFNA3 expression in some colorectal,
pancreatic and non-small cell lung tumors, with elevated expression
in the TIC subpopulations of these tumors. Specifically, EFNA3
expression was elevated in TIC subpopulations (TPC and TProg)
versus NTG cells isolated from several human colorectal tumors
(SCRx-CRL1 and -CR33; FIG. 3A). This expression was independent of
whether tumors had been exposed to standard of care
chemotherapeutic regimens such as FOLFIRI (i.e. 5-FU, oxaliplatin
and irinotecan). When pancreatic (SCRx-PA3, PA4 and PA6; FIG. 3B)
and non-small cell lung tumor samples (FIG. 3C) were analyzed by
SOLiD3 whole-transcriptome sequencing, EFNA3 gene expression was
similarly elevated in TPC versus NTG cells, and in non-small cell
lung tumor subpopulations versus normal lung (FIG. 3C), in most
patients, as defined using CD46.sup.+/CD324.sup.+ cell populations
as described herein. Furthermore, EFNA3 gene expression was
maintained or increased in human pancreatic TIC subpopulations
isolated from xenograft tumor bearing mice that had been exposed to
the standard of care chemotherapeutic agent, gemcitibine (FIG.
3B).
[0322] The observations detailed above show that EFNA1 and/or EFNA3
expression is generally elevated in TPC populations and suggests
that these membrane-tethered ligands may play an important role in
tumorigenesis and tumor maintenance, thus constituting excellent
targets for novel therapeutic approaches.
Example 3
Real-Time PCR Analysis of Ephrin-A Ligands in Enriched Tumor
Initiating Cell Populations
[0323] To validate the differential ephrin-A ligand expression
observed by whole transcriptome sequencing in TPC populations
versus TProg and NTG cells in colorectal cancer, and TG versus NTG
cells in pancreatic cancer, TaqMan.RTM. quantitative real-time PCR
was used to measure gene expression levels in respective cell
populations isolated from various NTX lines as set forth above. It
will be appreciated that such real-time PCR analysis allows for a
more direct and rapid measurement of gene expression levels for
discrete targets using primers and probe sets specific to a
particular gene of interest. TaqMan.RTM. real-time quantitative PCR
was performed on an Applied Biosystems 7900HT Machine (Life
Technologies), which was used to measure EFNA1 and EFNA3 gene
expression in multiple patient-derived NTX line cell populations
and corresponding controls. Moreover, the analysis was conducted as
specified in the instructions supplied with the TaqMan System and
using commercially available EFNA1 and EFNA3 primer/probe sets
(Life Technologies).
[0324] As seen in FIGS. 4A and 4B, quantitative real-time PCR
interrogation of gene expression in NTG and TPC populations
isolated from distinct colorectal NTX tumor lines (SCRx-CR2, CR4,
& CR14) showed that EFNA1 and EFNA3 gene expression is elevated
more than 1.5-fold in the TPC subpopulations versus NTG cells.
Likewise, pancreatic NTX line (SCRx-PA14) showed that EFNA3 gene
expression was elevated 1.3-fold in the TPC subpopulation versus
the NTG cells. The observation of elevated EFNA1 and EFNA3
expression in NTX TPC cell preparations as compared with NTG cell
controls from both colorectal and pancreatic patient-derived NTX
tumors using the more widely accepted methodology of real-time
quantitative PCR confirms the more sensitive SOLiD3 whole
transcriptome sequencing data of the previous Example, and supports
the observed association between EFNA1 and EFNA3 expressing cells
underlying tumorigenesis, resistance to therapy and recurrence.
Example 4
Expression of Ephrin-A Ligands in Unfractionated Colorectal Tumor
Specimens
[0325] In light of the fact that ephrin-A ligand gene expression
was found to be elevated in TPC populations from colorectal tumors
when compared with TProg and NTG cells from the same tumors,
experiments were conducted to determine whether elevated ephrin-A
ligand (i.e., EFNA1 and EFNA3) expression was also detectable in
unfractionated colorectal tumor samples versus normal adjacent
tissue (NAT). To further assess EFNA1 and EFNA3 gene expression in
additional colorectal cancer patient tumor samples and tumor
specimens from patients diagnosed with 1 of 17 other different
solid tumor types, Taqman qRT-PCR was performed using
TissueScan.TM. qPCR (Origene Technologies) 384-well arrays, to
determine how the expression of EFNA1 and EFNA3 in tumors compares
with levels in normal tissue samples. More particularly, using the
procedures detailed in Example 3 and the same EFNA1 and EFNA3
specific primer/probe sets, TaqMan real-time quantitative PCR was
performed in the wells of the Origene plates.
[0326] FIGS. 5A and 5B show the relative gene expression levels,
respectively, of human EFNA1 (FIG. 5A) and EFNA3 (FIG. 5B) in whole
tumor specimens (grey dots) or matched normal adjacent tissue (NAT;
white dots) from patients with one of eighteen different solid
tumor types. Data is normalized against mean gene expression in NAT
for each tumor type analyzed. Specimens not amplified were assigned
a Ct value of 45, which represents the last cycle of amplification
in the experimental protocol. Each dot represents an individual
tissue specimen, with the mean value represented as a black
line.
[0327] Using the Origene Array, it was observed that the majority
of patients diagnosed with prostate, ovarian, cervical, colon,
endometrial and bladder cancer, EFNA1 is overexpressed. EFNA3 is
overexpressed in endometrial, uterine, prostate, lung, bladder,
colon, breast, cervical, kidney and stomach cancer. This data
suggests that EFNA1 and EFNA3 might play a role in tumorigenesis
and/or progression in these tumors.
Example 6
Generation of Anti-EFNA Antibodies Using EFNA Immunogens
[0328] EFNA modulators in the form of murine antibodies were
produced in accordance with the teachings herein through
inoculation with soluble immunogens hEFNA1-ECD-His
(NP.sub.--004419.2--EFNA3 precursor), and hEFNA3-ECD-Fc
(NP.sub.--004943.1). Immunogens were all prepared using
commercially available starting materials (e.g., recombinant human
Ephrin-A1/EFNA1 Sino Biological Inc #10882-H08H) and/or techniques
well known to those skilled in the art.
[0329] More particularly murine antibodies were generated by
immunizing 9 female mice (3 each: Balb/c, CD-1, FVB) with various
preparations of EFNA1 or EFNA3 antigen. Immunogens included the
aforementioned Fc or His constructs comprising at least part of the
extracellular domain of human EFNA1 and human EFNA3. Mice were
immunized via footpad route for all injections. 10 .mu.g of EFNA1
or EFNA3 immunogen emulsified with an equal volume of TITERMAX.TM.
or alum adjuvant were used for immunization. After immunization
mice were euthanized, and draining lymph nodes (popliteal and
inguinal, if enlarged) were dissected out and used as a source for
antibody producing cells. Lymphocytes were released by mechanical
disruption of the lymph nodes using a tissue grinder.
[0330] Electrofusion was then performed followed by growth of the
hybridoma library in bulk and single cell deposition of the
hybridomas with a subsequent screen of the clones. To that end a
single cell suspension of harvested B cells were fused with
non-secreting P3x63Ag8.653 myeloma cells at a ratio of 1:1.
Electrofusion was performed using the Hybrimune System, model
47-0300, (BTX.RTM. Harvard Apparatus). Fused cells were resuspended
in hybridoma selection medium supplemented with Azaserine (Sigma
#A9666) (DMEM (Cellgro cat#15-017-CM) medium containing, 15% Fetal
Clone I serum (Hyclone), 10% BM Condimed (Roche Applied Sciences),
1 mM sodium pyruvate, 4 mM L-glutamine, 100 IU
Penicillin-Streptomycin, 50 .mu.M 2-mercaptoethanol, and 100 .mu.M
hypoxanthine) and then plated in four T225 flasks at 90 ml
selection medium per flask. The flasks are then placed in a
humidified 37.degree. C. incubator containing 5% CO.sub.2 and 95%
air for 6-7 days.
[0331] At 6-7 days of growth the library is plated at 1 cell per
well in 48 Falcon 96 well U-bottom plates using the Aria I cell
sorter. Briefly culture medium containing 15% Fetal Clone I serum
(Hyclone), 10% BM-Condimed (Roche Applied Sciences), 1 mM sodium
pyruvate, 4 mM L-glutamine, 100 IU Penecillin-Streptamycin, 50
.mu.M 2-mercaptoethanol, and 100 .mu.M hypoxanthine is plated at
200 ul per well in 48 Falcon 96 well U-bottom plates. Viable
hybridomas are placed at 1 cell per well using the Aria I cell
sorter and cultured for 10-11 days and the supernatants are assayed
for antibodies reactive by FACS or ELISA for EFNA1 or EFNA3.
[0332] For the ELISA screening microtiter plates were coated with
purified recombinant EFNA1 or EFNA3 His fusion proteins from
transfected 293 cells at 100 ng/well in carbonate buffer. Plates
incubated at 4.degree. C. overnight than blocked with 200
.mu.l/well of 3% BSA in PBS/Tween (0.05%). Supernatant from
hybridoma plates were added to each well and incubated for 1-2
hours at ambient temperature. The plates were washed with PBS/Tween
and than incubated with Goat anti mouse IgG, Fc Fragment Specific
conjugated with horseradish peroxidase (HRP) Jackson
ImmunoResearch) for one hour at room temperature. After washing,
the plates were developed with TMB substrate (Thermo Scientific
34028) and analyzed by spectrophotometer at OD 450.
[0333] Selected EFNA1 and EFNA3 secreting hybridomas from positive
wells were rescreened and subcloned by limited dilution or single
cell FACS sorting.
[0334] Sub cloning was performed on selected antigen-positive wells
using limited dilution plating. Plates were visually inspected for
the presence of single colony growth and supernatants from single
colony wells then screened by antigen-specific ELISAs described
above and FACS confirmation as described below. The resulting
clonal populations were expanded and cryopreserved in freezing
medium (90% FBS, 10% DMSO) and stored in liquid nitrogen. Both the
fusions from mice immunized with EFNA1 and mice immunized with
EFNA3 yielded numerous murine monoclonal antibodies reactive for
the respective antigen as determined using the ELISA protocol
described above.
[0335] As indicated selected growth positive hybridoma wells
secreting mouse immunoglobulins were also screened for human EFNA1
or EFNA3 specificity using a FACS assay as follows. Briefly
1.times.10.sup.5 per well Jurkat cells expressing human EFNA1 or
EFNA3 were incubated for 30 minutes with 25-100 ul hybridoma
supernatant. Cells were washed PBS/2% FCS twice and then incubated
with 50 ul per sample DyeLight 649 labeled goat-anti-mouse IgG, Fc
fragment specific secondary diluted 1:200 in PBS/2% FCS. After a 15
minute incubation cells were washed 2 times with PBS/2% FCS and
re-suspended in PBS/2% FCS with DAPI and analyzed by FACS Canto II
(BD Biosciences) under standard conditions and using the HTS
attachment. The resulting specific clonal hybridomas were expanded
and cryopreserved in CS-10 freezing medium (Biolife Solutions) and
stored in liquid nitrogen. The FACS analysis confirmed that
purified antibody from most or all of these hybridomas bind EFNA1
or EFNA3 in a concentration-dependent manner.
Example 7
Sequencing of Ephrin-A Ligand Modulators
[0336] Based on the foregoing, a number of exemplary distinct
monoclonal antibodies that bind immobilized human EFNA1 or EFNA3
with apparently high affinity were selected. As shown in FIGS. 6
and 7 sequence analysis of the DNA encoding mAbs from Example 6
confirmed that many had unique VDJ rearrangements and displayed
novel complementarity determining regions.
[0337] For initiation of sequencing TRIZOL reagent was purchased
from Invitrogen (Life Technologies). One step RT PCR kit and
QIAquick PCR Purification Kit were purchased from Qiagen, Inc. with
RNasin were from Promega. Custom oligonucleotides were purchased
from Integrated DNA Technologies.
