U.S. patent application number 11/927069 was filed with the patent office on 2008-05-01 for compositions and methods for treatment of non-hodgkin's lymphoma.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Avi J. Ashkenazi, Dylan L. Daniel, Theresa Shek.
Application Number | 20080102468 11/927069 |
Document ID | / |
Family ID | 35519991 |
Filed Date | 2008-05-01 |
United States Patent
Application |
20080102468 |
Kind Code |
A1 |
Ashkenazi; Avi J. ; et
al. |
May 1, 2008 |
COMPOSITIONS AND METHODS FOR TREATMENT OF NON-HODGKIN'S
LYMPHOMA
Abstract
The present invention is directed to compositions of matter
useful for the treatment of non-Hodgkin's lymphoma in mammals and
to methods of using those compositions for the same.
Inventors: |
Ashkenazi; Avi J.; (San
Mateo, CA) ; Daniel; Dylan L.; (San Francisco,
CA) ; Shek; Theresa; (San Mateo, CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
94080
|
Family ID: |
35519991 |
Appl. No.: |
11/927069 |
Filed: |
October 29, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11172193 |
Jun 30, 2005 |
|
|
|
11927069 |
Oct 29, 2007 |
|
|
|
60585132 |
Jul 2, 2004 |
|
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Current U.S.
Class: |
435/6.16 ;
435/7.1 |
Current CPC
Class: |
G01N 33/57407 20130101;
C12Q 2600/158 20130101; C12Q 1/6886 20130101; G01N 2500/10
20130101; C12Q 2600/136 20130101; C07K 16/3061 20130101; C12Q
2600/106 20130101; A61P 35/00 20180101 |
Class at
Publication: |
435/006 ;
435/007.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/53 20060101 G01N033/53 |
Claims
1. A method of diagnosing a non-Hodgkin's lymphoma that
overexpresses UNQ733, the method comprising determining the level
of UNQ733 in a test sample of tissue cells obtained from a
non-Hodgkin's lymphoma, wherein a higher level of UNQ733 in the
test sample, as compared to a control sample, is indicative of the
presence of a non-Hodgkin's lymphoma that overexpresses UNQ733.
2. The method of claim 1, wherein the level of UNQ733 is determined
based on the level of UNQ733 RNA in the test sample.
3. The method of claim 1, wherein the level of UNQ733 is determined
based on the level of UNQ733 polypeptide in the test sample.
4. The method of claim 1, wherein the non-Hodgkin's lymphoma that
overexpresses UNQ733 is a B cell lymphoma, diffuse large B cell
lymphoma, follicular lymphoma, small lymphocytic lymphoma,
malignant lymphoma, malignant T cell lymphoma, anaplastic large
cell lymphoma or mucosal associated lymphoid tissue lymphoma.
5. The method of claim 1, wherein the non-Hodgkin's lymphoma that
overexpresses UNQ733 is a T-cell lymphoma.
6. The method of claim 1, wherein the non-Hodgkin's lymphoma that
overexpresses UNQ733 is a B-cell lymphoma.
7. The method of claim 1, wherein determining the level of UNQ733
in the test sample comprises exposing the test sample to an
antibody that binds to a UNQ733 polypeptide and detecting binding
of the antibody to a UNQ733 polypeptide in the test sample.
8. The method of claim 7 wherein the antibody is detectably labeled
and/or attached to a solid support.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/172,193, filed Jun. 30, 2005, which claims the benefit of
U.S. Provisional Application No. 60/585,132, filed Jul. 2, 2004,
the contents of which are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention is directed to compositions of matter
useful for the treatment of hematopoietic tumor in mammals and to
methods of using those compositions of matter for the same.
BACKGROUND OF THE INVENTION
[0003] Malignant tumors (cancers) are the second leading cause of
death in the United States, after heart disease (Boring et al., C A
Cancel J. Clin. 43:7 (1993)). Cancer is characterized by the
increase in the number of abnormal, or neoplastic, cells derived
from a normal tissue which proliferate to form a tumor mass, the
invasion of adjacent tissues by these neoplastic tumor cells, and
the generation of malignant cells which eventually spread via the
blood or lymphatic system to regional lymph nodes and to distant
sites via a process called metastasis. In a cancerous state, a cell
proliferates under conditions in which normal cells would not grow.
Cancer manifests itself in a wide variety of forms, characterized
by different degrees of invasiveness and aggressiveness.
[0004] Cancers which involve cells generated during hematopoiesis,
a process by which cellular elements of blood, such as lymphocytes,
leukocytes, platelets, erythrocytes and natural killer cells are
generated are referred to as hematopoietic cancers. Lymphocytes
which can be found in blood and lymphatic tissue and are critical
for immune response are categorized into two main classes of
lymphocytes: B lymphocytes (B cells) and T lymphocytes (T cells),
which mediate humoral and cell mediated immunity, respectively.
[0005] B cells mature within the bone marrow and leave the marrow
expressing an antigen-binding antibody on their cell surface. When
a naive B cell first encounters the antigen for which its
membrane-bound antibody is specific, the cell begins to divide
rapidly and its progeny differentiate into memory B cells and
effector cells called "plasma cells". Memory B cells have a longer
life span and continue to express membrane-bound antibody with the
same specificity as the original parent cell. Plasma cells do not
produce membrane-bound antibody but instead produce the antibody in
a form that can be secreted. Secreted antibodies are the major
effector molecule of humoral immunity.
[0006] T cells mature within the thymus which provides an
environment for the proliferation and differentiation of immature T
cells. During T cell maturation, the T cells undergo the gene
rearrangements that produce the T-cell receptor and the positive
and negative selection which helps determine the cell-surface
phenotype of the mature T cell. Characteristic cell surface markers
of mature T cells are the CD3:T-cell receptor complex and one of
the coreceptors, CD4 or CD8.
[0007] In attempts to discover effective cellular targets for
cancer therapy, researchers have sought to identify transmembrane
or otherwise membrane-associated polypeptides that are specifically
expressed on the surface of one or more particular type(s) of
cancer cell as compared to on one or more normal non-cancerous
cell(s). Often, such membrane-associated polypeptides are more
abundantly expressed on the surface of the cancer cells as compared
to on the surface of the non-cancerous cells. The identification of
such tumor-associated cell surface antigen polypeptides has given
rise to the ability to specifically target cancer cells for
destruction via antibody-based therapies. In this regard, it is
noted that antibody-based therapy has proved very effective in the
treatment of certain cancers. For example, HERCEPTIN.RTM. and
RITUXAN.RTM. (both from Genentech Inc., South San Francisco,
Calif.) are antibodies that have been used successfully to treat
breast cancer and non-Hodgkin's lymphoma, respectively. More
specifically, HERCEPTIN.RTM. is a recombinant DNA-derived humanized
monoclonal antibody that selectively binds to the extracellular
domain of the human epidermal growth factor receptor 2 (HER2)
proto-oncogene. HER2 protein overexpression is observed in 25-30%
of primary breast cancers. RITUXAN.RTM. is a genetically engineered
chimeric murine/human monoclonal antibody directed against the CD20
antigen found on the surface of normal and malignant B lymphocytes.
Both these antibodies are recombinantly produced in CHO cells.
[0008] In other attempts to discover effective cellular targets for
cancer therapy, researchers have sought to identify (1)
non-membrane-associated polypeptides that are specifically produced
by one or more particular type(s) of cancer cell(s) as compared to
by one or more particular type(s) of non-cancerous normal cell(s),
(2) polypeptides that are produced by cancer cells at an expression
level that is significantly higher than that of one or more normal
non-cancerous cell(s), or (3) polypeptides whose expression is
specifically limited to only a single (or very limited number of
different) tissue type(s) in both the cancerous and non-cancerous
state (e.g., normal prostate and prostate tumor tissue). Such
polypeptides may remain intracellularly located or may be secreted
by the cancer cell. Moreover, such polypeptides may be expressed
not by the cancer cell itself, but rather by cells which produce
and/or secrete polypeptides having a potentiating or
growth-enhancing effect on cancer cells. Such secreted polypeptides
are often proteins that provide cancer cells with a growth and/or
survival advantage over normal cells and include such things as,
for example, angiogenic factors, cellular adhesion factors, growth
factors, and the like. Identification of antagonists of such
non-membrane associated polypeptides would be expected to serve as
effective therapeutic agents for the treatment of such cancers.
Furthermore, identification of the expression pattern of such
polypeptides would be useful for the diagnosis of particular
cancers in mammals.
[0009] Despite the above-identified advances in mammalian cancer
therapy, there is a great need for additional therapeutic agents
capable of detecting the presence of tumor in a mammal and for
effectively inhibiting neoplastic cell growth and/or survival,
respectively. The present invention discloses a polypeptide that is
expressed at abnormally high levels in a great variety of
non-Hodgkin's lymphomas, and provides methods and compositions for
using this polypeptide as a diagnostic and/or therapeutic target in
diagnosing and treating non-Hodgkin's lymphomas.
DISCLOSURE OF THE INVENTION
[0010] The present invention relates to the identification of a
cellular polypeptide (and its encoding nucleic acid, variant or
fragments thereof) which is specifically, differentially and/or
highly expressed by non-Hodgkin's tumors compared to normal
counterpart cell types. Interestingly and significantly, this
cellular polypeptide is highly expressed across a wide spectrum of
non-Hodgkin's lymphomas, including B and T-cell lymphomas. The
invention is also in part based on substantial insight (as
described herein) into the differential regulation of the
polypeptide with respect to specific subsets of non-Hodgkin's
lymphoma types, which provides significant advantages in tailoring
therapeutic approaches, if and where necessary, to selected
subset(s) within the wide array of tumor types that collectively
constitute the broad category of non-Hodgkin's lymphoma. This
polypeptide is herein referred to as UNQ733 polypeptide and is
expected to serve as an effective target for cancer therapy in
mammals.
[0011] Accordingly, in one aspect, the invention provides a method
for inhibiting the growth of a non-Hodgkin's lymphoma cell, wherein
the method comprises contacting the cell with a UNQ733 antagonist
which causes inhibition of the growth of the cell. In one
embodiment, the UNQ733 antagonist binds to UNQ733 polypeptide. In
one embodiment, the UNQ733 antagonist binds to UNQ733 on a
non-Hodgkin's lymphoma cell. In one embodiment, the UNQ733
antagonist causes death of a cell expressing and/or responsive to
UNQ733. In one embodiment, the UNQ733 antagonist is an antibody, an
oligopeptide, inorganic or organic small molecule. In one
embodiment, binding of the antibody, oligopeptide, inorganic or
organic small molecule to the UNQ733 polypeptide causes inhibition
of the growth of a cell expressing and/or responsive to the
polypeptide. In one embodiment, the cell is a cancer cell and the
UNQ733 antagonist causes death of the cell expressing and/or
responsive to the UNQ733 polypeptide. In one embodiment, the cell
is a cancer cell and binding of the antagonist to UNQ733
polypeptide causes death of the cell expressing and/or responsive
to the polypeptide. In one embodiment, the UNQ733 antagonist is an
antibody, oligopeptide, inorganic or organic small molecule and
binding of the antibody, oligopeptide, inorganic or organic small
molecule to the UNQ733 polypeptide causes death of the cell
expressing the polypeptide. In one embodiment, a UNQ733 antagonist
inhibits activation of UNQ733, e.g. by modulating enzymatic
processing of UNQ733 protein (e.g., by inhibiting a proprotein
convertase that cleaves UNQ733). In one embodiment, an antibody is
a monoclonal antibody, antibody fragment, chimeric antibody,
humanized antibody, human antibody, multi-specific antibody or
single-chain antibody. Antibodies, UNQ733 binding oligopeptides and
UNQ733 binding inorganic or organic small molecules employed in the
methods of the invention may optionally be conjugated to a growth
inhibitory agent or cytotoxic agent such as a toxin, including, for
example, a maytansinoid or calicheamicin, an antibiotic, a
radioactive isotope, a nucleolytic enzyme, or the like. In some
embodiments of methods of the invention, a chemotherapeutic agent
is also administered to the subject. The antibodies and UNQ733
binding oligopeptides employed in the methods of the invention may
be produced in any suitable host cell, including for example CHO
cells and bacterial cells.
[0012] In another aspect, the invention provides a method of
therapeutically treating a mammal having non-Hodgkin's lymphoma,
wherein the method comprises administering to the mammal a
therapeutically effective amount of a UNQ733 antagonist, thereby
resulting in the effective therapeutic treatment of the lymphoma.
In one embodiment, the UNQ733 antagonist is an antibody, an
oligopeptide, inorganic or organic small molecule. In one
embodiment, the UNQ733 antagonist binds to UNQ733 polypeptide,
thereby resulting in the effective therapeutic treatment of the
tumor. In one embodiment, a UNQ733 antagonist inhibits activation
of UNQ733, for example by modulating enzymatic processing of UNQ733
protein (e.g., by inhibiting a proprotein convertase that cleaves
UNQ733). In one embodiment, an antibody is a monoclonal antibody,
antibody fragment, chimeric antibody, humanized antibody, human
antibody, multi-specific antibody or single-chain antibody.
Antibodies, UNQ733 binding oligopeptides and UNQ733 binding
inorganic or organic small molecules employed in the methods of the
invention may optionally be conjugated to a growth inhibitory agent
or cytotoxic agent such as a toxin, including, for example, a
maytansinoid or calicheamicin, an antibiotic, a radioactive
isotope, a nucleolytic enzyme, or the like. In some embodiments of
methods of the invention, a chemotherapeutic agent is also
administered to the subject. The antibodies and UNQ733 binding
oligopeptides employed in the methods of the invention may be
produced in any suitable host cell, including for example CHO cells
and bacterial cells.
[0013] In one aspect, the invention provides a method of inhibiting
the growth of a non-Hodgkin's lymphoma comprising administering a
UNQ733 antagonist to the lymphoma, whereby growth of the lymphoma
is inhibited. In one aspect, the invention provides a method of
inhibiting the growth of a non-Hodgkin's lymphoma comprising
administering a UNQ733 antagonist to a cell that expresses and/or
is responsive to UNQ733, whereby growth of the lymphoma is
inhibited. In one aspect, the invention provides a method of
inhibiting growth of a non-Hodgkin's lymphoma comprising
administering a UNQ733 antagonist to a cell present in and/or
adjacent to the lymphoma, whereby growth of the lymphoma is
inhibited. In one embodiment, said cell is not a non-Hodgkin's
lymphoma cell (e.g., it is not a T or B cell); for example, said
cell may be a stromal cell. In one embodiment, the UNQ733
antagonist binds to UNQ733 polypeptide on a non-Hodgkin's lymphoma
cell. In one embodiment, the UNQ733 antagonist causes death of a
non-Hodgkin's lymphoma cell expressing and/or responsive to UNQ733.
In one embodiment, the UNQ733 antagonist is an antibody, an
oligopeptide, inorganic or organic small molecule. In one
embodiment, binding of the antibody, oligopeptide, inorganic or
organic small molecule to the UNQ733 polypeptide causes inhibition
of the growth of a non-Hodgkin's lymphoma cell expressing and/or
responsive to the polypeptide. In one embodiment, the UNQ733
antagonist is an antibody, oligopeptide, inorganic or organic small
molecule and binding of the antibody, oligopeptide or organic small
molecule to the UNQ733 polypeptide causes death of the
non-Hodgkin's lymphoma cell expressing and/or responsive to the
polypeptide. Optionally, the antibody is a monoclonal antibody,
antibody fragment, chimeric antibody, humanized antibody, human
antibody, multi-specific antibody or single-chain antibody.
Antibodies, UNQ733 binding oligopeptides and UNQ733 binding
inorganic or organic small molecules employed in the methods of the
invention may optionally be conjugated to a growth inhibitory agent
or cytotoxic agent such as a toxin, including, for example, a
maytansinoid or calicheamicin, an antibiotic, a radioactive
isotope, a nucleolytic enzyme, or the like. In one embodiment, a
UNQ733 antagonist inhibits activation of UNQ733, for example by
modulating enzymatic processing of UNQ733 protein (e.g., by
inhibiting a proprotein convertase that cleaves UNQ733). In some
embodiments of methods of the invention, a chemotherapeutic agent
is also administered. The antibodies and UNQ733 binding
oligopeptides employed in the methods of the invention may be
produced in any suitable host cell, including for example CHO cells
and bacterial cells.
[0014] In another aspect, the invention provides a method for
treating or preventing a cell proliferative disorder associated
with increased expression or activity of UNQ733, the method
comprising administering to a subject in need of such treatment an
effective amount of a UNQ733 antagonist. In one embodiment, the
cell proliferative disorder is cancer and the UNQ733 antagonist is
an anti-UNQ733 antibody, UNQ733 binding oligopeptide, UNQ733
binding inorganic or organic small molecule. In one embodiment, a
UNQ733 antagonist inhibits activation of UNQ733, e.g. by modulating
enzymatic processing of UNQ733 protein (e.g., by inhibiting a
proprotein convertase that cleaves UNQ733). Effective treatment or
prevention of the cell proliferative disorder may be a result of
direct killing or growth inhibition of cells that express and/or
are responsive to UNQ733 polypeptide or by antagonizing the cell
growth potentiating activity of UNQ733 polypeptide. In one
embodiment, the cell proliferative disorder is a non-Hodgkin's
lymphoma. In one embodiment, the cell proliferative disorder is
hyperplasia, which in one embodiment is diseased, non-malignant
tonsilar tissue.
[0015] In one aspect the invention provides a method of diagnosing
a non-Hodgkin's lymphoma of interest comprising determining the
level of UNQ733 in a test sample of tissue cells comprising cells
of origin for the lymphoma of interest and in a control sample of
known cells of the same tissue origin, wherein a higher level of
UNQ733 in the test sample, as compared to the control sample, is
indicative of the presence of the lymphoma. In another aspect, the
invention provides a method of determining whether an individual is
at risk for non-Hodgkin's lymphoma comprising determining the level
of UNQ733 in a test sample of tissue cells comprising cells of
origin for the lymphoma of interest from said individual and in a
control sample of known cells of the same tissue origin, wherein a
higher level of UNQ733 in the test sample, as compared to the
control sample, is an indication that the individual is at risk for
the lymphoma. In one embodiment of methods of the invention, the
level of UNQ733 is determined based on level of UNQ733 RNA in the
test sample. In one embodiment of methods of the invention, the
level of UNQ733 is determined based on level of UNQ733 polypeptide
in the test sample. In one embodiment of methods of the invention,
the level of UNQ733 is determined based on the level of UNQ733
protein activity in the sample. In some embodiments, the method
comprises exposing the sample to an antibody, oligopeptide,
inorganic or organic small molecule that binds to the UNQ733
polypeptide and determining binding of the antibody, oligopeptide,
inorganic or organic small molecule to the UNQ733 polypeptide in
the sample. An antibody, UNQ733 binding oligopeptide, or UNQ733
binding inorganic or organic small molecule employed in the method
may optionally be detectably labeled, attached to a solid support,
or the like.
[0016] In one aspect, the invention provides a method for screening
for biologically active agents for the treatment of non-Hodgkin's
lymphoma comprising combining a candidate agent with a transgenic
mammal having a genome comprising an integrated transgene encoding
UNQ733 operably linked to a promoter, wherein said transgene
results in said mammal acquiring non-Hodgkin's lymphoma and
determining the effect of said agent on non-Hodgkin's lymphoma in
said mammal. In yet another aspect, the invention provides a method
for screening for biologically active agents for the treatment of
non-Hodgkin's lymphoma comprising combining a candidate agent with
a transgenic mammal cell culture, each cell of said culture
comprising an integrated transgene encoding UNQ733 operably linked
to a promoter, wherein said transgene results in said mammal
acquiring non-Hodgkin's lymphoma, and determining the effect of
said candidate agent on the transgenic mammal cell culture. In one
embodiment of these methods, the candidate agent is an antibody or
fragment thereof, an oligopeptide, or an inorganic or organic small
molecule.
[0017] Yet another aspect of the present invention is directed to a
method of binding an antibody, oligopeptide, inorganic or organic
small molecule to a non-Hodgkin's lymphoma cell that expresses
and/or is responsive to UNQ733, wherein the method comprises
contacting said cell with said antibody, oligopeptide, inorganic or
organic small molecule under conditions which are suitable for
binding of the antibody, oligopeptide, or inorganic or organic
small molecule to UNQ733 polypeptide and allowing binding
therebetween. In one embodiment, binding of said antibody,
oligopeptide, inorganic or organic small molecule to UNQ733 on the
cell inhibits a UNQ733 biological function. In one embodiment, said
antibody, oligopeptide, inorganic or organic small molecule does
not compete with UNQ733 for binding to the cell. In one embodiment,
said antibody, oligopeptide, inorganic or organic small molecule
does not inhibit binding of UNQ733 to the cell. In one embodiment,
said antibody, oligopeptide, inorganic or organic small molecule
binds to UNQ733 bound on the cell and inhibits binding of free
(unbound) UNQ733 to the cell. In one embodiment, the UNQ733 binding
molecule is an antibody. In one embodiment, the antibody is
designated 3E7.9.20 (ATCC deposit no. PTA-6026), 3F10.11.2 (ATCC
deposit no. PTA-6027), 3H1.4.8 (ATCC deposit no. PTA-6028),
4A9.12.12 (ATCC deposit no. PTA-6029), 5A8.11.6 (ATCC deposit no.
PTA-6030), 5F2.6.14 (ATCC deposit no. PTA-6031), 9D6.11.15 (ATCC
deposit no. PTA-6032), 10G10.15.16 (ATCC deposit no. PTA-6033) or
12H4.11.3 (ATCC deposit no. PTA-6034). In one embodiment, the
antibody binds to the same epitope on UNQ733 as the antibody
designated 3E7.9.20 (ATCC deposit no. PTA-6026), 3F10.11.2 (ATCC
deposit no. PTA-6027), 3H1.4.8 (ATCC deposit no. PTA-6028),
4A9.12.12 (ATCC deposit no. PTA-6029), 5A8.11.6 (ATCC deposit no.
PTA-6030), 5F2.6.14 (ATCC deposit no. PTA-6031), 9D6.11.15 (ATCC
deposit no. PTA-6032), 10G10.15.16 (ATCC deposit no. PTA-6033)
and/or 12H4.11.3 (ATCC deposit no. PTA-6034). In one embodiment,
the antibody competes with the antibody designated 3E7.9.20 (ATCC
deposit no. PTA-6026), 3F10.11.2 (ATCC deposit no. PTA-6027),
3H1.4.8 (ATCC deposit no. PTA-6028), 4A9.12.12 (ATCC deposit no.
PTA-6029), 5A8.11.6 (ATCC deposit no. PTA-6030), 5F2.6.14 (ATCC
deposit no. PTA-6031), 9D6.11.15 (ATCC deposit no. PTA-6032),
10G10.15.16 (ATCC deposit no. PTA-6033) and/or 12H4.11.3 (ATCC
deposit no. PTA-6034) for binding to UNQ733 (e.g., where UNQ733 is
located in a cell free environment, is a secreted protein in vivo
or in vitro, or is bound to a cell (in vitro or in vivo)).
[0018] In one aspect, the invention provides a method of targeting
a therapeutic agent to a non-Hodgkin's lymphoma in a host, the
method comprising administering to the host said therapeutic agent
in a form that is linked to a molecule that binds UNQ733, whereby
the agent is targeted to the lymphoma in the host. The molecule
that binds UNQ733 can be any molecule capable of specifically
binding to UNQ733 protein in vivo, for example an antibody, an
oligopeptide, an inorganic or organic small molecule. In one
embodiment, the molecule that binds UNQ733 is a molecule capable of
specifically binding to UNQ733 located on a cell (either in vitro
or in vivo), for example where UNQ733 is bound to the surface of a
non-Hodgkin's lymphoma cell. In one embodiment, the molecule is an
antibody. In one embodiment, the antibody is designated 3E7.9.20
(ATCC deposit no. PTA-6026), 3F10.11.2 (ATCC deposit no. PTA-6027),
3H1.4.8 (ATCC deposit no. PTA-6028), 4A9.12.12 (ATCC deposit no.
PTA-6029), 5A8.11.6 (ATCC deposit no. PTA-6030), 5F2.6.14 (ATCC
deposit no. PTA-6031), 9D6.11.15 (ATCC deposit no. PTA-6032),
10G10.15.16 (ATCC deposit no. PTA-6033) or 12H4.11.3 (ATCC deposit
no. PTA-6034). In one embodiment, the antibody binds to the same
epitope on UNQ733 as the antibody designated 3E7.9.20 (ATCC deposit
no. PTA-6026), 3F10.11.2 (ATCC deposit no. PTA-6027), 3H1.4.8 (ATCC
deposit no. PTA-6028), 4A9.12.12 (ATCC deposit no. PTA-6029),
5A8.11.6 (ATCC deposit no. PTA-6030), 5F2.6.14 (ATCC deposit no.
PTA-6031), 9D6.11.15 (ATCC deposit no. PTA-6032), 10G10.15.16 (ATCC
deposit no. PTA-6033) and/or 12H4.11.3 (ATCC deposit no. PTA-6034).
In one embodiment, the antibody competes with the antibody
designated 3E7.9.20 (ATCC deposit no. PTA-6026), 3F10.11.2 (ATCC
deposit no. PTA-6027), 3H1.4.8 (ATCC deposit no. PTA-6028),
4A9.12.12 (ATCC deposit no. PTA-6029), 5A8.11.6 (ATCC deposit no.
PTA-6030), 5F2.6.14 (ATCC deposit no. PTA-6031), 9D6.11.15 (ATCC
deposit no. PTA-6032), 10G10.15.16 (ATCC deposit no. PTA-6033)
and/or 12H4.11.3 (ATCC deposit no. PTA-6034) for binding to UNQ733
(e.g., where UNQ733 is located in a cell free environment, is a
secreted protein in vivo or in vitro, or is bound to a cell (in
vitro or in vivo)). In one embodiment, the therapeutic agent is
linked to the molecule that binds UNQ733 in the form of a
multi-specific (e.g., bi-specific) antibody.
[0019] Other embodiments of the invention are directed to the use
of (a) a UNQ733 polypeptide (processed or unprocessed by a
proprotein convertase, singly or in combination), (b) a nucleic
acid encoding a processed or unprocessed UNQ733 polypeptide or a
vector or host cell comprising that nucleic acid, (c) an
anti-UNQ733 polypeptide antibody, (d) a UNQ733 binding
oligopeptide, (e) a UNQ733 binding inorganic or organic small
molecule, or (f) an inhibitor of an in vivo enzyme (such as a
proprotein convertase) that cleaves UNQ733 protein, in the
preparation of a medicament useful for the therapeutic treatment of
a non-Hodgkin's lymphoma.
[0020] In one aspect, the invention provides a UNQ733 binding
molecule useful in a method of the invention as herein described.
For example, a UNQ733 binding molecule can be a molecule (such as
an antibody) that binds to UNQ733 and inhibits binding of UNQ733 to
its receptor. In another example, a UNQ733 binding molecule can be
a molecule (such as an antibody) that binds UNQ733 that is bound to
a cell. In one embodiment, the binding molecule binds UNQ733 that
is bound to a cell but does not inhibit UNQ733 binding to a cell.
In yet another example, a UNQ733 binding molecule is a molecule
(such as an antibody) that binds UNQ733 only when said UNQ733 is
bound to a cell. In one embodiment, the binding molecule binds a
first UNQ733 polypeptide only when said first polypeptide is bound
to a cell but does not inhibit binding of a second UNQ733
polypeptide to a cell. In some embodiments, it is advantageous for
a UNQ733 binding molecule of the invention to be conjugated to a
therapeutic agent (e.g., a toxin, etc. as described in greater
detail hereinbelow). In one aspect, the invention provides a
composition comprising a UNQ733 antagonist, wherein the composition
is suitable for administration to a subject having non-Hodgkin's
lymphoma. In one embodiment, the composition comprises a
therapeutically effective amount of the UNQ733 antagonist. In one
embodiment, the composition further comprises a carrier which in
some embodiments is a pharmaceutically acceptable carrier. In one
embodiment, the UNQ733 antagonist is an antibody. In one
embodiment, the antibody is designated 3E7.9.20 (ATCC deposit no.
PTA-6026), 3F10.11.2 (ATCC deposit no. PTA-6027), 3H1.4.8 (ATCC
deposit no. PTA-6028), 4A9.12.12 (ATCC deposit no. PTA-6029),
5A8.11.6 (ATCC deposit no. PTA-6030), 5F2.6.14 (ATCC deposit no.
PTA-6031), 9D6.11.15 (ATCC deposit no. PTA-6032), 10G10.15.16 (ATCC
deposit no. PTA-6033) or 12H4.11.3 (ATCC deposit no. PTA-6034). In
one embodiment, the antibody binds to the same epitope on UNQ733 as
the antibody designated 3E7.9.20 (ATCC deposit no. PTA-6026),
3F10.11.2 (ATCC deposit no. PTA-6027), 3H1.4.8 (ATCC deposit no.
PTA-6028), 4A9.12.12 (ATCC deposit no. PTA-6029), 5A8.11.6 (ATCC
deposit no. PTA-6030), 5F2.6.14 (ATCC deposit no. PTA-6031),
9D6.11.15 (ATCC deposit no. PTA-6032), 10G10.15.16 (ATCC deposit
no. PTA-6033) and/or 12H4.11.3 (ATCC deposit no. PTA-6034). In one
embodiment, the antibody competes with the antibody designated
3E7.9.20 (ATCC deposit no. PTA-6026), 3F10.11.2 (ATCC deposit no.
PTA-6027), 3H1.4.8 (ATCC deposit no. PTA-6028), 4A9.12.12 (ATCC
deposit no. PTA-6029), 5A8.11.6 (ATCC deposit no. PTA-6030),
5F2.6.14 (ATCC deposit no. PTA-6031), 9D6.11.15 (ATCC deposit no.
PTA-6032), 10G10.15.16 (ATCC deposit no. PTA-6033) and/or 12H4.11.3
(ATCC deposit no. PTA-6034) for binding to UNQ733 (e.g., where
UNQ733 is located in a cell free environment, is a secreted protein
in vivo or in vitro, or is bound to a cell (in vitro or in
vivo)).
[0021] In yet another aspect, the invention provides an article of
manufacture comprising a container and a composition contained
within the container, wherein the composition comprises a UNQ733
antagonist as described herein. The article may further optionally
comprise a label affixed to the container, and/or a package insert
included with the container, that refers to the use of the
composition for therapeutic treatment of non-Hodgkin's lymphoma. In
one embodiment, the invention provides a kit comprising a
composition comprising a UNQ733 antagonist as described herein,
optionally further comprising instruction for using the composition
for treatment of non-Hodgkin's lymphoma.
[0022] In one aspect, the invention provides a composition
comprising a UNQ733 polypeptide binding molecule, wherein the
composition is suitable for detecting UNQ733 polypeptide in a test
sample from an individual suspected of having non-Hodgkin's
lymphoma. In one embodiment, the binding molecule is an antibody,
oligopeptide, inorganic or organic small molecule. In one
embodiment, the binding molecule is an antibody, wherein the
antibody is capable of binding to UNQ733 in a detection assay such
as western blot, immunoprecipitation and/or immunostaining. In one
embodiment, the molecule is an antibody. In one embodiment, the
antibody is designated 3E7.9.20 (ATCC deposit no. PTA-6026),
3F10.11.2 (ATCC deposit no. PTA-6027), 3H1.4.8 (ATCC deposit no.
PTA-6028), 4A9.12.12 (ATCC deposit no. PTA-6029), 5A8.11.6 (ATCC
deposit no. PTA-6030), 5F2.6.14 (ATCC deposit no. PTA-6031),
9D6.11.15 (ATCC deposit no. PTA-6032), 10G10.15.16 (ATCC deposit
no. PTA-6033) or 12H4.11.3 (ATCC deposit no. PTA-6034). In one
embodiment, the antibody binds to the same epitope on UNQ733 as the
antibody designated 3E7.9.20 (ATCC deposit no. PTA-6026), 3F10.11.2
(ATCC deposit no. PTA-6027), 3H1.4.8 (ATCC deposit no. PTA-6028),
4A9.12.12 (ATCC deposit no. PTA-6029), 5A8.11.6 (ATCC deposit no.
PTA-6030), 5F2.6.14 (ATCC deposit no. PTA-6031), 9D6.11.15 (ATCC
deposit no. PTA-6032), 10G10.15.16 (ATCC deposit no. PTA-6033)
and/or 12H4.11.3 (ATCC deposit no. PTA-6034). In one embodiment,
the antibody competes with the antibody designated 3E7.9.20 (ATCC
deposit no. PTA-6026), 3F10.11.2 (ATCC deposit no. PTA-6027),
3H1.4.8 (ATCC deposit no. PTA-6028), 4A9.12.12 (ATCC deposit no.
