U.S. patent application number 12/119183 was filed with the patent office on 2008-12-25 for compositions and methods for the diagnosis and treatment of tumor.
Invention is credited to Gretchen Frantz, Paul Polakis, Susan D. Spencer, Thomas D. Wu, Zemin Zhang.
Application Number | 20080318239 12/119183 |
Document ID | / |
Family ID | 29255342 |
Filed Date | 2008-12-25 |
United States Patent
Application |
20080318239 |
Kind Code |
A1 |
Frantz; Gretchen ; et
al. |
December 25, 2008 |
COMPOSITIONS AND METHODS FOR THE DIAGNOSIS AND TREATMENT OF
TUMOR
Abstract
The present invention is directed to compositions of matter
useful for the diagnosis and treatment of tumor in mammals and to
methods of using those compositions of matter for the same.
Inventors: |
Frantz; Gretchen; (San
Francisco, CA) ; Polakis; Paul; (Burlingame, CA)
; Spencer; Susan D.; (Tiburon, CA) ; Wu; Thomas
D.; (San Francisco, CA) ; Zhang; Zemin;
(Foster City, CA) |
Correspondence
Address: |
Goodwin Procter LLP;Attn: Patent Administrator
135 Commonwealth Drive
Menlo Park
CA
94025-1105
US
|
Family ID: |
29255342 |
Appl. No.: |
12/119183 |
Filed: |
May 12, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11538745 |
Oct 4, 2006 |
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12119183 |
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10953264 |
Sep 29, 2004 |
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11538745 |
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10411010 |
Apr 10, 2003 |
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10953264 |
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60404809 |
Aug 19, 2002 |
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60378885 |
May 8, 2002 |
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60373160 |
Apr 16, 2002 |
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Current U.S.
Class: |
435/6.16 |
Current CPC
Class: |
C07K 16/30 20130101;
C07K 16/2803 20130101; A61K 47/6843 20170801; A61K 2039/505
20130101; A61K 47/6809 20170801; C07K 16/18 20130101; A61K 47/6803
20170801; C12N 2799/026 20130101; A61K 47/6851 20170801 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1-20. (canceled)
21. A method of diagnosing the presence of a tumor in a mammal,
said method comprising determining the level of expression of a
gene encoding a protein comprising the amino acid sequence of SEQ
ID NO:30, in a test sample of tissue cells obtained from said
mammal and in a control sample of known normal cells of the same
tissue origin, wherein a higher level of expression of said protein
in the test sample, as compared to the control sample, is
indicative of the presence of tumor in the mammal from which the
test sample was obtained.
22. The method of claim 21, wherein the step of determining the
level of expression of a gene encoding said protein comprises
employing an oligonucleotide in an in situ hybridization or RT-PCR
analysis.
23. The method of claim 21, wherein said tumor is an ovarian tumor.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to compositions of matter
useful for the diagnosis and treatment of tumor in mammals and to
methods of using those compositions of matter for the same.
BACKGROUND OF THE INVENTION
[0002] Malignant tumors (cancers) are the second leading cause of
death in the United States, after heart disease (Boring et al., CA
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.
[0003] In attempts to discover effective cellular targets for
cancer diagnosis and 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.
[0004] In other attempts to discover effective cellular targets for
cancer diagnosis and 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
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.
[0005] Despite the above identified advances in mammalian cancer
therapy, there is a great need for additional diagnostic and
therapeutic agents capable of detecting the presence of tumor in a
mammal and for effectively inhibiting neoplastic cell growth,
respectively. Accordingly, it is an objective of the present
invention to identify: (I) cell membrane-associated polypeptides
that are more abundantly expressed on one or more type(s) of cancer
cell(s) as compared to on normal cells or on other different cancer
cells, (2) non-membrane-associated polypeptides that are
specifically produced by one or more particular type(s) of cancer
cell(s) (or by other cells that produce polypeptides having a
potentiating effect on the growth of cancer cells) as compared to
by one or more particular type(s) of non-cancerous normal cell(s),
(3) non-membrane-associated 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 (4)
polypeptides whose expression is specifically limited to only a
single (or very limited number of different) tissue type(s) in both
a cancerous and non-cancerous state (e.g., normal prostate and
prostate tumor tissue), and to use those polypeptides, and their
encoding nucleic acids, to produce compositions of matter useful in
the therapeutic treatment and diagnostic detection of cancer in
mammals. It is also an objective of the present invention to
identify cell membrane-associated, secreted or intracellular
polypeptides whose expression is limited to a single or very
limited number of tissues, and to use those polypeptides, and their
encoding nucleic acids, to produce compositions of matter useful in
the therapeutic treatment and diagnostic detection of cancer in
mammals.
SUMMARY OF THE INVENTION
A. Embodiments
[0006] In the present specification, Applicants describe for the
first time the identification of various cellular polypeptides (and
their encoding nucleic acids or fragments thereof) which are
expressed to a greater degree on the surface of or by one or more
types of cancer cell(s) as compared to on the surface of or by one
or more types of normal non-cancer cells. Alternatively, such
polypeptides are expressed by cells which produce and/or secrete
polypeptides having a potentiating or growth-enhancing effect on
cancer cells. Again alternatively, such polypeptides may not be
overexpressed by tumor cells as compared to normal cells of the
same tissue type, but rather may be specifically expressed by both
tumor cells and normal cells of only a single or very limited
number of tissue types (preferably tissues which are not essential
for life, e.g., prostate, etc.). All of the above polypeptides are
herein referred to as Tumor-associated Antigenic Target
polypeptides ("TAT" polypeptides) and are expected to serve as
effective targets for cancer therapy and diagnosis in mammals.
[0007] Accordingly, in one embodiment of the present invention, the
invention provides an isolated nucleic acid molecule having a
nucleotide sequence that encodes a tumor-associated antigenic
target polypeptide or fragment thereof (a "TAT" polypeptide).
[0008] In certain aspects, the isolated nucleic acid molecule
comprises a nucleotide sequence having 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%, 99% or 100% nucleic acid sequence identity, to (a) a DNA
molecule encoding a full-length TAT polypeptide having an amino
acid sequence as disclosed herein, a TAT polypeptide amino acid
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a transmembrane TAT polypeptide, with or
without the signal peptide, as disclosed herein or any other
specifically defined fragment of a full-length TAT polypeptide
amino acid sequence as disclosed herein, or (b) the complement of
the DNA molecule of (a).
[0009] In other aspects, the isolated nucleic acid molecule
comprises a nucleotide sequence having 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%, 99% or 100% nucleic acid sequence identity, to (a) a DNA
molecule comprising the coding sequence of a full-length TAT
polypeptide cDNA as disclosed herein, the coding sequence of a TAT
polypeptide lacking the signal peptide as disclosed herein, the
coding sequence of an extracellular domain of a transmembrane TAT
polypeptide, with or without the signal peptide, as disclosed
herein or the coding sequence of any other specifically defined
fragment of the full-length TAT polypeptide amino acid sequence as
disclosed herein, or (b) the complement of the DNA molecule of
(a).
[0010] In further aspects, the invention concerns an isolated
nucleic acid molecule comprising a nucleotide sequence having 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%, 99% or 100% nucleic acid
sequence identity, to (a) a DNA molecule that encodes the same
mature polypeptide encoded by the full-length coding region of any
of the human protein cDNAs deposited with the ATCC as disclosed
herein, or (b) the complement of the DNA molecule of (a).
[0011] Another aspect of the invention provides an isolated nucleic
acid molecule comprising a nucleotide sequence encoding a TAT
polypeptide which is either transmembrane domain-deleted or
transmembrane domain-inactivated, or is complementary to such
encoding nucleotide sequence, wherein the transmembrane domain(s)
of such polypeptide(s) are disclosed herein. Therefore, soluble
extracellular domains of the herein described TAT polypeptides are
contemplated.