[0338] Hybridoma cells were lysed in TRIZOL reagent for RNA
preparation. Between 10.sup.4 .mu.L and 10.sup.5 cells were
resuspended in 1 ml TRIZOL. Tubes were shaken vigorously after
addition of 200 .mu.l of chloroform. Samples were centrifuged at
4.degree. C. for 10 minutes. The aqueous phase was transferred to a
fresh microfuge tube and an equal volume of isopropanol was added.
Tubes were shaken vigorously and allowed to incubate at room
temperature for 10 minutes. Samples were then centrifuged at
4.degree. C. for 10 minutes. The pellets were washed once with 1 ml
of 70% ethanol and dried briefly at room temperature. The RNA
pellets were resuspended with 40 .mu.L of DEPC-treated water. The
quality of the RNA preparations was determined by fractionating 3
.mu.L in a 1% agarose gel. The RNA was stored in a -80.degree. C.
freezer until used.
[0339] The variable DNA sequences of the hybridoma amplified with
consensus primer sets specific for murine immunoglobulin heavy
chains and kappa light chains were obtained using a mix of variable
domain primers. One step RT-PCR kit was used to amplify the V.sub.H
and V.sub.K gene segments from each RNA sample. The Qiagen One-Step
RT-PCR Kit provides a blend of Sensiscript and Omniscript Reverse
Transcriptases, HotStarTaq DNA Polymerase, Qiagen OneStep RT-PCR
Buffer, a dNTP mix, and Q-Solution, a novel additive that enables
efficient amplification of "difficult" (e.g., GC-rich)
templates.
[0340] Reaction mixtures were prepared that included 3 .mu.L of
RNA, 0.5 of 100 .mu.M of either heavy chain or kappa light chain
primers 5 .mu.L, of 5.times.RT-PCR buffer, 1 .mu.L dNTPs, 1 .mu.L
of enzyme mix containing reverse transcriptase and DNA polymerase,
and 0.4 .mu.L of ribonuclease inhibitor RNasin (1 unit). The
reaction mixture contains all of the reagents required for both
reverse transcription and PCR. The thermal cycler program was RT
step 50.degree. C. for 30 minutes 95.degree. C. for 15 minutes
followed by 30 cycles of (95.degree. C. for 30 seconds, 48.degree.
C. for 30 seconds, 72.degree. C. for 1.0 minutes). There was then a
final incubation at 72.degree. C. for 10 minutes.
[0341] To prepare the PCR products for direct DNA sequencing, they
were purified using the QIAquick.TM. PCR Purification Kit according
to the manufacturer's protocol. The DNA was eluted from the spin
column using 50 .mu.L of sterile water and then sequenced directly
from both strands. PCR fragments were sequenced directly and DNA
sequences were analyzed using VBASE2 (Retter et al., Nucleic Acid
Res. 33; 671-674, 2005--data not shown).
[0342] As briefly alluded to above, the nucleic acid and
corresponding amino acid sequences of murine heavy and light chain
variable regions comprising exemplary modulators of the instant
invention are set forth in FIGS. 6 and 7. More specifically FIGS.
6A-6J provide the variable region sequences (SEQ ID NOS: 6-45) of
exemplary antibodies that react with EFNA1 while FIGS. 7A-7N
provide the variable region sequences (SEQ ID NOS: 46-101) of
exemplary antibodies that react with EFNA3. Note that for the
purposes of the instant disclosure antibody modulators that
primarily react with EFNA1 are designated SC9.xx while antibody
modulators that primarily react with EFNA3 are designated SC11.xx
where xx refers to the particular clone number.
Example 8
Characteristics of EFNA Modulators
[0343] Various methods were used to analyze the immunochemical
characteristics of selected EFNA1 and EFNA3 modulators generated as
set forth above. Specifically, a number of these antibodies were
characterized as to affinity, kinetics, binning, and cross
reactivity with regard to cynomolgus and mouse homologs (e.g., by
ForteBio). The reactivity of the modulators was also measured by
Western blot using reduced and non-reduced samples to provide some
indication as to whether the epitopes were linear or not. In
addition, the antibodies were tested for their ability to
neutralize (e.g., block receptor ligand interaction as per Example
11), and were benchmarked for their relative EC.sub.50 of killing
by in vitro cytotoxicity assay (e.g., as per Examples 12-14). The
results of this characterization are set forth in tabular form in
FIG. 8 for modulators that primarily react with EFNA1 and in FIG. 9
for modulators that primarily react with EFNA3. Affinities and
kinetic constants k.sub.on and k.sub.off of the selected modulators
were measured using bio-layer interferometry analysis on a ForteBio
RED (ForteBio, Inc.) with a standard antigen concentration series.
In general, the selected modulators exhibited relatively high
affinities in the nanomolar range.
[0344] As to antibody binning, ForteBio was used per manufacturer's
instructions to identify antibodies, which bound to the same or
different bins. Briefly, an antibody (Ab1) was captured onto an
anti-mouse capture chip, a high concentration of nonbinding
antibody was then used to block the chip and a baseline was
collected. Monomeric, recombinant ephrin-A1-His or ephrinA3-His was
then captured by the specific antibody (Ab1) and the tip was dipped
into a well with either the same antibody (Ab1) as a control or
into a well with a different antibody (Ab2). If additional binding
was observed with a new antibody, then Ab1 and Ab2 were determined
to be in a different bin. If no further binding occurred, similar
to the control Ab1, then Ab2 was determined to be in the same bin.
This process can be expanded to screen large libraries of unique
antibodies using a full row of antibodies representing unique bins
in a 96-well plate. This experiment showed the screened antibodies
bound to at least three different bins or epitopes on the ephrin-A1
and ephrin-A3 proteins, respectively. This number of bins is
consistent with an antigen less than 30 kDa.
[0345] In order to determine whether the epitope recognized by the
ephrin-A1 and ephrin-A3 modulator comprises contiguous amino acids
or is formed by noncontiguous amino acids juxtaposed by secondary
structure of the antigen, reduced and alkylated ELISA were run.
More particularly, using 0.5M DTT to reduce recombinantly expressed
protein and 0.25M iodoacetamide techniques well known in the art,
ephrin-A1 and ephrin-A3 antigen was then used to coat an ELISA
plate under alkali conditions. The respective modulators were then
exposed to the plate, washed and developed with an anti-mouse IgG
antibody conjugated to a developing agent. As detailed in FIG. 8
and FIG. 9, many ephrin-A1 and ephrin-A3 modulators substantially
reacted with both denatured and reduced protein. The antibodies
that were identified by binding to reduced and alkylated antigen on
ELISA were used in western blot to test for target expression in
cancer and normal tissues following verification on naive and
overexpressing cell lines.
[0346] Finally, cross-reactivity with regard to cynomolgus
ephrin-A1 homologs were evaluated in ForteBio using a concentration
series with recombinantly expressed, monomeric ephrin-A1 antigens.
This analysis was not completed for ephrin-A3 because the mature
protein found in cynomolgus monkey is identical in sequence and so
reactivity between human and cynomolgus protein would be identical.
As listed in FIG. 8 the selected modulators were reactive with any
number of the homologs. In particular, SC9.105 was cross-reactive
with mouse EFNA1, while all antibodies cross-reacted with the
highly similar cynomolgus EFNA1. ND in the tables indicates that
the data was not determined.
Example 9
Ephrin-A Ligand Modulators Demonstrate Cell Surface Binding
[0347] Supernatants from hybridomas producing antibodies raised
against EFNA1 or EFNA3 as set forth above were screened for cell
surface binding as measured in a flow cytometric assay. To
demonstrate the binding properties of selected modulators three
tumor cell lines known to express moderate levels of EFNA1
(HEK293Td, Z138, PC3) and a control (G401) were analyzed by FACS
using the EFNA1 antibody SC9.121. More specifically, fifty thousand
cells of each type were incubated with 10 .mu.g/ml purified
modulator for 60 minutes at 4.degree. C. The cells were washed once
with PBS containing 2% FBS, 2 mM EDTA and 0.05% sodium azide (wash
buffer) and then stained for 60 minutes at 4.degree. C. in the dark
with a Fc-region specific F(ab)2 fragment of Goat-anti-mouse IgG
polyclonal antibody conjugated to DyLight649 (Jackson Immuno
Research). Cells were washed twice with wash buffer, and
counterstained with 2 .mu.g/ml DAPI. Samples were collected on a
FACS Canto II (BD Biosciences) under standard conditions and using
the HTS attachment. FIG. 10A shows histograms of single live cells
stained with modulator SC9.121 (open histograms) or isotype control
antibody (shaded histograms) demonstrating moderate EFNA1
expression by HEK293Td, Z138 and PC3 cell lines.
[0348] Using a similar protocol and the same apparatus assays were
run to demonstrate that exemplary modulators which associate with
EFNA1 or EFNA3 bind to cells expressing the respective ligand. In
this case HEK293T cells were engineered by means of retroviral
transduction to express markedly higher levels of EFNA1 or EFNA3
than is expressed endogenously by the wild type parent. Binding of
the selected EFNA1 and EFNA3 modulators is shown, respectively, in
FIG. 10B and FIG. 10C wherein IgG2a is used as a negative control
and the measurements are depicted as mean fluorescence intensity
(MFI) of the chosen fluorescent channel. The graphs illustrate that
the modulators of the invention readily associate with ligands
expressed on the surface of cells.
Example 10
Ephrin-A Ligands Interact Selectively with Multiple EphA
Receptors
[0349] As discussed herein ephrin-A ligands are characterized as
promiscuous as they are known to bind to various EphA receptors. To
explore which EphA receptors have the potential to interact with
EFNA1 and EFNA3, a flow cytometric binding assay similar to the one
described in Example 9 was developed. More particularly soluble
EphA receptors expressed as human IgG1 Fc fusion constructs (10
.mu.g/ml; obtained commercially or generated in-house) were added
to fifty thousand HEK293T cells per well (FIG. 11A) or HEK293T
cells overexpressing EFNA1 (FIG. 11B) or EFNA3 (FIG. 11C) by means
of retroviral transduction for 1 hour in staining buffer at
4.degree. C. After washing, a secondary anti-human IgG polyclonal
antibody conjugated to DyeLight 649 (Jackson Immuno Research) was
added for one hour. After washing twice, samples were resuspended
in staining buffer containing 2 .mu.g/ml DAPI and analyzed on a
FACS Canto II (BD Biosciences) under standard conditions using the
HTS attachment. FIG. 11A demonstrates that EphA1, EphA3, EphA4,
EphA6, EphA7 and EphA10, but not EphA2, apparently bind to the low
levels of endogenously expressed ephrin-A ligands on parental
HEK293T cells. Engineering the cells to express elevated levels of
either EFNA1 (FIG. 11B) or EFNA3 (FIG. 11C) resulted in the
substantial binding of all tested EphA receptors in a dose
dependent manner albeit to varying degrees. These multiple
interactions again point to the advantages and potential
multifaceted points of action inherent in modulators of the instant
invention.
Example 11
EFNA Modulators Block Binding of EFNA to EphA Receptors
[0350] As seen in Example 10 most EphA receptors associate to some
degree with EFNA1 and EFNA3 ligands expressed on cell surfaces.
This binding can be inhibited using the ephrin-A modulators of the
instant invention and, in particular, through the use of monoclonal
antibodies that associate with EFNA1 or EFNA3. To illustrate this
aspect of the invention fifty thousand HEK293T cells overexpressing
EFNA1 or EFNA3 were deposited per well and incubated with 20
.mu.g/ml of the exemplary modulator in wash buffer for 1 hr at
4.degree. C. Mouse IgG isotypes and no antibody (data not shown)
serve as negative controls. Various EphA-Fc constructs (EphA1,
EphA2, EphA4 and EphA7 in the case of EFNA1 and EphA2, EphA4, EphA7
and EphA10 in the case of EFNA3) were then added to the cells at 10
.mu.g/ml in wash buffer for an additional 1 hr at 4.degree. C. The
cells were then washed twice with wash buffer, counterstained with
2 .mu.g/ml DAPI, and analyzed on a FACS Canto II (BD Biosciences)
under standard conditions using the HTS attachment. Results are
presented as mean fluorescence intensity (MFI) of the chosen
fluorescent channel.