PTA-6029), 5A8.11.6 (ATCC deposit no. PTA-6030), 5F2.6.14 (ATCC
deposit no. PTA-6031), 9D6.11.15 (ATCC deposit no. PTA-6032),
10G10.15.16 (ATCC deposit no. PTA-6033) and/or 12H4.11.3 (ATCC
deposit no. PTA-6034) for binding to UNQ733 (e.g., where UNQ733 is
located in a cell free environment, is a secreted protein in vivo
or in vitro, or is bound to a cell (in vitro or in vivo)). In one
embodiment, the composition further comprises a carrier.
[0023] In yet another aspect, the invention provides an article of
manufacture comprising a container and a composition contained
within the container, wherein the composition comprises a UNQ733
polypeptide binding molecule and is suitable for detecting UNQ733
polypeptide in a test sample from an individual suspected of having
non-Hodgkin's lymphoma. The article may further optionally comprise
a label affixed to the container, and/or a package insert included
with the container, that refers to the use of the composition for
detection of non-Hodgkin's lymphoma. In one embodiment, the
invention provides a kit comprising a composition comprising a
UNQ733 polypeptide binding molecule, optionally further comprising
instruction for using the composition for detecting non-Hodgkin's
lymphoma. In one embodiment, the binding molecule is an antibody,
wherein the antibody is capable of binding to UNQ733 in a detection
assay such as western blot, immunoprecipitation and/or
immunostaining. In one embodiment, the antibody is In one
embodiment, the molecule is an antibody. In one embodiment, the
antibody is designated 3E7.9.20 (ATCC deposit no. PTA-6026),
3F10.11.2 (ATCC deposit no. PTA-6027), 3H1.4.8 (ATCC deposit no.
PTA-6028), 4A9.12.12 (ATCC deposit no. PTA-6029), 5A8.11.6 (ATCC
deposit no. PTA-6030), 5F2.6.14 (ATCC deposit no. PTA-6031),
9D6.11.15 (ATCC deposit no. PTA-6032), 10G10.15.16 (ATCC deposit
no. PTA-6033) or 12H4.11.3 (ATCC deposit no. PTA-6034). In one
embodiment, the antibody binds to the same epitope on UNQ733 as the
antibody designated 3E7.9.20 (ATCC deposit no. PTA-6026), 3F10.11.2
(ATCC deposit no. PTA-6027), 3H1.4.8 (ATCC deposit no. PTA-6028),
4A9.12.12 (ATCC deposit no. PTA-6029), 5A8.11.6 (ATCC deposit no.
PTA-6030), 5F2.6.14 (ATCC deposit no. PTA-6031), 9D6.11.15 (ATCC
deposit no. PTA-6032), 10G10.15.16 (ATCC deposit no. PTA-6033)
and/or 12H4.11.3 (ATCC deposit no. PTA-6034). In one embodiment,
the antibody competes with the antibody designated 3E7.9.20 (ATCC
deposit no. PTA-6026), 3F10.11.2 (ATCC deposit no. PTA-6027),
3H1.4.8 (ATCC deposit no. PTA-6028), 4A9.12.12 (ATCC deposit no.
PTA-6029), 5A8.11.6 (ATCC deposit no. PTA-6030), 5F2.6.14 (ATCC
deposit no. PTA-6031), 9D6.11.15 (ATCC deposit no. PTA-6032),
10G10.15.16 (ATCC deposit no. PTA-6033) and/or 12H4.11.3 (ATCC
deposit no. PTA-6034) for binding to UNQ733 (e.g., where UNQ733 is
located in a cell free environment, is a secreted protein in vivo
or in vitro, or is bound to a cell (in vitro or in vivo)).
[0024] As described herein, an antibody of the invention can be in
any suitable form for use in a method of the invention. For
example, an antibody of the invention can be human, humanized or
chimeric. In one embodiment, an antibody of the invention is the
humanized or chimeric form of the antibody designated 3E7.9.20
(ATCC deposit no. PTA-6026), 3F10.11.2 (ATCC deposit no. PTA-6027),
3H1.4.8 (ATCC deposit no. PTA-6028), 4A9.12.12 (ATCC deposit no.
PTA-6029), 5A8.11.6 (ATCC deposit no. PTA-6030), 5F2.6.14 (ATCC
deposit no. PTA-6031), 9D6.11.15 (ATCC deposit no. PTA-6032),
10G10.15.16 (ATCC deposit no. PTA-6033) or 12H4.11.3 (ATCC deposit
no. PTA-6034). In one embodiment, an antibody of the invention is a
humanized, chimeric or human antibody that binds to the same
epitope on UNQ733 as the antibody designated 3E7.9.20 (ATCC deposit
no. PTA-6026), 3F10.11.2 (ATCC deposit no. PTA-6027), 3H1.4.8 (ATCC
deposit no. PTA-6028), 4A9.12.12 (ATCC deposit no. PTA-6029),
5A8.11.6 (ATCC deposit no. PTA-6030), 5F2.6.14 (ATCC deposit no.
PTA-6031), 9D6.11.15 (ATCC deposit no. PTA-6032), 10G10.15.16 (ATCC
deposit no. PTA-6033) and/or 12H4.11.3 (ATCC deposit no. PTA-6034).
In one embodiment, an antibody of the invention is a humanized,
chimeric or human antibody that competes with the antibody
designated 3E7.9.20 (ATCC deposit no. PTA-6026), 3F10.11.2 (ATCC
deposit no. PTA-6027), 3H1.4.8 (ATCC deposit no. PTA-6028),
4A9.12.12 (ATCC deposit no. PTA-6029), 5A8.11.6 (ATCC deposit no.
PTA-6030), 5F2.6.14 (ATCC deposit no. PTA-6031), 9D6.11.15 (ATCC
deposit no. PTA-6032), 10G10.15.16 (ATCC deposit no. PTA-6033)
and/or 12H4.11.3 (ATCC deposit no. PTA-6034) for binding to UNQ733
(e.g., where UNQ733 is located in a cell free environment, is a
secreted protein in vivo or in vitro, or is bound to a cell (in
vitro or in vivo)). In one embodiment, an antibody of the invention
comprises one, two, three, four, five or all of the hypervariable
regions, hypervariable loops and/or complementarity determining
region (CDR) sequences of the antibody designated 3E7.9.20 (ATCC
deposit no. PTA-6026), 3F10.11.2 (ATCC deposit no. PTA-6027),
3H1.4.8 (ATCC deposit no. PTA-6028), 4A9.12.12 (ATCC deposit no.
PTA-6029), 5A8.11.6 (ATCC deposit no. PTA-6030), 5F2.6.14 (ATCC
deposit no. PTA-6031), 9D6.11.15 (ATCC deposit no. PTA-6032),
10G10.15.16 (ATCC deposit no. PTA-6033) or 12H4.11.3 (ATCC deposit
no. PTA-6034). In one embodiment, an antibody of the invention
comprises one or both variable domains, or portion thereof, of the
antibody designated 3E7.9.20 (ATCC deposit no. PTA-6026), 3F10.11.2
(ATCC deposit no. PTA-6027), 3H1.4.8 (ATCC deposit no. PTA-6028),
4A9.12.12 (ATCC deposit no. PTA-6029), 5A8.11.6 (ATCC deposit no.
PTA-6030), 5F2.6.14 (ATCC deposit no. PTA-6031), 9D6.11.15 (ATCC
deposit no. PTA-6032), 10G10.15.16 (ATCC deposit no. PTA-6033) or
12H4.11.3 (ATCC deposit no. PTA-6034).
[0025] In some embodiments, methods and compositions of the
invention are directed to a non-Hodgkin's lymphoma that is a B-cell
lymphoma (not-otherwise-specified--"NOS"), diffuse large B cell
lymphoma, follicular lymphoma, small lymphocytic lymphoma,
malignant lymphoma (NOS), malignant T cell lymphoma, anaplastic
large cell lymphoma or mucosal associated lymphoid tissue
lymphoma.
[0026] Yet further embodiments of the present invention will be
evident to the skilled artisan upon a reading of the present
specification.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 depicts GeneLogic microarray expression data of the
UNQ733 mRNA.
[0028] FIG. 2 depicts UNQ733 mRNA overexpression profile in
non-Hodgkin's lymphomas assessed by in situ hybridization on tissue
microarray.
[0029] FIG. 3 depicts UNQ733 mRNA expression profile in normal
human tissues as assessed by in situ hybridization.
[0030] FIGS. 4A-D depict UNQ733 mRNA expression in germinal centers
and crypts.
[0031] FIG. 5 depicts UNQ733 mRNA expression profile in neoplastic
tissues.
[0032] FIGS. 6A-C depict cellular localization of UNQ733 mRNA in
malignant cells of non-Hodgkin's lymphoma.
[0033] FIG. 7 depicts Western blot analysis of UNQ733 protein.
[0034] FIG. 8 depicts immunoprecipitation data for UNQ733
protein.
[0035] FIG. 9 depicts immunohistochemistry data for UNQ733 protein.
Expression is observed in a subset of cells in tonsil crypt
(arrows) and in germinal centers (GC), most consisted with a
follicular dendritic cell.
[0036] FIG. 10 depicts flow cytometry data.
[0037] FIG. 11 depicts the effect of a proprotein convertase on
UNQ733. Lanes marked "v" refer to samples from vector mock
transfectants. Lanes marked "733" refer to samples from UNQ733
transfectants.
[0038] FIG. 12 depicts proportions of various UNQ733 protein
species obtained when cells were contacted with or without furin
inhibitor.
[0039] FIGS. 13A-B depict flow cytometry data for UNQ733 protein
binding to lymphoma cells.
[0040] FIG. 14 depicts flow cytometry data for detection of UNQ733
protein bound to non-Hodgkin's lymphoma cells.
[0041] FIGS. 15A-B depict binding of UNQ733 protein to C1R B
lymphoblast cells in a dose-dependent manner (A) and competition
binding data indicating specificy of binding of UNQ733 to these
cells (B).
[0042] FIG. 16 depicts UNQ733 binding on normal B cells that are
unstimulated or stimulated with agonistic antibody to CD40 and
recombinant IL-4.
[0043] FIG. 17 shows a nucleotide sequence (SEQ ID NO:1) of a
UNQ733 cDNA. Underlined codons in bold are the predicted start and
stop codons, respectively.
[0044] FIG. 18 shows the amino acid sequences (SEQ ID NOs:2-4) of
native and variant UNQ733 polypeptides.
MODES FOR CARRYING OUT THE INVENTION
I. Definitions
[0045] The terms "UNQ733 polypeptide" and "UNQ733 protein" as used
herein encompass native sequence polypeptides, polypeptide variants
and fragments of a native sequence polypeptide and polypeptide
variants (which are further defined herein). The UNQ733 polypeptide
described herein may be that which is isolated from a variety of
sources, such as from human tissue types or from another source, or
prepared by recombinant or synthetic methods. The terms "UNQ733
polypeptide" and "UNQ733 protein" also include variants of a UNQ733
polypeptide as disclosed herein.
[0046] A "native sequence UNQ733 polypeptide" comprises a
polypeptide having the same amino acid sequence as the
corresponding UNQ733 polypeptide derived from nature. In one
embodiment, a native sequence UNQ733 polypeptide comprises the
amino acid sequence of SEQ ID NO:2 (see FIG. 18). In another
embodiment, a native sequence UNQ733 polypeptide comprises an amino
acid sequence lacking the signal peptide. In one embodiment, a
native sequence UNQ733 polypeptide comprises the amino acid
sequence of SEQ ID NO:3 (see FIG. 18). In yet another embodiment, a
native sequence UNQ733 polypeptide comprises an amino acid sequence
resulting from enzymatic cleavage of the sequence of SEQ ID NO:2
with a proprotein convertase. In one embodiment, a native sequence
UNQ733 polypeptide comprises the amino acid sequence of SEQ ID NO:4
(see FIG. 18). Such native sequence UNQ733 polypeptide can be
isolated from nature or can be produced by recombinant or synthetic
means. The term "native sequence UNQ733 polypeptide" specifically
encompasses naturally-occurring truncated or secreted forms of the
specific UNQ733 polypeptide (e.g., an extracellular domain
sequence), naturally-occurring variant forms (e.g., alternatively
spliced forms) and naturally-occurring allelic variants of the
polypeptide. Native sequence UNQ733 is also reported in Marshall et
al, J. Immunol. (2002), 169:2381-2389; PCT Pub. No. WO9931117; and
PCT Pub. No. WO02/08288.
[0047] "UNQ733 polypeptide variant" means a UNQ733 polypeptide,
preferably an active UNQ733 polypeptide, as defined herein having
at least about 80% amino acid sequence identity with any of the
native sequence UNQ733 polypeptide sequences as disclosed herein.
Such UNQ733 polypeptide variants include, for instance, UNQ733
polypeptides wherein one or more amino acid residues are added, or
deleted, at the N- or C-terminus of a native amino acid sequence.
Ordinarily, a UNQ733 polypeptide variant will have at least about
80% amino acid sequence identity, alternatively at least about 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to a
native sequence UNQ733 polypeptide sequence as disclosed herein.
Ordinarily, UNQ733 variant polypeptides are at least about 10 amino
acids in length, alternatively at least about 20, 30, 40, 50, 60,
70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,
210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330,
340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460,
470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,
600 amino acids in length, or more. Optionally, UNQ733 variant
polypeptides will have no more than one conservative amino acid
substitution as compared to a native UNQ733 polypeptide sequence,
alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10
conservative amino acid substitution as compared to the native
UNQ733 polypeptide sequence.
[0048] "Percent (%) amino acid sequence identity" with respect to
the UNQ733 polypeptide sequences identified herein is defined as
the percentage of amino acid residues in a candidate sequence that
are identical with the amino acid residues in the specific UNQ733
polypeptide sequence, after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent sequence
identity, and not considering any conservative substitutions as
part of the sequence identity. Alignment for purposes of
determining percent amino acid sequence identity can be achieved in
various ways that are within the skill in the art, for instance,
using publicly available computer software such as BLAST, BLAST-2,
ALIGN or Megalign (DNASTAR) software. Those skilled in the art can
determine appropriate parameters for measuring alignment, including
any algorithms needed to achieve maximal alignment over the full
length of the sequences being compared. For purposes herein,
however, % amino acid sequence identity values are generated using
the sequence comparison computer program ALIGN-2, wherein the
complete source code for the ALIGN-2 program is provided in Table 1
below. The ALIGN-2 sequence comparison computer program was
authored by Genentech, Inc. and the source code shown in Table 1
below has been filed with user documentation in the U.S. Copyright
Office, Washington D.C., 20559, where it is registered under U.S.
Copyright Registration No. TXU510087. The ALIGN-2 program is
publicly available through Genentech, Inc., South San Francisco,
Calif. or may be compiled from the source code provided in Table 1
below. The ALIGN-2 program should be compiled for use on a UNIX
operating system, preferably digital UNIX V4.0D. All sequence
comparison parameters are set by the ALIGN-2 program and do not
vary.
[0049] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows: 100 times the fraction X/Y where X is
the number of amino acid residues scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of A and B, and where Y is the total number of amino acid residues
in B. It will be appreciated that where the length of amino acid
sequence A is not equal to the length of amino acid sequence B, the
% amino acid sequence identity of A to B will not equal the % amino
acid sequence identity of B to A. As examples of % amino acid
sequence identity calculations using this method, Tables 2 and 3
demonstrate how to calculate the % amino acid sequence identity of
the amino acid sequence designated "Comparison Protein" to the
amino acid sequence designated "UNQ733", wherein "UNQ733"
represents the amino acid sequence of a hypothetical UNQ733
polypeptide of interest, "Comparison Protein" represents the amino
acid sequence of a polypeptide against which the "UNQ733"
polypeptide of interest is being compared, and "X, "Y" and "Z" each
represent different hypothetical amino acid residues. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0050] "UNQ733 variant polynucleotide" or "UNQ733 variant nucleic
acid sequence" means a nucleic acid molecule which encodes a UNQ733
polypeptide, preferably an active UNQ733 polypeptide, as defined
herein and which has at least about 80% nucleic acid sequence
identity with a nucleotide acid sequence encoding a native sequence
UNQ733 polypeptide sequence as disclosed herein. Ordinarily, a
UNQ733 variant polynucleotide will have at least about 80% nucleic
acid sequence identity, alternatively at least about 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, or 99% nucleic acid sequence identity with a nucleic acid
sequence encoding a native sequence UNQ733 polypeptide sequence as
disclosed herein. Variants do not encompass the native nucleotide
sequence.
[0051] Ordinarily, UNQ733 variant polynucleotides are at least
about 5 nucleotides in length, alternatively at least about 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155,
160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240,
250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370,
380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500,
510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630,
640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760,
770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890,
900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000
nucleotides in length, wherein in this context the term "about"
means the referenced nucleotide sequence length plus or minus 10%
of that referenced length.
[0052] "Percent (%) nucleic acid sequence identity" with respect to
a UNQ733 nucleic acid sequence (encoding a native sequence UNQ733
polypeptide, for example the nucleic acid sequence of SEQ ID NO:1
in FIG. 17) disclosed herein is defined as the percentage of
nucleotides in a candidate sequence that are identical with the
nucleotides in the UNQ733 nucleic acid sequence of interest, after
aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity. Alignment for
purposes of determining percent nucleic acid sequence identity can
be achieved in various ways that are within the skill in the art,
for instance, using publicly available computer software such as
BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For purposes
herein, however, % nucleic acid sequence identity values are
generated using the sequence comparison computer program ALIGN-2,
wherein the complete source code for the ALIGN-2 program is
provided in Table 1 below. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc. and the source code shown
in Table 1 below has been filed with user documentation in the U.S.
Copyright Office, Washington D.C., 20559, where it is registered
under U.S. Copyright Registration No. TXU510087. The ALIGN-2
program is publicly available through Genentech, Inc., South San
Francisco, Calif. or may be compiled from the source code provided
in Table 1 below. The ALIGN-2 program should be compiled for use on
a UNIX operating system, preferably digital UNIX V4.0D. All
sequence comparison parameters are set by the ALIGN-2 program and
do not vary.
[0053] In situations where ALIGN-2 is employed for nucleic acid
sequence comparisons, the % nucleic acid sequence identity of a
given nucleic acid sequence C to, with, or against a given nucleic
acid sequence D (which can alternatively be phrased as a given
nucleic acid sequence C that has or comprises a certain % nucleic
acid sequence identity to, with, or against a given nucleic acid
sequence D) is calculated as follows: 100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of C and D, and where Z is the total number of nucleotides in D. It
will be appreciated that where the length of nucleic acid sequence
C is not equal to the length of nucleic acid sequence D, the %
nucleic acid sequence identity of C to D will not equal the %
nucleic acid sequence identity of D to C. As examples of % nucleic
acid sequence identity calculations, Tables 4 and 5, demonstrate
how to calculate the % nucleic acid sequence identity of the
nucleic acid sequence designated "Comparison DNA" to the nucleic
acid sequence designated "UNQ733 DNA", wherein "UNQ733 DNA"
represents a hypothetical UNQ733 nucleic acid sequence of interest,
"Comparison DNA" represents the nucleotide sequence of a nucleic
acid molecule against which the "UNQ733 DNA" nucleic acid molecule
of interest is being compared, and "N", "L" and "V" each represent
different hypothetical nucleotides. Unless specifically stated
otherwise, all % nucleic acid sequence identity values used herein
are obtained as described in the immediately preceding paragraph
using the ALIGN-2 computer program.
[0054] In other embodiments, UNQ733 variant polynucleotides are
nucleic acid molecules that encode a UNQ733 polypeptide and which
are capable of hybridizing, preferably under stringent
hybridization and wash conditions, to nucleotide sequences encoding
a UNQ733 polypeptide as disclosed herein. UNQ733 variant
polypeptides may be those that are encoded by a UNQ733 variant
polynucleotide.
[0055] "Isolated," means a molecule/compound (such as a
polypeptide) 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
typically interfere with therapeutic uses for the molecule/compound
(such as a polypeptide), and may include enzymes, hormones, and
other proteinaceous or non-proteinaceous solutes. In some
embodiments, a polypeptide or oligopeptide will be purified (1) to
a degree sufficient to obtain at least 10-15 residues of N-terminal
or internal amino acid sequence by use of a spinning cup
sequenator, or (2) to homogeneity by gel electrophoresis, for
example SDS-PAGE under non-reducing or reducing conditions using,
for example, Coomassie blue or silver stain. Isolated polypeptide
includes polypeptide in situ within recombinant cells, since at
least one component of the UNQ733 polypeptide natural environment
will not be present. Ordinarily, however, isolated polypeptide will
be prepared by at least one purification step.
[0056] An "isolated" polynucleotide is a polypeptide or
oligopeptide-encoding nucleic acid molecule that is identified and
separated from at least one contaminant nucleic acid molecule with
which it is ordinarily associated in the natural source of the
polypeptide or oligopeptide-encoding nucleic acid. An isolated
polypeptide or oligopeptide-encoding nucleic acid molecule is other
than in the form or setting in which it is found in nature.
Isolated polypeptide or oligopeptide-encoding nucleic acid
molecules therefore are distinguished from the specific polypeptide
or oligopeptide-encoding nucleic acid molecule as it exists in
natural cells. However, an isolated polypeptide or
oligopeptide-encoding nucleic acid molecule includes polypeptide or
oligopeptide-encoding nucleic acid molecules contained in cells
that ordinarily express the polypeptide or oligopeptide where, for
example, the nucleic acid molecule is in a chromosomal or
extrachromosomal location different from that of natural cells.
[0057] The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0058] Nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
that participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, "operably linked" means that the
DNA sequences being linked are contiguous, and, in the case of a
secretory leader, contiguous and in reading phase. However,
enhancers do not have to be contiguous. Linking is accomplished by
ligation at convenient restriction sites. If such sites do not
exist, the synthetic oligonucleotide adaptors or linkers are used
in accordance with conventional practice.
[0059] "Stringency" of hybridization reactions is readily
determinable by one of ordinary skill in the art, and generally is
an empirical calculation dependent upon probe length, washing
temperature, and salt concentration. In general, longer probes
require higher temperatures for proper annealing, while shorter
probes need lower temperatures. Hybridization generally depends on
the ability of denatured DNA to reanneal when complementary strands
are present in an environment below their melting temperature. The
higher the degree of desired homology between the probe and
hybridizable sequence, the higher the relative temperature which
can be used. As a result, it follows that higher relative
temperatures would tend to make the reaction conditions more
stringent, while lower temperatures less so. For additional details
and explanation of stringency of hybridization reactions, see
Ausubel et al., Current Protocols in Molecular Biology, Wiley
Interscience Publishers, (1995).
[0060] "Stringent conditions" or "high stringency conditions", as
defined herein, may be identified by those that: (1) employ low
ionic strength and high temperature for washing, for example 0.015
M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl
sulfate at 50.degree. C.; (2) employ during hybridization a
denaturing agent, such as formamide, for example, 50% (v/v)
formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1%
polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with
750 mM sodium chloride, 75 mM sodium citrate at 42.degree. C.; or
(3) overnight hybridization in a solution that employs 50%
formamide, 5.times.SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM
sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate,
5.times.Denhardt's solution, sonicated salmon sperm DNA (50
.mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42.degree. C., with
a 10 minute wash at 42.degree. C. in 0.2.times.SSC (sodium
chloride/sodium citrate) followed by a 10 minute high-stringency
wash consisting of 0.1.times.SSC containing EDTA at 55.degree.
C.
[0061] "Moderately stringent conditions" may be identified as
described by Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Press, 1989, and include the
use of washing solution and hybridization conditions (e.g.,
temperature, ionic strength and % SDS) less stringent that those
described above. An example of moderately stringent conditions is
overnight incubation at 37.degree. C. in a solution comprising: 20%
formamide, 5.times.SSC (150 mM NaCl, 15 mM trisodium citrate), 50
mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,
followed by washing the filters in 1.times.SSC at about
37-50.degree. C. The skilled artisan will recognize how to adjust
the temperature, ionic strength, etc. as necessary to accommodate
factors such as probe length and the like.
[0062] The term "epitope tagged" when used herein refers to a
chimeric polypeptide comprising a UNQ733 polypeptide or anti-UNQ733
antibody fused to a "tag polypeptide". The tag polypeptide has
enough residues to provide an epitope against which an antibody can
be made, yet is short enough such that it does not interfere with
activity of the polypeptide to which it is fused. The tag
polypeptide preferably also is fairly unique so that the antibody
does not substantially cross-react with other epitopes. Suitable
tag polypeptides generally have at least six amino acid residues
and usually between about 8 and 50 amino acid residues (preferably,
between about 10 and 20 amino acid residues). Examples of tag
polypeptides include detectable markers such as polyhistidine and
human placenta alkaline phosphatase.
[0063] "Active" or "activity" for the purposes herein refers to
form(s) of a UNQ733 polypeptide which retain a biological and/or an
immunological activity of native or naturally-occurring UNQ733
polypeptide, wherein "biological" activity refers to a biological
function (either inhibitory or stimulatory) caused by a native or
naturally-occurring UNQ733 polypeptide other than the ability to
induce the production of an antibody against an antigenic epitope
possessed by a native or naturally-occurring UNQ733 polypeptide and
an "immunological" activity refers to the ability to induce the
production of an antibody against an antigenic epitope possessed by
a native or naturally-occurring UNQ733 polypeptide.
[0064] The term "antagonist" is used in the broadest sense, and
includes any molecule that partially or fully blocks, inhibits, or
neutralizes a biological activity of a native UNQ733 polypeptide
disclosed herein. Suitable antagonist molecules specifically
include antagonist antibodies or antibody fragments, fragments or
amino acid sequence variants of native UNQ733 polypeptides,
peptides, antisense oligonucleotides, small organic or inorganic
small molecules, etc. Methods for identifying antagonists of a
UNQ733 polypeptide may comprise contacting a UNQ733 polypeptide
with a candidate antagonist molecule and measuring a detectable
change in one or more biological activities normally associated
with the UNQ733 polypeptide.
[0065] "Treating" or "treatment" or "alleviation" refers to both
therapeutic treatment and prophylactic or preventative measures,
wherein the object is to prevent or slow down (lessen) the targeted
pathologic condition or disorder. Those in need of treatment
include those already with the disorder as well as those prone to
have the disorder or those in whom the disorder is to be prevented.
A subject or mammal is successfully "treated" for a non-Hodgkin's
lymphoma if, after receiving a therapeutic amount of a UNQ733
antagonist according to the methods of the invention, the patient
shows observable and/or measurable reduction in or absence of one
or more of the following: reduction in the number of cancer cells
or absence of the cancer cells; reduction in the tumor size;
inhibition (i.e., slow to some extent and preferably stop) of
cancer cell infiltration into peripheral organs including the
spread of cancer into soft tissue and bone; inhibition (i.e., slow
to some extent and preferably stop) of tumor metastasis;
inhibition, to some extent, of tumor growth; and/or relief to some
extent, one or more of the symptoms associated with the specific
cancer; reduced morbidity and mortality, and improvement in quality
of life issues. To the extent the UNQ733 antagonist may prevent
growth and/or kill existing cancer cells, it may be cytostatic
and/or cytotoxic. Reduction of these signs or symptoms may also be
felt by the patient.
[0066] The above parameters for assessing successful treatment and
improvement in the disease are readily measurable by routine
procedures familiar to a physician. For cancer therapy, efficacy
can be measured, for example, by assessing the time to disease
progression (TTP) and/or determining the response rate (RR).
Metastasis can be determined by staging tests and by bone scan and
tests for calcium level and other enzymes to determine spread to
the bone. CT scans can also be done to look for spread to the
pelvis and lymph nodes in the area. Chest X-rays and measurement of
liver enzyme levels by known methods are used to look for
metastasis to the lungs and liver, respectively. Other routine
methods for monitoring the disease include transrectal
ultrasonography (TRUS) and transrectal needle biopsy (TRNB).
[0067] "Chronic" administration refers to administration of the
agent(s) in a continuous mode as opposed to an acute mode, so as to
maintain the initial therapeutic effect (activity) for an extended
period of time. "Intermittent" administration is treatment that is
not consecutively done without interruption, but rather is cyclic
in nature.
[0068] "Mammal" for purposes of the treatment of, alleviating the
symptoms of a cancer refers to any animal classified as a mammal,
including humans, domestic and farm animals, and zoo, sports, or
pet animals, such as dogs, cats, cattle, horses, sheep, pigs,
goats, rabbits, etc. Preferably, the mammal is human.
[0069] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0070] "Carriers" as used herein include pharmaceutically
acceptable carriers, excipients, or stabilizers which are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH buffered solution. Examples of
physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptide; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
TWEEN.RTM., polyethylene glycol (PEG), and PLURONICS.RTM..
[0071] By "solid phase" or "solid support" is meant a non-aqueous
matrix to which an antibody, UNQ733 polypeptide binding
oligopeptide or UNQ733 polypeptide binding organic or inorganic
small molecule of the invention can adhere or attach. Examples of
solid phases encompassed herein include those formed partially or
entirely of glass (e.g., controlled pore glass), polysaccharides
(e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol
and silicones. In certain embodiments, depending on the context,
the solid phase can comprise the well of an assay plate; in others
it is a purification column (e.g., an affinity chromatography
column). This term also includes a discontinuous solid phase of
discrete particles, such as those described in U.S. Pat. No.
4,275,149.
[0072] A "liposome" is a small vesicle composed of various types of
lipids, phospholipids and/or surfactant which is useful for
delivery of a drug (such as a UNQ733 polypeptide, an antibody
thereto or a UNQ733 polypeptide binding oligopeptide) to a mammal.
The components of the liposome are commonly arranged in a bilayer
formation, similar to the lipid arrangement of biological
membranes.
[0073] A "small" molecule or "small" organic small molecule is
defined herein to have a molecular weight below about 500
Daltons.
[0074] An "effective amount" of a UNQ733 antagonist as disclosed
herein is an amount sufficient to carry out a specifically stated
purpose. An "effective amount" may be determined empirically and in
a routine manner, in relation to the stated purpose.
[0075] The term "therapeutically effective amount" refers to an
amount of a UNQ733 antagonist effective to "treat" a disease or
disorder in a subject or mammal. In the case of cancer, the
therapeutically effective amount of the antagonist may reduce the
number of cancer cells; reduce the tumor size; inhibit (i.e., slow
to some extent and preferably stop) cancer cell infiltration into
peripheral organs; inhibit (i.e., slow to some extent and
preferably stop) tumor metastasis; inhibit, to some extent, tumor
growth; and/or relieve to some extent one or more of the symptoms
associated with the cancer. See the definition herein of
"treating". To the extent the antagonist may prevent growth and/or
kill existing cancer cells, it may be cytostatic and/or
cytotoxic.
[0076] A "growth inhibitory amount" of a UNQ733 antagonist is an
amount capable of inhibiting the growth of a cell, especially
tumor, e.g., cancer cell, either in vitro or in vivo. A "growth
inhibitory amount" of a UNQ733 antagonist for purposes of
inhibiting neoplastic cell growth may be determined empirically and
in a routine manner.
[0077] A "cytotoxic amount" of a UNQ733 antagonist is an amount
capable of causing the destruction of a cell, especially tumor,
e.g., cancer cell, either in vitro or in vivo. A "cytotoxic amount"
of a UNQ733 antagonist for purposes of inhibiting neoplastic cell
growth may be determined empirically and in a routine manner.
[0078] The term "antibody" is used in the broadest sense and
specifically covers, for example, single anti-UNQ733 polypeptide
monoclonal antibodies (including antagonist, binding and/or
neutralizing antibodies), anti-UNQ733 polypeptide antibody
compositions with polyepitopic specificity, polyclonal antibodies,
single chain anti-UNQ733 polypeptide antibodies, and fragments of
anti-UNQ733 polypeptide antibodies (see below) as long as they
exhibit the desired biological or immunological activity. The term
"immunoglobulin" (Ig) is used interchangeable with antibody
herein.
[0079] An antibody useful in methods of the invention is one which
has been identified and separated and/or recovered from a component
of its natural environment. Contaminant components of its natural
environment are materials which would interfere with therapeutic
uses for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by gel electrophoresis such as SDS-PAGE under reducing or
nonreducing conditions using, for example, Coomassie blue or silver
stain.