[0012] In other aspects, the present invention is directed to
isolated nucleic acid molecules which hybridize to (a) a nucleotide
sequence encoding a TAT polypeptide having a full-length amino acid
sequence as disclosed herein, a TAT polypeptide amino acid sequence
lacking the signal peptide as disclosed herein, an extracellular
domain of a transmembrane TAT polypeptide, with or without the
signal peptide, as disclosed herein or any other specifically
defined fragment of a full-length TAT polypeptide amino acid
sequence as disclosed herein, or (b) the complement of the
nucleotide sequence of (a). In this regard, an embodiment of the
present invention is directed to fragments of a full-length TAT
polypeptide coding sequence, or the complement thereof, as
disclosed herein, that may find use as, for example, hybridization
probes useful as, for example, diagnostic probes, antisense
oligonucleotide probes, or for encoding fragments of a full-length
TAT polypeptide that may optionally encode a polypeptide comprising
a binding site for an anti-TAT polypeptide antibody, a TAT binding
oligopeptide or other small organic molecule that binds to a TAT
polypeptide. Such nucleic acid fragments are usually 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. It is noted that novel fragments of a TAT
polypeptide-encoding nucleotide sequence may be determined in a
routine manner by aligning the TAT polypeptide-encoding nucleotide
sequence with other known nucleotide sequences using any of a
number of well known sequence alignment programs and determining
which TAT polypeptide-encoding nucleotide sequence fragment(s) are
novel. All of such novel fragments of TAT polypeptide-encoding
nucleotide sequences are contemplated herein. Also contemplated are
the TAT polypeptide fragments encoded by these nucleotide molecule
fragments, preferably those TAT polypeptide fragments that comprise
a binding site for an anti-TAT antibody, a TAT binding oligopeptide
or other small organic molecule that binds to a TAT
polypeptide.
[0013] In another embodiment, the invention provides isolated TAT
polypeptides encoded by any of the isolated nucleic acid sequences
hereinabove identified.
[0014] In a certain aspect, the invention concerns an isolated TAT
polypeptide, comprising an amino acid sequence having 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%, 99% or 100% amino acid sequence
identity, to a TAT polypeptide having a full-length amino acid
sequence as disclosed herein, a TAT polypeptide amino acid sequence
lacking the signal peptide as disclosed herein, an extracellular
domain of a transmembrane TAT polypeptide protein, with or without
the signal peptide, as disclosed herein, an amino acid sequence
encoded by any of the nucleic acid sequences disclosed herein or
any other specifically defined fragment of a full-length TAT
polypeptide amino acid sequence as disclosed herein.
[0015] In a further aspect, the invention concerns an isolated TAT
polypeptide comprising an amino acid sequence having 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 an
amino acid sequence encoded by any of the human protein cDNAs
deposited with the ATCC as disclosed herein.
[0016] In a specific aspect, the invention provides an isolated TAT
polypeptide without the N-terminal signal sequence and/or without
the initiating methionine and is encoded by a nucleotide sequence
that encodes such an amino acid sequence as hereinbefore described.
Processes for producing the same are also herein described, wherein
those processes comprise culturing a host cell comprising a vector
which comprises the appropriate encoding nucleic acid molecule
under conditions suitable for expression of the TAT polypeptide and
recovering the TAT polypeptide from the cell culture.
[0017] Another aspect of the invention provides an isolated TAT
polypeptide which is either transmembrane domain-deleted or
transmembrane domain-inactivated. Processes for producing the same
are also herein described, wherein those processes comprise
culturing a host cell comprising a vector which comprises the
appropriate encoding nucleic acid molecule under conditions
suitable for expression of the TAT polypeptide and recovering the
TAT polypeptide from the cell culture.
[0018] In other embodiments of the present invention, the invention
provides vectors comprising DNA encoding any of the herein
described polypeptides. Host cells comprising any such vector are
also provided. By way of example, the host cells may be CHO cells,
E. coli cells, or yeast cells. A process for producing any of the
herein described polypeptides is further provided and comprises
culturing host cells under conditions suitable for expression of
the desired polypeptide and recovering the desired polypeptide from
the cell culture.
[0019] In other embodiments, the invention provides isolated
chimeric polypeptides comprising any of the herein described TAT
polypeptides fused to a heterologous (non-TAT) polypeptide. Example
of such chimeric molecules comprise any of the herein described TAT
polypeptides fused to a heterologous polypeptide such as, for
example, an epitope tag sequence or a Fc region of an
immunoglobulin.
[0020] In another embodiment, the invention provides an antibody
which binds, preferably specifically, to any of the above or below
described polypeptides. Optionally, the antibody is a monoclonal
antibody, antibody fragment, chimeric antibody, humanized antibody,
single-chain antibody or antibody that competitively inhibits the
binding of an anti-TAT polypeptide antibody to its respective
antigenic epitope. Antibodies of the present 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. The antibodies of the present invention may
optionally be produced in CHO cells or bacterial cells and
preferably induce death of a cell to which they bind. For
diagnostic purposes, the antibodies of the present invention may be
delectably labeled, attached to a solid support, or the like.
[0021] In other embodiments of the present invention, the invention
provides vectors comprising DNA encoding any of the herein
described antibodies. Host cell comprising any such vector are also
provided. By way of example, the host cells may be CHO cells, E.
coli cells, or yeast cells. A process for producing any of the
herein described antibodies is further provided and comprises
culturing host cells under conditions suitable for expression of
the desired antibody and recovering the desired antibody from the
cell culture.
[0022] In another embodiment, the invention provides oligopeptides
("TAT binding oligopeptides") which bind, preferably specifically,
to any of the above or below described TAT polypeptides.
Optionally, the TAT binding oligopeptides of the present invention
may 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. The TAT binding oligopeptides of the present
invention may optionally be produced in CHO cells or bacterial
cells and preferably induce death of a cell to which they bind. For
diagnostic purposes, the TAT binding oligopeptides of the present
invention may be detectably labeled, attached to a solid support,
or the like.
[0023] In other embodiments of the present invention, the invention
provides vectors comprising DNA encoding any of the herein
described TAT binding oligopeptides. Host cell comprising any such
vector are also provided. By way of example, the host cells may be
CHO cells, E. coli cells, or yeast cells. A process for producing
any of the herein described TAT binding oligopeptides is further
provided and comprises culturing host cells under conditions
suitable for expression of the desired oligopeptide and recovering
the desired oligopeptide from the cell culture.
[0024] In another embodiment, the invention provides small organic
molecules ("TAT binding organic molecules") which bind, preferably
specifically, to any of the above or below described TAT
polypeptides. Optionally, the TAT binding organic molecules of the
present invention may 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. The TAT binding organic
molecules of the present invention preferably induce death of a
cell to which they bind. For diagnostic purposes, the TAT binding
organic molecules of the present invention may be detectably
labeled, attached to a solid support, or the like.
[0025] In a still further embodiment, the invention concerns a
composition of matter comprising a TAT polypeptide as described
herein, a chimeric TAT polypeptide as described herein, an anti-TAT
antibody as described herein, a TAT binding oligopeptide as
described herein, or a TAT binding organic molecule as described
herein, in combination with a carrier. Optionally, the carrier is a
pharmaceutically acceptable carrier.
[0026] In yet another embodiment, the invention concerns an article
of manufacture comprising a container and a composition of matter
contained within the container, wherein the composition of matter
may comprise a TAT polypeptide as described herein, a chimeric TAT
polypeptide as described herein, an anti-TAT antibody as described
herein, a TAT binding oligopeptide as described herein, or a TAT
binding organic molecule as described herein. The article may
further optionally comprise a label affixed to the container, or a
package insert included with the container, that refers to the use
of the composition of matter for the therapeutic treatment or
diagnostic detection of a tumor.