[0351] The results graphically represented in FIG. 12A depict
blocking of EphA receptor binding to EFNA1 ligand by EFNA1
modulators, and those in FIG. 12B depict blocking of EphA receptor
binding to EFNA3 ligand. A review of FIG. 12A shows that modulators
SC9.20, SC9.92, SC9.98, SC9.120, SC9.140 and SC9.141 substantially
inhibit the binding of all or some of the tested EphA 1, EphA2,
EphA4 and EphA7 receptors to EFNA 1 whereas modulators SC9.52,
SC9.66, SC9.96, SC9.116, SC9.121 and SC9.122 exhibit relatively
less inhibition. Some of these modulators selectively enhance
binding of EphA4 and EphA7-Fc. A review of FIG. 12B shows that
modulators SC11.18, SC11.27, SC11.30, SC11.32 and SC11.34
substantially inhibit the binding of all or some of the tested
EphA2, EphA4, EphA7 and EphA10 receptors to EFNA3 whereas
modulators SC11.9, SC11.19, SC11.37, SC11.47, SC11.51, SC11.53,
SC11.54, SC11.55, SC11.57, SC11.67 and SC11.112 exhibit relatively
less inhibition. Again some of these modulators selectively enhance
binding of EphA7 and EphA10-Fc. These data, when combined with the
results of the other Examples herein, suggest that these
modulators' ability to agonize or antagonize the binding of various
receptors may be significant in providing the observed therapeutic
effects of the instant invention.
Example 12
EFNA1 Modulators as Targeting Moieties
[0352] Targeting of a cytotoxic drug stably linked to an antibody
represents an approach that might have great therapeutic benefit
for patients with solid tumors. To determine whether the
EFNA1-specific antibodies described above were able to mediate the
delivery of a cytotoxic agent to live cells, an in vitro cell
killing assay was performed wherein an Anti-Mouse IgG Fab fragment
conjugated to the ribosome-inactivating protein Saporin (referred
to as FAB-ZAP.TM.) was added together with purified EFNA1
antibodies to target cells, and the ability of these Saporin
complexes to internalize and kill cells was measured 72 hours later
by measuring cell viability.
[0353] Specifically, 500 cells per well of the following cell types
were plated into 96 well tissue culture plates in their respective
culture media one day before the addition of antibodies and toxin
conjugate: parental HEK293T cells (FIG. 13A), and HEK293T
overexpressing EFNA1 cells (FIG. 13B). Purified mouse monoclonal
antibodies at various concentrations and a fixed concentration of
10 nM Anti-Mouse IgG Fab fragment covalently linked to Saporin
(Advanced Targeting Systems, #IT-48) were added to the cultures and
allowed to incubate for 72 hours. Viable cell numbers were
enumerated using CellTiter-Glo.RTM. (Promega Corp.) per the
manufacturer's protocol. Raw luminescence counts using cultures
containing cells with the Saporin Fab fragment (but no modulator)
were set as 100% reference values and all other counts calculated
accordingly (referred to as "Normalized RLU").
[0354] Using this assay it was demonstrated that, except for
SC9.105, all tested EFNA1 antibodies (but not isotype control
antibodies) are able to effectively mediate the killing of the
engineered target cells (FIG. 13B). This assay demonstrates that
internalization occurs upon binding of the EFNA1 antibody to the
cell surface without the need for additional cross-linking and that
cells expressing certain levels of EFNA are killed by EFNA
modulator mediated cytotoxicity. In this case parental HEK293T
cells expressing a low number of EFNA1 on their cell surface were
not killed while engineered HEK293T cells expressing the ligand
strongly (e.g., as per Example 9) were terminated in a modulator
dose dependent manner. These data clearly demonstrate the
effectiveness of the disclosed modulators when acting as vectors
for the selective internalization of cytotoxic payloads in cells
expressing EFNA 1.
Example 13
EFNA3 Modulators as Targeting Moieties
[0355] Using the protocol essentially as set forth in Example 12
the ability of EFNA3 modulators to mediate the killing of cells in
accordance with the present invention was demonstrated. In this
regard parental HEK293T cells (FIG. 14A) and engineered HEK293T
cells expressing EFNA3 (FIG. 14B) were deposited at approximately
500 cells per well in appropriate culture media. Again, purified
modulators comprising murine monoclonal antibodies at various
concentrations and a fixed concentration of 10 nM Anti-Mouse IgG
Fab fragment covalently linked to Saporin were added to the wells
and incubated in culture for 72 hours. Again viable cell numbers
were enumerated using CellTiter-Glo per the manufacturer's protocol
and reference RLU was established using wells comprising a
non-reactive control antibody.
[0356] This assay again demonstrated that the disclosed modulators,
in this case EFNA3 modulators, could effectively be used to mediate
cytotoxic induced killing of cells expressing certain levels of
ephrin-A ligand (FIG. 14B). Such findings support the observation
that, in accordance with the instant invention, EFNA modulators may
be used to effectively deliver toxic payloads into the cell through
association with cell surface ephrin-A ligand.
Example 14
EFNA1 Modulators as Targeting Moieties for Cancer Stem Cells
[0357] Based on the unexpected results detailed above an assay was
devised to confirm the findings obtained with engineered cells and
demonstrate that the disclosed modulators can effectively mediate
delivery of cytotoxic agents to tumor initiating cells expressing
ephrin-A ligand. More particularly the instant Example demonstrates
that the disclosed modulators may be used to promote toxin
internalization and cell killing of murine lineage-depleted NTX
cells (i.e. human tumor initiating cells propagated as low-passage
xenografts in immunocompromised mice).
[0358] In this respect NTX tumors, representing lung and ovarian
tumor specimens, were dissociated into a single cell suspension and
plated, at 2,500 cells per well, on BD Primaria.TM. plates (BD
Biosciences) in growth factor supplemented serum free media. LU50
(FIG. 15A) was initially isolated from a 76 year old male non-small
cell lung cancer patient and was passaged in mice three times prior
to in vitro culture as discussed in Example 1 above. OV26 (FIG.
15B) was derived from a 69 year old female ovarian cancer patient
and was similarly passaged twice in mice prior to in vitro culture.
After 3-5 days of culture at 37.degree. C./5% CO.sub.2/5% O.sub.2,
cells were contacted with a control (a non-reactive IgG1a or a
murine EFNA1 modulator (SC9.7, SC9.66, or SC9.96 at 100 pM or 10
pM) and Fab-ZAP (at 4 nM) as per the previous Examples.
Modulator-mediated saporin cytotoxicity was then assessed by
quantifying the remaining number of cells using CellTiter-Glo 5-7
days later.
[0359] As seen in FIGS. 15A and 15B exposure to EFNA1 modulators,
particularly at the 100 pM concentration, resulted in reduced LU50
and OV26 cell numbers indicating that the cytotoxic payload had
been internalized via EFNA1 binding and eliminated cancer stem
cells. In contrast, the IgG2a isotype control antibody did not
substantially impact the number of live cells after treatment.
These data clearly demonstrate that the disclosed modulators
effectively bind to ephrin-A ligands expressed on the surface of
cancer stem cell populations and can facilitate the delivery of a
cytotoxic payload (e.g., via internalization) resulting in
tumorigenic cell death (i.e., a reduction in tumor cell
frequency).
[0360] Those skilled in the art will further appreciate that the
present invention may be embodied in other specific forms without
departing from the spirit or central attributes thereof. In that
the foregoing description of the present invention discloses only
exemplary embodiments thereof, it is to be understood that other
variations are contemplated as being within the scope of the
present invention. Accordingly, the present invention is not
limited to the particular embodiments that have been described in
detail herein. Rather, reference should be made to the appended
claims as indicative of the scope and content of the invention.
Sequence CWU 1
1
10211590DNAHomo sapiens 1gccagatctg tgagcccagc gctgactgcg
ccgcggagaa agccagtggg aacccagacc 60cataggagac ccgcgtcccc gctcggcctg
gccaggcccc gcgctatgga gttcctctgg 120gcccctctct tgggtctgtg
ctgcagtctg gccgctgctg atcgccacac cgtcttctgg 180aacagttcaa
atcccaagtt ccggaatgag gactacacca tacatgtgca gctgaatgac
240tacgtggaca tcatctgtcc gcactatgaa gatcactctg tggcagacgc
tgccatggag 300cagtacatac tgtacctggt ggagcatgag gagtaccagc
tgtgccagcc ccagtccaag 360gaccaagtcc gctggcagtg caaccggccc
agtgccaagc atggcccgga gaagctgtct 420gagaagttcc agcgcttcac
acctttcacc ctgggcaagg agttcaaaga aggacacagc 480tactactaca
tctccaaacc catccaccag catgaagacc gctgcttgag gttgaaggtg
540actgtcagtg gcaaaatcac tcacagtcct caggcccatg acaatccaca
ggagaagaga 600cttgcagcag atgacccaga ggtgcgggtt ctacatagca
tcggtcacag tgctgcccca 660cgcctcttcc cacttgcctg gactgtgctg
ctccttccac ttctgctgct gcaaaccccg 720tgaaggtgta tgccacacct
ggccttaaag agggacaggc tgaagagagg gacaggcact 780ccaaacctgt
cttggggcca ctttcagagc ccccagccct gggaaccact cccaccacag
840gcataagcta tcacctagca gcctcaaaac gggtcagtat taaggttttc
aaccggaagg 900aggccaacca gcccgacagt gccatcccca ccttcacctc
ggagggatgg agaaagaagt 960ggagacagtc ctttcccacc attcctgcct
ttaagccaaa gaaacaagct gtgcaggcat 1020ggtcccttaa ggcacagtgg
gagctgagct ggaaggggcc acgtggatgg gcaaagcttg 1080tcaaagatgc
cccctccagg agagagccag gatgcccaga tgaactgact gaaggaaaag
1140caagaaacag tttcttgctt ggaagccagg tacaggagag gcagcatgct
tgggctgacc 1200cagcatctcc cagcaagacc tcatctgtgg agctgccaca
gagaagtttg tagccaggta 1260ctgcattctc tcccatcctg gggcagcact
ccccagagct gtgccagcag gggggctgtg 1320ccaacctgtt cttagagtgt
agctgtaagg gcagtgccca tgtgtacatt ctgcctagag 1380tgtagcctaa
agggcagggc ccacgtgtat agtatctgta tataagttgc tgtgtgtctg
1440tcctgatttc tacaactgga gtttttttat acaatgttct ttgtctcaaa
ataaagcaat 1500gtgttttttc ggacatgctt ttctgccact ccatattaaa
acatatgacc attgagtccc 1560tgctaaaaaa aaaaaaaaaa aaaaaaaaaa
15902219PRTHomo sapiens 2Met Glu Phe Leu Trp Ala Pro Leu Leu Gly
Leu Cys Cys Ser Leu Ala 1 5 10 15 Ala Ala Asp Arg His Thr Val Phe