[0080] The basic 4-chain antibody unit is a heterotetrameric
glycoprotein composed of two identical light (L) chains and two
identical heavy (H) chains (an IgM antibody consists of 5 of the
basic heterotetramer unit along with an additional polypeptide
called J chain, and therefore contain 10 antigen binding sites,
while secreted IgA antibodies can polymerize to form polyvalent
assemblages comprising 2-5 of the basic 4-chain units along with J
chain). In the case of IgGs, the 4-chain unit is generally about
150,000 daltons. Each L chain is linked to a H chain by one
covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain
isotype. Each H and L chain also has regularly spaced intrachain
disulfide bridges. Each H chain has at the N-terminus, a variable
domain (V.sub.H) followed by three constant domains (C.sub.H) for
each of the .alpha. and .gamma. chains and four C.sub.H domains for
.mu. and .epsilon. isotypes. Each L chain has at the N-terminus, a
variable domain (V.sub.L) followed by a constant domain (C.sub.L)
at its other end. The V.sub.L is aligned with the V.sub.H and the
C.sub.L is aligned with the first constant domain of the heavy
chain (C.sub.H1). Particular amino acid residues are believed to
form an interface between the light chain and heavy chain variable
domains. The pairing of a V.sub.H and V.sub.L together forms a
single antigen-binding site. For the structure and properties of
the different classes of antibodies, see, e.g., Basic and Clinical
Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and
Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn.,
1994, page 71 and Chapter 6.
[0081] The L chain from any vertebrate species can be assigned to
one of two clearly distinct types, called kappa and lambda, based
on the amino acid sequences of their constant domains. Depending on
the amino acid sequence of the constant domain of their heavy
chains (C.sub.H), immunoglobulins can be assigned to different
classes or isotypes. There are five classes of immunoglobulins:
IgA, IgD, IgE, IgG, and IgM, having heavy chains designated
.alpha., .delta., .epsilon., .gamma., and .mu., respectively. The
.gamma. and .alpha. classes are further divided into subclasses on
the basis of relatively minor differences in C.sub.H sequence and
function, e.g., humans express the following subclasses: IgG1,
IgG2, IgG3, IgG4, IgA1, and IgA2.
[0082] The term "variable" refers to the fact that certain segments
of the variable domains differ extensively in sequence among
antibodies. The V domain mediates antigen binding and define
specificity of a particular antibody for its particular antigen.
However, the variability is not evenly distributed across the
110-amino acid span of the variable domains. Instead, the V regions
consist of relatively invariant stretches called framework regions
(FRs) of 15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-12 amino
acids long. The variable domains of native heavy and light chains
each comprise four FRs, largely adopting a .beta.-sheet
configuration, connected by three hypervariable regions, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The hypervariable regions in each chain are
held together in close proximity by the FRs and, with the
hypervariable regions from the other chain, contribute to the
formation of the antigen-binding site of antibodies (see Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). The constant domains are not involved directly in binding
an antibody to an antigen, but exhibit various effector functions,
such as participation of the antibody in antibody dependent
cellular cytotoxicity (ADCC).
[0083] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g. around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3)
in the V.sub.L, and around about 1-35 (H1), 50-65 (H2) and 95-102
(H3) in the V.sub.H; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2)
and 91-96 (L3) in the V.sub.L, and 26-32 (H1), 53-55 (H2) and
96-101 (H3) in the V.sub.H; Chothia and Lesk J. Mol. Biol.
196:901-917 (1987)).
[0084] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigenic site. Furthermore, in contrast to polyclonal antibody
preparations which include different antibodies directed against
different determinants (epitopes), each monoclonal antibody is
directed against a single determinant on the antigen. In addition
to their specificity, the monoclonal antibodies are advantageous in
that they may be synthesized uncontaminated by other antibodies.
The modifier "monoclonal" is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies useful in the present invention may be
prepared by the hybridoma methodology first described by Kohler et
al., Nature, 256:495 (1975), or may be made using recombinant DNA
methods in bacterial, eukaryotic animal or plant cells (see, e.g.,
U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature, 352:624-628 (1991) and Marks
et al., J. Mol. Biol., 222:581-597 (1991), for example.
[0085] The monoclonal antibodies herein include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl.
Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of
interest herein include "primatized" antibodies comprising variable
domain antigen-binding sequences derived from a non-human primate
(e.g. Old World Monkey, Ape etc), and human constant region
sequences.
[0086] An "intact" antibody is one which comprises an
antigen-binding site as well as a C.sub.L and at least heavy chain
constant domains, C.sub.H1, C.sub.H2 and C.sub.H3. The constant
domains may be native sequence constant domains (e.g. human native
sequence constant domains) or amino acid sequence variant thereof.
Preferably, the intact antibody has one or more effector
functions.
[0087] "Antibody fragments" comprise a portion of an intact
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, and Fv fragments; diabodies; linear antibodies (see
U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng.
8(10): 1057-1062 [1995]); single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments.
[0088] Papain digestion of antibodies produces two identical
antigen-binding fragments, called "Fab" fragments, and a residual
"Fc" fragment, a designation reflecting the ability to crystallize
readily. The Fab fragment consists of an entire L chain along with
the variable region domain of the H chain (V.sub.H), and the first
constant domain of one heavy chain (C.sub.H1). Each Fab fragment is
monovalent with respect to antigen binding, i.e., it has a single
antigen-binding site. Pepsin treatment of an antibody yields a
single large F(ab').sub.2 fragment which roughly corresponds to two
disulfide linked Fab fragments having divalent antigen-binding
activity and is still capable of cross-linking antigen. Fab'
fragments differ from Fab fragments by having additional few
residues at the carboxy terminus of the 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 a free thiol group. F(ab').sub.2 antibody
fragments originally were produced as pairs of Fab' fragments which
have hinge cysteines between them. Other chemical couplings of
antibody fragments are also known.
[0089] The Fc fragment comprises the carboxy-terminal portions of
both H chains held together by disulfides. The effector functions
of antibodies are determined by sequences in the Fc region, which
region is also the part recognized by Fc receptors (FcR) found on
certain types of cells.
[0090] "Fv" is the minimum antibody fragment which contains a
complete antigen-recognition and -binding site. This fragment
consists of a dimer of one heavy- and one light-chain variable
region domain in tight, non-covalent association. From the folding
of these two domains emanate six hypervariable loops (3 loops each
from the H and L chain) that contribute the amino acid residues for
antigen binding and 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 at a lower affinity than
the entire binding site.
[0091] "Single-chain Fv" also abbreviated as "sFv" or "scFv" are
antibody fragments that comprise the V.sub.H and V.sub.L antibody
domains connected into a single polypeptide chain. Preferably, the
sFv polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains which enables the sFv to form the
desired structure for antigen binding. For a review of sFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,
Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315
(1994); Borrebaeck 1995, infra.
[0092] The term "diabodies" refers to small antibody fragments
prepared by constructing sFv fragments (see preceding paragraph)
with short linkers (about 5-10 residues) between the V.sub.H and
V.sub.L domains such that inter-chain but not intra-chain pairing
of the V domains is achieved, resulting in a bivalent fragment,
i.e., fragment having two antigen-binding sites. Bispecific
diabodies are heterodimers of two "crossover" sFv fragments in
which the V.sub.H and V.sub.L domains of the two antibodies are
present on different polypeptide chains. Diabodies are described
more fully in, for example, EP 404,097; WO 93/11161; and Hollinger
et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).
[0093] "Humanized" forms of non-human (e.g., rodent) antibodies are
chimeric antibodies that contain minimal sequence derived from the
non-human antibody. For the most part, humanized antibodies are
human immunoglobulins (recipient antibody) in which residues from a
hypervariable region of the recipient are replaced by residues from
a hypervariable region of a non-human species (donor antibody) such
as mouse, rat, rabbit or non-human primate having the desired
antibody specificity, affinity, and capability. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FRs
are those of a human immunoglobulin sequence. The humanized
antibody optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0094] A "species-dependent antibody," e.g., a mammalian anti-human
IgE antibody, is an antibody which has a stronger binding affinity
for an antigen from a first mammalian species than it has for a
homologue of that antigen from a second mammalian species.
Normally, the species-dependent antibody "bind specifically" to a
human antigen (i.e., has a binding affinity (Kd) value of no more
than about 1.times.10.sup.-7 M, preferably no more than about
1.times.10.sup.-8 and most preferably no more than about
1.times.10.sup.-9 M) but has a binding affinity for a homologue of
the antigen from a second non-human mammalian species which is at
least about 50 fold, or at least about 500 fold, or at least about
1000 fold, weaker than its binding affinity for the human antigen.
The species-dependent antibody can be of any of the various types
of antibodies as defined above, but preferably is a humanized or
human antibody.
[0095] A "UNQ733 polypeptide binding oligopeptide" is an
oligopeptide that binds, preferably specifically, to a UNQ733
polypeptide as described herein. UNQ733 polypeptide binding
oligopeptides may be chemically synthesized using known
oligopeptide synthesis methodology or may be prepared and purified
using recombinant technology. UNQ733 polypeptide binding
oligopeptides are usually at least about 5 amino acids in length,
alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100 amino acids in length or more, wherein such oligopeptides are
capable of binding, preferably specifically, to a UNQ733
polypeptide as described herein. UNQ733 polypeptide binding
oligopeptides may be identified without undue experimentation using
well known techniques. In this regard, it is noted that techniques
for screening oligopeptide libraries for oligopeptides that are
capable of specifically binding to a polypeptide target are well
known in the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373,
4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143;
PCT Publication Nos. WO 84/03506 and WO84/03564; Geysen et al.,
Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al.,
Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., in
Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J.
Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol.,
140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad.
Sci. USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry,
30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D.
et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991)
Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991)
Current Opin. Biotechnol., 2:668).
[0096] A "UNQ733 polypeptide binding organic or inorganic small
molecule" is an organic or inorganic small molecule other than an
oligopeptide or antibody as defined herein that binds, preferably
specifically, to a UNQ733 polypeptide as described herein. A small
molecule UNQ733 antagonist is preferably an organic small molecule.
UNQ733 polypeptide binding organic may be identified and chemically
synthesized using known methodology (see, e.g., PCT Publication
Nos. WO00/00823 and WO00/39585). UNQ733 polypeptide binding organic
small molecules are usually less than about 2000 daltons in size,
alternatively less than about 1500, 750, 500, 250 or 200 daltons in
size, wherein such organic small molecules that are capable of
binding, preferably specifically, to a UNQ733 polypeptide as
described herein may be identified without undue experimentation
using well known techniques. In this regard, it is noted that
techniques for screening organic small molecule libraries for
molecules that are capable of binding to a polypeptide target are
well known in the art (see, e.g., PCT Publication Nos. WO00/00823
and WO00/39585).
[0097] An antibody, oligopeptide, or other organic or inorganic
small molecule "which binds" an antigen of interest, e.g. a UNQ733
polypeptide, is one that binds the antigen with sufficient affinity
such that the antibody, oligopeptide, or other organic or inorganic
small molecule is useful as a therapeutic agent in targeting a cell
or tissue expressing the antigen, and does not significantly
cross-react with other proteins. In such embodiments, the extent of
binding of the antibody, oligopeptide, or other organic or
inorganic small molecule to a "non-target" protein will be less
than about 10% of the binding of the antibody, oligopeptide, or
other organic or inorganic small molecule to its particular target
protein as determined by fluorescence activated cell sorting (FACS)
analysis or radioimmunoprecipitation (RIA). With regard to the
binding of an antibody, oligopeptide, or other organic or inorganic
small molecule to a target molecule, the term "specific binding" or
"specifically binds to" or is "specific for" a particular
polypeptide or an epitope on a particular polypeptide target means
binding that is measurably different from a non-specific
interaction. Specific binding can be measured, for example, by
determining binding of a molecule compared to binding of a control
molecule, which generally is a molecule of similar structure that
does not have binding activity. For example, specific binding can
be determined by competition with a control molecule that is
similar to the target, for example, an excess of non-labeled
target. In this case, specific binding is indicated if the binding
of the labeled target to a probe is competitively inhibited by
excess unlabeled target. The term "specific binding" or
"specifically binds to" or is "specific for" a particular
polypeptide or an epitope on a particular polypeptide target as
used herein can be exhibited, for example, by a molecule having a
Kd for the target of at least about 10.sup.-4 M, alternatively at
least about 10.sup.-5 M, alternatively at least about 10.sup.-6 M,
alternatively at least about 10.sup.-7 M, alternatively at least
about 10.sup.-8 M, alternatively at least about 10.sup.-9 M,
alternatively at least about 10.sup.-10 M, alternatively at least
about 10.sup.-11 M, alternatively at least about 10.sup.-12 M, or
greater. In one embodiment, the term "specific binding" refers to
binding where a molecule binds to a particular polypeptide or
epitope on a particular polypeptide without substantially binding
to any other polypeptide or polypeptide epitope.
[0098] An antibody, oligopeptide, or other organic or inorganic
small molecule that "inhibits the growth of tumor cells expressing
UNQ733 polypeptide" or a "growth inhibitory" antibody,
oligopeptide, or other organic or inorganic small molecule is one
which results in measurable growth inhibition of non-Hodgkin's
lymphoma cells expressing or overexpressing the UNQ733 polypeptide.
The UNQ733 polypeptide may be bound to the surface of a cell or may
be in an extracellular environment. Preferred growth inhibitory
anti-UNQ733 polypeptide antibodies, oligopeptides, or organic or
inorganic small molecules inhibit growth of UNQ733
polypeptide-expressing tumor cells by preferably greater than about
20%, preferably from about 20% to about 50%, preferably by greater
than 50% (e.g., from about 50% to about 100%) as compared to the
appropriate control, the control typically being tumor cells not
treated with the antibody, oligopeptide, or other organic or
inorganic small molecule being tested. In one embodiment, growth
inhibition can be measured at an antibody concentration of about
0.1 to 30 .mu.g/ml or about 0.5 nM to 200 nM in cell culture, where
the growth inhibition is determined 1-10 days after exposure of the
tumor cells to the antibody. The antibody is growth inhibitory in
vivo if administration of the anti-UNQ733 polypeptide antibody at
about 1 .mu.g/kg to about 100 mg/kg body weight results in
reduction in tumor size or tumor cell proliferation within about 5
days to 3 months from the first administration of the antibody, or
within about 5 to 30 days.
[0099] A UNQ733 antagonist which "induces apoptosis" is one which
induces programmed cell death as determined by binding of annexin
V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic
reticulum, cell fragmentation, and/or formation of membrane
vesicles (called apoptotic bodies). The cell is usually one which
overexpresses UNQ733 polypeptide. Preferably the cell is a tumor
cell of a non-Hodgkin's lymphoma. Various methods are available for
evaluating the cellular events associated with apoptosis. For
example, phosphatidyl serine (PS) translocation can be measured by
annexin binding; DNA fragmentation can be evaluated through DNA
laddering; and nuclear/chromatin condensation along with DNA
fragmentation can be evaluated by any increase in hypodiploid
cells. Preferably, the UNQ733 antagonist which induces apoptosis is
one which results in about 2 to 50 fold, preferably about 5 to 50
fold, preferably about 10 to 50 fold, induction of annexin binding
relative to untreated cell in an annexin binding assay.
[0100] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody,
and vary with the antibody isotype. Examples of antibody effector
functions include: C1q binding and complement dependent
cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated
cytotoxicity (ADCC); phagocytosis; down regulation of cell surface
receptors (e.g., B cell receptor); and B cell activation.
[0101] "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) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The antibodies "arm" the cytotoxic cells and are absolutely
required for such killing. The primary cells for mediating ADCC, NK
cells, express Fc.gamma.RIII only, whereas monocytes express
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII. FcR expression on
hematopoietic cells is summarized in Table 3 on page 464 of Ravetch
and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess ADCC
activity of a molecule of interest, an in vitro ADCC assay, such as
that described in U.S. Pat. No. 5,500,362 or 5,821,337 may be
performed. Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in a animal model such as
that disclosed in Clynes et al. (USA) 95:652-656 (1998).
[0102] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. The preferred FcR is a native
sequence human FcR. Moreover, a preferred FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of these
receptors. Fc.gamma.RII 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
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (see review M. in Daeron,
Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in
Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (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 also includes the neonatal receptor, FcRn, which 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)).
[0103] "Human effector cells" are leukocytes which express one or
more FcRs and perform effector functions. Preferably, the cells
express at least Fc.gamma.RIII and perform ADCC effector function.
Examples of human leukocytes which mediate ADCC include peripheral
blood mononuclear cells (PBMC), natural killer (NK) cells,
monocytes, cytotoxic T cells and neutrophils; with PBMCs and NK
cells being preferred. The effector cells may be isolated from a
native source, e.g., from blood.
[0104] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass) which are bound to their cognate antigen.
To assess complement activation, a CDC assay, e.g., as described in
Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), may be
performed.
[0105] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated increase in cell number (generally referred to
herein as cell growth), which can be due to abnormal increase in
cell proliferation, abnormal decrease of cell death, or an
imbalance of amounts of cell proliferation and cell death. Examples
of cancer include, but are not limited to, hematopoietic cancers or
blood-related cancers, such as lymphoma, leukemia, myeloma or
lymphoid malignancies, but also cancers of the spleen and cancers
of the lymph nodes.
[0106] The term "non-Hodgkin's lymphoma" or "NHL", as used herein,
refers to a cancer of the lymphatic system other than Hodgkin's
lymphomas. Hodgkin's lymphomas can generally be distinguished from
non-Hodgkin's lymphomas by the presence of Reed-Sternberg cells in
Hodgkin's lymphomas and the absence of said cells in non-Hodgkin's
lymphomas. Examples of non-Hodgkin's lymphomas encompassed by the
term as used herein include any that would be identified as such by
one skilled in the art (e.g., an oncologist or pathologist) in
accordance with classification schemes known in the art, such as
the Revised European-American Lymphoma (REAL) scheme as described
in Color Atlas of Clinical Hematology, Third Edition; A. Victor
Hoffbrand and John E. Pettit (eds.) (Harcourt Publishers Limited
2000) (see, in particular FIG. 11.57, 11.58 and/or 11.59). More
specific examples include, but are not limited to, relapsed or
refractory NHL, front line low grade NHL, Stage III/IV NHL,
chemotherapy resistant NHL, precursor B lymphoblastic leukemia
and/or lymphoma, small lymphocytic lymphoma, B cell chronic
lymphacytic leukemia and/or prolymphocytic leukemia and/or small
lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma
and/or lymphoplasmacytic lymphoma, marginal zone B cell lymphoma,
splenic marginal zone lymphoma, extranodal marginal zone--MALT
lymphoma, nodal marginal zone lymphoma, hairy cell leukemia,
plasmacytoma and/or plasma cell myeloma, low grade/follicular
lymphoma, intermediate grade/follicular NHL, mantle cell lymphoma,
follicle center lymphoma (follicular), intermediate grade diffuse
NHL, diffuse large B-cell lymphoma, aggressive NHL (including
aggressive front-line NHL and aggressive relapsed NHL), NHL
relapsing after or refractory to autologous stem cell
transplantation, primary mediastinal large B-cell lymphoma, primary
effusion lymphoma, high grade immunoblastic NHL, high grade
lymphoblastic NHL, high grade small non-cleaved cell NHL, bulky
disease NHL, Burkitt's lymphoma, precursor (peripheral) T-cell
lymphoblastic leukemia and/or lymphoma, adult T-cell lymphoma
and/or leukemia, T cell chronic lymphocytic leukemia and/or
prolymphacytic leukemia, large granular lymphocytic leukemia,
mycosis fungoides and/or Sezary syndrome, extranodal natural
killer/T-cell (nasal type) lymphoma, enteropathy type T-cell
lymphoma, hepatosplenic T-cell lymphoma, subcutaneous panniculitis
like T-cell lymphoma, skin (cutaneous) lymphomas, anaplastic large
cell lymphoma, angiocentric lymphoma, intestinal T cell lymphoma,
peripheral T-cell (not otherwise specified) lymphoma and
angioimmunoblastic T-cell lymphoma.
[0107] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one embodiment, the cell
proliferative disorder is cancer.
[0108] "Tumor", as used herein, refers to all neoplastic cell
growth and proliferation, whether malignant or benign, and all
pre-cancerous and cancerous cells and tissues. The terms "cancer",
"cancerous", "cell proliferative disorder", "proliferative
disorder" and "tumor" are not mutually exclusive as referred to
herein.
[0109] An antibody, oligopeptide or other organic small molecule
which "induces cell death" is one which causes a viable cell to
become nonviable. The cell is one which expresses a UNQ733
polypeptide and is of a cell type which specifically expresses or
overexpresses a UNQ733 polypeptide. The cell may be cancerous or
normal cells of the particular cell type. The UNQ733 polypeptide
may be a transmembrane polypeptide expressed on the surface of a
cancer cell or may be a polypeptide that is produced and secreted
by a cancer cell. The cell may be a cancer cell, e.g., a B cell or
T cell. Cell death in vitro may be determined in the absence of
complement and immune effector cells to distinguish cell death
induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or
complement dependent cytotoxicity (CDC). Thus, the assay for cell
death may be performed using heat inactivated serum (i.e., in the
absence of complement) and in the absence of immune effector cells.
To determine whether the antibody, oligopeptide or other organic
small molecule is able to induce cell death, loss of membrane
integrity as evaluated by uptake of propidium iodide (PI), trypan
blue (see Moore et al. Cytotechnology 17:1-11 (1995)) or 7AAD can
be assessed relative to untreated cells. Preferred cell
death-inducing antibodies, oligopeptides or other organic small
molecules are those which induce PI uptake in the PI uptake assay
in BT474 cells.
[0110] A "UNQ733 polypeptide-expressing cell" is a cell which
expresses an endogenous or transfected UNQ733 polypeptide, for
example in a secreted form. UNQ733 polypeptide expression may be
determined in a detection or prognostic assay by evaluating levels
of the UNQ733 protein present in and/or on the surface of a cell,
and/or secreted by the cell (e.g., via an immunohistochemistry
assay using anti-UNQ733 polypeptide antibodies prepared against an
isolated UNQ733 polypeptide which may be prepared using recombinant
DNA technology from an isolated nucleic acid encoding the UNQ733
polypeptide; FACS analysis, etc.). Alternatively, or additionally,
one may measure levels of UNQ733 polypeptide-encoding nucleic acid
or mRNA in the cell, e.g., via fluorescent in situ hybridization
using a nucleic acid based probe corresponding to a UNQ733
polypeptide-encoding nucleic acid or the complement thereof; (FISH;
see WO98/45479 published October, 1998), Southern blotting,
Northern blotting, or polymerase chain reaction (PCR) techniques,
such as real time quantitative PCR (RT-PCR). One may also study
UNQ733 polypeptide expression by measuring shed antigen in a
biological fluid such as serum, e.g., using antibody-based assays
(see also, e.g., U.S. Pat. No. 4,933,294 issued Jun. 12, 1990;
WO91/05264 published Apr. 18, 1991; U.S. Pat. No. 5,401,638 issued
Mar. 28, 1995; and Sias et al., J. Immunol. Methods 132:73-80
(1990)). Aside from the above assays, various in vivo assays are
available to the skilled practitioner. For example, one may expose
cells within the body of the patient to an antibody which is
optionally labeled with a detectable label, e.g., a radioactive
isotope, and binding of the antibody to cells in the patient can be
evaluated, e.g., by external scanning for radioactivity or by
analyzing a biopsy taken from a patient previously exposed to the
antibody.
[0111] As used herein, the term "immunoadhesin" designates
antibody-like molecules which combine the binding specificity of a
heterologous protein (an "adhesin") with the effector functions of
immunoglobulin constant domains. Structurally, the immunoadhesins
comprise a fusion of an amino acid sequence with the desired
binding specificity which is other than the antigen recognition and
binding site of an antibody (i.e., is "heterologous"), and an
immunoglobulin constant domain sequence. The adhesin part of an
immunoadhesin molecule typically is a contiguous amino acid
sequence comprising at least the binding site of a receptor or a
ligand. The immunoglobulin constant domain sequence in the
immunoadhesin may be obtained from any immunoglobulin, such as
IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and
IgA-2), IgE, IgD or IgM.
[0112] The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the antibody, oligopeptide, or other organic or inorganic small
molecule so as to generate a "labeled" antibody, oligopeptide, or
other organic or inorganic small molecule. The label may be
detectable by itself (e.g. radioisotope labels or fluorescent
labels) or, in the case of an enzymatic label, may catalyze
chemical alteration of a substrate compound or composition which is
detectable.
[0113] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents e.g.
methotrexate, adriamicin, vinca alkaloids (vincristine,
vinblastine, etoposide), doxorubicin, melphalan, mitomycin C,
chlorambucil, daunorubicin or other intercalating agents, enzymes
and fragments thereof such as nucleolytic enzymes, antibiotics, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof, and the various antitumor or anticancer
agents disclosed below. Other cytotoxic agents are described below.
A tumoricidal agent causes destruction of tumor cells.
[0114] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,
MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic
acid; a camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e.g., calicheamicin, especially calicheamicin
gamma1I and calicheamicin omegaI1 (see, e.g., Agnew, Chem. Intl.
Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A;
an esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antibiotic chromophores), aclacinomysins,
actinomycin, authramycin, azaserine, bleomycins, cactinomycin,
carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; eflornithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;
procarbazine; PSK.RTM. polysaccharide complex (JHS Natural
Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine
(ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
("Ara-C"); thiotepa; taxoids, e.g., TAXOL.RTM. paclitaxel
(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE.TM.
Cremophor-free, albumin-engineered nanoparticle formulation of
paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),
and TAXOTERE.RTM. doxetaxel (Rhone-Poulenc Rorer, Antony, France);
chloranbucil; gemcitabine (GEMZAR.RTM.); 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine (VELBAN.RTM.); platinum; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN.RTM.);
oxaliplatin; leucovovin; vinorelbine (NAVELBINE.RTM.); novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid; capecitabine (XELODA.RTM.); pharmaceutically
acceptable salts, acids or derivatives of any of the above; as well
as combinations of two or more of the above such as CHOP, an
abbreviation for a combined therapy of cyclophosphamide,
doxorubicin, vincristine, and prednisolone, and FOLFOX, an
abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovovin.
[0115] Also included in this definition are anti-hormonal agents
that act to regulate, reduce, block, or inhibit the effects of
hormones that can promote the growth of cancer, and are often in
the form of systemic, or whole-body treatment. They may be hormones
themselves. Examples include anti-estrogens and selective estrogen
receptor modulators (SERMs), including, for example, tamoxifen
(including NOLVADEX.RTM. tamoxifen), EVISTA.RTM. raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and FARESTON.RTM. toremifene; anti-progesterones;
estrogen receptor down-regulators (ERDs); agents that function to
suppress or shut down the ovaries, for example, leutinizing
hormone-releasing hormone (LHRH) agonists such as LUPRON.RTM. and
ELIGARD.RTM. leuprolide acetate, goserelin acetate, buserelin
acetate and tripterelin; other anti-androgens such as flutamide,
nilutamide and bicalutamide; and aromatase inhibitors that inhibit
the enzyme aromatase, which regulates estrogen production in the
adrenal glands, such as, for example, 4(5)-imidazoles,
aminoglutethimide, MEGASE.RTM. megestrol acetate, AROMASIN.RTM.
exemestane, formestanie, fadrozole, RIVISOR.RTM. vorozole,
FEMARA.RTM. letrozole, and ARIMIDEX.RTM. anastrozole. In addition,
such definition of chemotherapeutic agents includes bisphosphonates
such as clodronate (for example, BONEFOS.RTM. or OSTAC.RTM.),
DIDROCAL.RTM. etidronate, NE-58095, ZOMETA.RTM. zoledronic
acid/zoledronate, FOSAMAX.RTM. alendronate, AREDIA.RTM.
pamidronate, SKELID.RTM. tiludronate, or ACTONEL.RTM. risedronate;
as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine
analog); antisense oligonucleotides, particularly those that
inhibit expression of genes in signaling pathways implicated in
abherant cell proliferation, such as, for example, PKC-alpha, Raf,
H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such
as THERATOPE.RTM. vaccine and gene therapy vaccines, for example,
ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM.
vaccine; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM.
rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase
small-molecule inhibitor also known as GW572016); and
pharmaceutically acceptable salts, acids or derivatives of any of
the above.
[0116] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a UNQ733
polypeptide-expressing non-Hodgkin's lymphoma cell, either in vitro
or in vivo. Thus, the growth inhibitory agent may be one which
significantly reduces the percentage of UNQ733
polypeptide-expressing cells in S phase. Examples of growth
inhibitory agents include agents that block cell cycle progression
(at a place other than S phase), such as agents that induce G1
arrest and M-phase arrest. Classical M-phase blockers include the
vincas (vincristine and vinblastine), taxanes, and topoisomerase II
inhibitors such as doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin. Those agents that arrest G1 also spill
over into S-phase arrest, for example, DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,
methotrexate, 5-fluorouracil, and ara-C. Further information can be
found in The Molecular Basis of Cancer, Mendelsohn and Israel,
eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and
antineoplastic drugs" by Murakami et al. (WB Saunders:
Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel and
docetaxel) are anticancer drugs both derived from the yew tree.
Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer), derived from the
European yew, is a semisynthetic analogue of paclitaxel
(TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and docetaxel
promote the assembly of microtubules from tubulin dimers and
stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
[0117] "Doxorubicin" is an anthracycline antibiotic. The full
chemical name of doxorubicin is
(8S-cis)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexapyranosyl)oxy]-7,-
8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-napht-
hacenedione.
[0118] The term "cytokine" is a generic term for proteins released
by one cell population which act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines,
and traditional polypeptide hormones. Included among the cytokines
are growth hormone such as human growth hormone, N-methionyl human
growth hormone, and bovine growth hormone; parathyroid hormone;
thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein
hormones such as follicle stimulating hormone (FSH), thyroid
stimulating hormone (TSH), and luteinizing hormone (LH); hepatic
growth factor; fibroblast growth factor; prolactin; placental
lactogen; tumor necrosis factor-.alpha. and -.beta.;
mullerian-inhibiting substance; mouse gonadotropin-associated
peptide; inhibin; activin; vascular endothelial growth factor
(VEGF); integrin; thrombopoietin (TPO); nerve growth factors such
as NGF-.beta.; platelet-growth factor; transforming growth factors
(TGFs) such as TGF-.alpha. and TGF-.beta.; insulin-like growth
factor-I and -II; erythropoietin (EPO); osteoinductive factors;
interferons such as interferon-.alpha., -.beta., and -.gamma.;
colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);
granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);
interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-11, IL-12; a tumor necrosis factor such
as TNF-.alpha. or TNF-.beta.; and other polypeptide factors
including LIF and kit ligand (KL). As used herein, the term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
[0119] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications and/or warnings
concerning the use of such therapeutic products. TABLE-US-00001
TABLE 2 UNQ733 XXXXXXXXXXXXXXX (Length = 15 polypeptide amino
acids) Comparison XXXXXYYYYYYY (Length = 12 Protein amino acids) %
amino acid sequence identity = (the number of identically matching
amino acid residues between the two polypeptide sequences as
determined by ALIGN-2) divided by (the total number of amino acid
residues of the UNQ733 polypeptide) = 5 divided by 15 = 33.3%
[0120] TABLE-US-00002 TABLE 3 UNQ733 XXXXXXXXXX (Length = 10
polypeptide amino acids) Comparison XXXXXYYYYYYZZYZ (Length = 15
Protein amino acids) % amino acid sequence identity = (the number
of identically matching amino acid residues between the two
polypeptide sequences as determined by ALIGN-2) divided by (the
total number of amino acid residues of the UNQ733 polypeptide) = 5
divided by 10 = 50%
[0121] TABLE-US-00003 TABLE 4 UNQ733 NNNNNNNNNNNNNN (Length = 14
DNA nucleotides) Comparison NNNNNNLLLLLLLLLL (Length = 16 DNA
nucleotides) % nucleic acid sequence identity = (the number of
identically matching nucleotides between the two nucleic acid
sequences as determined by ALIGN-2) divided by (the total number of
nucleotides of the UNQ733 polypeptide DNA sequence) = 6 divided by
14 = 42.9%
[0122] TABLE-US-00004 TABLE 5 UNQ733 DNA NNNNNNNNNNNN (Length = 12
nucleotides) Comparison DNA NNNNLLLVV (Length = 9 nucleotides) %
nucleic acid sequence identity = (the number of identically
matching nucleotides between the two nucleic acid sequences as
determined by ALIGN-2) divided by (the total number of nucleotides
of the UNQ733 polypeptide DNA sequence) = 4 divided by 12 =
33.3%
II. Compositions and Methods of the Invention
[0123] A. Anti-UNQ733 Antibodies
[0124] In one embodiment, the present invention provides
anti-UNQ733 polypeptide antibodies which may find use herein as
therapeutic and/or diagnostic agents. Exemplary antibodies include
polyclonal, monoclonal, humanized, bispecific, and heteroconjugate
antibodies.
[0125] 1. Polyclonal Antibodies
[0126] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen and an adjuvant. It may be useful to conjugate
the relevant antigen (especially when synthetic peptides are used)
to a protein that is immunogenic in the species to be immunized.