[0027] Another embodiment of the present invention is directed to
the use of a TAT polypeptide as described herein, a chimeric TAT
polypeptide as described herein, an anti-TAT polypeptide antibody
as described herein, a TAT binding oligopeptide as described
herein, or a TAT binding organic molecule as described herein, for
the preparation of a medicament useful in the treatment of a
condition which is responsive to the TAT polypeptide, chimeric TAT
polypeptide, anti-TAT polypeptide antibody, TAT binding
oligopeptide, or TAT binding organic molecule.
B. Additional Embodiments
[0028] Another embodiment of the present invention is directed to a
method for inhibiting the growth of a cell that expresses a TAT
polypeptide, wherein the method comprises contacting the cell with
an antibody, an oligopeptide or a small organic molecule that binds
to the TAT polypeptide, and wherein the binding of the antibody,
oligopeptide or organic molecule to the TAT polypeptide causes
inhibition of the growth of the cell expressing the TAT
polypeptide. In preferred embodiments, the cell is a cancer cell
and binding of the antibody, oligopeptide or organic molecule to
the TAT polypeptide causes death of the cell expressing the TAT
polypeptide. Optionally, the antibody is a monoclonal antibody,
antibody fragment, chimeric antibody, humanized antibody, or
single-chain antibody. Antibodies, TAT binding oligopeptides and
TAT binding organic molecules employed in the methods of the
present 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. The
antibodies and TAT binding oligopeptides employed in the methods of
the present invention may optionally be produced in CHO cells or
bacterial cells.
[0029] Yet another embodiment of the present invention is directed
to a method of therapeutically treating a mammal having a cancerous
tumor comprising cells that express a TAT polypeptide, wherein the
method comprises administering to the mammal a therapeutically
effective amount of an antibody, an oligopeptide or a small organic
molecule that binds to the TAT polypeptide, thereby resulting in
the effective therapeutic treatment of the tumor. Optionally, the
antibody is a monoclonal antibody, antibody fragment, chimeric
antibody, humanized antibody, or single-chain antibody. Antibodies,
TAT binding oligopeptides and TAT binding organic molecules
employed in the methods of the present 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. The antibodies and oligopeptides employed in the methods of
the present invention may optionally be produced in CHO cells or
bacterial cells.
[0030] Yet another embodiment of the present invention is directed
to a method of determining the presence of a TAT polypeptide in a
sample suspected of containing the TAT polypeptide, wherein the
method comprises exposing the sample to an antibody, oligopeptide
or small organic molecule that binds to the TAT polypeptide and
determining binding of the antibody, oligopeptide or organic
molecule to the TAT polypeptide in the sample, wherein the presence
of such binding is indicative of the presence of the TAT
polypeptide in the sample. Optionally, the sample may contain cells
(which may be cancer cells) suspected of expressing the TAT
polypeptide. The antibody, TAT binding oligopeptide or TAT binding
organic molecule employed in the method may optionally be
detectably labeled, attached to a solid support, or the like.
[0031] A further embodiment of the present invention is directed to
a method of diagnosing the presence of a tumor in a mammal, wherein
the method comprises detecting the level of expression of a gene
encoding a TAT polypeptide (a) in a test sample of tissue cells
obtained from said mammal, and (b) in a control sample of known
normal non-cancerous cells of the same tissue origin or type,
wherein a higher level of expression of the TAT polypeptide in the
test sample, as compared to the control sample, is indicative of
the presence of tumor in the mammal from which the test sample was
obtained.
[0032] Another embodiment of the present invention is directed to a
method of diagnosing the presence of a tumor in a mammal, wherein
the method comprises (a) contacting a test sample comprising tissue
cells obtained from the mammal with an antibody, oligopeptide or
small organic molecule that binds to a TAT polypeptide and (b)
detecting the formation of a complex between the antibody,
oligopeptide or small organic molecule and the TAT polypeptide in
the test sample, wherein the formation of a complex is indicative
of the presence of a tumor in the mammal. Optionally, the antibody,
TAT binding oligopeptide or TAT binding organic molecule employed
is detectably labeled, attached to a solid support, or the like,
and/or the test sample of tissue cells is obtained from an
individual suspected of having a cancerous tumor.
[0033] Yet another embodiment of the present invention is directed
to a method for treating or preventing a cell proliferative
disorder associated with altered, preferably increased, expression
or activity of a TAT polypeptide, the method comprising
administering to a subject in need of such treatment an effective
amount of an antagonist of a TAT polypeptide. Preferably, the cell
proliferative disorder is cancer and the antagonist of the TAT
polypeptide is an anti-TAT polypeptide antibody, TAT binding
oligopeptide, TAT binding organic molecule or antisense
oligonucleotide. Effective treatment or prevention of the cell
proliferative disorder may be a result of direct killing or growth
inhibition of cells that express a TAT polypeptide or by
antagonizing the cell growth potentiating activity of a TAT
polypeptide.
[0034] Yet another embodiment of the present invention is directed
to a method of binding an antibody, oligopeptide or small organic
molecule to a cell that expresses a TAT polypeptide, wherein the
method comprises contacting a cell that expresses a TAT polypeptide
with said antibody, oligopeptide or small organic molecule under
conditions which are suitable for binding of the antibody,
oligopeptide or small organic molecule to said TAT polypeptide and
allowing binding therebetween.
[0035] Other embodiments of the present invention are directed to
the use of (a) a TAT polypeptide, (b) a nucleic acid encoding a TAT
polypeptide or a vector or host cell comprising that nucleic acid,
(c) an anti-TAT polypeptide antibody, (d) a TAT-binding
oligopeptide, or (e) a TAT-binding small organic molecule in the
preparation of a medicament useful for (i) the therapeutic
treatment or diagnostic detection of a cancer or tumor, or (ii) the
therapeutic treatment or prevention of a cell proliferative
disorder.
[0036] Another embodiment of the present invention is directed to a
method for inhibiting the growth of a cancer cell, wherein the
growth of said cancer cell is at least in part dependent upon the
growth potentiating effect(s) of a TAT polypeptide (wherein the TAT
polypeptide may be expressed either by the cancer cell itself or a
cell that produces polypeptide(s) that have a growth potentiating
effect on cancer cells), wherein the method comprises contacting
the TAT polypeptide with an antibody, an oligopeptide or a small
organic molecule that binds to the TAT polypeptide, thereby
antagonizing the growth-potentiating activity of the TAT
polypeptide and, in turn, inhibiting the growth of the cancer cell.
Preferably the growth of the cancer cell is completely inhibited.
Even more preferably, binding of the antibody, oligopeptide or
small organic molecule to the TAT polypeptide induces the death of
the cancer cell. Optionally, the antibody is a monoclonal antibody,
antibody fragment, chimeric antibody, humanized antibody, or
single-chain antibody. Antibodies, TAT binding oligopeptides and
TAT binding organic molecules employed in the methods of the
present 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. The
antibodies and TAT binding oligopeptides employed in the methods of
the present invention may optionally be produced in CHO cells or
bacterial cells.
[0037] Yet another embodiment of the present invention is directed
to a method of therapeutically treating a tumor in a mammal,
wherein the growth of said tumor is at least in part dependent upon
the growth potentiating effect(s) of a TAT polypeptide, wherein the
method comprises administering to the mammal a therapeutically
effective amount of an antibody, an oligopeptide or a small organic
molecule that binds to the TAT polypeptide, thereby antagonizing
the growth potentiating activity of said TAT polypeptide and
resulting in the effective therapeutic treatment of the tumor.
Optionally, the antibody is a monoclonal antibody, antibody
fragment, chimeric antibody, humanized antibody, or single-chain
antibody. Antibodies, TAT binding oligopeptides and TAT binding
organic molecules employed in the methods of the present 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. The antibodies and
oligopeptides employed in the methods of the present invention may
optionally be produced in CHO cells or bacterial cells.