Trp Asn Ser Ser Asn Pro Lys Phe 20 25 30 Arg Asn Glu Asp Tyr Thr
Ile His Val Gln Leu Asn Asp Tyr Val Asp 35 40 45 Ile Ile Cys Pro
His Tyr Glu Asp His Ser Val Ala Val Gln Leu Asn 50 55 60 Asp Tyr
Val Asp Ile Ile Cys Pro His Tyr Glu Asp His Ser Val Ala 65 70 75 80
Gln Pro Gln Ser Lys Asp Gln Val Arg Trp Gln Cys Asn Arg Pro Ser 85
90 95 Ala Lys His Gly Pro Glu Lys Leu Ser Glu Lys Phe Gln Arg Phe
Thr 100 105 110 Pro Phe Thr Leu Gly Lys Glu Phe Lys Glu Gly His Ser
Tyr Tyr Tyr 115 120 125 Ile Ser Lys Pro Ile His Gln His Glu Asp Arg
Cys Leu Arg Leu Lys 130 135 140 Val Thr Val Ser Gly Lys Ile Thr His
Pro Gln Ala His Asp Asn Pro 145 150 155 160 Gln Glu Lys Arg Leu Ala
Ala Asp Asp Pro Glu Val Arg Val Leu His 165 170 175 Ser Ile Gly His
Ser Ala Ala Pro Arg Leu Phe Pro Leu Ala Trp Thr 180 185 190 Val Leu
Leu Leu Pro Leu Leu His Ser Ala Ala Pro Arg Leu Phe Pro 195 200 205
Leu Ala Trp Thr Val Leu Leu Leu Pro Leu Leu 210 215 3183PRTHomo
sapiens 3Met Glu Phe Leu Trp Ala Pro Leu Leu Gly Leu Cys Cys Ser
Leu Ala 1 5 10 15 Ala Ala Asp Arg His Thr Val Phe Trp Asn Ser Ser
Asn Pro Lys Phe 20 25 30 Arg Asn Glu Asp Tyr Thr Ile His Val Gln
Leu Asn Asp Tyr Val Asp 35 40 45 Ile Ile Cys Pro His Tyr Glu Asp
His Ser Val Ala Asp Ala Ala Met 50 55 60 Glu Gln Tyr Ile Leu Tyr
Leu Val Glu His Glu Glu Tyr Gln Leu Cys 65 70 75 80 Gln Pro Gln Ser
Lys Asp Gln Val Arg Trp Gln Cys Asn Arg Pro Ser 85 90 95 Ala Lys
His Gly Pro Glu Lys Leu Ser Glu Lys Phe Gln Arg Phe Thr 100 105 110
Pro Phe Thr Leu Gly Lys Glu Phe Lys Glu Gly His Ser Tyr Tyr Tyr 115
120 125 Ile Ser His Ser Pro Gln Ala His Asp Asn Pro Gln Glu Lys Arg
Leu 130 135 140 Ala Ala Asp Asp Pro Glu Val Arg Val Leu His Ser Ile
Gly His Ser 145 150 155 160 Ala Ala Pro Arg Leu Phe Pro Leu Ala Trp
Thr Val Leu Leu Leu Pro 165 170 175 Leu Leu Leu Leu Gln Thr Pro 180
4238PRTHomo sapiens 4Met Ala Ala Ala Pro Leu Leu Leu Leu Leu Leu
Leu Val Pro Val Pro 1 5 10 15 Leu Leu Pro Leu Leu Ala Gln Gly Pro
Gly Gly Ala Leu Gly Asn Arg 20 25 30 His Ala Val Tyr Trp Asn Ser
Ser Asn Gln His Leu Arg Arg Glu Gly 35 40 45 Tyr Thr Val Gln Val
Asn Val Asn Asp Tyr Leu Asp Ile Tyr Cys Pro 50 55 60 His Tyr Asn
Ser Ser Gly Val Gly Pro Gly Ala Gly Pro Gly Pro Gly 65 70 75 80 Gly
Gly Ala Glu Gln Tyr Val Leu Tyr Met Val Ser Arg Asn Gly Tyr 85 90
95 Arg Thr Cys Asn Ala Ser Gln Gly Phe Lys Arg Trp Glu Cys Asn Arg
100 105 110 Pro His Ala Pro His Ser Pro Ile Lys Phe Ser Glu Lys Phe
Gln Arg 115 120 125 Tyr Ser Ala Phe Ser Leu Gly Tyr Glu Phe His Ala
Gly His Glu Tyr 130 135 140 Tyr Tyr Ile Ser Thr Pro Thr His Asn Leu
His Trp Lys Cys Leu Arg 145 150 155 160 Met Lys Val Phe Val Cys Cys
Ala Ser Thr Ser His Ser Gly Glu Lys 165 170 175 Pro Val Pro Thr Leu
Pro Gln Phe Thr Met Gly Pro Asn Val Lys Ile 180 185 190 Asn Val Leu
Glu Asp Phe Glu Gly Glu Asn Pro Gln Val Pro Lys Leu 195 200 205 Glu
Lys Ser Ile Ser Gly Thr Ser Pro Lys Arg Glu His Leu Pro Leu 210 215
220 Ala Val Gly Ile Ala Phe Phe Leu Met Thr Phe Leu Ala Ser 225 230
235 51782DNAHomo sapiens 5ggagctggga agcggagaag ccgggagcgc
ggggctcagt cggggggcgg cggcggcggc 60ggctccgggg atggcggcgg ctccgctgct
gctgctgctg ctgctcgtgc ccgtgccgct 120gctgccgctg ctggcccaag
ggcccggagg ggcgctggga aaccggcatg cggtgtactg 180gaacagctcc
aaccagcacc tgcggcgaga gggctacacc gtgcaggtga acgtgaacga
240ctatctggat atttactgcc cgcactacaa cagctcgggg gtgggccccg
gggcgggacc 300ggggcccgga ggcggggcag agcagtacgt gctgtacatg
gtgagccgca acggctaccg 360cacctgcaac gccagccagg gcttcaagcg
ctgggagtgc aaccggccgc acgccccgca 420cagccccatc aagttctcgg
agaagttcca gcgctacagc gccttctctc tgggctacga 480gttccacgcc
ggccacgagt actactacat ctccacgccc actcacaacc tgcactggaa
540gtgtctgagg atgaaggtgt tcgtctgctg cgcctccaca tcgcactccg
gggagaagcc 600ggtccccact ctcccccagt tcaccatggg ccccaatgtg
aagatcaacg tgctggaaga 660ctttgaggga gagaaccctc aggtgcccaa
gcttgagaag agcatcagcg ggaccagccc 720caaacgggaa cacctgcccc
tggccgtggg catcgccttc ttcctcatga cgttcttggc 780ctcctagctc
tgccccctcc cctggggggg gagagatggg gcggggcttg gaaggagcag
840ggagcctttg gcctctccaa gggaagccta gtgggcctag acccctcctc
ccatggctag 900aagtggggcc tgcaccatac atctgtgtcc gccccctcta
ccccttcccc ccacgtaggg 960cactgtagtg gaccaagcac ggggacagcc
atgggtcccg ggcggccttg tggctctggt 1020aatgtttggt accaaacttg
ggggccaaaa agggcagtgc tcaggactcc ctggcccctg 1080gtacctttcc
ctgactcctg gtgccctctc cctttgtccc cccagagaga catatgcccc
1140cagagagagc aaatcgaagc gtgggaggca cccccattgc tctcctccag
gggcagaaca 1200tggggagggg actagatggg caaggggcag cactgcctgc
tgcttccttc ccctgtttac 1260agcaataagc acgtcctcct cccccactcc
cacttccagg attgtggttt ggattgaaac 1320caagtttaca agtagacacc
cctggggggg cgggcagtgg acaaggatgg caaggggtgg 1380gcattggggt
gccaggcagg catgtacaga ctctatatct ctatatataa tgtacagaca
1440gacagagtcc cttccctctt taaccccctg acctttcttg acttcccctt
cagcttcaga 1500ccccttcccc accaggctag gccccccaca cctgggggac
cccctggccc ctcttttgtc 1560ttctgtgaag acaggaccta tgcaacgcac
agacactttt ggagaccgta aaacaacaac 1620gccccctccc ttccagccct
gagccgggaa ccatctccca ggaccttgcc ctgctcaccc 1680tatgtggtcc
cacctatcct cctgggcctt tttcaagtgc tttggctgtg actttcatac
1740tctgctctta gtctaaaaaa aataaactgg agataaaaat aa 17826351DNAMus
sp. 6gaggtccagc tgcaacagtt tggagctgag ctggtgaagc ctggggcttc
agtaaagata 60tcctgcaagg cttctggcta cgcattcact gactacaaca tagactgggt
gaaacagagc 120catggaagga gccttgagtg gattggagat attaatccta
attatgaaag tactcgctac 180aaccggaagt tcatgggaaa ggccacattg
actgtagaca agtcctccaa cacagcctac 240atggatctcc gcagcctgac
atctgaggac actgcagtct attactgtac aagagatggt 300tcctatgcta
tggactactg gggtcaagga acctcagtca ccgtctcctc a 3517117PRTMus sp.
7Glu Val Gln Leu Gln Gln Phe Gly Ala Glu Leu Val Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asp
Tyr 20 25 30 Asn Ile Asp Trp Val Lys Gln Ser His Gly Arg Ser Leu
Glu Trp Ile 35 40 45 Gly Asp Ile Asn Pro Asn Tyr Glu Ser Thr Arg
Tyr Asn Arg Lys Phe 50 55 60 Met Gly Lys Ala Thr Leu Thr Val Asp
Lys Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Asp Leu Arg Ser Leu Thr
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Gly Ser
Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val
Ser Ser 115 8340DNAMus sp. 8gacattgtga tgacacagtc tccatcctcc
ctgagtgtgt cagcaggaga gaaggtcact 60ctgagctgca agtccagtca gagtctgtta
aacagtggac atcaaaagaa ctacttggcc 120tggtaccagc agaaaccagg
gcagcctcct aaactgttga tctacggggc atccactagg 180gaatctgggg
tccctgatcg cttcacaggc agtggatctg gaaccgattt cactcttacc
240atcagcagtg tgcaggctga agacctggca gtttattact gtcagaatga
tcataggtat 300cctctcacgt tcggtgctgg gaccaagctg gagctgaaac
3409113PRTMus sp. 9Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser
Val Ser Ala Gly 1 5 10 15 Glu Lys Val Thr Leu Ser Cys Lys Ser Ser
Gln Ser Leu Leu Asn Ser 20 25 30 Gly His Gln Lys Asn Tyr Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile
Tyr Gly Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe
Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser
Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn 85 90 95
Asp His Arg Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100
105 110 Lys 10360DNAMus sp. 10gaggtccagc tgcagcagtc tggacctgaa
ctagtgaaga ctggggcttc agtgaagata 60tcctgcaagg cttctggtta ctcattcagt
ggttactaca tgcactgggt caagcagagc 120cgaggaaaga gccttgagtg
gattggatat attcgttctt acaatggtgc tactagctac 180aaccagaagt
tcaagggcaa ggccaccttt actgtagaca catcctccag cacagcctac
240atgcagttca acagcctgac atctgaagac tctgcggtct atttctgtgc
aagagagggg 300aattactacg gtagtagcct tgacttctgg ggccaaggca
ccactctcac agtctcctca 36011120PRTMus sp. 11Glu Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Lys Thr Gly Ala 1 5 10 15 Ser Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Ser Phe Ser Gly Tyr 20 25 30 Tyr Met
His Trp Val Lys Gln Ser Arg Gly Lys Ser Leu Glu Trp Ile 35 40 45
Gly Tyr Ile Arg Ser Tyr Asn Gly Ala Thr Ser Tyr Asn Gln Lys Phe 50
55 60 Lys Gly Lys Ala Thr Phe Thr Val Asp Thr Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met Gln Phe Asn Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Phe Cys 85 90 95 Ala Arg Glu Gly Asn Tyr Tyr Gly Ser Ser Leu
Asp Phe Trp Gly Gln 100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115
120 12322DNAMus sp. 12gatgtccaga taacccagtc tccatcttat cttgctgcat
ctcctggaga aatcattact 60attaattgca gggcaagtaa gagcattagc aaatttttag
cctggtatca agcgaaacct 120gggaaaacta ataagcttct tatccactct
ggatccactt tgcaatctgg aattccatca 180aggttcagtg gcagtggatc
tggtacagat ttcactctca ccatcagtag cctggagcct 240gaagattttg
caatgtatta ctgtcaacag cataatgaat acccgtggac gttcggtgga
300ggcaccaagt tggagatcaa ac 32213107PRTMus sp. 13Asp Val Gln Ile
Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly 1 5 10 15 Glu Ile
Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Ser Lys Phe 20 25 30
Leu Ala Trp Tyr Gln Ala Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile 35
40 45 His Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln His
Asn Glu Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 105 14354DNAMus sp. 14tctgatgtac agcttcagga gtcaggacct
ggcctcgtga aaccttctca gtctctgtct 60ctcacctgct ctgtcactgg ctactccatc
accagtggtt attactggaa ctggatccgg 120cagtttccag gaaacagact
ggaatggatg gcctacataa gttacgacgg tagcaatgac 180tacaacccat
ctctcaaaaa tcgtatctcc atcactcgtg acacctctaa gaatcagttt
240ttcctgaagt tgaattctgt gactactgag gacacagcta catattactg
tgcaagaggt 300tacccgatcc tctttgctta ctggggccaa gggactctgg
tcactgtctc tgca 35415118PRTMus sp. 15Ser Asp Val Gln Leu Gln Glu
Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15 Gln Ser Leu Ser Leu
Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser 20 25 30 Gly Tyr Tyr
Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Arg Leu Glu 35 40 45 Trp
Met Ala Tyr Ile Ser Tyr Asp Gly Ser Asn Asp Tyr Asn Pro Ser 50 55
60 Leu Lys Asn Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe
65 70 75 80 Phe Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr
Tyr Tyr 85 90 95 Cys Ala Arg Gly Tyr Pro Ile Leu Phe Ala Tyr Trp
Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 16322DNAMus
sp. 16gacatcaaga tgacccagtc tccatcttcc atgaatgcat ctctaggaga
gagagtctct 60atcacttgca agacgagtca ggacattaat agctatttaa gctggctcca
gcagaaacca 120gggaaatctc caaagaccct gatctatcgt gcaaacagat
tggtagatgg ggtcccttca 180aggttcagtg gcagtggatc tgggcaagat
tattctctca ccatcagcag cctggagtat 240gaagatatgg gaatttatta
ttgtctacag tatgatgagt ttccgctcac gttcgggatt 300gggaccaagc