For example, the antigen can be conjugated to keyhole limpet
hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean
trypsin inhibitor, using a bifunctional or derivatizing agent,
e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation through
cysteine residues), N-hydroxysuccinimide (through lysine residues),
glutaraldehyde, succinic anhydride, SOCl.sub.2, or
R.sup.1N.dbd.C.dbd.NR, where R and R.sup.1 are different alkyl
groups.
[0127] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later, the
animals are boosted with 1/5 to 1/10 the original amount of peptide
or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later, the animals
are bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Conjugates also can be made in
recombinant cell culture as protein fusions. Also, aggregating
agents such as alum are suitably used to enhance the immune
response.
[0128] 2. Monoclonal Antibodies
[0129] Monoclonal antibodies may be made using the hybridoma method
first described by Kohler et al., Nature, 256:495 (1975), or may be
made by recombinant DNA methods (U.S. Pat. No. 4,816,567).
[0130] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as described above to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the protein used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
After immunization, lymphocytes are isolated and then fused with a
myeloma cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal
Antibodies: Principles and Practice, pp. 59-103 (Academic Press,
1986)).
[0131] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium which medium preferably contains one or
more substances that inhibit the growth or survival of the unfused,
parental myeloma cells (also referred to as fusion partner). For
example, if the parental myeloma cells lack the enzyme hypoxanthine
guanine phosphoribosyl transferase (HGPRT or HPRT), the selective
culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine (HAT medium), which
substances prevent the growth of HGPRT-deficient cells.
[0132] Preferred fusion partner myeloma cells are those that fuse
efficiently, support stable high-level production of antibody by
the selected antibody-producing cells, and are sensitive to a
selective medium that selects against the unfused parental cells.
Preferred myeloma cell lines are murine myeloma lines, such as
those derived from MOPC-21 and MPC-11 mouse tumors available from
the Salk Institute Cell Distribution Center, San Diego, Calif. USA,
and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the
American Type Culture Collection, Manassas, Va., USA. Human myeloma
and mouse-human heteromyeloma cell lines also have been described
for the production of human monoclonal antibodies (Kozbor, J.
Immunol., 133:3001 (1984); and Brodeur et al., Monoclonal Antibody
Production Techniques and Applications, pp. 51-63 (Marcel Dekker,
Inc., New York, 1987)).
[0133] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunosorbent assay
(ELISA).
[0134] The binding affinity of the monoclonal antibody can, for
example, be determined by the Scatchard analysis described in
Munson et al., Anal. Biochem., 107:220 (1980).
[0135] Once hybridoma cells that produce antibodies of the desired
specificity, affinity, and/or activity are identified, the clones
may be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal e.g., by i.p. injection of the cells
into mice.
[0136] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional antibody purification procedures such as, for
example, affinity chromatography (e.g., using protein A or protein
G-Sepharose) or ion-exchange chromatography, hydroxylapatite
chromatography, gel electrophoresis, dialysis, etc.
[0137] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transfected into host cells such as E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do
not otherwise produce antibody protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells. Review
articles on recombinant expression in bacteria of DNA encoding the
antibody include Skerra et al., Curr. Opinion in Immunol.,
5:256-262 (1993) and Pluckthun, Immunol. Revs. 130:151-188
(1992).
[0138] In a further embodiment, monoclonal antibodies or antibody
fragments can be isolated from antibody phage libraries generated
using the techniques described in McCafferty et al., Nature,
348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and
Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the
isolation of murine and human antibodies, respectively, using phage
libraries. Subsequent publications describe the production of high
affinity (nM range) human antibodies by chain shuffling (Marks et
al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0139] The DNA that encodes the antibody may be modified to produce
chimeric or fusion antibody polypeptides, for example, by
substituting human heavy chain and light chain constant domain
(C.sub.H and C.sub.L) sequences for the homologous murine sequences
(U.S. Pat. No. 4,816,567; and Morrison, et al., Proc. Natl. Acad.
Sci. USA, 81:6851 (1984)), or by fusing the immunoglobulin coding
sequence with all or part of the coding sequence for a
non-immunoglobulin polypeptide (heterologous polypeptide). The
non-immunoglobulin polypeptide sequences can substitute for the
constant domains of an antibody, or they are substituted for the
variable domains of one antigen-combining site of an antibody to
create a chimeric bivalent antibody comprising one
antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0140] 3. Human and Humanized Antibodies
[0141] The anti-UNQ733 antibodies of the invention may further
comprise humanized antibodies or human antibodies. Humanized forms
of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such
as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived
from non-human immunoglobulin. Humanized antibodies include human
immunoglobulins (recipient antibody) in which residues from a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. In some instances, Fv framework
residues of the human immunoglobulin are replaced by corresponding
non-human residues. Humanized antibodies may also comprise residues
which are found neither in the recipient antibody nor in the
imported CDR or framework sequences. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [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)].
[0142] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers [Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0143] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity and HAMA response (human anti-mouse antibody)
when the antibody is intended for human therapeutic use. According
to the so-called "best-fit" method, the sequence of the variable
domain of a rodent antibody is screened against the entire library
of known human variable domain sequences. The human V domain
sequence which is closest to that of the rodent is identified and
the human framework region (FR) within it accepted for the
humanized antibody (Sims et al., J. Immunol. 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework region derived from the consensus sequence
of all human antibodies of a particular subgroup of light or heavy
chains. The same framework may be used for several different
humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA,
89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).
[0144] It is further important that antibodies be humanized with
retention of high binding affinity for the antigen and other
favorable biological properties. To achieve this goal, according to
a preferred method, humanized antibodies are prepared by a process
of analysis of the parental sequences and various conceptual
humanized products using three-dimensional models of the parental
and humanized sequences. Three-dimensional immunoglobulin models
are commonly available and are familiar to those skilled in the
art. Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0145] Various forms of a humanized anti-UNQ733 polypeptide
antibody are contemplated. For example, the humanized antibody may
be an antibody fragment, such as a Fab, which is optionally
conjugated with one or more cytotoxic agent(s) in order to generate
an immunoconjugate. Alternatively, the humanized antibody may be an
intact antibody, such as an intact IgG1 antibody.
[0146] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array into such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno. 7:33
(1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of
GenPharm); 5,545,807; and WO 97/17852.
[0147] Alternatively, phage display technology (McCafferty et al.,
Nature 348:552-553 [1990]) can be used to produce human antibodies
and antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of
the B-cell. Phage display can be performed in a variety of formats,
reviewed in, e.g., Johnson, Kevin S, and Chiswell, David J.,
Current Opinion in Structural Biology 3:564-571 (1993). Several
sources of V-gene segments can be used for phage display. Clackson
et al., Nature, 352:624-628 (1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library
of V genes derived from the spleens of immunized mice. A repertoire
of V genes from unimmunized human donors can be constructed and
antibodies to a diverse array of antigens (including self-antigens)
can be isolated essentially following the techniques described by
Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al.,
EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and
5,573,905.
[0148] As discussed above, human antibodies may also be generated
by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610 and
5,229,275).
[0149] 4. Antibody Fragments
[0150] In certain circumstances there are advantages of using
antibody fragments, rather than whole antibodies. The smaller size
of the fragments allows for rapid clearance, and may lead to
improved access to solid tumors.
[0151] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
Fab, Fv and ScFv antibody fragments can all be expressed in and
secreted from E. coli, thus allowing the facile production of large
amounts of these fragments. Antibody fragments can be isolated from
the antibody phage libraries discussed above. Alternatively,
Fab'-SH fragments can be directly recovered from E. coli and
chemically coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Fab and F(ab').sub.2 fragment with increased in
vivo half-life comprising a salvage receptor binding epitope
residues are described in U.S. Pat. No. 5,869,046. Other techniques
for the production of antibody fragments will be apparent to the
skilled practitioner. In other embodiments, the antibody of choice
is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat.
No. 5,571,894; and U.S. Pat. No. 5,587,458. Fv and sFv are the only
species with intact combining sites that are devoid of constant
regions; thus, they are suitable for reduced nonspecific binding
during in vivo use. sFv fusion proteins may be constructed to yield
fusion of an effector protein at either the amino or the carboxy
terminus of an sFv. See Antibody Engineering, ed. Borrebaeck,
supra. The antibody fragment may also be a "linear antibody", e.g.,
as described in U.S. Pat. No. 5,641,870 for example. Such linear
antibody fragments may be monospecific or bispecific.
[0152] 5. Bispecific Antibodies
[0153] Bispecific antibodies are antibodies that have binding
specificities for at least two different epitopes. Exemplary
bispecific antibodies may bind to two different epitopes of a
UNQ733 polypeptide as described herein. Other such antibodies may
combine a UNQ733 polypeptide binding site with a binding site for
another polypeptide. Alternatively, an anti-UNQ733 polypeptide
antibody arm may be combined with an arm which binds to a
triggering molecule on a leukocyte such as a T-cell receptor
molecule (e.g. CD3), or Fc receptors for IgG (Fc.gamma.R), such as
Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.gamma.RIII (CD16),
so as to focus and localize cellular defense mechanisms to the
UNQ733 polypeptide-expressing and/or binding cell. Bispecific
antibodies may also be used to localize cytotoxic agents to cells
which express and/or bind UNQ733 polypeptide. These antibodies
possess a UNQ733 polypeptide binding arm and an arm which binds the
cytotoxic agent (e.g., saporin, anti-interferon-.alpha., vinca
alkaloid, ricin A chain, methotrexate or radioactive isotope
hapten). Bispecific antibodies can be prepared as full length
antibodies or antibody fragments (e.g., F(ab').sub.2 bispecific
antibodies).
[0154] WO 96/16673 describes a bispecific
anti-ErbB2/anti-Fc.gamma.RIII antibody and U.S. Pat. No. 5,837,234
discloses a bispecific anti-ErbB2/anti-Fc.gamma.RI antibody. A
bispecific anti-ErbB2/Fc.alpha. antibody is shown in WO98/02463.
U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3
antibody.
[0155] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the co-expression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829, and in Traunecker et al., EMBO J. 10:3655-3659
(1991).
[0156] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences.
Preferably, the fusion is with an Ig heavy chain constant domain,
comprising at least part of the hinge, C.sub.H2, and C.sub.H3
regions. It is preferred to have the first heavy-chain constant
region (CHI) containing the site necessary for light chain bonding,
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 cell. This
provides for greater 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 yield of the desired bispecific
antibody. It is, however, possible to insert the coding sequences
for two or all three polypeptide chains into a single expression
vector when the expression of at least two polypeptide chains in
equal ratios results in high yields or when the ratios have no
significant affect on the yield of the desired chain
combination.
[0157] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, 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.,
Methods in Enzymology 121:210 (1986).
[0158] According to another approach described in U.S. Pat. No.
5,731,168, the interface between a pair of antibody molecules can
be engineered to maximize the percentage of heterodimers which are
recovered from recombinant cell culture. The preferred interface
comprises at least a part of the C.sub.H3 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.
[0159] Bispecific antibodies 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.
[0160] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. For
example, bispecific antibodies can be prepared using chemical
linkage. Brennan et al., Science 229:81 (1985) describe a procedure
wherein intact antibodies are proteolytically cleaved to generate
F(ab').sub.2 fragments. These fragments are reduced in the presence
of the dithiol complexing agent, sodium arsenite, to stabilize
vicinal dithiols and prevent intermolecular disulfide formation.
The Fab' fragments generated are then converted to
thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB
derivatives is then reconverted to the Fab'-thiol by reduction with
mercaptoethylamine and is mixed with an equimolar amount of the
other Fab'-TNB derivative to form the bispecific antibody. The
bispecific antibodies produced can be used as agents for the
selective immobilization of enzymes.
[0161] Recent progress has facilitated the direct recovery of
Fab'-SH fragments from E. coli, which can be chemically coupled to
form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:
217-225 (1992) describe the production of a fully humanized
bispecific antibody F(ab').sub.2 molecule. Each Fab' fragment was
separately secreted from E. coli and subjected to directed chemical
coupling in vitro to form the bispecific antibody. The bispecific
antibody thus formed was able to bind to cells overexpressing the
ErbB2 receptor and normal human T cells, as well as trigger the
lytic activity of human cytotoxic lymphocytes against human breast
tumor targets. Various techniques for making and isolating
bispecific antibody fragments directly from recombinant cell
culture have also been described. For example, bispecific
antibodies have been produced using leucine zippers. Kostelny et
al., J. Immunol. 148(5):1547-1553 (1992). The leucine zipper
peptides from the Fos and Jun proteins were linked to the Fab'
portions of two different antibodies by gene fusion. The antibody
homodimers were reduced at the hinge region to form monomers and
then re-oxidized to form the antibody heterodimers. This method can
also be utilized for the production of antibody homodimers. The
"diabody" technology described by Hollinger et al., Proc. Natl.
Acad. Sci. USA 90:6444-6448 (1993) has provided an alternative
mechanism for making bispecific antibody fragments. The fragments
comprise a V.sub.H connected to a V.sub.L by a linker which is too
short to allow pairing between the two domains on the same chain.
Accordingly, the V.sub.H and V.sub.L domains of one fragment are
forced to pair with the complementary V.sub.L and V.sub.H domains
of another fragment, thereby forming two antigen-binding sites.
Another strategy for making bispecific antibody fragments by the
use of single-chain Fv (sFv) dimers has also been reported. See
Gruber et al., J. Immunol., 152:5368 (1994).
[0162] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
[0163] 6. Heteroconjugate Antibodies
[0164] Heteroconjugate antibodies are also within the scope of the
present invention. Heteroconjugate antibodies are composed of two
covalently joined antibodies. 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; EP 03089]. It is contemplated that the
antibodies may be prepared in vitro using known methods in
synthetic protein chemistry, including those involving crosslinking
agents. For example, immunotoxins may be constructed using a
disulfide exchange reaction or by forming a thioether bond.
Examples of suitable reagents for this purpose include
iminothiolate and methyl-4-mercaptobutyrimidate and those
disclosed, for example, in U.S. Pat. No. 4,676,980.
[0165] 7. Multivalent Antibodies
[0166] A multivalent antibody may be internalized (and/or
catabolized) faster than a bivalent antibody by a cell expressing
an antigen to which the antibodies bind. The antibodies of the
present invention can be multivalent antibodies (which are other
than of the IgM class) with three or more antigen binding sites
(e.g. tetravalent antibodies), which can be readily produced by
recombinant expression of nucleic acid encoding the polypeptide
chains of the antibody. The multivalent antibody can comprise a
dimerization domain and three or more antigen binding sites. The
preferred dimerization domain comprises (or consists of) an Fc
region or a hinge region. In this scenario, the antibody will
comprise an Fc region and three or more antigen binding sites
amino-terminal to the Fc region. The preferred multivalent antibody
herein comprises (or consists of) three to about eight, but
preferably four, antigen binding sites. The multivalent antibody
comprises at least one polypeptide chain (and preferably two
polypeptide chains), wherein the polypeptide chain(s) comprise two
or more variable domains. For instance, the polypeptide chain(s)
may comprise VD1-(X1).sub.n-VD2-(X2).sub.n-Fc, wherein VD1 is a
first variable domain, VD2 is a second variable domain, Fc is one
polypeptide chain of an Fc region, X1 and X2 represent an amino
acid or polypeptide, and n is 0 or 1. For instance, the polypeptide
chain(s) may comprise: VH-CH1-flexible linker-VH-CH1-Fc region
chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody
herein preferably further comprises at least two (and preferably
four) light chain variable domain polypeptides. The multivalent
antibody herein may, for instance, comprise from about two to about
eight light chain variable domain polypeptides. The light chain
variable domain polypeptides contemplated here comprise a light
chain variable domain and, optionally, further comprise a CL
domain.
[0167] 8. Effector Function Engineering
[0168] It may be desirable to modify the antibody of the invention
with respect to effector function, e.g., so as to enhance
antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B.
J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.,
Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can
be engineered which has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design 3:219-230 (1989). To increase the
serum half life of the antibody, one may incorporate a salvage
receptor binding epitope into the antibody (especially an antibody
fragment) as described in U.S. Pat. No. 5,739,277, for example. As
used herein, the term "salvage receptor binding epitope" refers to
an epitope of the Fc region of an IgG molecule (e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, or IgG.sub.4) that is responsible for
increasing the in vivo serum half-life of the IgG molecule.
[0169] 9. Immunoconjugates
[0170] The invention also pertains to immunoconjugates, or
antibody-drug conjugates (ADC), comprising an antibody conjugated
to a cytotoxic agent such as a chemotherapeutic agent, a drug, a
growth inhibitory agent, a toxin (e.g., an enzymatically active
toxin of bacterial, fungal, plant, or animal origin, or fragments
thereof), or a radioactive isotope (i.e., a radioconjugate).
[0171] The use of antibody-drug conjugates for the local delivery
of cytotoxic or cytostatic agents, i.e. drugs to kill or inhibit
tumor cells in the treatment of cancer (Syrigos and Epenetos (1999)
Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997)
Adv. Drg Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278)
theoretically allows targeted delivery of the drug moiety to
tumors, and intracellular accumulation therein, where systemic
administration of these unconjugated drug agents may result in
unacceptable levels of toxicity to normal cells as well as the
tumor cells sought to be eliminated (Baldwin et al., (1986) Lancet
pp. (Mar. 15, 1986):603-05; Thorpe, (1985) "Antibody Carriers Of
Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal
Antibodies'84: Biological And Clinical Applications, A. Pinchera et
al. (ed.s), pp. 475-506). Maximal efficacy with minimal toxicity is
sought thereby. Both polyclonal antibodies and monoclonal
antibodies have been reported as useful in these strategies
(Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87).
Drugs used in these methods include daunomycin, doxorubicin,
methotrexate, and vindesine (Rowland et al., (1986) supra). Toxins
used in antibody-toxin conjugates include bacterial toxins such as
diphtheria toxin, plant toxins such as ricin, small molecule toxins
such as geldanamycin (Mandler et al (2000) Jour. of the Nat. Cancer
Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.
Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.
13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc.
Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al
(1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res.
53:3336-3342). The toxins may effect their cytotoxic and cytostatic
effects by mechanisms including tubulin binding, DNA binding, or
topoisomerase inhibition. Some cytotoxic drugs tend to be inactive
or less active when conjugated to large antibodies or protein
receptor ligands.
[0172] ZEVALIN.RTM. (ibritumomab tiuxetan, Biogen/Idec) is an
antibody-radioisotope conjugate composed of a murine IgG1 kappa
monoclonal antibody directed against the CD20 antigen found on the
surface of normal and malignant B lymphocytes and .sup.111In or
.sup.90Y radioisotope bound by a thiourea linker-chelator (Wiseman
et al (2000) Eur. Jour. Nucl. Med. 27(7):766-77; Wiseman et al
(2002) Blood 99(12):4336-42; Witzig et al (2002) J. Clin. Oncol.
20(10):2453-63; Witzig et al (2002) J. Clin. Oncol.
20(15):3262-69). Although ZEVALIN has activity against B-cell
non-Hodgkin's Lymphoma (NHL), administration results in severe and
prolonged cytopenias in most patients. MYLOTARG.TM. (gemtuzumab
ozogamicin, Wyeth Pharmaceuticals), an antibody drug conjugate
composed of a hu CD33 antibody linked to calicheamicin, was
approved in 2000 for the treatment of acute myeloid leukemia by
injection (Drugs of the Future (2000) 25(7):686; U.S. Pat. Nos.
4,970,198; 5,079,233; 5,585,089; 5,606,040; 5,693,762; 5,739,116;
5,767,285; 5,773,001). Cantuzumab mertansine (Immunogen, Inc.), an
antibody drug conjugate composed of the huC242 antibody linked via
the disulfide linker SPP to the maytansinoid drug moiety, DM1, is
advancing into Phase II trials for the treatment of cancers that
express CanAg, such as colon, pancreatic, gastric, and others.
MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), an
antibody drug conjugate composed of the anti-prostate specific
membrane antigen (PSMA) monoclonal antibody linked to the
maytansinoid drug moiety, DM1, is under development for the
potential treatment of prostate tumors. The auristatin peptides,
auristatin E (AE) and monomethylauristatin (MMAE), synthetic
analogs of dolastatin, were conjugated to chimeric monoclonal
antibodies cBR96 (specific to Lewis Y on carcinomas) and cAC10
(specific to CD30 on hematological malignancies) (Doronina et al
(2003) Nature Biotechnology 21(7):778-784) and are under
therapeutic development.
[0173] Chemotherapeutic agents useful in the generation of such
immunoconjugates have been described above. Enzymatically active
toxins and fragments thereof that can be used include diphtheria A
chain, nonbinding active fragments of diphtheria toxin, exotoxin A
chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugated
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y, and .sup.186Re. Conjugates of the antibody and cytotoxic
agent are made using a variety of bifunctional protein-coupling
agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate
(SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters
(such as dimethyl adipimidate HCl), active esters (such as
disuccinimidyl suberate), aldehydes (such as glutaraldehyde),
bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine),
bis-diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
toluene 2,6-diisocyanate), and bis-active fluorine compounds (such
as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin
immunotoxin can be prepared as described in Vitetta et al.,
Science, 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0174] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, a trichothecene,
and CC1065, and the derivatives of these toxins that have toxin
activity, are also contemplated herein.
Maytansine and Maytansinoids
[0175] In one embodiment, an anti-UNQ733 polypeptide antibody (full
length or fragments) of the invention is conjugated to one or more
maytansinoid molecules.
[0176] Maytansinoids are mitototic inhibitors which act by
inhibiting tubulin polymerization. Maytansine was first isolated
from the east African shrub Maytenus serrata (U.S. Pat. No.
3,896,111). Subsequently, it was discovered that certain microbes
also produce maytansinoids, such as maytansinol and C-3 maytansinol
esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and
derivatives and analogues thereof are disclosed, for example, in
U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, the
disclosures of which are hereby expressly incorporated by
reference.
Maytansinoid-Antibody Conjugates
[0177] In an attempt to improve their therapeutic index, maytansine
and maytansinoids have been conjugated to antibodies specifically
binding to tumor cell antigens. Immunoconjugates containing
maytansinoids and their therapeutic use are disclosed, for example,
in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425
235 B1, the disclosures of which are hereby expressly incorporated
by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623
(1996) described immunoconjugates comprising a maytansinoid
designated DM1 linked to the monoclonal antibody C242 directed
against human colorectal cancer. The conjugate was found to be
highly cytotoxic towards cultured colon cancer cells, and showed
antitumor activity in an in vivo tumor growth assay. Chari et al.,
Cancer Research 52:127-131 (1992) describe immunoconjugates in
which a maytansinoid was conjugated via a disulfide linker to the
murine antibody A7 binding to an antigen on human colon cancer cell
lines, or to another murine monoclonal antibody TA. 1 that binds
the HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansonoid
conjugate was tested in vitro on the human breast cancer cell line
SK-BR-3, which expresses 3.times.10.sup.5 HER-2 surface antigens
per cell. The drug conjugate achieved a degree of cytotoxicity
similar to the free maytansinoid drug, which could be increased by
increasing the number of maytansinoid molecules per antibody
molecule. The A7-maytansinoid conjugate showed low systemic
cytotoxicity in mice.
Anti-UNQ733 Polypeptide Antibody-Maytansinoid Conjugates
(Immunoconjugates)
[0178] Anti-UNQ733 polypeptide antibody-maytansinoid conjugates are
prepared by chemically linking an anti-UNQ733 polypeptide antibody
to a maytansinoid molecule without significantly diminishing the
biological activity of either the antibody or the maytansinoid
molecule. An average of 3-4 maytansinoid molecules conjugated per
antibody molecule has shown efficacy in enhancing cytotoxicity of
target cells without negatively affecting the function or
solubility of the antibody, although even one molecule of
toxin/antibody would be expected to enhance cytotoxicity over the
use of naked antibody. Maytansinoids are well known in the art and
can be synthesized by known techniques or isolated from natural
sources. Suitable maytansinoids are disclosed, for example, in U.S.
Pat. No. 5,208,020 and in the other patents and nonpatent
publications referred to hereinabove. Preferred maytansinoids are
maytansinol and maytansinol analogues modified in the aromatic ring
or at other positions of the maytansinol molecule, such as various
maytansinol esters.
[0179] There are many linking groups known in the art for making
antibody-maytansinoid conjugates, including, for example, those
disclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, and
Chari et al., Cancer Research 52:127-131 (1992). The linking groups
include disulfide groups, thioether groups, acid labile groups,
photolabile groups, peptidase labile groups, or esterase labile
groups, as disclosed in the above-identified patents, disulfide and
thioether groups being preferred.
[0180] Conjugates of the antibody and maytansinoid may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et
al., Biochem. J. 173:723-737 [1978]) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a
disulfide linkage.
[0181] The linker may be attached to the maytansinoid molecule at
various positions, depending on the type of the link. For example,
an ester linkage may be formed by reaction with a hydroxyl group
using conventional coupling techniques. The reaction may occur at
the C-3 position having a hydroxyl group, the C-14 position
modified with hydroxymethyl, the C-15 position modified with a
hydroxyl group, and the C-20 position having a hydroxyl group. In a
preferred embodiment, the linkage is formed at the C-3 position of
maytansinol or a maytansinol analogue.
Calicheamicin
[0182] Another immunoconjugate of interest comprises an anti-UNQ733
polypeptide antibody conjugated to one or more calicheamicin
molecules. The calicheamicin family of antibiotics are capable of
producing double-stranded DNA breaks at sub-picomolar
concentrations. For the preparation of conjugates of the
calicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586,
5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296
(all to American Cyanamid Company). Structural analogues of
calicheamicin which may be used include, but are not limited to,
.gamma..sub.1.sup.I, .alpha..sub.2.sup.I, .alpha..sub.3.sup.I,
N-acetyl-.gamma..sub.1.sup.I, PSAG and .theta..sup.I.sub.1 (Hinman
et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer
Research 58:2925-2928 (1998) and the aforementioned U.S. patents to
American Cyanamid). Another anti-tumor drug that the antibody can
be conjugated is QFA which is an antifolate. Both calicheamicin and
QFA have intracellular sites of action and do not readily cross the
plasma membrane. Therefore, cellular uptake of these agents through
antibody mediated internalization greatly enhances their cytotoxic
effects.
Other Cytotoxic Agents
[0183] Other antitumor agents that can be conjugated to the
anti-UNQ733 polypeptide antibodies of the invention include BCNU,
streptozoicin, vincristine and 5-fluorouracil, the family of agents
known collectively LL-E33288 complex described in U.S. Pat. Nos.
5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.
5,877,296).
[0184] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor,
curcin, crotin, sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0185] The present invention further contemplates an
immunoconjugate formed between an antibody and a compound with
nucleolytic activity (e.g., a ribonuclease or a DNA endonuclease
such as a deoxyribonuclease; DNase).
[0186] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
anti-UNQ733 antibodies. Examples include At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, P.sup.212 and radioactive isotopes of Lu.
When the conjugate is used for detection, it may comprise a
radioactive atom for scintigraphic studies, for example tc.sup.99m
or I.sup.123, or a spin label for nuclear magnetic resonance (NMR)
imaging (also known as magnetic resonance imaging, mri), such as
iodine-123 again, iodine-131, indium-11, fluorine-19, carbon-13,
nitrogen-15, oxygen-17, gadolinium, manganese or iron.
[0187] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-119 in place of hydrogen. Labels such as
tc.sup.99m or I.sup.123, Re.sup.186, Re.sup.188 and In.sup.111 can
be attached via a cysteine residue in the peptide. Yttrium-90 can
be attached via a lysine residue. The IODOGEN method (Fraker et al
(1978) Biochem. Biophys. Res. Commun. 80: 49-57 can be used to
incorporate iodine-123. "Monoclonal Antibodies in
Immunoscintigraphy" (Chatal, CRC Press 1989) describes other
methods in detail.
[0188] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(Mx-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of the cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Research 52:127-131 (1992); U.S. Pat.
No. 5,208,020) may be used.
[0189] The compounds of the invention expressly contemplate, but
are not limited to, ADC prepared with cross-linker reagents: BMPS,
EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB,
SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,
sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidyl-(4-vinylsulfone)benzoate) which are commercially
available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill.,
U.S.A). See pages 467-498, 2003-2004 Applications Handbook and
Catalog.
Preparation of Antibody Drug Conjugates
[0190] In the antibody drug conjugates (ADC) of the invention, an
antibody (Ab) is conjugated to one or more drug moieties (D), e.g.
about 1 to about 20 drug moieties per antibody, through a linker
(L). The ADC of Formula I may be prepared by several routes,
employing organic chemistry reactions, conditions, and reagents
known to those skilled in the art, including: (1) reaction of a
nucleophilic group of an antibody with a bivalent linker reagent,
to form Ab-L, via a covalent bond, followed by reaction with a drug
moiety D; and (2) reaction of a nucleophilic group of a drug moiety
with a bivalent linker reagent, to form D-L, via a covalent bond,
followed by reaction with the nucleophilic group of an antibody.
Ab-(L-D).sub.p I
[0191] Nucleophilic groups on antibodies include, but are not
limited to: (i) N-terminal amine groups, (ii) side chain amine
groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine,
and (iv) sugar hydroxyl or amino groups where the antibody is
glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic
and capable of reacting to form covalent bonds with electrophilic
groups on linker moieties and linker reagents including: (i) active
esters such as NHS esters, HOBt esters, haloformates, and acid
halides; (ii) alkyl and benzyl halides such as haloacetamides;
(iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain
antibodies have reducible interchain disulfides, i.e. cysteine
bridges. Antibodies may be made reactive for conjugation with
linker reagents by treatment with a reducing agent such as DTT
(dithiothreitol). Each cysteine bridge will thus form,
theoretically, two reactive thiol nucleophiles. Additional
nucleophilic groups can be introduced into antibodies through the
reaction of lysines with 2-iminothiolane (Traut's reagent)
resulting in conversion of an amine into a thiol.
[0192] Antibody drug conjugates of the invention may also be
produced by modification of the antibody to introduce electrophilic
moieties, which can react with nucleophilic substituents on the
linker reagent or drug. The sugars of glycosylated antibodies may
be oxidized, e.g. with periodate oxidizing reagents, to form
aldehyde or ketone groups which may react with the amine group of
linker reagents or drug moieties. The resulting imine Schiff base
groups may form a stable linkage, or may be reduced, e.g. by
borohydride reagents to form stable amine linkages. In one
embodiment, reaction of the carbohydrate portion of a glycosylated
antibody with either galactose oxidase or sodium meta-periodate may
yield carbonyl (aldehyde and ketone) groups in the protein that can
react with appropriate groups on the drug (Hermanson, Bioconjugate
Techniques). In another embodiment, proteins containing N-terminal
serine or threonine residues can react with sodium meta-periodate,
resulting in production of an aldehyde in place of the first amino
acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146;
U.S. Pat. No. 5,362,852). Such aldehyde can be reacted with a drug
moiety or linker nucleophile.
[0193] Likewise, nucleophilic groups on a drug moiety include, but
are not limited to: amine, thiol, hydroxyl, hydrazide, oxime,
hydrazine, thiosemicarbazone, hydrazine carboxylate, and
acrylhydrazide groups capable of reacting to form covalent bonds
with electrophilic groups on linker moieties and linker reagents
including: (i) active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; (ii) alkyl and benzyl halides such
as haloacetamides; (iii) aldehydes, ketones, carboxyl, and
maleimide groups.
[0194] Alternatively, a fusion protein comprising the anti-UNQ733
polypeptide antibody and cytotoxic agent may be made, e.g., by
recombinant techniques or peptide synthesis. The length of DNA may
comprise respective regions encoding the two portions of the
conjugate either adjacent one another or separated by a region
encoding a linker peptide which does not destroy the desired
properties of the conjugate.
[0195] In yet another embodiment, the antibody may be conjugated to
a "receptor" (such streptavidin) for utilization in tumor
pre-targeting wherein the antibody-receptor conjugate is
administered to the patient, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g., avidin) which is conjugated to
a cytotoxic agent (e.g., a radionucleotide).
[0196] 10. Immunoliposomes
[0197] The anti-UNQ733 polypeptide antibodies disclosed herein may
also be formulated as immunoliposomes. A "liposome" is a small
vesicle composed of various types of lipids, phospholipids and/or
surfactant which is useful for delivery of a drug to a mammal. The
components of the liposome are commonly arranged in a bilayer
formation, similar to the lipid arrangement of biological
membranes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc.
Natl. Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and
4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with
enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556.
[0198] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et al., J. National Cancer Inst.
81(19):1484 (1989).