C. Further Additional Embodiments
[0038] In yet further embodiments, the invention is directed to the
following set of potential claims for this application:
[0039] 1. Isolated nucleic acid having a nucleotide sequence that
has at least 80% nucleic acid sequence identity to:
[0040] (a) a DNA molecule encoding the amino acid sequence shown in
any one of FIGS. 17-32 (SEQ ID NOS:17-32);
[0041] (b) a DNA molecule encoding the amino acid sequence shown in
any one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its associated
signal peptide;
[0042] (c) a DNA molecule encoding an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
with its associated signal peptide;
[0043] (d) a DNA molecule encoding an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
lacking its associated signal peptide;
[0044] (e) the nucleotide sequence shown in any one of FIGS. 1-16
(SEQ ID NOS:1-16);
[0045] (f) the full-length coding region of the nucleotide sequence
shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0046] (g) the complement of (a), (b), (c), (d), (e) or (f).
[0047] 2. Isolated nucleic acid having:
[0048] (a) a nucleotide sequence that encodes the amino acid
sequence shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32);
[0049] (b) a nucleotide sequence that encodes the amino acid
sequence shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
lacking its associated signal peptide;
[0050] (c) a nucleotide sequence that encodes an extracellular
domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32), with its associated signal peptide;
[0051] (d) a nucleotide sequence that encodes an extracellular
domain of the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32), lacking its associated signal peptide;
[0052] (e) the nucleotide sequence shown in any one of FIGS. 1-16
(SEQ ID NOS:1-16);
[0053] (f) the full-length coding region of the nucleotide sequence
shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0054] (g) the complement of (a), (b), (c), (d), (e) or (f).
[0055] 3. Isolated nucleic acid that hybridizes to:
[0056] (a) a nucleic acid that encodes the amino acid sequence
shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32);
[0057] (b) a nucleic acid that encodes the amino acid sequence
shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its
associated signal peptide;
[0058] (c) a nucleic acid that encodes an extracellular domain of
the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
with its associated signal peptide;
[0059] (d) a nucleic acid that encodes an extracellular domain of
the polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
lacking its associated signal peptide;
[0060] (e) the nucleotide sequence shown in any one of FIGS. 1-16
(SEQ ID NOS:1-16);
[0061] (f) the full-length coding region of the nucleotide sequence
shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0062] (g) the complement of (a), (b), (c), (d), (e) or (f).
[0063] 4. The nucleic acid of Claim 3, wherein the hybridization
occurs under stringent conditions.
[0064] 5. The nucleic acid of Claim 3 which is at least about 5
nucleotides in length.
[0065] 6. An expression vector comprising the nucleic acid of Claim
1, 2 or 3.
[0066] 7. The expression vector of Claim 6, wherein said nucleic
acid is operably linked to control sequences recognized by a host
cell transformed with the vector.
[0067] 8. A host cell comprising the expression vector of Claim
7.
[0068] 9. The host cell of Claim 8 which is a CHO cell, an E. Coli
cell or a yeast cell.
[0069] 10. A process for producing a polypeptide comprising
culturing the host cell of Claim 8 under conditions suitable for
expression of said polypeptide and recovering said polypeptide from
the cell culture.
[0070] 11. An isolated polypeptide having at least 80% amino acid
sequence identity to:
[0071] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32);
[0072] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32), lacking its associated signal peptide;
[0073] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), with its associated signal
peptide;
[0074] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its associated
signal peptide;
[0075] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0076] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID
NOS:1-16).
[0077] 12. An isolated polypeptide having:
[0078] (a) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32);
[0079] (b) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32), lacking its associated signal peptide
sequence;
[0080] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
with its associated signal peptide sequence;
[0081] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
lacking its associated signal peptide sequence;
[0082] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0083] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-16
(SEQ ID NOS:1-16).
[0084] 13. A chimeric polypeptide comprising the polypeptide of
Claim 11 or 12 fused to a heterologous polypeptide.
[0085] 14. The chimeric polypeptide of Claim 13, wherein said
heterologous polypeptide is an epitope tag sequence or an Fc region
of an immunoglobulin.
[0086] 15. An isolated antibody that binds to a polypeptide having
at least 80% amino acid sequence identity to:
[0087] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32);
[0088] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32), lacking its associated signal peptide;
[0089] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), with its associated signal
peptide;
[0090] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its associated
signal peptide;
[0091] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0092] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID
NOS:1-16).
[0093] 16. An isolated antibody that binds to a polypeptide
having:
[0094] (a) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32);
[0095] (b) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32), lacking its associated signal peptide
sequence;
[0096] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
with its associated signal peptide sequence;
[0097] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
lacking its associated signal peptide sequence;
[0098] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0099] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-16
(SEQ ID NOS:1-16).
[0100] 17. The antibody of Claim 15 or 16 which is a monoclonal
antibody.
[0101] 18. The antibody of Claim 15 or 16 which is an antibody
fragment.
[0102] 19. The antibody of Claim 15 or 16 which is a chimeric or a
humanized antibody.
[0103] 20. The antibody of Claim 15 or 16 which is conjugated to a
growth inhibitory agent.
[0104] 21. The antibody of Claim 15 or 16 which is conjugated to a
cytotoxic agent.
[0105] 22. The antibody of Claim 21, wherein the cytotoxic agent is
selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
[0106] 23. The antibody of Claim 21, wherein the cytotoxic agent is
a toxin.
[0107] 24. The antibody of Claim 23, wherein the toxin is selected
from the group consisting of maytansinoid and calicheamicin.
[0108] 25. The antibody of Claim 23, wherein the toxin is a
maytansinoid.
[0109] 26. The antibody of Claim 15 or 16 which is produced in
bacteria.
[0110] 27. The antibody of Claim 15 or 16 which is produced in CHO
cells.
[0111] 28. The antibody of Claim 15 or 16 which induces death of a
cell to which it binds.
[0112] 29. The antibody of Claim 15 or 16 which is detectably
labeled.
[0113] 30. An isolated nucleic acid having a nucleotide sequence
that encodes the antibody of Claim 15 or 16.
[0114] 31. An expression vector comprising the nucleic acid of
Claim 30 operably linked to control sequences recognized by a host
cell transformed with the vector.
[0115] 32. A host cell comprising the expression vector of Claim
31.
[0116] 33. The host cell of Claim 32 which is a CHO cell, an E.
coli cell or a yeast cell.
[0117] 34. A process for producing an antibody comprising culturing
the host cell of Claim 32 under conditions suitable for expression
of said antibody and recovering said antibody from the cell
culture.
[0118] 35. An isolated oligopeptide that binds to a polypeptide
having at least 80% amino acid sequence identity to:
[0119] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32);
[0120] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32), lacking its associated signal peptide;
[0121] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), with its associated signal
peptide;
[0122] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its associated
signal peptide;
[0123] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0124] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID
NOS:1-16).
[0125] 36. An isolated oligopeptide that binds to a polypeptide
having:
[0126] (a) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32);
[0127] (b) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32), lacking its associated signal peptide
sequence;
[0128] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
with its associated signal peptide sequence;
[0129] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
lacking its associated signal peptide sequence;
[0130] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0131] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-16
(SEQ ID NOS:1-16).
[0132] 37. The oligopeptide of Claim 35 or 36 which is conjugated
to a growth inhibitory agent.
[0133] 38. The oligopeptide of Claim 35 or 36 which is conjugated
to a cytotoxic agent.
[0134] 39. The oligopeptide of Claim 38, wherein the cytotoxic
agent is selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
[0135] 40. The oligopeptide of Claim 38, wherein the cytotoxic
agent is a toxin.
[0136] 41. The oligopeptide of Claim 40, wherein the toxin is
selected from the group consisting of maytansinoid and
calicheamicin.
[0137] 42. The oligopeptide of Claim 40, wherein the toxin is a
maytansinoid.
[0138] 43. The oligopeptide of Claim 35 or 36 which induces death
of a cell to which it binds.
[0139] 44. The oligopeptide of Claim 35 or 36 which is detectably
labeled.