tggagctgaa ac 32217107PRTMus sp. 17Asp Ile Lys Met Thr Gln Ser Pro
Ser Ser Met Asn Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Ser Ile Thr
Cys Lys Thr Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Ser Trp Leu
Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Arg
Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr 65
70 75 80 Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe
Pro Leu 85 90 95 Thr Phe Gly Ile Gly Thr Lys Leu Glu Leu Lys 100
105 18354DNAMus sp. 18gaggtgcagc tggtggagtc tgggggaggc ttagtgaagc
ctggagggtc cctgaaactc 60tcctgtgcag cctctggatt cactttcagt agctatggca
tgtcttgggt tcgccagact 120ccggagaaga ggctggagtg ggtcgcagcc
attaatatta atggtggtat cacctactat 180ccagacactg tgaagggccg
attcaccatc tccagagaca atgccaagaa caccctgtcc 240ctgcaaatga
gcagtctgag gtctgaggac acagccttct attactgtgc aagagacatc
300tcgggctatg ctatggacta ctggggtcaa ggaacctcgg tcaccgtctc ctca
35419118PRTMus sp. 19Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser
Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg
Leu Glu Trp Val 35 40 45 Ala Ala Ile Asn Ile Asn Gly Gly Ile Thr
Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Thr Leu Ser 65 70 75 80 Leu Gln Met Ser Ser Leu
Arg Ser Glu Asp Thr Ala Phe Tyr Tyr Cys 85 90 95 Ala Arg Asp Ile
Ser Gly Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Ser Val
Thr Val Ser Ser 115 20322DNAMus sp. 20gatatccaga tgacacagac
tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60atcagttgca gggcaagtca
ggacattagc aattatttaa actggtatca gcagaaacca 120gatggaactg
ttaaactcct gatctcctac agatcaagat taccctcagg agtcccatca
180aggttcagtg gcagtgggtc tggaacatat tattctctca ccattagcaa
cctggcgcaa 240gaagattttg ccacttactt ttgccaacag ggtcatacgc
ttccgtggac gttcggtgga 300ggcaccaagc tgaaaatcaa ac 32221107PRTMus
sp. 21Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu
Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile
Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr
Val Lys Leu Leu Ile 35 40 45 Ser Tyr Arg Ser Arg Leu Pro Ser Gly
Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Tyr Tyr
Ser Leu Thr Ile Ser Asn Leu Ala Gln 65 70 75 80 Glu Asp Phe Ala Thr
Tyr Phe Cys Gln Gln Gly His Thr Leu Pro Trp 85 90 95 Thr Phe Gly
Gly Gly Thr Lys Leu Lys Ile Lys 100 105 22357DNAMus sp.
22gaggtccagc tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg
60tcctgcaagg cttctggata cacattcact agttatgtta tgcactgggt gaagcagaag
120cctgggcagg gccttgagtg gattggattt attaatcctc acaatgaggg
tactaagtac 180aatgagaagt tcaaaggcaa ggccacactg acttcagaca
aatcctccac cacagccttc 240atggagctca gcagcctgac ctctgaggac
tctgcggtct attactgtgc aagaacgtgg 300gtcccttacg acggccttgc
tgactggggc caagggactc tgatcactgt ctctgaa 35723119PRTMus sp. 23Glu
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45 Gly Phe Ile Asn Pro His Asn Glu Gly Thr Lys Tyr
Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys
Ser Ser Thr Thr Ala Phe 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Trp Val Pro
Tyr Asp Gly Leu Ala Asp Trp Gly Gln Gly 100 105 110 Thr Leu Ile Thr
Val Ser Glu 115 24323DNAMus sp. 24gacatcttgc tgactcagtc tccagccatc
ctgtctgtta gtccagggga aagagtcagt 60ttctcctgca gggccagtca gagcattggc
acaagcatac actggtatca gcaaagaaca 120actggttctc caaggcttct
cataaaggat gcttctgagt ctatctctgg gatcccttcc 180aggtttagtg
gcagtggaac agggacagat tttactctta ctatcaacag tgtggagtct
240gaagatattg cagattatta ttgtcaacaa agtaatagct ggccatacac
gttcggcggg 300gggaccaagc tggaaataaa acg 32325108PRTMus sp. 25Asp
Ile Leu Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10
15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Ser
20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Thr Gly Ser Pro Arg Leu
Leu Ile 35 40 45 Lys Asp Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Thr Gly Thr Asp Phe Thr Leu Thr
Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys
Gln Gln Ser Asn Ser Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 26357DNAMus sp. 26gaggtccagc
tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg 60tcctgcaagg
cttctggata cacgttcact agttatgtta tgcactgggt gaagcagaag
120cctgggcagg gccttgagtg gattggattt attaatcctc acaatgaggg
tactaagtac 180aatgagaagt tcaagggcaa ggccacactg acttcagaca
aatcctccac cacagcctac 240atggagctca gcagcctgac ctctgaggac
tctgcggtct attactgtgc aagaacgagg 300gtcccttacg acggccttgc
ttactggggc caagggactc tggtcactgt ctctgca 35727119PRTMus sp. 27Glu
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10
15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu
Trp Ile 35 40 45 Gly Phe Ile Asn Pro His Asn Glu Gly Thr Lys Tyr
Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys
Ser Ser Thr Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr Ser
Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Arg Val Pro
Tyr Asp Gly Leu Ala Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr
Val Ser Ala 115 28323DNAMus sp. 28gacatcttcc tgactcagtc tccagccatc
ctgtctgtta gtccaggaga aagagtcagt 60ttctcctgca gggccagtca gagcattggc
acaagcttac actggtatca gcaaagaaca 120aatggttctc caagacttct
cataaaggat gcttctgagt ctatctctgg gatcccttcc 180aggtttagtg
gcagtggatc agggacagat tttactctca ccatcaacag tgtggagtct
240gaagatattg cagattatta ctgtcaacaa agtaataggt ggccatacac
attcggaggg 300gggaccaagc tggaaataaa acg 32329108PRTMus sp. 29Asp
Ile Phe Leu Thr Gln Ser Pro Ala Ile Leu Ser Val Ser Pro Gly 1 5 10
15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Ser
20 25 30 Leu His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu
Leu Ile 35 40 45 Lys Asp Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr Cys
Gln Gln Ser Asn Arg Trp Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 30351DNAMus sp. 30caggtgcagc
tggagcagtc aggacctggc ctagtgcagc cctcacagag cctgtccata 60acctgcacag
tctctggttt ctcattaatt agcgatggtg tacactgggt tcgccagtct
120ccaggaaagg gtctggagtg gctgggcgtg atatggaaaa gtggaagcac
agactacaat 180ggagctttca tgtccagact gagcatcacc aaggacaact
ccaagagcca agttttcttt 240gaaatgaaca gtctgcaatc tgatgacact
gccatgtact actgtgccat tcattcctac 300ggctacgtga ttgcttactg
gggccaaggg actctggtca ctgtctctgc a 35131117PRTMus sp. 31Gln Val Gln
Leu Glu Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser
Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Ile Ser Asp 20 25
30 Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45 Gly Val Ile Trp Lys Ser Gly Ser Thr Asp Tyr Asn Gly Ala
Phe Met 50 55 60 Ser Arg Leu Ser Ile Thr Lys Asp Asn Ser Lys Ser
Gln Val Phe Phe 65 70 75 80 Glu Met Asn Ser Leu Gln Ser Asp Asp Thr
Ala Met Tyr Tyr Cys Ala 85 90 95 Ile His Ser Tyr Gly Tyr Val Ile
Ala Tyr Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ala 115
32310DNAMus sp. 32gatatccaga tgacacagac tacatcctcc ctgtctgcct
ctctgggaga cagagtcacc 60atcagttgca gggcaagtca agacatttac aaatatttaa
actggtatca gcagaaacca 120gatggaactg ttaaactcct gatctactac
acatcaagat tacactcagg agtcccatca 180aggttcagtg gcagtgggtc
tggaacagat tattctctca ccattaccaa cctggagcaa 240gaagacattg
ccacttactt ttgccaacag ggtgatacgc ttccgtggac gttcggtggc
300ggcaccaagc 31033103PRTMus sp. 33Asp Ile Gln Met Thr Gln Thr Thr
Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser
Cys Arg Ala Ser Gln Asp Ile Tyr Lys Tyr 20 25 30 Leu Asn Trp Tyr
Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr
Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Thr Asn Leu Glu Gln 65
70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asp Thr Leu
Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys 100 34345DNAMus sp.
34gaagtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc
60tcctgtgcag cctctggatt cactttcagt aactttgcca tgtcttgggt tcgccagact
120ccggagaaga ggctggagtg ggtcgcaacc attactagtg gtggtactta
cacatacaat 180ccagacagtg tgaagggtcg attcaccatc tccagagaca
atgccaagaa cattttgtac 240ctgcaaatga gcagtctgag gtctgaggac
acggccatgt attactgtgc acgacaaaac 300tactttgact actggggcca
aggcaccatt ctcacagtct cctca 34535115PRTMus sp. 35Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu
Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Phe 20 25 30
Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35
40 45 Ala Thr Ile Thr Ser Gly Gly Thr Tyr Thr Tyr Asn Pro Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ile Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu Asp Thr
Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gln Asn Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr Ile Leu Thr 100 105 110 Val Ser Ser 115 36322DNAMus
sp. 36gaaaatgtgc tcacccagtc tccagcaatc atgtctgcat ctctagggga
gaaggtcacc 60atgagctgca gggccagctc aagtgtaaat tacatgtact ggtaccagca
gaagtcagat 120gcctccccca aactatggat ttatttcaca tccaacctgg
ctcctggagt cccaggtcgc 180ttcagtggca gtgggtctgg gaactcttat
tctctcacaa tcagcagcat ggagggtgaa 240gatgctgcca cttatttctg
ccagcagttt actagtcccc catccatcac gttcggtgct 300gggaccaagc
tggagcagaa ac 32237107PRTMus sp. 37Glu Asn Val Leu Thr Gln Ser Pro
Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Lys Val Thr Met Ser
Cys Arg Ala Ser Ser Ser Val Asn Tyr Met 20 25 30 Tyr Trp Tyr Gln
Gln Lys Ser Asp Ala Ser Pro Lys Leu Trp Ile Tyr 35 40 45 Phe Thr
Ser Asn Leu Ala Pro Gly Val Pro Gly Arg Phe Ser Gly Ser 50 55 60
Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Gly Glu 65
70 75 80 Asp Ala Ala Thr Tyr Phe Cys Gln Gln Phe Thr Ser Pro Pro
Ser Ile 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100
105 38357DNAMus sp. 38caggttcagc tgcagcagtc tggagctgag ctgatgaagc
ctggggcctc agtgaagata 60tcctgcaagg ctactggcta cacattcagt acctactgga
tagagtgggt aaaacagagg 120cctggacatg gccttgagtg gattggagag
attttacctg gaagtggtaa tattaagtac 180aatgagagat tcaagggcaa
ggccacattc actgcagata catcctccaa cacagcctac 240atgcaactca
gcagcctgac atctgaagac tctgccgtct attactgtgc aacgactacg
300gtagtatcta cgaactttga ctactggggc caaggcacca ctctcactgt ctcctca
35739119PRTMus sp. 39Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr
Gly Tyr Thr Phe Ser Thr Tyr 20 25 30 Trp Ile Glu Trp Val Lys Gln
Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro
Gly Ser Gly Asn Ile Lys Tyr Asn Glu Arg Phe 50 55 60 Lys Gly Lys
Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met
Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95 Ala Thr Thr Thr Val Val Ser Thr Asn Phe Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Thr Leu Thr Val Ser Ser 115 40322DNAMus sp.