[0199] B. UNQ733 Polypeptide Binding Oligopeptides
[0200] UNQ733 polypeptide binding oligopeptides of the invention
are oligopeptides that bind, preferably specifically, to a UNQ733
polypeptide as described herein. UNQ733 polypeptide binding
oligopeptides may be chemically synthesized using known
oligopeptide synthesis methodology or may be prepared and purified
using recombinant technology. UNQ733 polypeptide binding
oligopeptides are usually at least about 5 amino acids in length,
alternatively at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,
50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83,
84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or
100 amino acids in length or more, wherein such oligopeptides that
are capable of binding, preferably specifically, to a UNQ733
polypeptide as described herein. UNQ733 polypeptide binding
oligopeptides may be identified without undue experimentation using
well known techniques. In this regard, it is noted that techniques
for screening oligopeptide libraries for oligopeptides that are
capable of specifically binding to a polypeptide target are well
known in the art (see, e.g., U.S. Pat. Nos. 5,556,762, 5,750,373,
4,708,871, 4,833,092, 5,223,409, 5,403,484, 5,571,689, 5,663,143;
PCT Publication Nos. WO 84/03506 and WO84/03564; Geysen et al.,
Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984); Geysen et al.,
Proc. Natl. Acad. Sci. U.S.A., 82:178-182 (1985); Geysen et al., in
Synthetic Peptides as Antigens, 130-149 (1986); Geysen et al., J.
Immunol. Meth., 102:259-274 (1987); Schoofs et al., J. Immunol.,
140:611-616 (1988), Cwirla, S. E. et al. (1990) Proc. Natl. Acad.
Sci. USA, 87:6378; Lowman, H. B. et al. (1991) Biochemistry,
30:10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D.
et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al. (1991)
Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991)
Current Opin. Biotechnol., 2:668).
[0201] In this regard, bacteriophage (phage) display is one well
known technique which allows one to screen large oligopeptide
libraries to identify member(s) of those libraries which are
capable of specifically binding to a polypeptide target. Phage
display is a technique by which variant polypeptides are displayed
as fusion proteins to the coat protein on the surface of
bacteriophage particles (Scott, J. K. and Smith, G. P. (1990)
Science, 249: 386). The utility of phage display lies in the fact
that large libraries of selectively randomized protein variants (or
randomly cloned cDNAs) can be rapidly and efficiently sorted for
those sequences that bind to a target molecule with high affinity.
Display of peptide (Cwirla, S. E. et al. (1990) Proc. Natl. Acad.
Sci. USA, 87:6378) or protein (Lowman, H. B. et al. (1991)
Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352:
624; Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A.
S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363) libraries on
phage have been used for screening millions of polypeptides or
oligopeptides for ones with specific binding properties (Smith, G.
P. (1991) Current Opin. Biotechnol., 2:668). Sorting phage
libraries of random mutants requires a strategy for constructing
and propagating a large number of variants, a procedure for
affinity purification using the target receptor, and a means of
evaluating the results of binding enrichments. U.S. Pat. Nos.
5,223,409, 5,403,484, 5,571,689, and 5,663,143.
[0202] Although most phage display methods have used filamentous
phage, lambdoid phage display systems (WO 95/34683; U.S. Pat. No.
5,627,024), T4 phage display systems (Ren et al., Gene, 215: 439
(1998); Zhu et al., Cancer Research, 58(15): 3209-3214 (1998);
Jiang et al., Infection & Immunity, 65(11): 4770-4777 (1997);
Ren et al., Gene, 195(2):303-311 (1997); Ren, Protein Sci., 5: 1833
(1996); Efimov et al., Virus Genes, 10: 173 (1995)) and T7 phage
display systems (Smith and Scott, Methods in Enzymology, 217:
228-257 (1993); U.S. Pat. No. 5,766,905) are also known.
[0203] Many other improvements and variations of the basic phage
display concept have now been developed. These improvements enhance
the ability of display systems to screen peptide libraries for
binding to selected target molecules and to display functional
proteins with the potential of screening these proteins for desired
properties. Combinatorial reaction devices for phage display
reactions have been developed (WO 98/14277) and phage display
libraries have been used to analyze and control bimolecular
interactions (WO 98/20169; WO 98/20159) and properties of
constrained helical peptides (WO 98/20036). WO 97/35196 describes a
method of isolating an affinity ligand in which a phage display
library is contacted with one solution in which the ligand will
bind to a target molecule and a second solution in which the
affinity ligand will not bind to the target molecule, to
selectively isolate binding ligands. WO 97/46251 describes a method
of biopanning a random phage display library with an affinity
purified antibody and then isolating binding phage, followed by a
micropanning process using microplate wells to isolate high
affinity binding phage. The use of Staphlylococcus aureus protein A
as an affinity tag has also been reported (Li et al. (1998) Mol.
Biotech., 9:187). WO 97/47314 describes the use of substrate
subtraction libraries to distinguish enzyme specificities using a
combinatorial library which may be a phage display library. A
method for selecting enzymes suitable for use in detergents using
phage display is described in WO 97/09446. Additional methods of
selecting specific binding proteins are described in U.S. Pat. Nos.
5,498,538, 5,432,018, and WO 98/15833.
[0204] Methods of generating peptide libraries and screening these
libraries are also disclosed in U.S. Pat. Nos. 5,723,286,
5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018,
5,698,426, 5,763,192, and 5,723,323.
[0205] C. UNQ733 Polypeptide Binding Small Molecules
[0206] UNQ733 polypeptide binding small molecules are preferably
organic molecules other than oligopeptides or antibodies as defined
herein that bind, preferably specifically, to a UNQ733 polypeptide
as described herein. UNQ733 polypeptide binding organic small
molecules may be identified and chemically synthesized using known
methodology (see, e.g., PCT Publication Nos. WO00/00823 and
WO00/39585). UNQ733 polypeptide binding organic small molecules are
usually less than about 2000 daltons in size, alternatively less
than about 1500, 750, 500, 250 or 200 daltons in size, wherein such
organic small molecules that are capable of binding, preferably
specifically, to a UNQ733 polypeptide as described herein may be
identified without undue experimentation using well known
techniques. In this regard, it is noted that techniques for
screening organic small molecule libraries for molecules that are
capable of binding to a polypeptide target are well known in the
art (see, e.g., PCT Publication Nos. WO00/00823 and WO00/39585).
UNQ733 polypeptide binding organic small molecules may be, for
example, aldehydes, ketones, oximes, hydrazones, semicarbazones,
carbazides, primary amines, secondary amines, tertiary amines,
N-substituted hydrazines, hydrazides, alcohols, ethers, thiols,
thioethers, disulfides, carboxylic acids, esters, amides, ureas,
carbamates, carbonates, ketals, thioketals, acetals, thioacetals,
aryl halides, aryl sulfonates, alkyl halides, alkyl sulfonates,
aromatic compounds, heterocyclic compounds, anilines, alkenes,
alkynes, diols, amino alcohols, oxazolidines, oxazolines,
thiazolidines, thiazolines, enamines, sulfonamides, epoxides,
aziridines, isocyanates, sulfonyl chlorides, diazo compounds, acid
chlorides, or the like.
[0207] D. Screening for Anti-UNQ733 Polypeptide Antibodies, UNQ733
Polypeptide Binding Oligopeptides and UNQ733 Polypeptide Binding
Small Molecules with the Desired Properties
[0208] Techniques for generating antibodies, oligopeptides and
small molecules that bind to UNQ733 polypeptides have been
described above. One may further select antibodies, oligopeptides
or other small molecules with certain biological characteristics,
as desired.
[0209] The growth inhibitory effects of an anti-UNQ733 antibody,
oligopeptide or other small molecule of the invention may be
assessed by methods known in the art, e.g., using cells which
express a UNQ733 polypeptide either endogenously or following
transfection with the UNQ733 polypeptide gene. For example,
appropriate tumor cell lines and UNQ733 polypeptide-transfected
cells may be treated with an anti-UNQ733 polypeptide monoclonal
antibody, oligopeptide or other small molecule of the invention at
various concentrations for a few days (e.g., 2-7) days and stained
with crystal violet or MTT or analyzed by some other colorimetric
assay. Another method of measuring proliferation would be by
comparing .sup.3H-thymidine uptake by the cells treated in the
presence or absence an anti-UNQ733 polypeptide antibody, UNQ733
polypeptide binding oligopeptide or UNQ733 polypeptide binding
small molecule of the invention. After treatment, the cells are
harvested and the amount of radioactivity incorporated into the DNA
quantitated in a scintillation counter. Appropriate positive
controls include treatment of a selected cell line with a growth
inhibitory antibody known to inhibit growth of that cell line.
Growth inhibition of tumor cells in vivo can be determined in
various ways known in the art. The tumor cell may be one that
overexpresses a UNQ733 polypeptide. The anti-UNQ733 polypeptide
antibody, UNQ733 polypeptide binding oligopeptide or UNQ733
polypeptide binding organic small molecule will inhibit cell
proliferation of a UNQ733 polypeptide-expressing tumor cell in
vitro or in vivo by about 25-100% compared to the untreated tumor
cell, more preferably, by about 30-100%, and even more preferably
by about 50-100% or 70-100%, in one embodiment, at an antibody
concentration of about 0.5 to 30 .mu.g/ml. Growth inhibition can be
measured at an antibody concentration of about 0.5 to 30 .mu.g/ml
or about 0.5 nM to 200 nM in cell culture, where the growth
inhibition is determined 1-10 days after exposure of the tumor
cells to the antibody. The antibody is growth inhibitory in vivo if
administration of the anti-UNQ733 polypeptide antibody at about 1
.mu.g/kg to about 100 mg/kg body weight results in reduction in
tumor size or reduction of tumor cell proliferation within about 5
days to 3 months from the first administration of the antibody,
preferably within about 5 to 30 days.
[0210] To select for an anti-UNQ733 polypeptide antibody, UNQ733
polypeptide binding oligopeptide or UNQ733 polypeptide binding
organic small molecule which induces cell death, loss of membrane
integrity as indicated by, e.g., propidium iodide (PI), trypan blue
or 7AAD uptake may be assessed relative to control. A PI uptake
assay can be performed in the absence of complement and immune
effector cells. UNQ733 polypeptide-expressing tumor cells are
incubated with medium alone or medium containing the appropriate
anti-UNQ733 polypeptide antibody (e.g., at about 10 .mu.g/ml),
UNQ733 polypeptide binding oligopeptide or UNQ733 polypeptide
binding organic small molecule. The cells are incubated for a 3-day
time period. Following each treatment, cells are washed and
aliquoted into 35 mm strainer-capped 12.times.75 tubes (1 ml per
tube, 3 tubes per treatment group) for removal of cell clumps.
Tubes then receive PI (10 .mu.g/ml). Samples may be analyzed using
a FACSCAN.RTM. flow cytometer and FACSCONVERT.RTM. CellQuest
software (Becton Dickinson). Those anti-UNQ733 polypeptide
antibodies, UNQ733 polypeptide binding oligopeptides or UNQ733
polypeptide binding organic small molecules that induce
statistically significant levels of cell death as determined by PI
uptake may be selected as cell death-inducing anti-UNQ733
polypeptide antibodies, UNQ733 polypeptide binding oligopeptides or
UNQ733 polypeptide binding organic small molecules.
[0211] To screen for antibodies, oligopeptides or other organic
small molecules which bind to an epitope on a UNQ733 polypeptide
bound by an antibody of interest, a routine cross-blocking assay
such as that described in Antibodies, A Laboratory Manual, Cold
Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be
performed. This assay can be used to determine if a test antibody,
oligopeptide or other organic small molecule binds the same site or
epitope as a known anti-UNQ733 polypeptide antibody. Alternatively,
or additionally, epitope mapping can be performed by methods known
in the art. For example, the antibody sequence can be mutagenized
such as by alanine scanning, to identify contact residues. The
mutant antibody is initially tested for binding with polyclonal
antibody to ensure proper folding. In a different method, peptides
corresponding to different regions of a UNQ733 polypeptide can be
used in competition assays with the test antibodies or with a test
antibody and an antibody with a characterized or known epitope.
[0212] E. Antibody Dependent Enzyme Mediated Prodrug Therapy
(ADEPT)
[0213] The antibodies of the present invention may also be used in
ADEPT by conjugating the antibody to a prodrug-activating enzyme
which converts a prodrug (e.g., a peptidyl chemotherapeutic agent,
see WO81/01145) to an active anti-cancer drug. See, for example, WO
88/07378 and U.S. Pat. No. 4,975,278.
[0214] The enzyme component of the immunoconjugate useful for ADEPT
includes any enzyme capable of acting on a prodrug in such a way so
as to covert it into its more active, cytotoxic form.
[0215] Enzymes that are useful in the method of this invention
include, but are not limited to, alkaline phosphatase useful for
converting phosphate-containing prodrugs into free drugs;
arylsulfatase useful for converting sulfate-containing prodrugs
into free drugs; cytosine deaminase useful for converting non-toxic
5-fluorocytosine into the anti-cancer drug, 5-fluorouracil;
proteases, such as serratia protease, thermolysin, subtilisin,
carboxypeptidases and cathepsins (such as cathepsins B and L), that
are useful for converting peptide-containing prodrugs into free
drugs; D-alanylcarboxypeptidases, useful for converting prodrugs
that contain D-amino acid substituents; carbohydrate-cleaving
enzymes such as .beta.-galactosidase and neuraminidase useful for
converting glycosylated prodrugs into free drugs; .beta.-lactamase
useful for converting drugs derivatized with .beta.-lactams into
free drugs; and penicillin amidases, such as penicillin V amidase
or penicillin G amidase, useful for converting drugs derivatized at
their amine nitrogens with phenoxyacetyl or phenylacetyl groups,
respectively, into free drugs. Alternatively, antibodies with
enzymatic activity, also known in the art as "abzymes", can be used
to convert the prodrugs of the invention into free active drugs
(see, e.g., Massey, Nature 328:457-458 (1987)). Antibody-abzyme
conjugates can be prepared as described herein for delivery of the
abzyme to a tumor cell population.
[0216] The enzymes of this invention can be covalently bound to the
anti-UNQ733 antibodies by techniques well known in the art such as
the use of the heterobifunctional crosslinking reagents discussed
above. Alternatively, fusion proteins comprising at least the
antigen binding region of an antibody of the invention linked to at
least a functionally active portion of an enzyme of the invention
can be constructed using recombinant DNA techniques well known in
the art (see, e.g., Neuberger et al., Nature 312:604-608
(1984).
[0217] F. Anti-UNQ733 Polypeptide Antibody Variants
[0218] In addition to the anti-UNQ733 polypeptide antibodies
described herein, it is contemplated that anti-UNQ733 polypeptide
antibody variants can be prepared. Anti-UNQ733 polypeptide antibody
variants can be prepared by introducing appropriate nucleotide
changes into the encoding DNA, and/or by synthesis of the desired
antibody. Those skilled in the art will appreciate that amino acid
changes may alter post-translational processes of the anti-UNQ733
polypeptide antibody, such as changing the number or position of
glycosylation sites or altering the membrane anchoring
characteristics.
[0219] Variations in the anti-UNQ733 polypeptide antibodies
described herein can be made, for example, using any of the
techniques and guidelines for conservative and non-conservative
mutations set forth, for instance, in U.S. Pat. No. 5,364,934.
Variations may be a substitution, deletion or insertion of one or
more codons encoding the antibody that results in a change in the
amino acid sequence as compared with the native sequence antibody
or polypeptide. Optionally the variation is by substitution of at
least one amino acid with any other amino acid in one or more of
the domains of the anti-UNQ733 polypeptide antibody. Guidance in
determining which amino acid residue may be inserted, substituted
or deleted without adversely affecting the desired activity may be
found by comparing the sequence of the anti-UNQ733 polypeptide
antibody with that of homologous known protein molecules and
minimizing the number of amino acid sequence changes made in
regions of high homology. Amino acid substitutions can be the
result of replacing one amino acid with another amino acid having
similar structural and/or chemical properties, such as the
replacement of a leucine with a serine, i.e., conservative amino
acid replacements. Insertions or deletions may optionally be in the
range of about 1 to 5 amino acids. The variation allowed may be
determined by systematically making insertions, deletions or
substitutions of amino acids in the sequence and testing the
resulting variants for activity exhibited by the parent
sequence.
[0220] Anti-UNQ733 polypeptide antibody and UNQ733 polypeptide
fragments are provided herein. Such fragments may be truncated at
the N-terminus or C-terminus, or may lack internal residues, for
example, when compared with a full length native antibody or
protein. Certain fragments lack amino acid residues that are not
essential for a desired biological activity of the anti-UNQ733
antibody or UNQ733 polypeptide.
[0221] Anti-UNQ733 antibody and UNQ733 polypeptide fragments may be
prepared by any of a number of conventional techniques. Desired
peptide fragments may be chemically synthesized. An alternative
approach involves generating antibody or polypeptide fragments by
enzymatic digestion, e.g., by treating the protein with an enzyme
known to cleave proteins at sites defined by particular amino acid
residues, or by digesting the DNA with suitable restriction enzymes
and isolating the desired fragment. Yet another suitable technique
involves isolating and amplifying a DNA fragment encoding a desired
antibody or polypeptide fragment, by polymerase chain reaction
(PCR). Oligonucleotides that define the desired termini of the DNA
fragment are employed at the 5' and 3' primers in the PCR.
Preferably, anti-UNQ733 antibody and UNQ733 polypeptide fragments
share at least one biological and/or immunological activity with
the native anti-UNQ733 polypeptide antibody or UNQ733 polypeptide
disclosed herein.
[0222] In particular embodiments, conservative substitutions of
interest are shown in Table 6 under the heading of preferred
substitutions. If such substitutions result in a change in
biological activity, then more substantial changes, denominated
exemplary substitutions in Table 6, or as further described below
in reference to amino acid classes, are introduced and the products
screened. TABLE-US-00005 TABLE 6 Original Exemplary Preferred
Residue Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg
(R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D)
Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp;
Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu;
Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine; Ile; Val;
Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile
Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser
(S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp;
Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala;
Norleucine
[0223] Substantial modifications in function or immunological
identity of the anti-UNQ733 antibody or UNQ733 polypeptide are
accomplished by selecting substitutions that differ significantly
in their effect on maintaining (a) the structure of the polypeptide
backbone in the area of the substitution, for example, as a sheet
or helical conformation, (b) the charge or hydrophobicity of the
molecule at the target site, or (c) the bulk of the side chain.
Amino acids may be grouped according to similarities in the
properties of their side chains (in A. L. Lehninger, in
Biochemistry, second ed., pp. 73-75, Worth Publishers, New York
(1975)):
(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe
(F), Trp (W), Met (M)
(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y),
Asn (N), Gln (Q)
(3) acidic: Asp (D), Glu (E)
(4) basic: Lys (K), Arg (R), His (H)
[0224] Alternatively, naturally occurring residues may be divided
into groups based on common side-chain properties:
[0225] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0226] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0227] (3) acidic: Asp, Glu;
[0228] (4) basic: His, Lys, Arg;
[0229] (5) residues that influence chain orientation: Gly, Pro;
[0230] (6) aromatic: Trp, Tyr, Phe.
[0231] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class. Such substituted
residues also may be introduced into the conservative substitution
sites or, more preferably, into the remaining (non-conserved)
sites.
[0232] The variations can be made using methods known in the art
such as oligonucleotide-mediated (site-directed) mutagenesis,
alanine scanning, and PCR mutagenesis. Site-directed mutagenesis
[Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al.,
Nucl. Acids Res., 10:6487 (1987)], cassette mutagenesis [Wells et
al., Gene, 34:315 (1985)], restriction selection mutagenesis [Wells
et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or
other known techniques can be performed on the cloned DNA to
produce the anti-UNQ733 polypeptide antibody or UNQ733 polypeptide
variant DNA.
[0233] Scanning amino acid analysis can also be employed to
identify one or more amino acids along a contiguous sequence. Among
the preferred scanning amino acids are relatively small, neutral
amino acids. Such amino acids include alanine, glycine, serine, and
cysteine. Alanine is typically a preferred scanning amino acid
among this group because it eliminates the side-chain beyond the
beta-carbon and is less likely to alter the main-chain conformation
of the variant [Cunningham and Wells, Science, 244:1081-1085
(1989)]. Alanine is also typically preferred because it is the most
common amino acid. Further, it is frequently found in both buried
and exposed positions [Creighton, The Proteins, (W.H. Freeman &
Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. If alanine
substitution does not yield adequate amounts of variant, an
isometric amino acid can be used.
[0234] Any cysteine residue not involved in maintaining the proper
conformation of the anti-UNQ733 polypeptide antibody or UNQ733
polypeptide also may be substituted, generally with serine, to
improve the oxidative stability of the molecule and prevent
aberrant crosslinking. Conversely, cysteine bond(s) may be added to
the anti-UNQ733 antibody or UNQ733 polypeptide to improve its
stability (particularly where the antibody is an antibody fragment
such as an Fv fragment).
[0235] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody (e.g., a humanized or human antibody). Generally,
the resulting variant(s) selected for further development will have
improved biological properties relative to the parent antibody from
which they are generated. A convenient way for generating such
substitutional variants involves affinity maturation using phage
display. Briefly, several hypervariable region sites (e.g., 6-7
sites) are mutated to generate all possible amino substitutions at
each site. The antibody variants thus generated are displayed in a
monovalent fashion from filamentous phage particles as fusions to
the gene III product of M13 packaged within each particle. The
phage-displayed variants are then screened for their biological
activity (e.g., binding affinity) as herein disclosed. In order to
identify candidate hypervariable region sites for modification,
alanine scanning mutagenesis can be performed to identify
hypervariable region residues contributing significantly to antigen
binding. Alternatively, or additionally, it may be beneficial to
analyze a crystal structure of the antigen-antibody complex to
identify contact points between the antibody and human UNQ733
polypeptide. Such contact residues and neighboring residues are
candidates for substitution according to the techniques elaborated
herein. Once such variants are generated, the panel of variants is
subjected to screening as described herein and antibodies with
superior properties in one or more relevant assays may be selected
for further development.
[0236] Nucleic acid molecules encoding amino acid sequence variants
of the anti-UNQ733 antibody are prepared by a variety of methods
known in the art. These methods include, but are not limited to,
isolation from a natural source (in the case of naturally occurring
amino acid sequence variants) or preparation by
oligonucleotide-mediated (or site-directed) mutagenesis, PCR
mutagenesis, and cassette mutagenesis of an earlier prepared
variant or a non-variant version of the anti-UNQ733 antibody.
[0237] G. Modifications of Anti-UNQ733 Antibodies and UNQ733
Polypeptides
[0238] Covalent modifications of anti-UNQ733 polypeptide antibodies
and UNQ733 polypeptides are included within the scope of this
invention. One type of covalent modification includes reacting
targeted amino acid residues of an anti-UNQ733 antibody or UNQ733
polypeptide with an organic derivatizing agent that is capable of
reacting with selected side chains or the N- or C-terminal residues
of the anti-UNQ733 polypeptide antibody or UNQ733 polypeptide.
Derivatization with bifunctional agents is useful, for instance,
for crosslinking anti-UNQ733 antibody or UNQ733 polypeptide to a
water-insoluble support matrix or surface for use in the method for
purifying anti-UNQ733 antibodies, and vice-versa. Commonly used
crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0239] Other modifications include deamidation of glutaminyl and
asparaginyl residues to the corresponding glutamyl and aspartyl
residues, respectively, hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues,
methylation of the .alpha.-amino groups of lysine, arginine, and
histidine side chains [T. E. Creighton, Proteins: Structure and
Molecular Properties, W.H. Freeman & Co., San Francisco, pp.
79-86 (1983)], acetylation of the N-terminal amine, and amidation
of any C-terminal carboxyl group.
[0240] Another type of covalent modification of the anti-UNQ733
antibody or UNQ733 polypeptide included within the scope of this
invention comprises altering the native glycosylation pattern of
the antibody or polypeptide. "Altering the native glycosylation
pattern" is intended for purposes herein to mean deleting one or
more carbohydrate moieties found in native sequence anti-UNQ733
antibody or UNQ733 polypeptide (either by removing the underlying
glycosylation site or by deleting the glycosylation by chemical
and/or enzymatic means), and/or adding one or more glycosylation
sites that are not present in the native sequence anti-UNQ733
antibody or UNQ733 polypeptide. In addition, the phrase includes
qualitative changes in the glycosylation of the native proteins,
involving a change in the nature and proportions of the various
carbohydrate moieties present.
[0241] Glycosylation of antibodies and other polypeptides is
typically either N-linked or O-linked. N-linked refers to the
attachment of the carbohydrate moiety to the side chain of an
asparagine residue. The tripeptide sequences asparagine-X-serine
and asparagine-X-threonine, where X is any amino acid except
proline, are the recognition sequences for enzymatic attachment of
the carbohydrate moiety to the asparagine side chain. Thus, the
presence of either of these tripeptide sequences in a polypeptide
creates a potential glycosylation site. O-linked glycosylation
refers to the attachment of one of the sugars N-aceylgalactosamine,
galactose, or xylose to a hydroxyamino acid, most commonly serine
or threonine, although 5-hydroxyproline or 5-hydroxylysine may also
be used.
[0242] Addition of glycosylation sites to the anti-UNQ733 antibody
or UNQ733 polypeptide is conveniently accomplished by altering the
amino acid sequence such that it contains one or more of the
above-described tripeptide sequences (for N-linked glycosylation
sites). The alteration may also be made by the addition of, or
substitution by, one or more serine or threonine residues to the
sequence of the original anti-UNQ733 antibody or UNQ733 polypeptide
(for O-linked glycosylation sites). The anti-UNQ733 antibody or
UNQ733 polypeptide amino acid sequence may optionally be altered
through changes at the DNA level, particularly by mutating the DNA
encoding the anti-UNQ733 antibody or UNQ733 polypeptide at
preselected bases such that codons are generated that will
translate into the desired amino acids.
[0243] Another means of increasing the number of carbohydrate
moieties on the anti-UNQ733 antibody or UNQ733 polypeptide is by
chemical or enzymatic coupling of glycosides to the polypeptide.
Such methods are described in the art, e.g., in WO 87/05330
published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit. Rev.
Biochem., pp. 259-306 (1981).
[0244] Removal of carbohydrate moieties present on the anti-UNQ733
antibody or UNQ733 polypeptide may be accomplished chemically or
enzymatically or by mutational substitution of codons encoding for
amino acid residues that serve as targets for glycosylation.
Chemical deglycosylation techniques are known in the art and
described, for instance, by Hakimuddin, et al., Arch. Biochem.
Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118:131
(1981). Enzymatic cleavage of carbohydrate moieties on polypeptides
can be achieved by the use of a variety of endo- and
exo-glycosidases as described by Thotakura et al., Meth. Enzymol.,
138:350 (1987).
[0245] Another type of covalent modification of anti-UNQ733
antibody or UNQ733 polypeptide comprises linking the antibody or
polypeptide to one of a variety of nonproteinaceous polymers, e.g.,
polyethylene glycol (PEG), polypropylene glycol, or
polyoxyalkylenes, in the manner set forth in U.S. Pat. No.
4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
The antibody or polypeptide also may be entrapped in microcapsules
prepared, for example, by coacervation techniques or by interfacial
polymerization (for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively), in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules), or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A.,
Ed., (1980).
[0246] The anti-UNQ733 antibody or UNQ733 polypeptide of the
present invention may also be modified in a way to form chimeric
molecules comprising an anti-UNQ733 antibody or UNQ733 polypeptide
fused to another, heterologous polypeptide or amino acid
sequence.
[0247] In one embodiment, such a chimeric molecule comprises a
fusion of the anti-UNQ733 antibody or UNQ733 polypeptide with a tag
polypeptide which provides an epitope to which an anti-tag antibody
can selectively bind. The epitope tag is generally placed at the
amino- or carboxyl-terminus of the anti-UNQ733 antibody or UNQ733
polypeptide. The presence of such epitope-tagged forms of the
anti-UNQ733 antibody or UNQ733 polypeptide can be detected using an
antibody against the tag polypeptide. Also, provision of the
epitope tag enables the anti-UNQ733 antibody or UNQ733 polypeptide
to be readily purified by affinity purification using an anti-tag
antibody or another type of affinity matrix that binds to the
epitope tag. Various tag polypeptides and their respective
antibodies are well known in the art. Examples include
poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly)
tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et
al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the
8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al.,
Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes
Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et
al., Protein Engineering, 3(6):547-553 (1990)]. Other tag
polypeptides include the Flag-peptide [Hopp et al., BioTechnology,
6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al.,
Science, 255:192-194 (1992)]; an .alpha.-tubulin epitope peptide
[Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the
T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl.
Acad. Sci. USA, 87:6393-6397 (1990)].
[0248] In an alternative embodiment, the chimeric molecule may
comprise a fusion of the anti-UNQ733 antibody or UNQ733 polypeptide
with an immunoglobulin or a particular region of an immunoglobulin.
For a bivalent form of the chimeric molecule (also referred to as
an "immunoadhesin"), such a fusion could be to the Fc region of an
IgG molecule. The Ig fusions preferably include the substitution of
a soluble (transmembrane domain deleted or inactivated) form of an
anti-UNQ733 antibody or UNQ733 polypeptide in place of at least one
variable region within an Ig molecule. In a particularly preferred
embodiment, the immunoglobulin fusion includes the hinge, CH.sub.2
and CH.sub.3, or the hinge, CH.sub.1, CH.sub.2 and CH.sub.3 regions
of an IgG1 molecule. For the production of immunoglobulin fusions
see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0249] H. Preparation of Anti-UNQ733 Antibodies and UNQ733
Polypeptides
[0250] The description below relates primarily to production of
anti-UNQ733 antibodies and UNQ733 polypeptides by culturing cells
transformed or transfected with a vector containing anti-UNQ733
antibody- and UNQ733 polypeptide-encoding nucleic acid. It is, of
course, contemplated that alternative methods, which are well known
in the art, may be employed to prepare anti-UNQ733 antibodies and
UNQ733 polypeptides. For instance, the appropriate amino acid
sequence, or portions thereof, may be produced by direct peptide
synthesis using solid-phase techniques [see, e.g., Stewart et al.,
Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco,
Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)].
In vitro protein synthesis may be performed using manual techniques
or by automation. Automated synthesis may be accomplished, for
instance, using an Applied Biosystems Peptide Synthesizer (Foster
City, Calif.) using manufacturer's instructions. Various portions
of the anti-UNQ733 antibody or UNQ733 polypeptide may be chemically
synthesized separately and combined using chemical or enzymatic
methods to produce the desired anti-UNQ733 antibody or UNQ733
polypeptide.
[0251] 1. Isolation of DNA Encoding Anti-UNQ733 Antibody or UNQ733
Polypeptide
[0252] DNA encoding anti-UNQ733 antibody or UNQ733 polypeptide may
be obtained from a cDNA library prepared from tissue believed to
possess the anti-UNQ733 antibody or UNQ733 polypeptide mRNA and to
express it at a detectable level. Accordingly, human anti-UNQ733
antibody or UNQ733 polypeptide DNA can be conveniently obtained
from a cDNA library prepared from human tissue. The anti-UNQ733
antibody- or UNQ733 polypeptide-encoding gene may also be obtained
from a genomic library or by known synthetic procedures (e.g.,
automated nucleic acid synthesis).
[0253] Libraries can be screened with probes (such as
oligonucleotides of at least about 20-80 bases) designed to
identify the gene of interest or the protein encoded by it.
Screening the cDNA or genomic library with the selected probe may
be conducted using standard procedures, such as described in
Sambrook et al., Molecular Cloning: A Laboratory Manual (New York:
Cold Spring Harbor Laboratory Press, 1989). An alternative means to
isolate the gene encoding anti-UNQ733 antibody or UNQ733
polypeptide is to use PCR methodology [Sambrook et al., supra;
Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring
Harbor Laboratory Press, 1995)].
[0254] Techniques for screening a cDNA library are well known in
the art. The oligonucleotide sequences selected as probes should be
of sufficient length and sufficiently unambiguous that false
positives are minimized. The oligonucleotide is preferably labeled
such that it can be detected upon hybridization to DNA in the
library being screened. Methods of labeling are well known in the
art, and include the use of radiolabels like .sup.32P-labeled ATP,
biotinylation or enzyme labeling. Hybridization conditions,
including moderate stringency and high stringency, are provided in
Sambrook et al., supra.
[0255] Sequences identified in such library screening methods can
be compared and aligned to other known sequences deposited and
available in public databases such as GenBank or other private
sequence databases. Sequence identity (at either the amino acid or
nucleotide level) within defined regions of the molecule or across
the full-length sequence can be determined using methods known in
the art and as described herein.
[0256] Nucleic acid having protein coding sequence may be obtained
by screening selected cDNA or genomic libraries using the deduced
amino acid sequence disclosed herein for the first time, and, if
necessary, using conventional primer extension procedures as
described in Sambrook et al., supra, to detect precursors and
processing intermediates of mRNA that may not have been
reverse-transcribed into cDNA.