[0140] 45. A TAT binding organic molecule that binds to a
polypeptide having at least 80% amino acid sequence identity
to:
[0141] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32);
[0142] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32), lacking its associated signal peptide;
[0143] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), with its associated signal
peptide;
[0144] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its associated
signal peptide;
[0145] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0146] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID
NOS:1-16).
[0147] 46. The organic molecule of Claim 45 that binds to a
polypeptide having:
[0148] (a) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32);
[0149] (b) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32), lacking its associated signal peptide
sequence;
[0150] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
with its associated signal peptide sequence;
[0151] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
lacking its associated signal peptide sequence;
[0152] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0153] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-16
(SEQ ID NOS:1-16).
[0154] 47. The organic molecule of Claim 45 or 46 which is
conjugated to a growth inhibitory agent.
[0155] 48. The organic molecule of Claim 45 or 46 which is
conjugated to a cytotoxic agent.
[0156] 49. The organic molecule of Claim 48, wherein the cytotoxic
agent is selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
[0157] 50. The organic molecule of Claim 48, wherein the cytotoxic
agent is a toxin.
[0158] 51. The organic molecule of Claim 50, wherein the toxin is
selected from the group consisting of maytansinoid and
calicheamicin.
[0159] 52. The organic molecule of Claim 50, wherein the toxin is a
maytansinoid.
[0160] 53. The organic molecule of Claim 45 or 46 which induces
death of a cell to which it binds.
[0161] 54. The organic molecule of Claim 45 or 46 which is
detectably labeled.
[0162] 55. A composition of matter comprising:
[0163] (a) the polypeptide of Claim 11;
[0164] (b) the polypeptide of Claim 12;
[0165] (c) the chimeric polypeptide of Claim 13;
[0166] (d) the antibody of Claim 15;
[0167] (e) the antibody of Claim 16;
[0168] (f) the oligopeptide of Claim 35;
[0169] (g) the oligopeptide of Claim 36;
[0170] (h) the TAT binding organic molecule of Claim 45; or
[0171] (i) the TAT binding organic molecule of Claim 46; in
combination with a carrier.
[0172] 56. The composition of matter of Claim 55, wherein said
carrier is a pharmaceutically acceptable carrier.
[0173] 57. An article of manufacture comprising:
[0174] (a) a container; and
[0175] (b) the composition of matter of Claim 55 contained within
said container.
[0176] 58. The article of manufacture of Claim 57 further
comprising a label affixed to said container, or a package insert
included with said container, referring to the use of said
composition of matter for the therapeutic treatment of or the
diagnostic detection of a cancer.
[0177] 59. A method of inhibiting the growth of a cell that
expresses a protein having at least 80% amino acid sequence
identity to:
[0178] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32);
[0179] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32), lacking its associated signal peptide;
[0180] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), with its associated signal
peptide;
[0181] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its associated
signal peptide;
[0182] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0183] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID
NOS:1-16), said method comprising contacting said cell with an
antibody, oligopeptide or organic molecule that binds to said
protein, the binding of said antibody, oligopeptide or organic
molecule to said protein thereby causing an inhibition of growth of
said cell.
[0184] 60. The method of Claim 59, wherein said antibody is a
monoclonal antibody.
[0185] 61. The method of Claim 59, wherein said antibody is an
antibody fragment.
[0186] 62. The method of Claim 59, wherein said antibody is a
chimeric or a humanized antibody.
[0187] 63. The method of Claim 59, wherein said antibody,
oligopeptide or organic molecule is conjugated to a growth
inhibitory agent.
[0188] 64. The method of Claim 59, wherein said antibody,
oligopeptide or organic molecule is conjugated to a cytotoxic
agent.
[0189] 65. The method of Claim 64, wherein said cytotoxic agent is
selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
[0190] 66. The method of Claim 64, wherein the cytotoxic agent is a
toxin.
[0191] 67. The method of Claim 66, wherein the toxin is selected
from the group consisting of maytansinoid and calicheamicin.
[0192] 68. The method of Claim 66, wherein the toxin is a
maytansinoid.
[0193] 69. The method of Claim 59, wherein said antibody is
produced in bacteria.
[0194] 70. The method of Claim 59, wherein said antibody is
produced in CHO cells.
[0195] 71. The method of Claim 59, wherein said cell is a cancer
cell.
[0196] 72. The method of Claim 71, wherein said cancer cell is
further exposed to radiation treatment or a chemotherapeutic
agent.
[0197] 73. The method of Claim 71, wherein said cancer cell is
selected from the group consisting of a breast cancer cell, a
colorectal cancer cell, a lung cancer cell, an ovarian cancer cell,
a central nervous system cancer cell, a liver cancer cell, a
bladder cancer cell, a pancreatic cancer cell, a cervical cancer
cell, a melanoma cell and a leukemia cell.
[0198] 74. The method of Claim 71, wherein said protein is more
abundantly expressed by said cancer cell as compared to a normal
cell of the same tissue origin.
[0199] 75. The method of Claim 59 which causes the death of said
cell.
[0200] 76. The method of Claim 59, wherein said protein has:
[0201] (a) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32);
[0202] (b) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32), lacking its associated signal peptide
sequence;
[0203] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
with its associated signal peptide sequence;
[0204] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in anyone of FIGS. 17-32 (SEQ ID NOS:17-32),
lacking its associated signal peptide sequence;
[0205] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0206] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-16
(SEQ ID NOS:1-16).
[0207] 77. A method of therapeutically treating a mammal having a
cancerous tumor comprising cells that express a protein having at
least 80% amino acid sequence identity to:
[0208] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32);
[0209] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32), lacking its associated signal peptide;
[0210] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), with its associated signal
peptide;
[0211] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its associated
signal peptide;
[0212] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0213] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID
NOS:1-16), said method comprising administering to said mammal a
therapeutically effective amount of an antibody, oligopeptide or
organic molecule that binds to said protein, thereby effectively
treating said mammal.
[0214] 78. The method of Claim 77, wherein said antibody is a
monoclonal antibody.
[0215] 79. The method of Claim 77, wherein said antibody is an
antibody fragment.
[0216] 80. The method of Claim 77, wherein said antibody is a
chimeric or a humanized antibody.
[0217] 81. The method of Claim 77, wherein said antibody,
oligopeptide or organic molecule is conjugated to a growth
inhibitory agent.
[0218] 82. The method of Claim 77, wherein said antibody,
oligopeptide or organic molecule is conjugated to a cytotoxic
agent.
[0219] 83. The method of Claim 82, wherein said cytotoxic agent is
selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
[0220] 84. The method of Claim 82, wherein the cytotoxic agent is a
toxin.
[0221] 85. The method of Claim 84, wherein the toxin is selected
from the group consisting of maytansinoid and calicheamicin.
[0222] 86. The method of Claim 84, wherein the toxin is a
maytansinoid.
[0223] 87. The method of Claim 77, wherein said antibody is
produced in bacteria.
[0224] 88. The method of Claim 77, wherein said antibody is
produced in CHO cells.
[0225] 89. The method of Claim 77, wherein said tumor is further
exposed to radiation treatment or a chemotherapeutic agent.
[0226] 90. The method of Claim 77, wherein said tumor is a breast
tumor, a colorectal tumor, a lung tumor, an ovarian tumor, a
central nervous system tumor, a liver tumor, a bladder tumor, a
pancreatic tumor, or a cervical tumor.
[0227] 91. The method of Claim 77, wherein said protein is more
abundantly expressed by the cancerous cells of said tumor as
compared to a normal cell of the same tissue origin.
[0228] 92. The method of Claim 77, wherein said protein has:
[0229] (a) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32);
[0230] (b) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32), lacking its associated signal peptide
sequence;
[0231] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
with its associated signal peptide sequence;
[0232] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
lacking its associated signal peptide sequence;
[0233] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0234] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-16
(SEQ ID NOS:1-16).