40gacattgtga tgactcagtc tccagccacc ctgtctgtga ctccaggaga tagagtctct
60ctttcctgca gggccagcca gagtattagc gactacttac actggtatca acaaaaatca
120catgagtctc caaggcttct catcaaatat gcttcccaat ccatctctgg
gatcccctcc 180aggttcagtg gcagtggatc agggtcagat ttcactctca
gtatcaacag tatggaacct 240gaagatgttg gagtgtatta ctgtcaaaat
ggtcacagct ttcctcggac gttcggtgga 300ggcaccaagc tggaaatcaa ac
32241107PRTMus sp. 41Asp Ile Val Met Thr Gln Ser Pro Ala Thr Leu
Ser Val Thr Pro Gly 1 5 10 15 Asp Arg Val Ser Leu Ser Cys Arg Ala
Ser Gln Ser Ile Ser Asp Tyr 20 25 30 Leu His Trp Tyr Gln Gln Lys
Ser His Glu Ser Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln
Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Ser Asp Phe Thr Leu Ser Ile Asn Ser Met Glu Pro 65 70 75 80 Glu
Asp Val Gly Val Tyr Tyr Cys Gln Asn Gly His Ser Phe Pro Arg 85 90
95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 42342DNAMus
sp. 42gaggtgaagc ttttcgagtc tggaggtggc ctggtgcagc ctggaggatc
cctgaaactc 60tcctgtgcag cctcaggatt cgattttagt agatactgga tgacttgggt
ccggcaggct 120ccagggacag ggctagaatg gattggagaa attaatccag
atagcagtac gataaactat 180acgccatctc tgagggataa attcatcatc
tccagagaca acgccaaaaa tgcgctgaac 240ctgcaaatga gcaaagtgag
atctgaggac acagcccttt attactgtca ctcctatgct 300atggactact
ggggtcaagg aacctcagtc accgtctcct ca 34243114PRTMus sp. 43Glu Val
Lys Leu Phe Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20
25 30 Trp Met Thr Trp Val Arg Gln Ala Pro Gly Thr Gly Leu Glu Trp
Ile 35 40 45 Gly Glu Ile Asn Pro Asp Ser Ser Thr Ile Asn Tyr Thr
Pro Ser Leu 50 55 60 Arg Asp Lys Phe Ile Ile Ser Arg Asp Asn Ala
Lys Asn Ala Leu Asn 65 70 75 80 Leu Gln Met Ser Lys Val Arg Ser Glu
Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 His Ser Tyr Ala Met Asp Tyr
Trp Gly Gln Gly Thr Ser Val Thr Val 100 105 110 Ser Ser 44337DNAMus
sp. 44gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca
accagcctcc 60atctcttgca agtcaagtca gagcctctta gatagtgatg gaaagacata
tttgaattgg 120ttgttacaga ggccaggcca gtctccaaag cgcctaatct
atctggtgtc taaactggac 180tctggagtcc ctgacaggtt cactggcagt
ggatcaggga cagatttcac actgaaaatc 240agcagagtgg aggctgagga
tttgggagtt tattattgct ggcaaggtac acattttcct 300cagacgttcg
gtggaggcac caagctggaa atcaaac 33745112PRTMus sp. 45Asp Val Val Met
Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro
Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30
Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35
40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val
Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr
Tyr Cys Trp Gln Gly
85 90 95 Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys 100 105 110 46342DNAMus sp. 46gaggtgcagc ttgttgagtc
tggtggagga ttggtgcagc ctaaagggtc attgaaactc 60tcatgtgcag cctctggatt
caccttcgat acctacgcca tgaactgggt ccgccaggct 120ccaggaaagg
gtttggaatg ggttgctcgc ataagaagta aaagtaatga ttatgcaaca
180tattatgtcg attcagtgaa agacaggttc accatttcca gagatgattc
acaaaacatg 240ctctatctgc acatgaacaa cttgaaaact gaggacacag
ccatatatta ctgtatgatc 300tcttcgactt tttttgactg ttggggccaa
ggcacctctc tc 34247114PRTMus sp. 47Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Asp Thr Tyr 20 25 30 Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg
Ile Arg Ser Lys Ser Asn Asp Tyr Ala Thr Tyr Tyr Val Asp 50 55 60
Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Asn Met 65
70 75 80 Leu Tyr Leu His Met Asn Asn Leu Lys Thr Glu Asp Thr Ala
Ile Tyr 85 90 95 Tyr Cys Met Ile Ser Ser Thr Phe Phe Asp Cys Trp
Gly Gln Gly Thr 100 105 110 Ser Leu 48337DNAMus sp. 48gatgttgtga
tgacccagac tccactcact ttgtcggtta ccattggaca accagcctct 60atctcttgca
ggtcaagtca gagcctctta tatagtaatg gaaataccta tttgaattgg
120ttattacaga ggccaggcca gtctccaaag cgcctagtct atctggtgtc
taaactggac 180tctggagtcc ctgacaggtt cactggcact ggatcaggaa
cagattttac cctgaagatc 240agcagagtgg aggctgagga tttgggagtt
tattactgcg tgcaaggtac acattttcca 300ttcacgttcg gctcggggac
aaagttggaa ataaaac 33749112PRTMus sp. 49Asp Val Val Met Thr Gln Thr
Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile
Ser Cys Arg Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Asn
Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro
Lys Arg Leu Val Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55
60 Asp Arg Phe Thr Gly Thr Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Val
Gln Gly 85 90 95 Thr His Phe Pro Phe Thr Phe Gly Ser Gly Thr Lys
Leu Glu Ile Lys 100 105 110 50345DNAMus sp. 50gaggtccagt tgcaacagtc
tggacctgag ctaatgaagc ctggggcttc agtgaagatg 60tcctgcaaga cttctggata
cacattcact gattacaaca tacactgggt gaagcagaac 120caaggaaaga
gcctagagtg gatcggagaa attaatcctt acactggtgg tactggctac
180aaccagaaat tcacaggcaa ggccacattg actgtagaca agtcctccag
cacagcctac 240atggagctcc gcagcctaac atctgaggac tctgcagtct
attactgtgc aagagatggt 300tactttgact actggggcca aggcaccact
ctcacagtct cctca 34551115PRTMus sp. 51Glu Val Gln Leu Gln Gln Ser
Gly Pro Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser
Cys Lys Thr Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Asn Ile His
Trp Val Lys Gln Asn Gln Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly
Glu Ile Asn Pro Tyr Thr Gly Gly Thr Gly Tyr Asn Gln Lys Phe 50 55
60 Thr Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asp Gly Tyr Phe Asp Tyr Trp Gly Gln Gly
Thr Thr Leu Thr 100 105 110 Val Ser Ser 115 52312DNAMus sp.
52gacatccaga tgacccagtc tccatcctcc ttatctgcct ctctgggaga aagagtcagt
60ctcacttgtc gggcaagtca ggaaattagt gattacttaa gctggcttca gcagaaacca
120gatggaacta ttaaacgcct gatctacgcc gcatccactt tagattctgg
tgtcccaaaa 180aggttcagtg gcagtaggtc tgggtcagat tattctctca
ccatcagcag ccttgagtct 240gaagattttg cagactatta ctgtctacaa
tatgctagtt ctccgctcac gttcggtgct 300gggaccaagc tg 31253104PRTMus
sp. 53Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu
Gly 1 5 10 15 Glu Arg Val Ser Leu Thr Cys Arg Ala Ser Gln Glu Ile
Ser Asp Tyr 20 25 30 Leu Ser Trp Leu Gln Gln Lys Pro Asp Gly Thr
Ile Lys Arg Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Asp Ser Gly
Val Pro Lys Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Ser Asp Tyr
Ser Leu Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Phe Ala Asp
Tyr Tyr Cys Leu Gln Tyr Ala Ser Ser Pro Leu 85 90 95 Thr Phe Gly
Ala Gly Thr Lys Leu 100 54354DNAMus sp. 54gaggtccagc tgcaacagtc
tggacctgag ctggtgaagc ctggggcttc agtgaagatg 60tcctgcaagg cttctggata
cacattcact gactacagca tacactgggt gaagcagagc 120cttggaaaga
gccttgagtg gattggatat attaccccta acactggtgg cactaactac
180aaccagaact tcaaggacaa ggccatattg actgtaaaca agtcctccag
cacagcctac 240atggagctcc gcagcctgac atcggaggat tctgcagtct
attactgtgc aagaaactgg 300ggttttttcc tctttgacta ctggggccaa
ggcaccactc tcacagtctc ctca 35455118PRTMus sp. 55Glu Val Gln Leu Gln
Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys
Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Ser
Ile His Trp Val Lys Gln Ser Leu Gly Lys Ser Leu Glu Trp Ile 35 40
45 Gly Tyr Ile Thr Pro Asn Thr Gly Gly Thr Asn Tyr Asn Gln Asn Phe
50 55 60 Lys Asp Lys Ala Ile Leu Thr Val Asn Lys Ser Ser Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Trp Gly Phe Phe Leu Phe Asp
Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115
56319DNAMus sp. 56agtattgtga tgacccagac tcccaaattc ctgcctgtaa
cagcagaaga cagggttacc 60ataacctgca aggccactca gagtgtgagt aatgaagtag
cttggtacca acagaaggca 120gggcagtctc ctaaactgat gatatactat
gcatccaatc gctacactgg agtccctgat 180cgcttcactg gcagtggatc
tggcacggat ttcactttca ccatcagcag tgtgcaggtt 240gaagacctgg
cagtttattt ctgtcagcat cattacagtt ctcccacgtt cggtgctggg
300accaagctgg agctgaaac 31957106PRTMus sp. 57Ser Ile Val Met Thr
Gln Thr Pro Lys Phe Leu Pro Val Thr Ala Glu 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Lys Ala Thr Gln Ser Val Ser Asn Glu 20 25 30 Val
Ala Trp Tyr Gln Gln Lys Ala Gly Gln Ser Pro Lys Leu Met Ile 35 40
45 Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Val
Gln Val 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln His His Tyr
Ser Ser Pro Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105 58389DNAMus sp.modified_base(379)..(379)a, c, t, g, unknown
or other 58caggtccaac tgcagcagcc tggggctgaa attgtgaggc ctggggcttc
agtgaagctg 60tcctgcaagg cttctggcta cacctttacc gcctattgga tgcactgggt
gaaacagagg 120cctggacaag gccctgagtg gatcggagca attgatcctt
ctgatagtta tacttactac 180aatcaaaagt tcaagggcaa ggccacattg
actgtagaca catcctccaa ctcagcctac 240atgcagctca gcagcctgac
atctgaggac tctgcggtct atttctgtgc aagatgggat 300tactacgatg
gtaactgggg ccaaggcacc actctcacag tctcctcagc caaaacaaca
360cccccatccg tctatcccng ggcccctga 38959116PRTMus sp. 59Gln Val Gln
Leu Gln Gln Pro Gly Ala Glu Ile Val Arg Pro Gly Ala 1 5 10 15 Ser
Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ala Tyr 20 25
30 Trp Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Pro Glu Trp Ile
35 40 45 Gly Ala Ile Asp Pro Ser Asp Ser Tyr Thr Tyr Tyr Asn Gln
Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Thr Ser Ser
Asn Ser Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Trp Asp Tyr Tyr Asp Gly
Asn Trp Gly Gln Gly Thr Thr Leu 100 105 110 Thr Val Ser Ser 115
60338DNAMus sp. 60gatgttgtga tgacccaaac tccactctcc ctgcctgtca
gtcttggaga tcaagcctcc 60atctcttgca gatctagtca gagccttgta cacagtgatg
gaaacaccta tttacattgg 120tacctgcaga agccaggcca gtctccaaag
ctcctgatct acagagtttc caaccgcttt 180tctggggtcc cagacaggtt
cagtggcagt ggatcaggga cagatttcac actcaagatc 240agcagagtgg
aggctgagga tctgggagtt tatttctgct ctcaaagtac acatgttccg
300tacacgttcg gaggggggac caagctggaa ataaaacg 33861112PRTMus sp.
61Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1
5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His
Ser 20 25 30 Asp Gly Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg
Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp
Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro Tyr
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 62366DNAMus
sp. 62tctgatgtgc accttcagga gacgggacct ggcctggtga aaccttctca
gtctctgtcc 60gtcacctgca ctgtcactgg ttactcaatc accagtgatt atgcctggaa
ctggatccgg 120cagtttccag gaaacaaact ggagtggatg ggctacataa
gctacagtgg tggcactagg 180tacaacccat ctgtcaaaag tcgaatctct
atcactcgag acacatccaa gaaccagttc 240ttcctgcagt tgaattctgt
gactactgag gacacagcca catattactg tgcaagaggg 300ccctatgctg
gttaccccgc ctggtttgat tactggggcc cagggactct ggtcactgtc 360tctgca
36663122PRTMus sp. 63Ser Asp Val His Leu Gln Glu Thr Gly Pro Gly
Leu Val Lys Pro Ser 1 5 10 15 Gln Ser Leu Ser Val Thr Cys Thr Val
Thr Gly Tyr Ser Ile Thr Ser 20 25 30 Asp Tyr Ala Trp Asn Trp Ile
Arg Gln Phe Pro Gly Asn Lys Leu Glu 35 40 45 Trp Met Gly Tyr Ile
Ser Tyr Ser Gly Gly Thr Arg Tyr Asn Pro Ser 50 55 60 Val Lys Ser
Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Phe
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr 85 90
95 Cys Ala Arg Gly Pro Tyr Ala Gly Tyr Pro Ala Trp Phe Asp Tyr Trp
100 105 110 Gly Pro Gly Thr Leu Val Thr Val Ser Ala 115 120
64319DNAMus sp. 64gaaaatgtgc tcacccagtc tccagcaatc atgtctgcat
ctctagggga gaaggtcacc 60atgagctgca gggccaactc aagtataaat tacatgtact
ggtaccagca gaagtcagat 120gcctccccca aactatggat ttattacaca
tccaacctgg ctcctggagt cccagctcgc 180ttcagtggca gtgggtctgg
gaactcttat tctctcacaa tcagcagcat ggagggtgaa 240gatgctgcca
cttattactg ccagcagttt actagttccc cgtggacgtt cggtggaggc
300accaagctgg aaatcaaac 31965106PRTMus sp. 65Glu Asn Val Leu Thr
Gln Ser Pro Ala Ile Met Ser Ala Ser Leu Gly 1 5 10 15 Glu Lys Val
Thr Met Ser Cys Arg Ala Asn Ser Ser Ile Asn Tyr Met 20 25 30 Tyr
Trp Tyr Gln Gln Lys Ser Asp Ala Ser Pro Lys Leu Trp Ile Tyr 35 40
45 Tyr Thr Ser Asn Leu Ala Pro Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60 Gly Ser Gly Asn Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu
Gly Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Phe Thr Ser
Ser Pro Trp Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 66354DNAMus sp. 66gcggtgcagc ttgttgagtc tggtggagga
ttggtgcagc ctgaagggtc attgaaactc 60tcgtgtgcag cctctggatt caccttcaat
gcctacgcca tgaactgggt ccgccaggct 120ccaggaaagg gtttggaatg
ggttgctcgc ataagaagta aaagtaatga ttatgcaaca 180tattatgccg
attcagtgaa agacaggttc accatctcca gagatgattc acaaagcatg
240ctctatctgc aaatgaacaa cttgaaaact gaggacacag gcatgtatta
ctgtttgacc 300ttctctgtag atttgcacta ctggggccaa ggcaccactc
tcacagtctc ctca 35467118PRTMus sp. 67Ala Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Glu Gly 1 5 10 15 Ser Leu Lys Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asn Ala Tyr 20 25 30 Ala Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Arg Ile Arg Ser Lys Ser Asn Asp Tyr Ala Thr Tyr Tyr Ala Asp 50 55
60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met
65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Gly
Met Tyr 85 90 95 Tyr Cys Leu Thr Phe Ser Val Asp Leu His Tyr Trp
Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 68338DNAMus
sp. 68gatgttgtga tgacccagac tccactcact ttgtcggtta ccattggaca
accagcctct 60atctcttgca agtcaagtca gagcctctta tatagtaatg gaaagaccta
tttgaattgg 120ttattacaga ggccaggcca gtctccaaag cgcctaatct
atctgatgtc taaactggac 180tctggagtcc ctgacaggtt cactggcagt
ggatcaggaa cagattttac actgaaaatc 240agcagagtgg aggctgagga
tttgggaatt tattactgct tgcaaggtac acattttccg 300tacacgttcg
gaggggggac cgagctggaa attaaacg 33869113PRTMus sp. 69Asp Val Val Met
Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro
Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30
Asn Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35
40 45 Pro Lys Arg Leu Ile Tyr Leu Met Ser Lys Leu Asp Ser Gly Val
Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Ile Tyr
Tyr Cys Leu Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly
Gly Thr Glu Leu Glu Ile Lys 100 105 110 Arg 70354DNAMus sp.
70tcccgggggc ccctggagtc tgatggagga ttggtgcagc ctgaagggtc attgaaactc
60tcatgtgcag cctctggatt caccttcaat acctacgcca tgaactgggt ccgccaggct
120ccaggaaagg gtttggaatg ggttgctcgc ataagaagta aaagtaataa
ttatgtaaca 180tattatgccg attcagtgaa agacaggttc accgtctcca
gagatgattc acaaagcatg 240ctctatctgc aaatgaacaa cttgaaaact
gaggacacag gcatgtatta ctgtgtgacc 300ttctctgtgg atttgcacta
ttggggccaa ggcaccactc tcacagtctc ctca 35471118PRTMus sp. 71Ser Arg
Gly Pro Leu Glu Ser Asp Gly Gly Leu Val Gln Pro Glu Gly 1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20
25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ala Arg Ile Arg Ser Lys Ser Asn Asn Tyr Val Thr Tyr
Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Phe Thr Val Ser Arg Asp
Asp Ser Gln Ser Met 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys
Thr Glu Asp Thr Gly Met Tyr 85 90 95 Tyr Cys Val Thr Phe Ser Val
Asp Leu His Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser
Ser 115
72338DNAMus sp. 72gatgttgtga tgacccagac tccgctcact ttgtcggtta
ccattggaca accagcctct 60atctcttgca agtcaggtca gagcctctta tatggtaatg
gaaaaaccta tttgaattgg 120ttatttcaga ggccaggcca gtctccaaag
cgcctaatct atctggtgtc taaactggac 180tctggagtcc ctgacaggtt
cactggcagt ggatcaggaa cagattttac actgaaaatc 240agcagagtgg
aggctgagga tttgggagtt tattactgct tgcaaggtac acattttccg
300tacacgttcg gaggggggac cgagctggaa ataaaacg 33873113PRTMus sp.
73Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1
5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Gly Gln Ser Leu Leu Tyr
Gly 20 25 30 Asn Gly Lys Thr Tyr Leu Asn Trp Leu Phe Gln Arg Pro
Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu
Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp
Leu Gly Val Tyr Tyr Cys Leu Gln Gly 85 90 95 Thr His Phe Pro Tyr
Thr Phe Gly Gly Gly Thr Glu Leu Glu Ile Lys 100 105 110 Arg
74366DNAMus sp. 74gaggtccagc tacaacaatc tggacctgag ctggtgaagc
ctggggcttc agtgaagata 60tcctgtaagg cttctggata cacgttcact gactactaca
tgaactgggt gaaacagact 120catggaaaga gccttgagtg gattggagat
attactccta acagtggtgg tcctacctac 180aaccagaatt tcaagggcaa
ggccacattg actgttgaca ggtcctccac cacagccttc 240atggagctcc
gcagcctgac ctctgatgac tctgctgtct attactgtgt aagatcggct
300tattactacg gtactaacta cgactttgac tactggggcc aaggcaccac
tctcacagtc 360tcctca 36675122PRTMus sp. 75Glu Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met
Asn Trp Val Lys Gln Thr His Gly Lys Ser Leu Glu Trp Ile 35 40 45
Gly Asp Ile Thr Pro Asn Ser Gly Gly Pro Thr Tyr Asn Gln Asn Phe 50
55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Thr Thr Ala
Phe 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Asp Asp Ser Ala Val
Tyr Tyr Cys 85 90 95 Val Arg Ser Ala Tyr Tyr Tyr Gly Thr Asn Tyr
Asp Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Leu Thr Val Ser
Ser 115 120 76319DNAMus sp. 76caaattgttc tcacccagtc tccagcaatc
atgtctgcat ctccagggga gaaggtcacc 60atgacctgca gtgccagctc aagtgttact
tacatgtact ggtaccaaca gaagccagga 120tcctcaccca aaccctggat
ttatcgcaca tccaaccttg cttctggagt ccctactcgc 180ttcagtggca
gtgggtctgg gacctcttac tctctcacaa tcagcagcgt ggaggccgaa
240gatactgcca cttattactg ccagcagtac agtgattacc cgctcacgtt
cggtgctggg 300accaagctgg agctgaaac 31977106PRTMus sp. 77Gln Ile Val
Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu
Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Thr Tyr Met 20 25
30 Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr
35 40 45 Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Thr Arg Phe Ser
Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser
Val Glu Ala Glu 65 70 75 80 Asp Thr Ala Thr Tyr Tyr Cys Gln Gln Tyr
Ser Asp Tyr Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu
Leu Lys 100 105 78354DNAMus sp. 78gaggtgcagc ttgttgagtc tggtggagga
ttggtgcagc ctgaagggtc attgaaactc 60tcatgtgcag cctctggatt caccttcaat
acctacgcca tgaactgggt ccgccaggct 120ccaggaaagg gtttggaatg
ggttgctcgc atcagaagta aaagtaataa ttatgcaaca 180tattatgccg
attcagtgaa agacaggttc accatctcca gagatgattc acaaagtatg
240ctctatctgc aaatgaacaa cttgaaaact gaggacacag gcatgtatta
ctgtttgacc 300ttctctgtag atttgcacta ctggggccaa ggcaccgctc
tcacagtctc ctca 35479118PRTMus sp. 79Glu Val Gln Leu Val Glu Ser
Gly Gly Gly Leu Val Gln Pro Glu Gly 1 5 10 15 Ser Leu Lys Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala Met Asn
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala
Arg Ile Arg Ser Lys Ser Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55
60 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met
65 70 75 80 Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Gly
Met Tyr 85 90 95 Tyr Cys Leu Thr Phe Ser Val Asp Leu His Tyr Trp
Gly Gln Gly Thr 100 105 110 Ala Leu Thr Val Ser Ser 115 80322DNAMus
sp. 80gacatcttgc tgactcagtc tccagccatc ctgtctgtga gtccaggaga
aagagtcagt 60ttctcctgca gggccagtca gagcattggc acggacatac actggtttca
gctaaaaaca 120aatggttctc caagacttct cataaattat acttctgagt
ctatctctgt gatcccttcc 180aggcttagtg gcagtggatc agggacagat
tttactctta gcatcaacag tgtggagtct 240gaagattttg cagattgtca
ctgtcaacaa aataataact ggccgctcac gttcggtgct 300gggaccaagc
tggagctgaa ac 32281107PRTMus sp. 81Asp Ile Leu Leu Thr Gln Ser Pro
Ala Ile Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Ser Phe Ser
Cys Arg Ala Ser Gln Ser Ile Gly Thr Asp 20 25 30 Ile His Trp Phe
Gln Leu Lys Thr Asn Gly Ser Pro Arg Leu Leu Ile 35 40 45 Asn Tyr
Thr Ser Glu Ser Ile Ser Val Ile Pro Ser Arg Leu Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser 65