[0257] 2. Selection and Transformation of Host Cells
[0258] Host cells are transfected or transformed with expression or
cloning vectors described herein for anti-UNQ733 antibody or UNQ733
polypeptide production and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences. The culture conditions, such as media, temperature, pH
and the like, can be selected by the skilled artisan without undue
experimentation. In general, principles, protocols, and practical
techniques for maximizing the productivity of cell cultures can be
found in Mammalian Cell Biotechnology: a Practical Approach, M.
Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.
[0259] Methods of eukaryotic cell transfection and prokaryotic cell
transformation are known to the ordinarily skilled artisan, for
example, CaCl.sub.2, CaPO.sub.4, liposome-mediated and
electroporation. Depending on the host cell used, transformation is
performed using standard techniques appropriate to such cells. The
calcium treatment employing calcium chloride, as described in
Sambrook et al., supra, or electroporation is generally used for
prokaryotes. Infection with Agrobacterium tumefaciens is used for
transformation of certain plant cells, as described by Shaw et al.,
Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. For
mammalian cells without such cell walls, the calcium phosphate
precipitation method of Graham and van der Eb, Virology, 52:456-457
(1978) can be employed. General aspects of mammalian cell host
system transfections have been described in U.S. Pat. No.
4,399,216. Transformations into yeast are typically carried out
according to the method of Van Solingen et al., J. Bact., 130:946
(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829
(1979). However, other methods for introducing DNA into cells, such
as by nuclear microinjection, electroporation, bacterial protoplast
fusion with intact cells, or polycations, e.g., polybrene,
polyornithine, may also be used. For various techniques for
transforming mammalian cells, see Keown et al., Methods in
Enzymology, 185:527-537 (1990) and Mansour et al., Nature,
336:348-352 (1988).
[0260] Suitable host cells for cloning or expressing the DNA in the
vectors herein include prokaryote, yeast, or higher eukaryote
cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as E. coli. Various E. coli
strains are publicly available, such as E. coli K12 strain MM294
(ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110
(ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic
host cells include Enterobacteriaceae such as Escherichia, e.g., E.
coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. These examples are illustrative rather than limiting.
Strain W3110 is one particularly preferred host or parent host
because it is a common host strain for recombinant DNA product
fermentations. Preferably, the host cell secretes minimal amounts
of proteolytic enzymes. For example, strain W3110 may be modified
to effect a genetic mutation in the genes encoding proteins
endogenous to the host, with examples of such hosts including E.
coli W3110 strain 1A2, which has the complete genotype tonA; E.
coli W3110 strain 9E4, which has the complete genotype tonA ptr3;
E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete
genotype tonA ptr3 phoA E15 (argF-lac) 169 degP ompT kan.sup.r; E.
coli W3110 strain 37D6, which has the complete genotype tonA ptr3
phoA E15 (argF-lac) 169 degP ompT rbs 7 ilvG kan.sup.r; E. coli
W3110 strain 40B4, which is strain 37D6 with a non-kanamycin
resistant degP deletion mutation; and an E. coli strain having
mutant periplasmic protease disclosed in U.S. Pat. No. 4,946,783
issued 7 Aug. 1990. Alternatively, in vitro methods of cloning,
e.g., PCR or other nucleic acid polymerase reactions, are
suitable.
[0261] Full length antibody, antibody fragments, and antibody
fusion proteins can be produced in bacteria, in particular when
glycosylation and Fc effector function are not needed, such as when
the therapeutic antibody is conjugated to a cytotoxic agent (e.g.,
a toxin) and the immunoconjugate by itself shows effectiveness in
tumor cell destruction. Full-length antibodies have greater half
life in circulation. Production in E. coli is faster and more cost
efficient. For expression of antibody fragments and polypeptides in
bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et. al.), U.S.
Pat. No. 5,789,199 (Joly et al.), and U.S. Pat. No. 5,840,523
(Simmons et al.) which describes translation initiation regio (TIR)
and signal sequences for optimizing expression and secretion, these
patents incorporated herein by reference. After expression, the
antibody is isolated from the E. coli cell paste in a soluble
fraction and can be purified through, e.g., a protein A or G column
depending on the isotype. Final purification can be carried out
similar to the process for purifying antibody expressed e.g., in
CHO cells.
[0262] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for anti-UNQ733 antibody- or UNQ733 polypeptide-encoding vectors.
Saccharomyces cerevisiae is a commonly used lower eukaryotic host
microorganism. Others include Schizosaccharomyces pombe (Beach and
Nurse, Nature, 290: 140 [1981]; EP 139,383 published 2 May 1985);
Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,
Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis
(MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol.,
154(2):737-742 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus
(ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC
56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al.,
Bio/Technology, 8:135 (1990)), K. therotolerans, and K. marxianus;
yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et
al., J. Basic Microbiol., 28:265-278 [1988]); Candida; Trichoderma
reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl.
Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such as
Schwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990);
and filamentous fungi such as, e.g., Neurospora, Penicillium,
Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillus
hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res.
Commun., 112:284-289 [1983]; Tilburn et al., Gene, 26:205-221
[1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474
[1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479 [1985]).
Methylotropic yeasts are suitable herein and include, but are not
limited to, yeast capable of growth on methanol selected from the
genera consisting of Hansenula, Candida, Kloeckera, Pichia,
Saccharomyces, Torulopsis, and Rhodotorula. A list of specific
species that are exemplary of this class of yeasts may be found in
C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).
[0263] Suitable host cells for the expression of glycosylated
anti-UNQ733 antibody or UNQ733 polypeptide are derived from
multicellular organisms. Examples of invertebrate cells include
insect cells such as Drosophila S2 and Spodoptera Sf9, as well as
plant cells, such as cell cultures of cotton, corn, potato,
soybean, petunia, tomato, and tobacco. Numerous baculoviral strains
and variants and corresponding permissive insect host cells from
hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti
(mosquito), Aedes albopictus (mosquito), Drosophila melanogaster
(fruitfly), and Bombyx mori have been identified. A variety of
viral strains for transfection are publicly available, e.g., the
L-1 variant of Autographa californica NPV and the Bm-5 strain of
Bombyx mori NPV, and such viruses may be used as the virus herein
according to the present invention, particularly for transfection
of Spodoptera frugiperda cells.
[0264] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol.
36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl.
Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather,
Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL
70); African green monkey kidney cells (VERO-76, ATCC CRL-1587);
human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney
cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC
CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells
(Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);
TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982));
MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
[0265] Host cells are transformed with the above-described
expression or cloning vectors for anti-UNQ733 antibody or UNQ733
polypeptide production and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences.
[0266] 3. Selection and Use of a Replicable Vector
[0267] The nucleic acid (e.g., cDNA or genomic DNA) encoding
anti-UNQ733 antibody or UNQ733 polypeptide may be inserted into a
replicable vector for cloning (amplification of the DNA) or for
expression. Various vectors are publicly available. The vector may,
for example, be in the form of a plasmid, cosmid, viral particle,
or phage. The appropriate nucleic acid sequence may be inserted
into the vector by a variety of procedures. In general, DNA is
inserted into an appropriate restriction endonuclease site(s) using
techniques known in the art. Vector components generally include,
but are not limited to, one or more of a signal sequence, an origin
of replication, one or more marker genes, an enhancer element, a
promoter, and a transcription termination sequence. Construction of
suitable vectors containing one or more of these components employs
standard ligation techniques which are known to the skilled
artisan.
[0268] The UNQ733 polypeptide may be produced recombinantly not
only directly, but also as a fusion polypeptide with a heterologous
polypeptide, which may be a signal sequence or other polypeptide
having a specific cleavage site at the N-terminus of the mature
protein or polypeptide. In general, the signal sequence may be a
component of the vector, or it may be a part of the anti-UNQ733
antibody- or UNQ733 polypeptide-encoding DNA that is inserted into
the vector. The signal sequence may be a prokaryotic signal
sequence selected, for example, from the group of the alkaline
phosphatase, penicillinase, lpp, or heat-stable enterotoxin II
leaders. For yeast secretion the signal sequence may be, e.g., the
yeast invertase leader, alpha factor leader (including
Saccharomyces and Kluyveromyces .alpha.-factor leaders, the latter
described in U.S. Pat. No. 5,010,182), or acid phosphatase leader,
the C. albicans glucoamylase leader (EP 362,179 published 4 Apr.
1990), or the signal described in WO 90/13646 published 15 Nov.
1990. In mammalian cell expression, mammalian signal sequences may
be used to direct secretion of the protein, such as signal
sequences from secreted polypeptides of the same or related
species, as well as viral secretory leaders.
[0269] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Such sequences are well known for a variety of
bacteria, yeast, and viruses. The origin of replication from the
plasmid pBR322 is suitable for most Gram-negative bacteria, the
2.mu. plasmid origin is suitable for yeast, and various viral
origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for
cloning vectors in mammalian cells.
[0270] Expression and cloning vectors will typically contain a
selection gene, also termed a selectable marker. Typical selection
genes encode proteins that (a) confer resistance to antibiotics or
other toxins, e.g., ampicillin, neomycin, methotrexate, or
tetracycline, (b) complement auxotrophic deficiencies, or (c)
supply critical nutrients not available from complex media, e.g.,
the gene encoding D-alanine racemase for Bacilli.
[0271] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the anti-UNQ733 antibody- or UNQ733 polypeptide-encoding
nucleic acid, such as DHFR or thymidine kinase. An appropriate host
cell when wild-type DHFR is employed is the CHO cell line deficient
in DHFR activity, prepared and propagated as described by Urlaub et
al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitable
selection gene for use in yeast is the trp1 gene present in the
yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979);
Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157
(1980)]. The trp1 gene provides a selection marker for a mutant
strain of yeast lacking the ability to grow in tryptophan, for
example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12
(1977)].
[0272] Expression and cloning vectors usually contain a promoter
operably linked to the anti-UNQ733 antibody- or UNQ733
polypeptide-encoding nucleic acid sequence to direct mRNA
synthesis. Promoters recognized by a variety of potential host
cells are well known. Promoters suitable for use with prokaryotic
hosts include the .beta.-lactamase and lactose promoter systems
[Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature,
281:544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter
system [Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776], and
hybrid promoters such as the tac promoter [deBoer et al., Proc.
Natl. Acad. Sci. USA, 80:21-25 (1983)]. Promoters for use in
bacterial systems also will contain a Shine-Dalgarno (S.D.)
sequence operably linked to the DNA encoding anti-UNQ733 antibody
or UNQ733 polypeptide.
[0273] Examples of suitable promoting sequences for use with yeast
hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman
et al., J. Biol. Chem., 255:2073 (1980)] or other glycolytic
enzymes [Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland,
Biochemistry, 17:4900 (1978)], such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate
decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0274] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in EP
73,657.
[0275] Anti-UNQ733 antibody or UNQ733 polypeptide transcription
from vectors in mammalian host cells is controlled, for example, by
promoters obtained from the genomes of viruses such as polyoma
virus, fowlpox virus (UK 2,211,504 published 5 Jul. 1989),
adenovirus (such as Adenovirus 2), bovine papilloma virus, avian
sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and
Simian Virus 40 (SV40), from heterologous mammalian promoters,
e.g., the actin promoter or an immunoglobulin promoter, and from
heat-shock promoters, provided such promoters are compatible with
the host cell systems.
[0276] Transcription of a DNA encoding the anti-UNQ733 antibody or
UNQ733 polypeptide by higher eukaryotes may be increased by
inserting an enhancer sequence into the vector. Enhancers are
cis-acting elements of DNA, usually about from 10 to 300 bp, that
act on a promoter to increase its transcription. Many enhancer
sequences are now known from mammalian genes (globin, elastase,
albumin, .alpha.-fetoprotein, and insulin). Typically, however, one
will use an enhancer from a eukaryotic cell virus. Examples include
the SV40 enhancer on the late side of the replication origin (bp
100-270), the cytomegalovirus early promoter enhancer, the polyoma
enhancer on the late side of the replication origin, and adenovirus
enhancers. The enhancer may be spliced into the vector at a
position 5' or 3' to the anti-UNQ733 antibody or UNQ733 polypeptide
coding sequence, but is preferably located at a site 5' from the
promoter.
[0277] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human, or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3',
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain nucleotide segments transcribed as polyadenylated
fragments in the untranslated portion of the mRNA encoding
anti-UNQ733 antibody or UNQ733 polypeptide.
[0278] Still other methods, vectors, and host cells suitable for
adaptation to the synthesis of anti-UNQ733 antibody or UNQ733
polypeptide in recombinant vertebrate cell culture are described in
Gething et al., Nature, 293:620-625 (1981); Mantei et al., Nature,
281:40-46 (1979); EP 117,060; and EP 117,058.
[0279] 4. Culturing the Host Cells
[0280] The host cells used to produce the anti-UNQ733 antibody or
UNQ733 polypeptide of this invention may be cultured in a variety
of media. Commercially available media such as Ham's F10 (Sigma),
Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and
Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for
culturing the host cells. In addition, any of the media described
in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.
Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866;
4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or
U.S. Pat Re. 30,985 may be used as culture media for the host
cells. Any of these media may be supplemented as necessary with
hormones and/or other growth factors (such as insulin, transferrin,
or epidermal growth factor), salts (such as sodium chloride,
calcium, magnesium, and phosphate), buffers (such as HEPES),
nucleotides (such as adenosine and thymidine), antibiotics (such as
GENTAMYCIN.TM. drug), trace elements (defined as inorganic
compounds usually present at final concentrations in the micromolar
range), and glucose or an equivalent energy source. Any other
necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. The
culture conditions, such as temperature, pH, and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan.
[0281] 5. Detecting Gene Amplification/Expression
[0282] Gene amplification and/or expression may be measured in a
sample directly, for example, by conventional Southern blotting,
Northern blotting to quantitate the transcription of mRNA [Thomas,
Proc. Natl. Acad. Sci. USA, 77:5201-5205 (1980)], dot blotting (DNA
analysis), or in situ hybridization, using an appropriately labeled
probe, based on the sequences provided herein. Alternatively,
antibodies may be employed that can recognize specific duplexes,
including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes
or DNA-protein duplexes. The antibodies in turn may be labeled and
the assay may be carried out where the duplex is bound to a
surface, so that upon the formation of duplex on the surface, the
presence of antibody bound to the duplex can be detected.
[0283] Gene expression, alternatively, may be measured by
immunological methods, such as immunohistochemical staining of
cells or tissue sections and assay of cell culture or body fluids,
to quantitate directly the expression of gene product. Antibodies
useful for immunohistochemical staining and/or assay of sample
fluids may be either monoclonal or polyclonal, and may be prepared
in any mammal. Conveniently, the antibodies may be prepared against
a native sequence UNQ733 polypeptide or against a synthetic peptide
based on the DNA sequence provided herein or against exogenous
sequence fused to UNQ733 polypeptide DNA and encoding a specific
antibody epitope.
[0284] 6. Purification of Anti-UNQ733 Antibody and UNQ733
Polypeptide
[0285] Forms of anti-UNQ733 antibody and UNQ733 polypeptide may be
recovered from culture medium or from host cell lysates. If
membrane-bound, it can be released from the membrane using a
suitable detergent solution (e.g. Triton-X 100) or by enzymatic
cleavage. Cells employed in expression of anti-UNQ733 antibody and
UNQ733 polypeptide can be disrupted by various physical or chemical
means, such as freeze-thaw cycling, sonication, mechanical
disruption, or cell lysing agents.
[0286] It may be desired to purify anti-UNQ733 antibody and UNQ733
polypeptide from recombinant cell proteins or polypeptides. The
following procedures are exemplary of suitable purification
procedures: by fractionation on an ion-exchange column; ethanol
precipitation; reverse phase HPLC; chromatography on silica or on a
cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;
ammonium sulfate precipitation; gel filtration using, for example,
Sephadex G-75; protein A Sepharose columns to remove contaminants
such as IgG; and metal chelating columns to bind epitope-tagged
forms of the anti-UNQ733 antibody and UNQ733 polypeptide. Various
methods of protein purification may be employed and such methods
are known in the art and described for example in Deutscher,
Methods in Enzymology, 182 (1990); Scopes, Protein Purification
Principles and Practice, Springer-Verlag, New York (1982). The
purification step(s) selected will depend, for example, on the
nature of the production process used and the particular
anti-UNQ733 antibody or UNQ733 polypeptide produced.
[0287] When using recombinant techniques, the antibody can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed fragments, are removed, for example, by
centrifugation or ultrafiltration. Carter et al., Bio/Technology
10: 163-167 (1992) describe a procedure for isolating antibodies
which 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 min.
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.
[0288] The 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 antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2 or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., EMBO J. 5:15671575 (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.TM. 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.
[0289] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and contaminants may be
subjected to low pH hydrophobic interaction chromatography using an
elution buffer at a pH between about 2.5-4.5, preferably performed
at low salt concentrations (e.g., from about 0-0.25M salt).
[0290] I. Pharmaceutical Formulations
[0291] Therapeutic formulations of the anti-UNQ733 antibodies,
UNQ733 polypeptide binding oligopeptides, UNQ733 polypeptide
binding organic or inorganic small molecules and/or UNQ733
polypeptides used in accordance with the present invention are
prepared for storage by mixing the antibody, polypeptide,
oligopeptide or organic/inorganic small molecule having the desired
degree of purity with optional pharmaceutically acceptable
carriers, excipients or stabilizers (Remington's Pharmaceutical
Sciences 16th edition, Osol, A. Ed. (1980)), in the form of
lyophilized formulations or aqueous solutions. Acceptable carriers,
excipients, or stabilizers are nontoxic to recipients at the
dosages and concentrations employed, and include buffers such as
acetate, Tris, phosphate, citrate, and other organic acids;
antioxidants including ascorbic acid and methionine; preservatives
(such as octadecyldimethylbenzyl ammonium chloride; hexamethonium
chloride; benzalkonium chloride, benzethonium chloride; phenol,
butyl or benzyl alcohol; alkyl parabens such as methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and
m-cresol); low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, histidine,
arginine, or lysine; monosaccharides, disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; tonicifiers such as trehalose and sodium
chloride; sugars such as sucrose, mannitol, trehalose or sorbitol;
surfactant such as polysorbate; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.RTM., PLURONICS.RTM. or
polyethylene glycol (PEG). The antibody preferably comprises the
antibody at a concentration of between 5-200 mg/ml, preferably
between 10-100 mg/ml.
[0292] The formulations herein may also contain more than one
active compound as necessary for the particular indication being
treated, preferably those with complementary activities that do not
adversely affect each other. For example, in addition to an
anti-UNQ733 antibody, UNQ733 polypeptide binding oligopeptide, or
UNQ733 polypeptide binding organic or inorganic small molecule, it
may be desirable to include in the one formulation, an additional
antibody, e.g., a second anti-UNQ733 antibody which binds a
different epitope on the UNQ733 polypeptide, or an antibody to some
other target such as a growth factor that affects the growth of the
particular cancer. Alternatively, or additionally, the composition
may further comprise a chemotherapeutic agent, cytotoxic agent,
cytokine, growth inhibitory agent, anti-hormonal agent, and/or
cardioprotectant. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0293] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences,
16th edition, Osol, A. Ed. (1980).
[0294] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semi-permeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g., films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.RTM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid.
[0295] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0296] J. Treatment with Anti-UNQ733 Antibodies UNQ733 Polypeptide
Binding Oligopeptides and UNQ733 Polypeptide Binding
Organic/Inorganic Small Molecules
[0297] To determine UNQ733 polypeptide expression in the cancer,
various detection assays are available. In one embodiment, UNQ733
polypeptide overexpression may be analyzed by immunohistochemistry
(IHC). Parrafin embedded tissue sections from a tumor biopsy may be
subjected to the IHC assay and accorded a UNQ733 polypeptide
staining intensity criteria as follows:
[0298] Score 0--no staining is observed or membrane staining is
observed in less than 10% of tumor cells.
[0299] Score 1+--a faint/barely perceptible membrane staining is
detected in more than 10% of the tumor cells. The cells are only
stained in part of their membrane.
[0300] Score 2+--a weak to moderate complete membrane staining is
observed in more than 10% of the tumor cells.
[0301] Score 3+--a moderate to strong complete membrane staining is
observed in more than 10% of the tumor cells.
[0302] Those tumors with 0 or 1+ scores for UNQ733 polypeptide
expression may be characterized as not overexpressing UNQ733
polypeptide, whereas those tumors with 2+ or 3+ scores may be
characterized as overexpressing UNQ733 polypeptide.
[0303] Alternatively, or additionally, FISH assays such as the
INFORM.RTM. (sold by Ventana, Ariz.) or PATHVISION.RTM. (Vysis,
Ill.) may be carried out on formalin-fixed, paraffin-embedded tumor
tissue to determine the extent (if any) of UNQ733 polypeptide
overexpression in the tumor.
[0304] UNQ733 polypeptide overexpression or amplification may be
evaluated using an in vivo detection assay, e.g., by administering
a molecule (such as an antibody, oligopeptide or organic small
molecule) which binds the molecule to be detected and is tagged
with a detectable label (e.g., a radioactive isotope or a
fluorescent label) and externally scanning the patient for
localization of the label.
[0305] As described above, the anti-UNQ733 antibodies,
oligopeptides and organic small molecules of the invention have
various non-therapeutic applications. The anti-UNQ733 antibodies,
oligopeptides and organic/inorganic small molecules of the present
invention can be useful for staging of UNQ733
polypeptide-expressing cancers (e.g., in radioimaging). The
antibodies, oligopeptides and organic small molecules are also
useful for purification or immunoprecipitation of UNQ733
polypeptide from cells, for detection and quantitation of UNQ733
polypeptide in vitro, e.g., in an ELISA or a Western blot, to kill
and eliminate UNQ733 polypeptide-expressing cells from a population
of mixed cells as a step in the purification of other cells.
[0306] Currently, depending on the stage of the cancer, cancer
treatment involves one or a combination of the following therapies:
surgery to remove the cancerous tissue, radiation therapy, and
chemotherapy. Anti-UNQ733 antibody, oligopeptide or organic small
molecule therapy may be especially desirable in elderly patients
who do not tolerate the toxicity and side effects of chemotherapy
well and in metastatic disease where radiation therapy has limited
usefulness. The tumor targeting anti-UNQ733 antibodies,
oligopeptides and organic/inorganic small molecules of the
invention are useful to alleviate UNQ733 polypeptide-expressing
cancers upon initial diagnosis of the disease or during relapse.
For therapeutic applications, the anti-UNQ733 antibody,
oligopeptide or organic/inorganic small molecule can be used alone,
or in combination therapy with, e.g., hormones, antiangiogens, or
radiolabelled compounds, or with surgery, cryotherapy, and/or
radiotherapy. Anti-UNQ733 antibody, oligopeptide or
organic/inorganic small molecule treatment can be administered in
conjunction with other forms of conventional therapy, either
consecutively with, pre- or post-conventional therapy.
Chemotherapeutic drugs such as TAXOTERE.RTM. (docetaxel),
TAXOL.RTM. (palictaxel), estramustine and mitoxantrone are used in
treating cancer, in particular, in good risk patients. In the
present method of the invention for treating or alleviating cancer,
the cancer patient can be administered anti-UNQ733 antibody,
oligopeptide or organic/inorganic small molecule in conjunction
with treatment with the one or more of the preceding
chemotherapeutic agents. In particular, combination therapy with
palictaxel and modified derivatives (see, e.g., EP0600517) is
contemplated. The anti-UNQ733 antibody, oligopeptide or
organic/inorganic small molecule will be administered with a
therapeutically effective dose of the chemotherapeutic agent. In
another embodiment, the anti-UNQ733 antibody, oligopeptide or
organic/inorganic small molecule is administered in conjunction
with chemotherapy to enhance the activity and efficacy of the
chemotherapeutic agent, e.g., paclitaxel. The Physicians' Desk
Reference (PDR) discloses dosages of these agents that have been
used in treatment of various cancers. The dosing regimen and
dosages of these aforementioned chemotherapeutic drugs that are
therapeutically effective will depend on the particular cancer
being treated, the extent of the disease and other factors familiar
to the physician of skill in the art and can be determined by the
physician.
[0307] In one particular embodiment, a conjugate comprising an
anti-UNQ733 antibody, oligopeptide or organic/inorganic small
molecule conjugated with a cytotoxic agent is administered to the
patient. Preferably, the immunoconjugate bound to the UNQ733
protein is internalized by the cell, resulting in increased
therapeutic efficacy of the immunoconjugate in killing the cancer
cell to which it binds. In a preferred embodiment, the cytotoxic
agent targets or interferes with the nucleic acid in the cancer
cell. Examples of such cytotoxic agents are described above and
include maytansinoids, calicheamicins, ribonucleases and DNA
endonucleases.
[0308] The anti-UNQ733 antibodies, oligopeptides, organic/inorganic
small molecules or toxin conjugates thereof are administered to a
human patient, in accord with known methods, such as intravenous
administration, e.g., as a bolus or by continuous infusion over a
period of time, by intramuscular, intraperitoneal,
intracerobrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, oral, topical, or inhalation routes. Intravenous or
subcutaneous administration of the antibody, oligopeptide or
organic small molecule is preferred.
[0309] Other therapeutic regimens may be combined with the
administration of the anti-UNQ733 antibody, oligopeptide or
organic/inorganic small molecule. The combined administration
includes co-administration, using separate formulations or a single
pharmaceutical formulation, and consecutive administration in
either order, wherein preferably there is a time period while both
(or all) active agents simultaneously exert their biological
activities. Preferably such combined therapy results in a
synergistic therapeutic effect.
[0310] It may also be desirable to combine administration of the
anti-UNQ733 antibody or antibodies, oligopeptides or
organic/inorganic small molecules, with administration of an
antibody directed against another tumor antigen associated with the
particular cancer.
[0311] In another embodiment, the therapeutic treatment methods of
the present invention involves the combined administration of an
anti-UNQ733 antibody (or antibodies), oligopeptides or
organic/inorganic small molecules and one or more chemotherapeutic
agents or growth inhibitory agents, including co-administration of
cocktails of different chemotherapeutic agents. Chemotherapeutic
agents include estramustine phosphate, prednimustine, cisplatin,
5-fluorouracil, melphalan, cyclophosphamide, hydroxyurea and
hydroxyureataxanes (such as paclitaxel and doxetaxel) and/or
anthracycline antibiotics. Preparation and dosing schedules for
such chemotherapeutic agents may be used according to
manufacturers' instructions or as determined empirically by the
skilled practitioner. Preparation and dosing schedules for such
chemotherapy are also described in Chemotherapy Service Ed., M. C.
Perry, Williams & Wilkins, Baltimore, Md. (1992).
[0312] The antibody, oligopeptide or organic/inorganic small
molecule may be combined with an anti-hormonal compound; e.g., an
anti-estrogen compound such as tamoxifen; an anti-progesterone such
as onapristone (see, EP 616 812); or an anti-androgen such as
flutamide, in dosages known for such molecules. Where the cancer to
be treated is androgen independent cancer, the patient may
previously have been subjected to anti-androgen therapy and, after
the cancer becomes androgen independent, the anti-UNQ733 antibody,
oligopeptide or organic/inorganic small molecule (and optionally
other agents as described herein) may be administered to the
patient.
[0313] Sometimes, it may be beneficial to also co-administer a
cardioprotectant (to prevent or reduce myocardial dysfunction
associated with the therapy) or one or more cytokines to the
patient. In addition to the above therapeutic regimes, the patient
may be subjected to surgical removal of cancer cells and/or
radiation therapy, before, simultaneously with, or post antibody,
oligopeptide or organic/inorganic small molecule therapy. Suitable
dosages for any of the above co-administered agents are those
presently used and may be lowered due to the combined action
(synergy) of the agent and anti-UNQ733 antibody, oligopeptide or
organic/inorganic small molecule.
[0314] For the prevention or treatment of disease, the dosage and
mode of administration will be chosen by the physician according to
known criteria. The appropriate dosage of antibody, oligopeptide or
organic/inorganic small molecule will depend on the type of disease
to be treated, as defined above, the severity and course of the
disease, whether the antibody, oligopeptide or organic/inorganic
small molecule is administered for preventive or therapeutic
purposes, previous therapy, the patient's clinical history and
response to the antibody, oligopeptide or organic/inorganic small
molecule, and the discretion of the attending physician. The
antibody, oligopeptide or organic/inorganic small molecule is
suitably administered to the patient at one time or over a series
of treatments. Preferably, the antibody, oligopeptide or
organic/inorganic small molecule is administered by intravenous
infusion or by subcutaneous injections. Depending on the type and
severity of the disease, about 1 .mu.g/kg to about 50 mg/kg body
weight (e.g., about 0.1-15 mg/kg/dose) of antibody can be an
initial candidate dosage for administration to the patient,
whether, for example, by one or more separate administrations, or
by continuous infusion. A dosing regimen can comprise administering
an initial loading dose of about 4 mg/kg, followed by a weekly
maintenance dose of about 2 mg/kg of the anti-UNQ733 antibody.
However, other dosage regimens may be useful. A typical daily
dosage might range from about 1 .mu.g/kg to 100 mg/kg or more,
depending on the factors mentioned above. For repeated
administrations over several days or longer, depending on the
condition, the treatment is sustained until a desired suppression
of disease symptoms occurs. The progress of this therapy can be
readily monitored by conventional methods and assays and based on
criteria known to the physician or other persons of skill in the
art.
[0315] Aside from administration of the antibody protein to the
patient, the present application contemplates administration of the
antibody by gene therapy. Such administration of nucleic acid
encoding the antibody is encompassed by the expression
"administering a therapeutically effective amount of an antibody".
See, for example, WO96/07321 published Mar. 14, 1996 concerning the
use of gene therapy to generate intracellular antibodies.
[0316] There are two major approaches to getting the nucleic acid
(optionally contained in a vector) into the patient's cells; in
vivo and ex vivo. For in vivo delivery the nucleic acid is injected
directly into the patient, usually at the site where the antibody
is required. For ex vivo treatment, the patient's cells are
removed, the nucleic acid is introduced into these isolated cells
and the modified cells are administered to the patient either
directly or, for example, encapsulated within porous membranes
which are implanted into the patient (see, e.g., U.S. Pat. Nos.
4,892,538 and 5,283,187). There are a variety of techniques
available for introducing nucleic acids into viable cells. The
techniques vary depending upon whether the nucleic acid is
transferred into cultured cells in vitro, or in vivo in the cells
of the intended host. Techniques suitable for the transfer of
nucleic acid into mammalian cells in vitro include the use of
liposomes, electroporation, microinjection, cell fusion,
DEAE-dextran, the calcium phosphate precipitation method, etc. A
commonly used vector for ex vivo delivery of the gene is a
retroviral vector.
[0317] The currently preferred in vivo nucleic acid transfer
techniques include transfection with viral vectors (such as
adenovirus, Herpes simplex I virus, or adeno-associated virus) and
lipid-based systems (useful lipids for lipid-mediated transfer of
the gene are DOTMA, DOPE and DC-Chol, for example). For review of
the currently known gene marking and gene therapy protocols see
Anderson et al., Science 256:808-813 (1992). See also WO 93/25673
and the references cited therein.
[0318] The anti-UNQ733 antibodies of the invention can be in the
different forms encompassed by the definition of "antibody" herein.
Thus, the antibodies include full length or intact antibody,
antibody fragments, native sequence antibody or amino acid
variants, humanized, chimeric or fusion antibodies,
immunoconjugates, and functional fragments thereof. In fusion
antibodies an antibody sequence is fused to a heterologous
polypeptide sequence. The antibodies can be modified in the Fc
region to provide desired effector functions. As discussed in more
detail in the sections herein, with the appropriate Fc regions, the
naked antibody bound on the cell surface can induce cytotoxicity,
e.g., via antibody-dependent cellular cytotoxicity (ADCC) or by
recruiting complement in complement dependent cytotoxicity, or some
other mechanism. Alternatively, where it is desirable to eliminate
or reduce effector function, so as to minimize side effects or
therapeutic complications, certain other Fc regions may be
used.
[0319] In one embodiment, the antibody competes for binding or bind
substantially to, the same epitope as the antibodies of the
invention. Antibodies having the biological characteristics of the
present anti-UNQ733 antibodies of the invention are also
contemplated, specifically including the in vivo tumor targeting
and any cell proliferation inhibition or cytotoxic
characteristics.
[0320] Methods of producing the above antibodies are described in
detail herein.