[0235] 93. A method of determining the presence of a protein in a
sample suspected of containing said protein, wherein said protein
has at least 80% amino acid sequence identity to:
[0236] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32);
[0237] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32), lacking its associated signal peptide;
[0238] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), with its associated signal
peptide;
[0239] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its associated
signal peptide;
[0240] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0241] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID
NOS:1-16), said method comprising exposing said sample to an
antibody, oligopeptide or organic molecule that binds to said
protein and determining binding of said antibody, oligopeptide or
organic molecule to said protein in said sample, wherein binding of
the antibody, oligopeptide or organic molecule to said protein is
indicative of the presence of said protein in said sample.
[0242] 94. The method of Claim 93, wherein said sample comprises a
cell suspected of expressing said protein.
[0243] 95. The method of Claim 94, wherein said cell is a cancer
cell.
[0244] 96. The method of Claim 93, wherein said antibody,
oligopeptide or organic molecule is detectably labeled.
[0245] 97. The method of Claim 93, wherein said protein has:
[0246] (a) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32);
[0247] (b) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32), lacking its associated signal peptide
sequence;
[0248] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
with its associated signal peptide sequence;
[0249] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
lacking its associated signal peptide sequence;
[0250] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0251] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-16
(SEQ ID NOS:1-16).
[0252] 98. A method of diagnosing the presence of a tumor in a
mammal, said method comprising determining the level of expression
of a gene encoding a protein having at least 80% amino acid
sequence identity to:
[0253] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32);
[0254] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32), lacking its associated signal peptide;
[0255] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), with its associated signal
peptide;
[0256] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its associated
signal peptide;
[0257] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0258] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID
NOS:1-16), in a test sample of tissue cells obtained from said
mammal and in a control sample of known normal cells of the same
tissue origin, wherein a higher level of expression of said protein
in the test sample, as compared to the control sample, is
indicative of the presence of tumor in the mammal from which the
test sample was obtained.
[0259] 99. The method of Claim 98, wherein the step of determining
the level of expression of a gene encoding said protein comprises
employing an oligonucleotide in an in situ hybridization or RT-PCR
analysis.
[0260] 100. The method of Claim 98, wherein the step determining
the level of expression of a gene encoding said protein comprises
employing an antibody in an immunohistochemistry or Western blot
analysis.
[0261] 101. The method of Claim 98, wherein said protein has:
[0262] (a) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32);
[0263] (b) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32), lacking its associated signal peptide
sequence;
[0264] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
with its associated signal peptide sequence;
[0265] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
lacking its associated signal peptide sequence;
[0266] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0267] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-16
(SEQ ID NOS:1-16).
[0268] 102. A method of diagnosing the presence of a tumor in a
mammal, said method comprising contacting a test sample of tissue
cells obtained from said mammal with an antibody, oligopeptide or
organic molecule that binds to a protein having at least 80% amino
acid sequence identity to:
[0269] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32);
[0270] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32), lacking its associated signal peptide;
[0271] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), with its associated signal
peptide;
[0272] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its associated
signal peptide;
[0273] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0274] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID
NOS:1-16), and detecting the formation of a complex between said
antibody, oligopeptide or organic molecule and said protein in the
test sample, wherein the formation of a complex is indicative of
the presence of a tumor in said mammal.
[0275] 103. The method of Claim 102, wherein said antibody,
oligopeptide or organic molecule is detectably labeled.
[0276] 104. The method of Claim 102, wherein said test sample of
tissue cells is obtained from an individual suspected of having a
cancerous tumor.
[0277] 105. The method of Claim 102, wherein said protein has:
[0278] (a) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32);
[0279] (b) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32), lacking its associated signal peptide
sequence;
[0280] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
with its associated signal peptide sequence;
[0281] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
lacking its associated signal peptide sequence;
[0282] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0283] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-16
(SEQ ID NOS:1-16).
[0284] 106. A method for treating or preventing a cell
proliferative disorder associated with increased expression or
activity of a protein having at least 80% amino acid sequence
identity to:
[0285] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32);
[0286] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32), lacking its associated signal peptide;
[0287] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), with its associated signal
peptide;
[0288] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its associated
signal peptide;
[0289] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0290] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID
NOS:1-16), said method comprising administering to a subject in
need of such treatment an effective amount of an antagonist of said
protein, thereby effectively treating or preventing said cell
proliferative disorder.
[0291] 107. The method of Claim 106, wherein said cell
proliferative disorder is cancer.
[0292] 108. The method of Claim 106, wherein said antagonist is an
anti-TAT polypeptide antibody, TAT binding oligopeptide, TAT
binding organic molecule or antisense oligonucleotide.
[0293] 109. A method of binding an antibody, oligopeptide or
organic molecule to a cell that expresses a protein having at least
80% amino acid sequence identity to:
[0294] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32);
[0295] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32), lacking its associated signal peptide;
[0296] (c) all extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), with its associated signal
peptide;
[0297] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its associated
signal peptide;
[0298] (c) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0299] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID
NOS:1-16), said method comprising contacting said cell with an
antibody, oligopeptide or organic molecule that binds to said
protein and allowing the binding of the antibody, oligopeptide or
organic molecule to said protein to occur, thereby binding said
antibody, oligopeptide or organic molecule to said cell.
[0300] 110. The method of Claim 109, wherein said antibody is a
monoclonal antibody.
[0301] 111. The method of Claim 109, wherein said antibody is an
antibody fragment.
[0302] 112. The method of Claim 109, wherein said antibody is a
chimeric or a humanized antibody.
[0303] 113. The method of Claim 109, wherein said antibody,
oligopeptide or organic molecule is conjugated to a growth
inhibitory agent.
[0304] 114. The method of Claim 109, wherein said antibody,
oligopeptide or organic molecule is conjugated to a cytotoxic
agent.
[0305] 115. The method of Claim 114, wherein said cytotoxic agent
is selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
[0306] 116. The method of Claim 114, wherein the cytotoxic agent is
a toxin.
[0307] 117. The method of Claim 116, wherein the toxin is selected
from the group consisting of maytansinoid and calicheamicin.
[0308] 118. The method of Claim 116, wherein the toxin is a
maytansinoid.
[0309] 119. The method of Claim 109, wherein said antibody is
produced in bacteria.
[0310] 120. The method of Claim 109, wherein said antibody is
produced in CHO cells.
[0311] 121. The method of Claim 109, wherein said cell is a cancer
cell.
[0312] 122. The method of Claim 121, wherein said cancer cell is
further exposed to radiation treatment or a chemotherapeutic
agent.
[0313] 123. The method of Claim 121, wherein said cancer cell is
selected from the group consisting of a breast cancer cell, a
colorectal cancer cell, a lung cancer cell, an ovarian cancer cell,
a central nervous system cancer cell, a liver cancer cell, a
bladder cancer cell, a pancreatic cancer cell, a cervical cancer
cell, a melanoma cell and a leukemia cell.
[0314] 124. The method of Claim 123, wherein said protein is more
abundantly expressed by said cancer cell as compared to a normal
cell of the same tissue origin.
[0315] 125. The method of Claim 109 which causes the death of said
cell.
[0316] 126. Use of a nucleic acid as claimed in any of Claims 1 to
5 or 30 in the preparation of a medicament for the therapeutic
treatment or diagnostic detection of a cancer.
[0317] 127. Use of a nucleic acid as claimed in any of Claims 1 to
5 or 30 in the preparation of a medicament for treating a
tumor.
[0318] 128. Use of a nucleic acid as claimed in any of Claims 1 to
5 or 30 in the preparation of a medicament for treatment or
prevention of a cell proliferative disorder.
[0319] 129. Use of an expression vector as claimed in any of Claims
6, 7 or 31 in the preparation of a medicament for the therapeutic
treatment or diagnostic detection of a cancer.
[0320] 130. Use of an expression vector as claimed in any of Claims
6, 7 or 31 in the preparation of medicament for treating a
tumor.
[0321] 131. Use of an expression vector as claimed in any of Claims
6, 7 or 31 in the preparation of a medicament for treatment or
prevention of a cell proliferative disorder.