70 75 80 Glu Asp Phe Ala Asp Cys His Cys Gln Gln Asn Asn Asn Trp
Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100
105 82366DNAMus sp. 82gaggtccagc tgcaacaatc tggacctgag ctggtgaagc
ctggggcttc agtgaagata 60tcctgtaagg cttctggata cacgttcact gactactaca
tgaactgggt gaagcagagc 120catggaaaga gccttgagtg gattggagat
attaatccta acattggtgg tactaactac 180aaccagaagt tcaagggcaa
ggccacattg actgtagaca agtcctccag tacagcctac 240atggagctcc
gcagcctgac atctgaggac tctgcagtct attactgtgc aagaagctgg
300atctactatg gttacgaccc tgatatggac tactggggtc aaggaacatc
agtcaccgtc 360tcctca 36683122PRTMus sp. 83Glu Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met
Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45
Gly Asp Ile Asn Pro Asn Ile Gly Gly Thr Asn Tyr Asn Gln Lys Phe 50
55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Ser Trp Ile Tyr Tyr Gly Tyr Asp Pro
Asp Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Ser Val Thr Val Ser
Ser 115 120 84297DNAMus sp. 84caaattgttc tcacccagtc tccagcaatc
atgtctgcat ctccagggga gaaggtcacc 60atgacctgca gtgccagctc aagtctaagt
tacatgtact ggtaccagca gaagccagga 120tcctcaccca aaccctggat
ttatcgcaca tccaacctgg cttctggagt ccctactcgc 180ttcagtggca
gtgggtctgg gacctcttac tctctcacaa tcagcagcgt ggaggccgaa
240gatgctgcca cttattactg ccagcagtac agtaattacc cgctcacgtt cggtgct
2978599PRTMus sp. 85Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser
Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser
Ser Ser Leu Ser Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro Gly
Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Arg Thr Ser Asn Leu Ala
Ser Gly Val Pro Thr Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr
Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp Ala
Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Asn Tyr Pro Leu Thr 85 90 95
Phe Gly Ala 86360DNAMus sp. 86gaggtccagc tgcaacaatc tggacctgag
ctggtgaagc ctggggcttc agtgaagata 60tcctgtaagg cttctggata cacgttcact
gactattata tgaactgggt gagacagagc 120catggaaaga gccttgagtg
gattggagat gttcatccta acattggtac tattaactac 180aaccagaagt
tcaaggacaa ggccacattg actatagaca agtcctccag tacagcctac
240atggagctcc gcagcctgac atctgaagac tctgcagtct atttctgtgc
aagagaaggg 300gcctattatg gtcccccttt tgcttactgg ggccaaggga
ctctggtcac tgtctctgca 36087120PRTMus sp. 87Glu Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met
Asn Trp Val Arg Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45
Gly Asp Val His Pro Asn Ile Gly Thr Ile Asn Tyr Asn Gln Lys Phe 50
55 60 Lys Asp Lys Ala Thr Leu Thr Ile Asp Lys Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Phe Cys 85 90 95 Ala Arg Glu Gly Ala Tyr Tyr Gly Pro Pro Phe
Ala Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ala 115
120 88406DNAMus sp.modified_base(385)..(385)a, c, t, g, unknown or
other 88actggatggt gggaagatgg atacagttgg tgcagcatca gcccgtttaa
tttccaactt 60ggtcccccct ccgaacgtgg aggaatctcc caactgcgct gacagtcata
ttttgcagta 120tcctcctcct ccacaggatg gatgttgagg gtgcagtctg
tcccagaccc actgccactg 180aacctggcag ggaccccaga ttctaggttg
gatgcatact tgatgaggag cttgggtggc 240tgtcctggta tctgttggta
ccagtatata tgactatagc tagatgaatt gacactttgg 300ctggccctgc
atgagatggt ggccctctgc cccagagata cagctaagga agcaggagac
360tgtgtcagca caatgtcacc agtgnaacct ggaacccana gcagca
40689111PRTMus sp. 89Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu
Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Ala
Ser Gln Ser Val Asn Ser Ser 20 25 30 Ser Tyr Ser His Ile Tyr Trp
Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys
Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Cys Thr Leu Asn Ile His 65 70 75 80 Pro
Val Glu Glu Glu Asp Thr Ala Lys Tyr Asp Cys Gln Arg Ser Trp 85 90
95 Glu Ile Pro Pro Arg Ser Glu Gly Gly Pro Ser Trp Lys Leu Asn 100
105 110 90360DNAMus sp. 90gaggtccaac tgcaacaatc tggacctgag
ctggtgaagc ctggggcttc agtgaagata 60tcctgtaagg cttctggata cacgttcact
gactactaca tgaactgggt gaagcagagc 120catggaaaga gccttgagtg
gattggagat attcatccta acaatggtgg tgctaactac 180aaccagaagt
tcaagggcaa ggccacattg actgtagacc agtcctccag cacagcctac
240atggagctcc gcagcctgac atctgaggac tctgcagtct atttctgtgc
aagagagggg 300gattacggtg gtaactctat ggactactgg ggtcaaggaa
cctcagtcac cgtctcctca 36091120PRTMus sp. 91Glu Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met
Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45
Gly Asp Ile His Pro Asn Asn Gly Gly Ala Asn Tyr Asn Gln Lys Phe 50
55 60 Lys Gly Lys Ala Thr Leu Thr Val Asp Gln Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Phe Cys 85 90 95 Ala Arg Glu Gly Asp Tyr Gly Gly Asn Ser Met
Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115
120 92319DNAMus sp. 92caaattgttc tcacccagtc tccagcaatc atgtctgcat
ctccagggga gaaggtcgcc 60atgacctgca gtgccagctc aagtgtaact tacatgtact
ggtaccagca gaagccagga 120tcctcaccca aaccctggat ttatcgcaca
tccaacctgg cttctggagt ccctgctcgc 180ttcagtggca gtgggtctgg
gacctcttac tctctcacaa tcagcagcgt ggaggccgaa 240gatgctgcca
cttattactg ccagcagtac gataattacc cgctcacgtt cggtgctggg
300accaagctgg agctgaaac 31993106PRTMus sp. 93Gln Ile Val Leu Thr
Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val
Ala Met Thr Cys Ser Ala Ser Ser Ser Val Thr Tyr Met 20 25 30 Tyr
Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40
45 Arg Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu
Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn
Tyr Pro Leu Thr 85 90 95 Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys
100 105 94360DNAMus sp. 94gaggtccaac tgcaacaatc tggacctgag
ctggtgaagc ctggggcttc agtgaagata 60tcctgtaagg cttctggata cacgttcact
gactactata tgaactgggt gaagcagagc 120catggaaaga gccttgagtg
gattggagat attcatccta actatggtgg ttctaactac 180aaccagaagt
tcaagggcaa ggccacattg actgtagacc ggtcctccag cacagcctac
240atggagctcc gcagcctgac atctgaggac tctgcagtct atttctgtgc
aagagagggg 300gattacggtg gtagctctat ggactactgg ggtcaaggaa
cctcagtcac cgtctcctca 36095119PRTMus sp. 95Val Gln Leu Gln Gln Ser
Gly Pro Glu Leu Val Lys Pro Gly Ala Ser 1 5 10 15 Val Lys Ile Ser
Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Tyr 20 25 30 Met Asn
Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile Gly 35 40 45
Asp Ile His Pro Asn Tyr Gly Gly Ser Asn Tyr Asn Gln Lys Phe Lys 50
55 60 Gly Lys Ala Thr Leu Thr Val Asp Arg Ser Ser Ser Thr Ala Tyr
Met 65 70 75 80 Glu Leu Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Phe Cys Ala 85 90 95 Arg Glu Gly Asp Tyr Gly Gly Ser Ser Met Asp
Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val Thr Val Ser Ser 115
96307DNAMus sp. 96caaattgttc tcacccagtc tccagcaatc atgtctgcat
ctccagggga gaaggtcgcc 60atgacctgca gtgccagctc aagtgtaact tacatgtact
ggtaccagca gaagccagga 120tcctcaccca aaccctggat ttatcgcaca
tccaacctgg cttctggagt ccctgctcgc 180ttcagtggca gtgggtctgg
gacctcttac tctctcacaa tcagcagcgt ggaggccgaa 240gatgctgcca
cttattactg ccagcagtat gataattatc cgctcacgtt cggtgctggg 300accaagc
30797102PRTMus sp. 97Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met
Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Ala Met Thr Cys Ser Ala
Ser Ser Ser Val Thr Tyr Met 20 25 30 Tyr Trp Tyr Gln Gln Lys Pro
Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Arg Thr Ser Asn Leu
Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly
Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu Ala Glu 65 70 75 80 Asp
Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Tyr Pro Leu Thr 85 90
95 Phe Gly Ala Gly Thr Lys 100 98357DNAMus sp. 98caggtgcagc
tgaagcagtc aggacctggc cttgtgcagc cctcacagag cctgtccatc 60acctgcacag
tctctggttt ctcattatct aactatggtg tacactgggt tcgccagtct
120ccaggaaagg gtctggagtg gctgggagtg atatggagtg gtggaagcac
agactataat 180gcagctttca
tatccagact gaacatcaac aaggacaatt ccaagagcca agttttcttt
240aaaatgaaca gtctgcaatc tgatgacaca gccatatatt actgtgccga
ctactatgat 300tacgatgggg cctggtttgc ttactggggc caagggactc
tggtcactgt ctctgca 35799119PRTMus sp. 99Gln Val Gln Leu Lys Gln Ser
Gly Pro Gly Leu Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr
Cys Thr Val Ser Gly Phe Ser Leu Ser Asn Tyr 20 25 30 Gly Val His
Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly
Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile 50 55
60 Ser Arg Leu Asn Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe
65 70 75 80 Lys Met Asn Ser Leu Gln Ser Asp Asp Thr Ala Ile Tyr Tyr
Cys Ala 85 90 95 Asp Tyr Tyr Asp Tyr Asp Gly Ala Trp Phe Ala Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ala 115
100322DNAMus sp. 100gacattgtga tgacccagtc tcaaaaattc atgtccactt
cagtaggaga cagggtcagc 60gtcacctgca aggccagtca gaacgtgggt actaatgtag
cctggtttca acagaaacca 120gggcaatctc ctaaagcact gattcactcg
gcatcctacc ggtacagtgg agtccctgat 180cgcttcacag gcagtggatc
tgggacagat ttcactctca ccatcagcaa tgtgcagcct 240gaagacttgg
cagagtattt ctgtcagcaa cataacagct ttcctctcac gttcggtgct
300gggactaagc tggagctgaa ac 322101107PRTMus sp. 101Asp Ile Val Met
Thr Gln Ser Gln Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg
Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn 20 25 30
Val Ala Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Ala Leu Ile 35
40 45 His Ser Ala Ser Tyr Arg Tyr Ser Gly Val Pro Asp Arg Phe Thr
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn
Val Gln Pro 65 70 75 80 Glu Asp Leu Ala Glu Tyr Phe Cys Gln Gln His
Asn Ser Phe Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu
Leu Lys 100 105 1026PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic 6xHis tag" 102His His His His His
His 1 5
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