[0321] The present anti-UNQ733 antibodies, oligopeptides and
organic/inorganic small molecules are useful for treating a UNQ733
polypeptide-expressing lymphoma, specifically non-Hodgkin's
lymphoma, or alleviating one or more symptoms of the lymphoma in a
mammal. Methods of the invention encompass usage of UNQ733
antagonists in the treatment and/or alleviation of symptoms of
metastatic tumors associated with non-Hodgkin's lymphoma. The
antibody, oligopeptide or organic/inorganic small molecule UNQ733
antagonist is able to bind to at least a portion of the cancer
cells that express UNQ733 polypeptide in the mammal. In one
embodiment, the antibody, oligopeptide or organic/inorganic small
molecule is effective to destroy or kill UNQ733
polypeptide-expressing and/or -responsive tumor cells or inhibit
the growth of such tumor cells, in vitro or in vivo, upon binding
to UNQ733 polypeptide. Such an antibody includes a naked
anti-UNQ733 antibody (not conjugated to any agent). Naked
antibodies that have cytotoxic or cell growth inhibition properties
can be further harnessed with a cytotoxic agent to render them even
more potent in tumor cell destruction. Cytotoxic properties can be
conferred to an anti-UNQ733 antibody by, e.g., conjugating the
antibody with a cytotoxic agent, to form an immunoconjugate as
described herein. In some embodiments, the cytotoxic agent or a
growth inhibitory agent is a small molecule. In some embodiments,
toxins such as calicheamicin or a maytansinoid and analogs or
derivatives thereof, are used.
[0322] The invention provides a composition comprising an
anti-UNQ733 antibody, oligopeptide or organic/inorganic small
molecule of the invention, and a carrier. For the purposes of
treating cancer, compositions can be administered to the patient in
need of such treatment, wherein the composition can comprise one or
more anti-UNQ733 antibodies present as an immunoconjugate or as the
naked antibody. In a further embodiment, the compositions can
comprise these antibodies, oligopeptides or organic/inorganic small
molecules in combination with other therapeutic agents such as
cytotoxic or growth inhibitory agents, including chemotherapeutic
agents. The invention also provides formulations comprising an
anti-UNQ733 antibody, oligopeptide or organic/inorganic small
molecule of the invention, and a carrier. In one embodiment, the
formulation is a therapeutic formulation comprising a
pharmaceutically acceptable carrier.
[0323] Another aspect of the invention is isolated nucleic acids
encoding the anti-UNQ733 antibodies. Nucleic acids encoding both
the H and L chains and especially the hypervariable region
residues, chains which encode the native sequence antibody as well
as variants, modifications and humanized versions of the antibody,
are encompassed.
[0324] The invention also provides methods useful for treating
non-Hodgkin's lymphoma or alleviating one or more symptoms of the
lymphoma in a mammal, comprising administering a therapeutically
effective amount of an anti-UNQ733 antibody, oligopeptide or
organic/inorganic small molecule to the mammal. The antibody,
oligopeptide or organic/inorganic small molecule therapeutic
compositions can be administered short term (acute) or chronic, or
intermittent as directed by physician. Also provided are methods of
inhibiting the growth of, and killing a UNQ733
polypeptide-expressing and/or -responsive cell.
[0325] The invention also provides kits and articles of manufacture
comprising at least one anti-UNQ733 antibody, oligopeptide or
organic/inorganic small molecule. Kits containing anti-UNQ733
antibodies, oligopeptides or organic/inorganic small molecules find
use, e.g., for UNQ733 polypeptide cell killing assays, for
purification or immunoprecipitation of UNQ733 polypeptide from
cells. For example, for isolation and purification of UNQ733
polypeptide, the kit can contain an anti-UNQ733 antibody,
oligopeptide or organic/inorganic small molecule coupled to beads
(e.g., sepharose beads). Kits can be provided which contain the
antibodies, oligopeptides or organic/inorganic small molecules for
detection and quantitation of UNQ733 polypeptide in vitro, e.g., in
an ELISA or a Western blot. Such antibody, oligopeptide or
organic/inorganic small molecule useful for detection may be
provided with a label such as a fluorescent or radiolabel.
[0326] K. Articles of Manufacture and Kits
[0327] Another embodiment of the invention is an article of
manufacture containing materials useful for the treatment of a
UNQ733 polypeptide expressing cancer, such as a non-Hodgkin's
lymphoma. The article of manufacture 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 which is
effective for treating the cancer 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). At least one active agent in the composition is
an anti-UNQ733 antibody, oligopeptide or organic/inorganic small
molecule of the invention. The label or package insert indicates
that the composition is used for treating cancer. The label or
package insert will further comprise instructions for administering
the antibody, oligopeptide or organic/inorganic small molecule
composition to the cancer patient. Additionally, the article of
manufacture may further comprise a second container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0328] Kits are also provided that are useful for various purposes,
e.g., for UNQ733 polypeptide-expressing or cell killing assays, for
purification or immunoprecipitation of UNQ733 polypeptide from
cells. For isolation and purification of UNQ733 polypeptide, the
kit can contain an anti-UNQ733 antibody, oligopeptide or
organic/inorganic small molecule coupled to beads (e.g., sepharose
beads). Kits can be provided which contain the antibodies,
oligopeptides or organic/inorganic small molecules for detection
and quantitation of UNQ733 polypeptide in vitro, e.g., in an ELISA
or a Western blot. As with the article of manufacture, the kit
comprises a container and a label or package insert on or
associated with the container. The container holds a composition
comprising at least one anti-UNQ733 polypeptide antibody,
oligopeptide or organic/inorganic small molecule of the invention.
Additional containers may be included that contain, e.g., diluents
and buffers, control antibodies. The label or package insert may
provide a description of the composition as well as instructions
for the intended in vitro or detection use.
[0329] L. UNQ733 Polypeptides and UNQ733 Polypeptide-Encoding
Nucleic Acids--Specific Forms and Applications
[0330] Nucleotide sequences (or their complement) encoding UNQ733
polypeptides have various applications in the art of molecular
biology, including uses as hybridization probes, in chromosome and
gene mapping and in the generation of anti-sense RNA and DNA
probes. UNQ733 polypeptide-encoding nucleic acid will also be
useful for the preparation of UNQ733 polypeptides by the
recombinant techniques described herein, wherein those UNQ733
polypeptides may find use, for example, in the preparation of
anti-UNQ733 antibodies as described herein.
[0331] A full-length native sequence UNQ733 polypeptide gene, or
portions thereof, may be used as hybridization probes for a cDNA
library to isolate other cDNAs (for instance, those encoding
naturally-occurring variants of UNQ733 polypeptide or UNQ733
polypeptide from other species) which have a desired sequence
identity to a native UNQ733 polypeptide sequence disclosed herein.
Optionally, the length of the probes will be about 20 to about 50
bases. The hybridization probes may be derived from at least
partially novel regions of the full length native nucleotide
sequence wherein those regions may be determined without undue
experimentation or from genomic sequences including promoters,
enhancer elements and introns of native sequence UNQ733
polypeptide. By way of example, a screening method will comprise
isolating the coding region of the UNQ733 polypeptide gene using
the known DNA sequence to synthesize a selected probe of about 40
bases. Hybridization probes may be labeled by a variety of labels,
including radionucleotides such as .sup.32P or .sup.35S, or
enzymatic labels such as alkaline phosphatase coupled to the probe
via avidin/biotin coupling systems. Labeled probes having a
sequence complementary to that of the UNQ733 polypeptide gene of
the present invention can be used to screen libraries of human
cDNA, genomic DNA or mRNA to determine which members of such
libraries the probe hybridizes to. Hybridization techniques are
described in further detail in the Examples below. Any EST
sequences disclosed in the present application may similarly be
employed as probes, using the methods disclosed herein.
[0332] Other useful fragments of the UNQ733 polypeptide-encoding
nucleic acids include antisense or sense oligonucleotides
comprising a singe-stranded nucleic acid sequence (either RNA or
DNA) capable of binding to target UNQ733 polypeptide mRNA (sense)
or UNQ733 polypeptide DNA (antisense) sequences. Antisense or sense
oligonucleotides, according to the present invention, comprise a
fragment of the coding region of UNQ733 DNA. Such a fragment
generally comprises at least about 14 nucleotides, preferably from
about 14 to 30 nucleotides. The ability to derive an antisense or a
sense oligonucleotide, based upon a cDNA sequence encoding a given
protein is described in, for example, Stein and Cohen (Cancer Res.
48:2659, 1988) and van der Krol et al. (BioTechniques 6:958,
1988).
[0333] Binding of antisense or sense oligonucleotides to target
nucleic acid sequences results in the formation of duplexes that
block transcription or translation of the target sequence by one of
several means, including enhanced degradation of the duplexes,
premature termination of transcription or translation, or by other
means. Such methods are encompassed by the present invention. The
antisense oligonucleotides thus may be used to block expression of
a UNQ733 protein, wherein the UNQ733 protein may play a role in the
induction of cancer in mammals. Antisense or sense oligonucleotides
further comprise oligonucleotides having modified
sugar-phosphodiester backbones (or other sugar linkages, such as
those described in WO 91/06629) and wherein such sugar linkages are
resistant to endogenous nucleases. Such oligonucleotides with
resistant sugar linkages are stable in vivo (i.e., capable of
resisting enzymatic degradation) but retain sequence specificity to
be able to bind to target nucleotide sequences.
[0334] Preferred intragenic sites for antisense binding include the
region incorporating the translation initiation/start codon
(5'-AUG/5'-ATG) or termination/stop codon (5'-UAA, 5'-UAG and
5-UGA/5'-TAA, 5'-TAG and 5'-TGA) of the open reading frame (ORF) of
the gene. These regions refer to a portion of the mRNA or gene that
encompasses from about 25 to about 50 contiguous nucleotides in
either direction (i.e., 5' or 3') from a translation initiation or
termination codon. Other preferred regions for antisense binding
include: introns; exons; intron-exon junctions; the open reading
frame (ORF) or "coding region," which is the region between the
translation initiation codon and the translation termination codon;
the 5' cap of an mRNA which comprises an N7-methylated guanosine
residue joined to the 5'-most residue of the mRNA via a 5'-5'
triphosphate linkage and includes 5' cap structure itself as well
as the first 50 nucleotides adjacent to the cap; the 5'
untranslated region (5'UTR), the portion of an mRNA in the 5'
direction from the translation initiation codon, and thus including
nucleotides between the 5' cap site and the translation initiation
codon of an mRNA or corresponding nucleotides on the gene; and the
3' untranslated region (3'UTR), the portion of an mRNA in the 3'
direction from the translation termination codon, and thus
including nucleotides between the translation termination codon and
3' end of an mRNA or corresponding nucleotides on the gene.
[0335] Specific examples of preferred antisense compounds useful
for inhibiting expression of UNQ733 polypeptide include
oligonucleotides containing modified backbones or non-natural
internucleoside linkages. Oligonucleotides having modified
backbones include those that retain a phosphorus atom in the
backbone and those that do not have a phosphorus atom in the
backbone. For the purposes of this specification, and as sometimes
referenced in the art, modified oligonucleotides that do not have a
phosphorus atom in their internucleoside backbone can also be
considered to be oligonucleosides. Preferred modified
oligonucleotide backbones include, for example, phosphorothioates,
chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotri-esters, methyl and other alkyl phosphonates
including 3'-alkylene phosphonates, 5'-alkylene phosphonates and
chiral phosphonates, phosphinates, phosphoramidates including
3'-amino phosphoramidate and aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates,
thionoalkylphosphotriesters, selenophosphates and borano-phosphates
having normal 3'-5' linkages, 2'-5' linked analogs of these, and
those having inverted polarity wherein one or more internucleotide
linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Preferred
oligonucleotides having inverted polarity comprise a single 3' to
3' linkage at the 3'-most internucleotide linkage i.e. a single
inverted nucleoside residue which may be abasic (the nucleobase is
missing or has a hydroxyl group in place thereof). Various salts,
mixed salts and free acid forms are also included. Representative
United States patents that teach the preparation of
phosphorus-containing linkages include, but are not limited to,
U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243;
5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;
5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;
5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;
5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;
5,672,697 and 5,625,050, each of which is herein incorporated by
reference.
[0336] Preferred modified oligonucleotide backbones that do not
include a phosphorus atom therein have backbones that are formed by
short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; riboacetyl backbones; alkene containing backbones;
sulfamate backbones; methyleneimino and methylenehydrazino
backbones; sulfonate and sulfonamide backbones; amide backbones;
and others having mixed N, O, S and CH.sub.2 component parts.
Representative United States patents that teach the preparation of
such oligonucleosides include, but are not limited to, U.S. Pat.
Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;
5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677;
5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;
5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;
5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269 and
5,677,439, each of which is herein incorporated by reference.
[0337] In other preferred antisense oligonucleotides, both the
sugar and the internucleoside linkage, i.e., the backbone, of the
nucleotide units are replaced with novel groups. The base units are
maintained for hybridization with an appropriate nucleic acid
target compound. One such oligomeric compound, an oligonucleotide
mimetic that has been shown to have excellent hybridization
properties, is referred to as a peptide nucleic acid (PNA). In PNA
compounds, the sugar-backbone of an oligonucleotide is replaced
with an amide containing backbone, in particular an
aminoethylglycine backbone. The nucleobases are retained and are
bound directly or indirectly to aza nitrogen atoms of the amide
portion of the backbone. Representative United States patents that
teach the preparation of PNA compounds include, but are not limited
to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of
which is herein incorporated by reference. Further teaching of PNA
compounds can be found in Nielsen et al., Science, 1991, 254,
1497-1500.
[0338] Preferred antisense oligonucleotides incorporate
phosphorothioate backbones and/or heteroatom backbones, and in
particular --CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--]
described in the above referenced U.S. Pat. No. 5,489,677, and the
amide backbones of the above referenced U.S. Pat. No. 5,602,240.
Also preferred are antisense oligonucleotides having morpholino
backbone structures of the above-referenced U.S. Pat. No.
5,034,506.
[0339] Modified oligonucleotides may also contain one or more
substituted sugar moieties. Preferred oligonucleotides comprise one
of the following at the 2' position: OH; F; O-alkyl, S-alkyl, or
N-alkyl; alkenyl, S-alkeynyl, or N-alkenyl; O-alkynyl, S-alkynyl or
N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and
alkynyl may be substituted or unsubstituted C.sub.1 to C.sub.10
alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Particularly
preferred are O[(CH.sub.2).sub.nO].sub.mCH.sub.3,
O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m
are from 1 to about 10. Other preferred antisense oligonucleotides
comprise one of the following at the 2' position: C.sub.1 to
C.sub.10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl,
alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl,
Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3,
ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl,
heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted
silyl, an RNA cleaving group, a reporter group, an intercalator, a
group for improving the pharmacokinetic properties of an
oligonucleotide, or a group for improving the pharmacodynamic
properties of an oligonucleotide, and other substituents having
similar properties. A preferred modification includes
2'-methoxyethoxy(2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples hereinbelow, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).
[0340] A further preferred modification includes Locked Nucleic
Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or
4' carbon atom of the sugar ring thereby forming a bicyclic sugar
moiety. The linkage is preferably a methelyne (--CH.sub.2--).sub.n
group bridging the 2' oxygen atom and the 4' carbon atom wherein n
is 1 or 2. LNAs and preparation thereof are described in WO
98/39352 and WO 99/14226.
[0341] Other preferred modifications include
2'-methoxy(2'-O--CH.sub.3),
2'-aminopropoxy(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl
(2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl
(2'-O--CH.sub.2--CH.dbd.CH.sub.2) and 2'-fluoro (2'-F). The
2'-modification may be in the arabino (up) position or ribo (down)
position. A preferred 2'-arabino modification is 2'-F. Similar
modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides and the
5' position of 5' terminal nucleotide. Oligonucleotides may also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative United States patents that
teach the preparation of such modified sugar structures include,
but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;
5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;
5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747;
and 5,700,920, each of which is herein incorporated by reference in
its entirety.
[0342] Oligonucleotides may also include nucleobase (often referred
to in the art simply as "base") modifications or substitutions. As
used herein, "unmodified" or "natural" nucleobases include the
purine bases adenine (A) and guanine (G), and the pyrimidine bases
thymine (T), cytosine (C) and uracil (U). Modified nucleobases
include other synthetic and natural nucleobases such as
5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,
hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives
of adenine and guanine, 2-propyl and other alkyl derivatives of
adenine and guanine, 2-thiouracil, 2-thiothymine and
2-thiocytosine, 5-halouracil and cytosine, 5-propynyl
(--C.ident.C--CH.sub.3 or --CH.sub.2--C.ident.CH) uracil and
cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo
uracil, cytosine and thymine, 5-uracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and
other 8-substituted adenines and guanines, 5-halo particularly
5-bromo, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,
2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and
7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further
modified nucleobases include tricyclic pyrimidines such as
phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
phenothiazine cytidine
(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a
substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole
cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
Modified nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, those disclosed in The Concise Encyclopedia Of Polymer
Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John
Wiley & Sons, 1990, and those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613. Certain of
these nucleobases are particularly useful for increasing the
binding affinity of the oligomeric compounds of the invention.
These include 5-substituted pyrimidines, 6-azapyrimidines and N-2,
N-6 and O-6 substituted purines, including 2-aminopropyladenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions have been shown to increase nucleic acid duplex
stability by 0.6-1.2.degree. C. (Sanghvi et al, Antisense Research
and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are
preferred base substitutions, even more particularly when combined
with 2'-O-methoxyethyl sugar modifications. Representative United
States patents that teach the preparation of modified nucleobases
include, but are not limited to: U.S. Pat. No. 3,687,808, as well
as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273;
5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177;
5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617;
5,645,985; 5,830,653; 5,763,588; 6,005,096; 5,681,941 and
5,750,692, each of which is herein incorporated by reference.
[0343] Another modification of antisense oligonucleotides comprises
chemically linking to the oligonucleotide one or more moieties or
conjugates which enhance the activity, cellular distribution or
cellular uptake of the oligonucleotide. The compounds of the
invention can include conjugate groups covalently bound to
functional groups such as primary or secondary hydroxyl groups.
Conjugate groups of the invention include intercalators, reporter
molecules, polyamines, polyamides, polyethylene glycols,
polyethers, groups that enhance the pharmacodynamic properties of
oligomers, and groups that enhance the pharmacokinetic properties
of oligomers. Typical conjugates groups include cholesterols,
lipids, cation lipids, phospholipids, cationic phospholipids,
biotin, phenazine, folate, phenanthridine, anthraquinone, acridine,
fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance
the pharmacodynamic properties, in the context of this invention,
include groups that improve oligomer uptake, enhance oligomer
resistance to degradation, and/or strengthen sequence-specific
hybridization with RNA. Groups that enhance the pharmacokinetic
properties, in the context of this invention, include groups that
improve oligomer uptake, distribution, metabolism or excretion.
Conjugate moieties include but are not limited to lipid moieties
such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad.
Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al.,
Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,
hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,
660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3,
2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,
1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or
undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10,
1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;
Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,
e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety. Oligonucleotides of
the invention may also be conjugated to active drug substances, for
example, aspirin, warfarin, phenylbutazone, ibuprofen, suprofen,
fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,
dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid,
folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,
indomethicin, a barbiturate, a cephalosporin, a sulfa drug, an
antidiabetic, an antibacterial or an antibiotic.
Oligonucleotide-drug conjugates and their preparation are described
in U.S. patent application Ser. No. 09/334,130 (filed Jun. 15,
1999) and U.S. Pat. Nos.: 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;
5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,
5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;
5,599,928 and 5,688,941, each of which is herein incorporated by
reference.
[0344] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an oligonucleotide.
The present invention also includes antisense compounds which are
chimeric compounds. "Chimeric" antisense compounds or "chimeras,"
in the context of this invention, are antisense compounds,
particularly oligonucleotides, which contain two or more chemically
distinct regions, each made up of at least one monomer unit, i.e.,
a nucleotide in the case of an oligonucleotide compound. These
oligonucleotides typically contain at least one region wherein the
oligonucleotide is modified so as to confer upon the
oligonucleotide increased resistance to nuclease degradation,
increased cellular uptake, and/or increased binding affinity for
the target nucleic acid. An additional region of the
oligonucleotide may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is
a cellular endonuclease which cleaves the RNA strand of an RNA:DNA
duplex. Activation of RNase H, therefore, results in cleavage of
the RNA target, thereby greatly enhancing the efficiency of
oligonucleotide inhibition of gene expression. Consequently,
comparable results can often be obtained with shorter
oligonucleotides when chimeric oligonucleotides are used, compared
to phosphorothioate deoxyoligonucleotides hybridizing to the same
target region. Chimeric antisense compounds of the invention may be
formed as composite structures of two or more oligonucleotides,
modified oligonucleotides, oligonucleosides and/or oligonucleotide
mimetics as described above. Preferred chimeric antisense
oligonucleotides incorporate at least one 2' modified sugar
(preferably 2'-O--(CH.sub.2).sub.2--O--CH.sub.3) at the 3' terminal
to confer nuclease resistance and a region with at least 4
contiguous 2'-H sugars to confer RNase H activity. Such compounds
have also been referred to in the art as hybrids or gapmers.
Preferred gapmers have a region of 2' modified sugars (preferably
2'-O--(CH.sub.2).sub.2--O--CH.sub.3) at the 3'-terminal and at the
5' terminal separated by at least one region having at least 4
contiguous 2'-H sugars and preferably incorporate phosphorothioate
backbone linkages. Representative United States patents that teach
the preparation of such hybrid structures include, but are not
limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007;
5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;
5,652,355; 5,652,356; and 5,700,922, each of which is herein
incorporated by reference in its entirety.
[0345] The antisense compounds used in accordance with this
invention may be conveniently and routinely made through the
well-known technique of solid phase synthesis. Equipment for such
synthesis is sold by several vendors including, for example,
Applied Biosystems (Foster City, Calif.). Any other means for such
synthesis known in the art may additionally or alternatively be
employed. It is well known to use similar techniques to prepare
oligonucleotides such as the phosphorothioates and alkylated
derivatives. The compounds of the invention may also be admixed,
encapsulated, conjugated or otherwise associated with other
molecules, molecule structures or mixtures of compounds, as for
example, liposomes, receptor targeted molecules, oral, rectal,
topical or other formulations, for assisting in uptake,
distribution and/or absorption. Representative United States
patents that teach the preparation of such uptake, distribution
and/or absorption assisting formulations include, but are not
limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;
5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;
4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;
5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;
5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;
5,580,575; and 5,595,756, each of which is herein incorporated by
reference.
[0346] Other examples of sense or antisense oligonucleotides
include those oligonucleotides which are covalently linked to
organic moieties, such as those described in WO 90/10048, and other
moieties that increases affinity of the oligonucleotide for a
target nucleic acid sequence, such as poly-(L-lysine). Further
still, intercalating agents, such as ellipticine, and alkylating
agents or metal complexes may be attached to sense or antisense
oligonucleotides to modify binding specificities of the antisense
or sense oligonucleotide for the target nucleotide sequence.
[0347] Antisense or sense oligonucleotides may be introduced into a
cell containing the target nucleic acid sequence by any gene
transfer method, including, for example, CaPO.sub.4-mediated DNA
transfection, electroporation, or by using gene transfer vectors
such as Epstein-Barr virus. In a preferred procedure, an antisense
or sense oligonucleotide is inserted into a suitable retroviral
vector. A cell containing the target nucleic acid sequence is
contacted with the recombinant retroviral vector, either in vivo or
ex vivo. Suitable retroviral vectors include, but are not limited
to, those derived from the murine retrovirus M-MuLV, N2 (a
retrovirus derived from M-MuLV), or the double copy vectors
designated DCT5A, DCT5B and DCT5C (see WO 90/13641).
[0348] Sense or antisense oligonucleotides also may be introduced
into a cell containing the target nucleotide sequence by formation
of a conjugate with a ligand binding molecule, as described in WO
91/04753. Suitable ligand binding molecules include, but are not
limited to, cell surface receptors, growth factors, other
cytokines, or other ligands that bind to cell surface receptors.
Preferably, conjugation of the ligand binding molecule does not
substantially interfere with the ability of the ligand binding
molecule to bind to its corresponding molecule or receptor, or
block entry of the sense or antisense oligonucleotide or its
conjugated version into the cell.
[0349] Alternatively, a sense or an antisense oligonucleotide may
be introduced into a cell containing the target nucleic acid
sequence by formation of an oligonucleotide-lipid complex, as
described in WO 90/10448. The sense or antisense
oligonucleotide-lipid complex is preferably dissociated within the
cell by an endogenous lipase.
[0350] Antisense or sense RNA or DNA molecules are generally at
least about 5 nucleotides in length, alternatively at least about
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150,
155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230,
240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360,
370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490,
500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620,
630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750,
760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880,
890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000
nucleotides in length, wherein in this context the term "about"
means the referenced nucleotide sequence length plus or minus 10%
of that referenced length.
[0351] The probes may also be employed in PCR techniques to
generate a pool of sequences for identification of closely related
UNQ733 polypeptide coding sequences.
[0352] Nucleotide sequences encoding a UNQ733 polypeptide can also
be used to construct hybridization probes for mapping the gene
which encodes that UNQ733 polypeptide and for the genetic analysis
of individuals with genetic disorders. The nucleotide sequences
provided herein may be mapped to a chromosome and specific regions
of a chromosome using known techniques, such as in situ
hybridization, linkage analysis against known chromosomal markers,
and hybridization screening with libraries.
[0353] The UNQ733 polypeptide can be used in assays to identify
other proteins or molecules involved in a binding interaction with
the UNQ733 polypeptide. By such methods, inhibitors of the
receptor/ligand binding interaction can be identified. Proteins
involved in such binding interactions can also be used to screen
for peptide or small molecule inhibitors of the binding
interaction. Screening assays can be designed to find lead
compounds that mimic the biological activity of a native UNQ733
polypeptide or a receptor for UNQ733 polypeptide. Such screening
assays will include assays amenable to high-throughput screening of
chemical libraries, making them particularly suitable for
identifying small molecule drug candidates. Small molecules
contemplated include synthetic organic or inorganic compounds. The
assays can be performed in a variety of formats, including
protein-protein binding assays, biochemical screening assays,
immunoassays and cell based assays, which are well characterized in
the art.
[0354] Nucleic acids which encode UNQ733 polypeptide or its
modified forms can also be used to generate either transgenic
animals or "knock out" animals which, in turn, are useful in the
development and screening of therapeutically useful reagents. A
transgenic animal (e.g., a mouse or rat) is an animal having cells
that contain a transgene, which transgene was introduced into the
animal or an ancestor of the animal at a prenatal, e.g., an
embryonic stage. A transgene is a DNA which is integrated into the
genome of a cell from which a transgenic animal develops. In one
embodiment, cDNA encoding UNQ733 polypeptide can be used to clone
genomic DNA encoding UNQ733 polypeptide in accordance with
established techniques and the genomic sequences used to generate
transgenic animals that contain cells which express DNA encoding
UNQ733 polypeptide. Methods for generating transgenic animals,
particularly animals such as mice or rats, have become conventional
in the art and are described, for example, in U.S. Pat. Nos.
4,736,866 and 4,870,009. Typically, particular cells would be
targeted for UNQ733 polypeptide transgene incorporation with
tissue-specific enhancers. Transgenic animals that include a copy
of a transgene encoding UNQ733 polypeptide introduced into the germ
line of the animal at an embryonic stage can be used to examine the
effect of increased expression of DNA encoding UNQ733 polypeptide.
Such animals can be used as tester animals for reagents thought to
confer protection from, for example, pathological conditions
associated with its overexpression. In accordance with this facet
of the invention, an animal is treated with the reagent and a
reduced incidence of the pathological condition, compared to
untreated animals bearing the transgene, would indicate a potential
therapeutic intervention for the pathological condition.
[0355] Alternatively, non-human homologues of UNQ733 polypeptide
can be used to construct a UNQ733 gene "knock out" animal which has
a defective or altered gene encoding UNQ733 polypeptide as a result
of homologous recombination between the endogenous gene encoding
UNQ733 polypeptide and altered genomic DNA encoding UNQ733
polypeptide introduced into an embryonic stem cell of the animal.
For example, cDNA encoding UNQ733 polypeptide can be used to clone
genomic DNA encoding UNQ733 polypeptide in accordance with
established techniques. A portion of the genomic DNA encoding
UNQ733 polypeptide can be deleted or replaced with another gene,
such as a gene encoding a selectable marker which can be used to
monitor integration. Typically, several kilobases of unaltered
flanking DNA (both at the 5' and 3' ends) are included in the
vector [see e.g., Thomas and Capecchi, Cell, 51:503 (1987) for a
description of homologous recombination vectors]. The vector is
introduced into an embryonic stem cell line (e.g., by
electroporation) and cells in which the introduced DNA has
homologously recombined with the endogenous DNA are selected [see
e.g., Li et al., Cell, 69:915 (1992)]. The selected cells are then
injected into a blastocyst of an animal (e.g., a mouse or rat) to
form aggregation chimeras [see e.g., Bradley, in Teratocarcinomas
and Embryonic Stem Cells: A Practical Approach, E. J. Robertson,
ed. (IRL, Oxford, 1987), pp. 113-152]. A chimeric embryo can then
be implanted into a suitable pseudopregnant female foster animal
and the embryo brought to term to create a "knock out" animal.
Progeny harboring the homologously recombined DNA in their germ
cells can be identified by standard techniques and used to breed
animals in which all cells of the animal contain the homologously
recombined DNA. Knockout animals can be characterized for instance,
for their ability to defend against certain pathological conditions
and for their development of pathological conditions due to absence
of the UNQ733 polypeptide.
[0356] Nucleic acid encoding the UNQ733 polypeptides may also be
used in gene therapy. In gene therapy applications, genes are
introduced into cells in order to achieve in vivo synthesis of a
therapeutically effective genetic product, for example for
replacement of a defective gene. "Gene therapy" includes both
conventional gene therapy where a lasting effect is achieved by a
single treatment, and the administration of gene therapeutic
agents, which involves the one time or repeated administration of a
therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can
be used as therapeutic agents for blocking the expression of
certain genes in vivo. It has already been shown that short
antisense oligonucleotides can be imported into cells where they
act as inhibitors, despite their low intracellular concentrations
caused by their restricted uptake by the cell membrane. (Zamecnik
et al, Proc. Natl. Acad. Sci. USA 83:4143-4146 [1986]). The
oligonucleotides can be modified to enhance their uptake, e.g. by
substituting their negatively charged phosphodiester groups by
uncharged groups.
[0357] There are a variety of techniques available for introducing
nucleic acids into viable cells. The techniques vary depending upon
whether the nucleic acid is transferred into cultured cells in
vitro, or in vivo in the cells of the intended host. Techniques
suitable for the transfer of nucleic acid into mammalian cells in
vitro include the use of liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate
precipitation method, etc. The currently preferred in vivo gene
transfer techniques include transfection with viral (typically
retroviral) vectors and viral coat protein-liposome mediated
transfection (Dzau et al., Trends in Biotechnology 11, 205-210
[1993]). In some situations it is desirable to provide the nucleic
acid source with an agent that targets the target cells, such as an
antibody specific for a cell surface membrane protein or the target
cell, a ligand for a receptor on the target cell, etc. Where
liposomes are employed, proteins which bind to a cell surface
membrane protein associated with endocytosis may be used for
targeting and/or to facilitate uptake, e.g. capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, proteins that
target intracellular localization and enhance intracellular
half-life. The technique of receptor-mediated endocytosis is
described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432
(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414
(1990). For review of gene marking and gene therapy protocols see
Anderson et al., Science 256, 808-813 (1992).
[0358] The nucleic acid molecules encoding the UNQ733 polypeptides
or fragments thereof described herein are useful for chromosome
identification. In this regard, there exists an ongoing need to
identify new chromosome markers, since relatively few chromosome
marking reagents, based upon actual sequence data are presently
available. Each UNQ733 nucleic acid molecule of the present
invention can be used as a chromosome marker.
[0359] UNQ733 polypeptides and nucleic acid molecules of the
invention may be used diagnostically for tissue typing, wherein
UNQ733 polypeptides may be differentially expressed in one tissue
as compared to another, preferably in a diseased tissue as compared
to a normal tissue of the same tissue type. UNQ733 nucleic acid
molecules will find use for generating probes for PCR, Northern
analysis, Southern analysis and Western analysis.
[0360] This invention encompasses methods of screening compounds to
identify those that prevent the effect of the UNQ733 polypeptide
(antagonists). Screening assays for antagonist drug candidates are
designed to identify compounds that bind or complex with the UNQ733
polypeptides encoded by the genes identified herein, or otherwise
interfere with the interaction of the encoded polypeptides with
other cellular proteins, including e.g., inhibiting the expression
of UNQ733 polypeptide from cells. Such screening assays will
include assays amenable to high-throughput screening of chemical
libraries, making them particularly suitable for identifying small
molecule drug candidates.