[0322] 132. Use of a host cell as claimed in any of Claims 8, 9,
32, or 33 in the preparation of a medicament for the therapeutic
treatment or diagnostic detection of a cancer.
[0323] 133. Use of a host cell as claimed in any of Claims 8, 9, 32
or 33 in the preparation of a medicament for treating a tumor.
[0324] 134. Use of a host cell as claimed in any of Claims 8, 9, 32
or 33 in the preparation of a medicament for treatment or
prevention of a cell proliferative disorder.
[0325] 135. Use of a polypeptide as claimed in any of Claims 11 to
14 in the preparation of a medicament for the therapeutic treatment
or diagnostic detection of a cancer.
[0326] 136. Use of a polypeptide as claimed in any of Claims 11 to
14 in the preparation of a medicament for treating a tumor.
[0327] 137. Use of a polypeptide as claimed in any of Claims 11 to
14 in the preparation of a medicament for treatment or prevention
of a cell proliferative disorder.
[0328] 138. Use of an antibody as claimed in any of Claims 15 to 29
in the preparation of a medicament for the therapeutic treatment or
diagnostic detection of a cancer.
[0329] 139. Use of an antibody as claimed in any of Claims 15 to 29
in the preparation of a medicament for treating a tumor.
[0330] 140. Use of an antibody as claimed in any of Claims 15 to 29
in the preparation of a medicament for treatment or prevention of a
cell proliferative disorder.
[0331] 141. Use of an oligopeptide as claimed in any of Claims 35
to 44 in the preparation of a medicament for the therapeutic
treatment or diagnostic detection of a cancer.
[0332] 142. Use of an oligopeptide as claimed in any of Claims 35
to 44 in the preparation of a medicament for treating a tumor.
[0333] 143. Use of an oligopeptide as claimed in any of Claims 35
to 44 in the preparation of a medicament for treatment or
prevention of a cell proliferative disorder.
[0334] 144. Use of a TAT binding organic molecule as claimed in any
of Claims 45 to 54 in the preparation of a medicament for the
therapeutic treatment or diagnostic detection of a cancer.
[0335] 145. Use of a TAT binding organic molecule as claimed in any
of Claims 45 to 54 in the preparation of a medicament for treating
a tumor,
[0336] 146. Use of a TAT binding organic molecule as claimed in any
of Claims 45 to 54 in the preparation of a medicament for treatment
or prevention of a cell proliferative disorder.
[0337] 147. Use of a composition of matter as claimed in any of
Claims 55 or 56 in the preparation of a medicament for the
therapeutic treatment or diagnostic detection of a cancer.
[0338] 148. Use of a composition of matter as claimed in any of
Claims 55 or 56 in the preparation of a medicament for treating a
tumor.
[0339] 149. Use of a composition of matter as claimed in any of
Claims 55 or 56 in the preparation of a medicament for treatment or
prevention of a cell proliferative disorder.
[0340] 150. Use of an article of manufacture as claimed in any of
Claims 57 or 58 in the preparation of a medicament for the
therapeutic treatment or diagnostic detection of a cancer.
[0341] 151. Use of an article of manufacture as claimed in any of
Claims 57 or 58 in the preparation of a medicament for treating a
tumor.
[0342] 152. Use of an article of manufacture as claimed in any of
Claims 57 or 58 in the preparation of a medicament for treatment or
prevention of a cell proliferative disorder.
[0343] 153. A method for inhibiting the growth of a cell, wherein
the growth of said cell is at least in part dependent upon a growth
potentiating effect of a protein having at least 80% amino acid
sequence identity to:
[0344] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32);
[0345] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32), lacking its associated signal peptide;
[0346] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), with its associated signal
peptide;
[0347] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its associated
signal peptide;
[0348] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0349] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID
NOS:1-16), said method comprising contacting said protein with an
antibody, oligopeptide or organic molecule that binds to said
protein, there by inhibiting the growth of said cell.
[0350] 154. The method of Claim 153, wherein said cell is a cancer
cell.
[0351] 155. The method of Claim 153, wherein said protein is
expressed by said cell.
[0352] 156. The method of Claim 153, wherein the binding of said
antibody, oligopeptide or organic molecule to said protein
antagonizes a cell growth-potentiating activity of said
protein.
[0353] 157. The method of Claim 153, wherein the binding of said
antibody, oligopeptide or organic molecule to said protein induces
the death of said cell.
[0354] 158. The method of Claim 153, wherein said antibody is a
monoclonal antibody.
[0355] 159. The method of Claim 153, wherein said antibody is an
antibody fragment.
[0356] 160. The method of Claim 153, wherein said antibody is a
chimeric or a humanized antibody.
[0357] 161. The method of Claim 153, wherein said antibody,
oligopeptide or organic molecule is conjugated to a growth
inhibitory agent.
[0358] 162. The method of Claim 153, wherein said antibody,
oligopeptide or organic molecule is conjugated to a cytotoxic
agent.
[0359] 163. The method of Claim 162, wherein said cytotoxic agent
is selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
[0360] 164. The method of Claim 162, wherein the cytotoxic agent is
a toxin.
[0361] 165. The method of Claim 164, wherein the toxin is selected
from the group consisting of maytansinoid and calicheamicin.
[0362] 166. The method of Claim 164, wherein the toxin is a
maytansinoid.
[0363] 167. The method of Claim 153, wherein said antibody is
produced in bacteria.
[0364] 168. The method of Claim 153, wherein said antibody is
produced in CHO cells.
[0365] 169. The method of Claim 153, wherein said protein has:
[0366] (a) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32);
[0367] (b) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32), lacking its associated signal peptide
sequence;
[0368] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
with its associated signal peptide sequence;
[0369] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
lacking its associated signal peptide sequence;
[0370] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0371] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-16
(SEQ ID NOS:1-16).
[0372] 170. A method of therapeutically treating a tumor in a
mammal, wherein the growth of said tumor is at least in part
dependent upon a growth potentiating effect of a protein having at
least 80% amino acid sequence identity to:
[0373] (a) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32);
[0374] (b) the polypeptide shown in any one of FIGS. 17-32 (SEQ ID
NOS:17-32), lacking its associated signal peptide;
[0375] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), with its associated signal
peptide;
[0376] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 17-32 (SEQ ID NOS:17-32), lacking its associated
signal peptide;
[0377] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0378] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-16 (SEQ ID
NOS:1-16), said method comprising contacting said protein with an
antibody, oligopeptide or organic molecule that binds to said
protein, thereby effectively treating said tumor.
[0379] 171. The method of Claim 170, wherein said protein is
expressed by cells of said tumor.
[0380] 172. The method of Claim 170, wherein the binding of said
antibody, oligopeptide or organic molecule to said protein
antagonizes a cell growth-potentiating activity of said
protein.
[0381] 173. The method of Claim 170, wherein said antibody is a
monoclonal antibody.
[0382] 174. The method of Claim 170, wherein said antibody is an
antibody fragment.
[0383] 175. The method of Claim 170, wherein said antibody is a
chimeric or a humanized antibody.
[0384] 176. The method of Claim 170, wherein said antibody,
oligopeptide or organic molecule is conjugated to a growth
inhibitory agent.
[0385] 177. The method of Claim 170, wherein said antibody,
oligopeptide or organic molecule is conjugated to a cytotoxic
agent.
[0386] 178. The method of Claim 177, wherein said cytotoxic agent
is selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes.
[0387] 179. The method of Claim 177, wherein the cytotoxic agent is
a toxin.
[0388] 180. The method of Claim 179, wherein the toxin is selected
from the group consisting of maytansinoid and calicheamicin.
[0389] 181. The method of Claim 179, wherein the toxin is a
maytansinoid.
[0390] 182. The method of Claim 170, wherein said antibody is
produced in bacteria.
[0391] 183. The method of Claim 170, wherein said antibody is
produced in CHO cells.