[0361] The assays can be performed in a variety of formats,
including protein-protein binding assays, biochemical screening
assays, immunoassays, and cell-based assays, which are well
characterized in the art.
[0362] All assays for antagonists are common in that they call for
contacting the drug candidate with a UNQ733 polypeptide encoded by
a nucleic acid identified herein under conditions and for a time
sufficient to allow these two components to interact.
[0363] In binding assays, the interaction is binding and the
complex formed can be isolated or detected in the reaction mixture.
In a particular embodiment, the UNQ733 polypeptide or the drug
candidate is immobilized on a solid phase, e.g., on a microtiter
plate, by covalent or non-covalent attachments. Non-covalent
attachment generally is accomplished by coating the solid surface
with a solution of the UNQ733 polypeptide and drying.
Alternatively, an immobilized antibody, e.g., a monoclonal
antibody, specific for the UNQ733 polypeptide to be immobilized can
be used to anchor it to a solid surface. The assay is performed by
adding the non-immobilized component, which may be labeled by a
detectable label, to the immobilized component, e.g., the coated
surface containing the anchored component. When the reaction is
complete, the non-reacted components are removed, e.g., by washing,
and complexes anchored on the solid surface are detected. When the
originally non-immobilized component carries a detectable label,
the detection of label immobilized on the surface indicates that
complexing occurred. Where the originally non-immobilized component
does not carry a label, complexing can be detected, for example, by
using a labeled antibody specifically binding the immobilized
complex.
[0364] If the candidate compound interacts with but does not bind
to a UNQ733 polypeptide, its interaction with that polypeptide can
be assayed by methods well known for detecting protein-protein
interactions. Such assays include traditional approaches, such as,
e.g., cross-linking, co-immunoprecipitation, and co-purification
through gradients or chromatographic columns. In addition,
protein-protein interactions can be monitored by using a
yeast-based genetic system described by Fields and co-workers
(Fields and Song, Nature (London), 340:245-246 (1989); Chien et
al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) as disclosed
by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89: 5789-5793
(1991). Many transcriptional activators, such as yeast GAL4,
consist of two physically discrete modular domains, one acting as
the DNA-binding domain, the other one functioning as the
transcription-activation domain. The yeast expression system
described in the foregoing publications (generally referred to as
the "two-hybrid system") takes advantage of this property, and
employs two hybrid proteins, one in which the target protein is
fused to the DNA-binding domain of GAL4, and another, in which
candidate activating proteins are fused to the activation domain.
The expression of a GAL1-lacZ reporter gene under control of a
GAL4-activated promoter depends on reconstitution of GAL4 activity
via protein-protein interaction. Colonies containing interacting
polypeptides are detected with a chromogenic substrate for
.beta.-galactosidase. A complete kit (MATCHMAKER.TM.) for
identifying protein-protein interactions between two specific
proteins using the two-hybrid technique is commercially available
from Clontech. This system can also be extended to map protein
domains involved in specific protein interactions as well as to
pinpoint amino acid residues that are crucial for these
interactions.
[0365] Compounds that interfere with the interaction of a gene
encoding a UNQ733 polypeptide identified herein and other intra- or
extracellular components can be tested as follows: usually a
reaction mixture is prepared containing the product of the gene and
the intra- or extracellular component under conditions and for a
time allowing for the interaction and binding of the two products.
To test the ability of a candidate compound to inhibit binding, the
reaction is run in the absence and in the presence of the test
compound. In addition, a placebo may be added to a third reaction
mixture, to serve as positive control. The binding (complex
formation) between the test compound and the intra- or
extracellular component present in the mixture is monitored as
described hereinabove. The formation of a complex in the control
reaction(s) but not in the reaction mixture containing the test
compound indicates that the test compound interferes with the
interaction of the test compound and its reaction partner.
[0366] To assay for antagonists, the UNQ733 polypeptide may be
added to a cell along with the compound to be screened for a
particular activity and the ability of the compound to inhibit the
activity of interest in the presence of the UNQ733 polypeptide
indicates that the compound is an antagonist to the UNQ733
polypeptide. Alternatively, antagonists may be detected by
combining the UNQ733 polypeptide and a potential antagonist with
membrane-bound UNQ733 polypeptide receptors or encoded receptors
under appropriate conditions for a competitive inhibition assay.
The UNQ733 polypeptide can be labeled, such as by radioactivity,
such that the number of UNQ733 polypeptide molecules bound to the
receptor can be used to determine the effectiveness of the
potential antagonist. The gene encoding the receptor can be
identified by numerous methods known to those of skill in the art,
for example, ligand panning and FACS sorting. Coligan et al.,
Current Protocols in Immun., 1(2): Chapter 5 (1991). Preferably,
expression cloning is employed wherein polyadenylated RNA is
prepared from a cell responsive to the UNQ733 polypeptide and a
cDNA library created from this RNA is divided into pools and used
to transfect COS cells or other cells that are not responsive to
the UNQ733 polypeptide. Transfected cells that are grown on glass
slides are exposed to labeled UNQ733 polypeptide. The UNQ733
polypeptide can be labeled by a variety of means including
iodination or inclusion of a recognition site for a site-specific
protein kinase. Following fixation and incubation, the slides are
subjected to autoradiographic analysis. Positive pools are
identified and sub-pools are prepared and re-transfected using an
interactive sub-pooling and re-screening process, eventually
yielding a single clone that encodes the putative receptor.
[0367] As an alternative approach for receptor identification,
labeled UNQ733 polypeptide can be photoaffinity-linked with cell
membrane or extract preparations that express the receptor
molecule. Cross-linked material is resolved by PAGE and exposed to
X-ray film. The labeled complex containing the receptor can be
excised, resolved into peptide fragments, and subjected to protein
micro-sequencing. The amino acid sequence obtained from
micro-sequencing would be used to design a set of degenerate
oligonucleotide probes to screen a cDNA library to identify the
gene encoding the putative receptor.
[0368] In another assay for antagonists, mammalian cells or a
membrane preparation expressing the receptor would be incubated
with labeled UNQ733 polypeptide in the presence of the candidate
compound. The ability of the compound to enhance or block this
interaction could then be measured.
[0369] More specific examples of potential antagonists include an
oligonucleotide that binds to the fusions of immunoglobulin with
UNQ733 polypeptide, and, in particular, antibodies including,
without limitation, poly- and monoclonal antibodies and antibody
fragments, single-chain antibodies, anti-idiotypic antibodies, and
chimeric or humanized versions of such antibodies or fragments, as
well as human antibodies and antibody fragments. Alternatively, a
potential antagonist may be a closely related protein, for example,
a mutated form of the UNQ733 polypeptide that recognizes the
receptor but imparts no effect, thereby competitively inhibiting
the action of the UNQ733 polypeptide.
[0370] Another potential UNQ733 antagonist is an antisense RNA or
DNA construct prepared using antisense technology, where, e.g., an
antisense RNA or DNA molecule acts to block directly the
translation of mRNA by hybridizing to targeted mRNA and preventing
protein translation. Antisense technology can be used to control
gene expression through triple-helix formation or antisense DNA or
RNA, both of which methods are based on binding of a polynucleotide
to DNA or RNA. For example, the 5' coding portion of the
polynucleotide sequence, which encodes the mature UNQ733
polypeptides herein, can be used to design an antisense RNA
oligonucleotide of from about 10 to 40 base pairs in length. A DNA
oligonucleotide is designed to be complementary to a region of the
gene involved in transcription (triple helix--see Lee et al., Nucl.
Acids Res., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988);
Dervan et al., Science, 251:1360 (1991)), thereby preventing
transcription and the production of the UNQ733 polypeptide. The
antisense RNA oligonucleotide hybridizes to the mRNA in vivo and
blocks translation of the mRNA molecule into the UNQ733 polypeptide
(antisense-Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides
as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton,
Fla., 1988). The oligonucleotides described above can also be
delivered to cells such that the antisense RNA or DNA may be
expressed in vivo to inhibit production of the UNQ733 polypeptide.
When antisense DNA is used, oligodeoxyribonucleotides derived from
the translation-initiation site, e.g., between about -10 and +10
positions of the target gene nucleotide sequence, are
preferred.
[0371] Potential antagonists include small molecules that bind to
the active site, the receptor binding site, or growth factor or
other relevant binding site of the UNQ733 polypeptide, thereby
blocking the normal biological activity of the UNQ733 polypeptide.
Examples of small molecules include, but are not limited to, small
peptides or peptide-like molecules, preferably soluble peptides,
and synthetic non-peptidyl organic or inorganic compounds.
[0372] Ribozymes are enzymatic RNA molecules capable of catalyzing
the specific cleavage of RNA. Ribozymes act by sequence-specific
hybridization to the complementary target RNA, followed by
endonucleolytic cleavage. Specific ribozyme cleavage sites within a
potential RNA target can be identified by known techniques. For
further details see, e.g., Rossi, Current Biology, 4:469-471
(1994), and PCT publication No. WO 97/33551 (published Sep. 18,
1997).
[0373] Nucleic acid molecules in triple-helix formation used to
inhibit transcription should be single-stranded and composed of
deoxynucleotides. The base composition of these oligonucleotides is
designed such that it promotes triple-helix formation via Hoogsteen
base-pairing rules, which generally require sizeable stretches of
purines or pyrimidines on one strand of a duplex. For further
details see, e.g., PCT publication No. WO 97/33551, supra.
[0374] These small molecules can be identified by any one or more
of the screening assays discussed hereinabove and/or by any other
screening techniques well known for those skilled in the art.
[0375] Isolated UNQ733 polypeptide-encoding nucleic acid can be
used for recombinantly producing UNQ733 polypeptide using
techniques well known in the art and as described herein. In turn,
the produced UNQ733 polypeptides can be employed for generating
anti-UNQ733 antibodies using techniques well known in the art and
as described herein.
[0376] Antibodies specifically binding a UNQ733 polypeptide
identified herein, as well as other molecules identified by the
screening assays disclosed hereinbefore, can be administered for
the treatment of various disorders, including cancer, in the form
of pharmaceutical compositions.
[0377] If the UNQ733 polypeptide is intracellular and whole
antibodies are used as inhibitors, internalizing antibodies are
preferred. However, lipofections or liposomes can also be used to
deliver the antibody, or an antibody fragment, into cells. Where
antibody fragments are used, the smallest inhibitory fragment that
specifically binds to the binding domain of the target protein is
preferred. For example, based upon the variable-region sequences of
an antibody, peptide molecules can be designed that retain the
ability to bind the target protein sequence. Such peptides can be
synthesized chemically and/or produced by recombinant DNA
technology. See, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA,
90: 7889-7893 (1993).
[0378] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. Alternatively, or in addition, the
composition may comprise an agent that enhances its function, such
as, for example, a cytotoxic agent, cytokine, chemotherapeutic
agent, or growth-inhibitory agent. Such molecules are suitably
present in combination in amounts that are effective for the
purpose intended.
[0379] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0380] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
Materials & Methods
[0381] Cell Lines Chinese hamster ovary, DP12 (American Type
Culture Collection (ATCC, Manassa, Va.)) and PGSB (ATCC) mutants
were used for transient expression and production of recombinant
proteins. Jurkat T lymphoma, BJAB and Daudi B cell lymphomas, and
Reh B cell leukemia were obtained from ATCC.
[0382] RNA Expression Analysis: For the analysis of UNQ733 mRNA
expression in multiple human tumor and normal biopsy samples,
Affymetrix data were obtained from Gene Logic, Inc. (Gaithersberg,
Md.). A total of 150 samples [6 normal samples; 62 cancer samples;
and 82 non-cancer diseased samples] were analyzed. Gene Logic data
were normalized using global scaling with a target intensity of
100. The Affymetrix data for UNQ733 in FIG. 1 was generated from
the U133 probe set ID 229152_at. For in situ hybridization, a
368-bp .sup.33P-labeled antisense riboprobe was generated from a
UNQ733 specific PCR product using a primer with oligonucleotide
sequence 5'-CAG-GTG-ACC-AGG-TTT-ATC-GTG-3' (SEQ ID NO:5) and a
sense control riboprobe with the primer
5'-AAT-ATT-CCA-GGG-CCA-GTC-ACT-3' (SEQ ID NO:6). Representative
normal tissues and cancers including three lymphoma tissue
microarrays (Cybrdi, Inc.; Frederick, Md.) were also screened by in
situ hybridization.
[0383] Recombinant Proteins: UNQ733 cDNA was generated by PCR
amplification from a human multi-tissue cDNA library. The PCR
product was cloned into pSVID, a pRK based vector with a
His.sub.8-tag. PGSB cells (ATCC) were transfected using Fugene 6
(Roche; Indianapolis, Ind.) and conditioned media collected after 7
days. His.sub.8-tagged UNQ733 polypeptide was purified by
Ni-chromatography followed by dialysis into a 10 mM citrate, pH 3.0
buffer. An N-terminal UNQ733 human placental alkaline phosphatase
(UNQ733-AP) fusion protein was generated by inserting the entire
UNQ733 coding sequence into a pRK vector containing the AP gene.
CHO cells were transfected using Fugene 6 (Roche) and supernatant
allowed to condition for 6 days. All experiments used 733-AP
supernatant and a control supernatant (Relt-AP) that was normalized
for AP activity.
[0384] Monoclonal Anti-UNQ733 Antibodies: BALB/c mice were
immunized 15 times with 1.0 .mu.g of UNQ733-His8 resuspended in
monophosphoryl lipid A/trehalose dicorynomycolate adjuvant (Corixa;
Hamilton, Mont.) into each hind footpad at 3 to 4 days intervals.
Three days after final boost, popliteal lymph nodes were fused with
myeloma cell line P3X63Ag8.653 (ATCC, Manassa, Va.) using 50%
polyethylene glycol and cultured in 96 well tissue culture plates.
Culture supernatants were initially screened for their ability to
bind to UNQ733-His8. The selected hybridomas were then cloned by
limiting dilution.
[0385] Antibody clones were deposited with the ATCC under the
Budapest Treaty. American Type Culture Collection (ATCC) is located
at 10801 University Boulevard, Manassas, Va. 20110-2209. Details of
the deposits are as follows: TABLE-US-00006 Clone designation
Deposit Date ATCC Deposit No. 3E7.9.20 Jun. 2, 2004 PTA-6026
3F10.11.2 Jun. 2, 2004 PTA-6027 3H1.4.8 Jun. 2, 2004 PTA-6028
4A9.12.12 Jun. 2, 2004 PTA-6029 5A8.11.6 Jun. 2, 2004 PTA-6030
5F2.6.14 Jun. 2, 2004 PTA-6031 9D6.11.15 Jun. 2, 2004 PTA-6032
10G10.15.16 Jun. 2, 2004 PTA-6033 12H4.11.3 Jun. 2, 2004
PTA-6034
[0386] These deposits were made under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of a viable culture of the deposit for 30 years from the date of
deposit. The deposits will be made available by ATCC under the
terms of the Budapest Treaty, and subject to an agreement between
Genentech, Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of the culture of the deposit to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 USC .sctn. 122 and the
Commissioner's rules pursuant thereto (including 37 CFR .sctn. 1.14
with particular reference to 8860G 638).
[0387] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
[0388] Western blots and Immunoprecipitation: Purified
His.sub.8-tagged UNQ733 protein was subjected to 18% SDS-PAGE and
immunoblotting (Invitrogen; Carlsbad, Calif.). Blots were incubated
with 1 .mu.g/ml anti-UNQ733 Mab overnight at 4.degree. C. Rabbit
anti-mouse Ig horseradish peroxidase (Amersham; Piscataway, N.J.)
was used as a secondary antibody, and the blots were developed
using the SuperSignal West Pico Luminol/Enhancer Solution (Pierce;
Rockford, Ill.). Immunoprecipitation was done by adding 1 .mu.g
purified Mab to 50 ng of purified His.sub.8-tagged UNQ733 in TBS
[50 mM Tris, 150 mM NaCl], followed by protein A/G-agarose
(Pierce), and incubated overnight at 4.degree. C. The
immunoprecipitates were recovered, washed with TBS, and subjected
to 18% SDS-PAGE. Blots were incubated with India His Probe (1:5000;
Pierce) to detect His.sub.8-tagged UNQ733, washed in TBS+0.05%
Tween-20 and developed as described above.
[0389] Immunohistochemistry: Frozen human tonsil sections (5.mu.)
were fixed in -20.degree. C. acetone for 5 minutes followed by
removal of OCT in TBST (Tris-buffered saline with Tween 20).
Endogenous peroxidase, avidin and biotin were quenched using KPL
blocking buffer (KPL, 37-00-84; Gaithersburg, Md.) and Vector
avidin/biotin blocking kit (Vector, SP-2001; Burlingame, Calif.)
respectively, followed by TBST rinses. Slides were incubated for 30
minutes in 10% normal horse serum in 3% BSA/PBS for 30 minutes.
Slides were then incubated in 10 .mu.g/ml anti-UNQ733 (clone 3H1)
antibody for 60 minutes at room temperature. After washes in TBST,
slides were incubated with 2.5 .mu.g/ml biotinylated horse
anti-mouse secondary antibody (Vector, BA-2001) for 30 minutes at
room temperature. Following TBST washes slides were incubated with
Vectastain ABC Elite Reagents (Vector, PK-6100) for 30 minutes at
room temperature, slides were then rinsed with TBST, incubated with
Pierce Metal Enhanced DAB (Pierce, 1856090) for four minutes, and
rinsed in water. Slides were then counterstained with Mayer's
hematoxylin, dehydrated, mounted and coverslipped for bright field
viewing.
[0390] Flow Cytometry For analysis of UNQ733 expression in tonsil
cells, fresh human tonsils tissue was minced and digested in 50
mg/ml of collagenase IV and 50 U/ml of DNase I (Sigma-Aldrich; St.
Louis, Mo., USA). The resulting cell suspension was subjected to
negative selection of B cells with anti-CD19 microbeads on an
autoMACS separator (Miltenyi Biotec; Auburn, Calif.). The
CD19-fraction was blocked with Fc blocking reagent (Miltenyi
Biotec) followed by staining anti-CD14-FITC (clone M5E2; BD
Pharmingen; San Diego, Calif.) and anti-CD21-PE (clone B-ly4; BD
Pharmingen) in FACS wash [PBS+0.5% bovine serum albumin] for 30 min
at ambient temperature. Cells were washed in FACS wash and fixed
with FACSLyse solution (BD Pharmingen) for 20 minutes at ambient
temperature. Cells were washed with FACS wash and treated with FACS
Perm 2 solution (BD Pharmingen). Cells were washed in FACS wash and
incubated with 10 .mu.g/ml biotinylated UNQ733-specific Mab 12H4
for 30 min at ambient temperature followed by washing and
incubation with SA-APC (BD Pharmingen). Cells were analyzed on a BD
FACSCaliber flow cytometer. FDC were gated as CD14.sup.+CD21.sup.+
cells and monocytes gated as CD14.sup.+CD21.sup.- cells.
[0391] For analysis of His.sub.8-tagged UNQ733 binding to lymphoma
cell lines, cells were incubated with 50 .mu.g/ml (4.5 .mu.M) or 20
.mu.g/ml (1.8 .mu.M) His.sub.8-tagged UNQ733 diluted in FACS wash
at ambient temperature for 30 minutes. Cells were washed and bound
His.sub.8-tagged UNQ733 detected with 10 .mu.g/ml
anti-His.sub.6(C-term)-FITC (Invitrogen). Alternately,
His.sub.8-tagged UNQ733 bound to cells was detected with 10
.mu.g/ml biotinylated UNQ733-specific Mab 12H4 followed by SA-PE
(BD Pharmingen). For analysis of His.sub.8-tagged UNQ733 binding to
human peripheral blood B cells, peripheral blood from normal
healthy human donors was obtained. Peripheral blood mononuclear
cells were isolated using LSM lymphocyte separation medium (ICN;
Costa Mesa, Calif.). B cells were purified using anti-CD19
microbeads and a MACS separator (Miltenyi Biotec). B cells were
cultured in Iscove's modified Dulbecco's media+10% fetal bovine
serum in the presence or absence of 1 .mu.g/ml anti-CD40 (clone
5C3; BD Pharmingen) and 10 ng/ml rIL-4 (Preprotech) for 48 hours
before flow cytometric analysis as described above. B cells were
gated by staining with anti-CD19-APC (clone HIB19; BD
Pharmingen).
[0392] Protein Expression and Sequencing: PGSB cells were
transiently transfected with His.sub.8-tagged UNQ733 using Fugene 6
(Roche). The cultures were incubated in the presence or absence of
10 .mu.M decanoyl-Arg-Val-Lys-Arg-chloromethylketone (Bachem; King
of Prussia, Pa.), a proprotein convertase inhibitor, added to the
cultures on day one. After 6 days in culture, supernatants were
harvested, affinity precipitated with Ni-NTA (Qiagen; Valencia,
Calif.). The Ni-NTA bound protein was subjected to 18% SDS-PAGE and
transblotted to PVDF for N-terminal sequencing by Edman degradation
on a Procise N-Terminal Sequencer (ABI; Foster City, Calif.).
[0393] Protein Binding Assay: BJAB cells were incubated with serial
dilutions of UNQ733-AP supernatant or a single concentration of
Relt-AP control supernatant. After incubation for 1 hour at
4.degree. C., cells were washed three times with PBS, and
transferred to a 96-well microplate. Cells were heated to
50.degree. C. for 1 hour to inactivate endogenous alkaline
phosphatase activity. 1-Step PNPP substrate buffer (Pierce) was
added, and the reaction was allowed to proceed for 30 minutes at
ambient temperature in the dark. Cells were centrifuged and an
aliquot of substrate buffer was removed for analysis of absorbance
at 405 nm on a SpectraMax 250 microplate reader (Molecular Devices;
Sunnyvale, Calif.).
[0394] For competitive inhibition of UNQ733-AP binding, a fixed
concentration (0.5.times.) of UNQ733-AP supernatant was used and
simultaneously incubated with increasing concentrations of
His.sub.8-tagged UNQ733 or a vehicle control [10 mM citrate, pH 3.0
buffer].
Results
UNQ733 is Over-Expressed in Non-Hodgkin's Lymphoma and Hyperplastic
Tonsils
[0395] Oligonucleotide-based microarray expression data from over
4800 human biopsy samples were analyzed for genes that showed
elevated mRNA expression relative to normal tissue. One such gene
was designated UNQ733, and it was found to be over-expressed in
tumors of lymphoid origin (FIG. 1). Within the lymphoid tumors
analyzed, it was found significantly over-expressed in 29 of 62
(46.8%) non-Hodgkin's lymphomas. By this analysis over-expression
of UNQ733 was also observed in 75 of 82 (91%) diseased,
non-malignant tonsilar tissue designated hyperplastic by
histopathology.
[0396] To further assess the prevalence of UNQ733 overexpression in
non-Hodgkin's lymphoma, we obtained three lymphoma tissue
microarrays (TMA) containing 173 non-Hodgkin's lymphoma tissue
cores. In situ hybridization was carried out on the TMAs using a
UNQ733-specific probe. In 66 of 173 (38%) individual tumors, UNQ733
mRNA expression was detected in the malignant cells. Overexpression
of UNQ733 was observed in B cell lymphoma (NOS), diffuse large B
cell lymphoma, follicular lymphoma, small lymphocytic lymphoma,
malignant lymphoma (NOS), malignant T cell lymphoma, anaplastic
large cell lymphoma and mucosal associated lymphoid tissue
lymphoma.
[0397] In situ hybridization was carried out on normal and
neoplastic human tissue. UNQ733 mRNA expression was observed in the
germinal centers of all normal lymphoid tissues including the
spleen, lymph nodes, tonsils, and Peyer's patches (FIG. 3). In
tonsils, UNQ733 expression was detected in the germinal centers
(FIG. 4A, 4B) and crypts (FIG. 4C, 4D). Furthermore, UNQ733
expression was observed in the serous salivary glands (not shown).
In neoplastic tissue, expression was observed in the malignant
cells of 38% (3/8) of non-Hodgkin's lymphomas analyzed (FIGS. 5
& 6).
[0398] Monoclonal antibodies (MAb) against recombinant
His.sub.8-tagged UNQ733 were generated and characterized. A panel
of 9 MAbs with reactivity to recombinant UNQ733 by ELISA was
obtained and characterized in western blot assays,
immunoprecipitation assays and immunohistochemistry assays (IHC) to
assess binding to UNQ733 in cell-free and cell-associated
environments. Two of the MAbs were found to detect recombinant
UNQ733 in a western blot assay, 10G10 and 12H4 (FIG. 7). These
antibodies were able to detect recombinant His.sub.8-tagged UNQ733
in the western blot. Most of the monoclonal antibodies were able to
immunoprecipitate UNQ733 (FIG. 8). The MAb 3H1 was tested for its
ability to bind UNQ733 in frozen tonsilar tissue by IHC. The
pattern of staining observed was identical to the localization of
UNQ733 mRNA by in situ hybridization. Strong staining was observed
in the crypts and weak staining in the germinal centers (FIG. 9).
The pattern of staining observed was consistent with a follicular
dendritic cell (FDC).
[0399] To ascertain the identity of the cell type expressing UNQ733
in the tonsil, fresh tonsil tissue from surgical resections was
obtained and used in flow cytometry to delineate the FDCs and
monocytes by CD14 and CD21 expression. Biotinylated-12H4 MAb was
used to detect intracellular UNQ733. Expression of UNQ733 protein
was detected in the FDC population, but not in the monocytes (FIG.
10), a finding consistent with the immunohistochemistry data.
UNQ733 is Processed by a Proprotein Convertase
[0400] Proprotein convertases (PCs) are a family of proteolytic
enzymes that cleave biologically inactive precursor proteins into
their mature active forms. Members of this family of proteases
recognize amino acid sequences with the motif (K/R)-X-X-(K/R)/.
UNQ733 contains a putative PC cleavage site R-E-K-R/S.sup.30. Two
protein species are typically observed when recombinant
UNQ733-His.sub.8 was analyzed by western blot (FIG. 7). N-terminal
sequencing of the proteins confirmed the presence of two species,
the first beginning with the sequence .sup.18FPVSQDQE, which was
consistent with the predicted "full-length" protein after signal
sequence cleavage. The second protein began with the sequence
.sup.30SISDSDEL that would be the predicted product of PC cleavage
of UNQ733. To confirm that the activity of a PC was responsible for
the observed cleavage event, UNQ733-His.sub.8 was expressed in the
presence and absence of a PC inhibitor,
decanoyl-RVKR-chloromethylketone (FIG. 11). Multiple species were
still observed, but the proportion of "full-length" UNQ733 was
substantially increased in the PC inhibitor treated supernatants
(FIG. 12). This indicates that UNQ733 is processed by a PC.
UNQ733 Binds to Lymphoma Cell Lines and Primary B Cells
[0401] UNQ733 binding to lymphoma cell lines was assessed by flow
cytometry. Binding of UNQ733-His.sub.8 to both BJAB cells, a human
B cell lymphoma (FIG. 13A), and Jurkat cells, a human T cell
lymphoma (FIG. 13B) was detected by using a FITC conjugated
anti-His.sub.6 detecting antibody (Invitrogen). Antibody binding
was observed for both cell lines, with the BJAB cells showing
higher binding of the UNQ733-His.sub.8 than the Jurkat cells. These
studies were extended to test the panel of MAbs for the ability to
detect binding of UNQ733 on a subset of human B cell
lymphoma/leukemia cell lines. Several MAbs, including 3H1 and
10G10, were able to detect bound UNQ733-His.sub.8 (not shown); 12H4
showed the best signal to noise ratio in detecting UNQ733-His.sub.8
bound to BJAB, Reh and Daudi cells (FIG. 14). To evaluate the
specificity of the binding observed by the UNQ733-His.sub.8
molecule, a UNQ733-human placenta alkaline phosphatase (UNQ733-AP)
fusion protein was used to detect bound UNQ733, and
UNQ733-His.sub.8 used to function as a competitor. The UNQ733-AP
protein in tissue culture supernatants was found to bind to the C1R
B lymphoblast cell line in a dose-dependent manner while a control
AP fusion protein, RELT-AP, showed no binding (FIG. 15A). When
tested in a competition assay, UNQ733-His.sub.8 was observed to
inhibit the binding of UNQ733-AP to C1R cells (FIG. 15B). This
indicated that the binding of UNQ733 to B lymphomas is
specific.
[0402] UNQ733 is expressed in follicular dendritic cells in the
germinal centers of secondary lymphoid organs. Because germinal
centers are a site of B cell selection, class-switch recombination,
and somatic hypermutation, the role of UNQ733 in non-neoplastic B
cell biology was also ascertained. Peripheral blood mononuclear
cells were obtained from normal healthy human donors, and the B
cells were purified and analyzed for UNQ733 binding (FIG. 16). It
was observed that "resting" peripheral blood B cells from multiple
human donors bound UNQ733. When those PB B cells were first
stimulated with an agonistic MAb specific to CD40 and recombinant
IL-4, the binding of UNQ733 was found to increase 2-5 fold
depending on the donor (FIG. 16). It appears that stimulation of
the B cells with anti-CD40 results in increased surface expression
of the receptor for UNQ733.
Sequence CWU 1
1
11 1 537 DNA Homo sapiens 1 taaaacagct acaatattcc agggccagtc
acttgccatt tctcataaca 50 gcgtcagaga gaaagaactg actgaaacgt
ttgagatgaa gaaagttctc 100 ctcctgatca cagccatctt ggcagtggct
gttggtttcc cagtctctca 150 agaccaggaa cgagaaaaaa gaagtatcag
tgacagcgat gaattagctt 200 cagggttttt tgtgttccct tacccatatc
catttcgccc acttccacca 250 attccatttc caagatttcc atggtttaga
cgtaattttc ctattccaat 300 acctgaatct gcccctacaa ctccccttcc
tagcgaaaag taaacaagaa 350 ggataagtca cgataaacct ggtcacctga
aattgaaatt gagccacttc 400 cttgaagaat caaaattcct gttaataaaa
gaaaaacaaa tgtaattgaa 450 atagcacaca gcattctcta gtcaatatct
ttagtgatct tctttaataa 500 acatgaaagc aaagattttg gtttcttaat ttccaca
537 2 85 PRT Homo sapiens 2 Met Lys Lys Val Leu Leu Leu Ile Thr Ala
Ile Leu Ala Val Ala 1 5 10 15 Val Gly Phe Pro Val Ser Gln Asp Gln
Glu Arg Glu Lys Arg Ser 20 25 30 Ile Ser Asp Ser Asp Glu Leu Ala
Ser Gly Phe Phe Val Phe Pro 35 40 45 Tyr Pro Tyr Pro Phe Arg Pro
Leu Pro Pro Ile Pro Phe Pro Arg 50 55 60 Phe Pro Trp Phe Arg Arg
Asn Phe Pro Ile Pro Ile Pro Glu Ser 65 70 75 Ala Pro Thr Thr Pro
Leu Pro Ser Glu Lys 80 85 3 68 PRT Rana catesbeiana 3 Phe Pro Val
Ser Gln Asp Gln Glu Arg Glu Lys Arg Ser Ile Ser 1 5 10 15 Asp Ser
Asp Glu Leu Ala Ser Gly Phe Phe Val Phe Pro Tyr Pro 20 25 30 Tyr
Pro Phe Arg Pro Leu Pro Pro Ile Pro Phe Pro Arg Phe Pro 35 40 45
Trp Phe Arg Arg Asn Phe Pro Ile Pro Ile Pro Glu Ser Ala Pro 50 55
60 Thr Thr Pro Leu Pro Ser Glu Lys 65 4 56 PRT Homo sapiens 4 Ser
Ile Ser Asp Ser Asp Glu Leu Ala Ser Gly Phe Phe Val Phe 1 5 10 15
Pro Tyr Pro Tyr Pro Phe Arg Pro Leu Pro Pro Ile Pro Phe Pro 20 25
30 Arg Phe Pro Trp Phe Arg Arg Asn Phe Pro Ile Pro Ile Pro Glu 35
40 45 Ser Ala Pro Thr Thr Pro Leu Pro Ser Glu Lys 50 55 5 21 DNA
Artificial sequence Oligonucleotide 5 caggtgacca ggtttatcgtg 21 6
21 DNA Artificial sequence Oligonucleotide 6 aatattccag ggccagtcact
21 7 4 PRT Artificial sequence Synthetic inhibitor 7 Arg Val Lys
Arg 1 8 4 PRT Artificial sequence Cleavage site 8 Arg Glu Lys Arg 1
9 4 PRT Artificial sequence Cleavage site 9 Arg Glu Lys Ser 1 10 8
PRT Homo sapiens 10 Phe Pro Val Ser Gln Asp Gln Glu 1 5 11 8 PRT
Homo sapiens 11 Ser Ile Ser Asp Ser Asp Glu Leu 1 5
* * * * *