[0392] 184. The method of Claim 170, wherein said protein has:
[0393] (a) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32);
[0394] (b) the amino acid sequence shown in any one of FIGS. 17-32
(SEQ ID NOS:17-32), lacking its associated signal peptide
sequence;
[0395] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
with its associated signal peptide sequence;
[0396] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 17-32 (SEQ ID NOS:17-32),
lacking its associated signal peptide sequence;
[0397] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-16 (SEQ ID NOS:1-16); or
[0398] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-16
(SEQ ID NOS:1-16).
[0399] 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
[0400] FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a TAT293
cDNA, wherein SEQ ID NO:1 is a clone designated herein as
"DNA293550".
[0401] FIG. 2 shows a nucleotide sequence (SEQ ID NO:2) of a TAT294
cDNA, wherein SEQ ID NO:2 is a clone designated herein as
"DNA150813".
[0402] FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a TAT295
cDNA, wherein SEQ ID NO:3 is a clone designated herein as
"DNA225785".
[0403] FIG. 4 shows a nucleotide sequence (SEQ ID NO:4) of a TAT296
cDNA, wherein SEQ ID NO:4 is a clone designated herein as
"DNA226313".
[0404] FIG. 5 shows a nucleotide sequence (SEQ ID NO:5) of a TAT297
cDNA, wherein SEQ ID NO:5 is a clone designated herein as
"DNA103365".
[0405] FIG. 6 shows a nucleotide sequence (SEQ ID NO:6) of a TAT298
cDNA, wherein SEQ ID NO:6 is a clone designated herein as
"DNA88158".
[0406] FIG. 7 shows a nucleotide sequence (SEQ ID NO:7) of a TAT347
cDNA, wherein SEQ ID NO:7 is a clone designated herein as
"DNA225800".
[0407] FIG. 8 shows a nucleotide sequence (SEQ ID NO:8) of a TAT299
cDNA, wherein SEQ ID NO:8 is a clone designated herein as
"DNA103386".
[0408] FIG. 9 shows a nucleotide sequence (SEQ ID NO:9) of a TAT351
cDNA, wherein SEQ ID NO:9 is a clone designated herein as
DNA273438'.
[0409] FIG. 10 shows a nucleotide sequence (SEQ ID NO:10) of a
TAT300 cDNA, wherein SEQ ID NO:10 is a clone designated herein as
"DNA225865".
[0410] FIG. 11 shows a nucleotide sequence (SEQ ID NO:11) of a
TAT301 cDNA, wherein SEQ ID NO:11 is a clone designated herein as
"DNA196665".
[0411] FIG. 12 shows a nucleotide sequence (SEQ ID NO:12) of a
TAT352 cDNA wherein SEQ ID NO:12 is a clone designated herein as
"DNA287183".
[0412] FIGS. 13A-B show a nucleotide sequence (SEQ ID NO:13) of a
TAT302 cDNA, wherein SEQ ID NO:13 is a clone designated herein as
"DNA226403".
[0413] FIG. 14 shows a nucleotide sequence (SEQ ID NO:14) of a
TAT303 cDNA, wherein SEQ ID NO:14 is a clone designated herein as
"DNA73736".
[0414] FIG. 15 shows a nucleotide sequence (SEQ ID NO:15) of a
TAT304 cDNA, wherein SEQ ID NO:15 is a clone designated herein as
"DNA299884".
[0415] FIG. 16 shows a nucleotide sequence (SEQ ID NO:16) of a
TAT305 cDNA, wherein SEQ ID NO:16 is a clone designated herein as
"DNA299885".
[0416] FIG. 17 shows the amino acid sequence (SEQ ID NO:17) derived
from the coding sequence of SEQ ID NO:1 shown in FIG. 1.
[0417] FIG. 18 shows the amino acid sequence (SEQ ID NO:18) derived
from the coding sequence of SEQ ID NO:2 shown in FIG. 2.
[0418] FIG. 19 shows the amino acid sequence (SEQ ID NO:19) derived
from the coding sequence of SEQ ID NO:3 shown in FIG. 3.
[0419] FIG. 20 shows the amino acid sequence (SEQ ID NO:20) derived
from the coding sequence of SEQ ID NO:4 shown in FIG. 4.
[0420] FIG. 21 shows the amino acid sequence (SEQ ID NO:21) derived
from the coding sequence of SEQ ID NO:5 shown in FIG. 5.
[0421] FIG. 22 shows the amino acid sequence (SEQ ID NO:22) derived
from the coding sequence of SEQ ID NO:6 shown in FIG. 6.
[0422] FIG. 23 shows the amino acid sequence (SEQ ID NO:23) derived
from the coding sequence of SEQ ID NO:7 shown in FIG. 7.
[0423] FIG. 24 shows the amino acid sequence (SEQ ID NO:24) derived
from the coding sequence of SEQ ID NO:8 shown in FIG. 8.
[0424] FIG. 25 shows the amino acid sequence (SEQ ID NO:25) derived
from the coding sequence of SEQ ID NO:9 shown in FIG. 9.
[0425] FIG. 26 shows the amino acid sequence (SEQ ID NO:26) derived
from the coding sequence of SEQ ID NO:10 shown in FIG. 10.
[0426] FIG. 27 shows the amino acid sequence (SEQ ID NO:27) derived
from the coding sequence of SEQ ID NO:11 shown in FIG. 11.
[0427] FIG. 28 shows the amino acid sequence (SEQ ID NO:28) derived
from the coding sequence of SEQ ID NO:12 shown in FIG. 12.
[0428] FIG. 29 shows the amino acid sequence (SEQ ID NO:29) derived
from the coding sequence of SEQ ID NO:13 shown in FIGS. 13A-B.
[0429] FIG. 30 shows the amino acid sequence (SEQ ID NO:30) derived
from the coding sequence of SEQ ID NO:14 shown in FIG. 14.
[0430] FIG. 31 shows the amino acid sequence (SEQ ID NO:31) derived
from the coding sequence of SEQ ID NO:15 shown in FIG. 15.
[0431] FIG. 32 shows the amino acid sequence (SEQ ID NO:32) derived
from the coding sequence of SEQ ID NO:16 shown in FIG. 16.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
1. Definitions
[0432] The terms "TAT polypeptide" and "TAT" as used herein and
when immediately followed by a numerical designation, refer to
various polypeptides, wherein the complete designation (i.e.,
TAT/number) refers to specific polypeptide sequences as described
herein. The terms "TAT/number polypeptide" and "TAT/number" wherein
the term "number" is provided as an actual numerical designation as
used herein encompass native sequence polypeptides, polypeptide
variants and fragments of native sequence polypeptides and
polypeptide variants (which are further defined herein). The TAT
polypeptides described herein may be isolated from a variety of
sources, such as from human tissue types or from another source, or
prepared by recombinant or synthetic methods. The term "TAT
polypeptide" refers to each individual TAT/number polypeptide
disclosed herein. All disclosures in this specification which refer
to the "TAT polypeptide" refer to each of the polypeptides
individually as well as jointly. For example, descriptions of the
preparation of, purification of, derivation of, formation of
antibodies to or against, formation of TAT binding oligopeptides to
or against, formation of TAT binding organic molecules to or
against, administration of, compositions containing, treatment of a
disease with, etc., pertain to each polypeptide of the invention
individually. The term "TAT polypeptide" also includes variants of
the TAT/number polypeptides disclosed herein.
[0433] A "native sequence TAT polypeptide" comprises a polypeptide
having the same amino acid sequence as the corresponding TAT
polypeptide derived from nature. Such native sequence TAT
polypeptides can be isolated from nature or can be produced by
recombinant or synthetic means. The term "native sequence TAT
polypeptide" specifically encompasses naturally-occurring truncated
or secreted forms of the specific TAT polypeptide (e.g., an
extracellular domain sequence), naturally-occurring variant forms
(e.g., alternatively spliced forms) and naturally-occurring allelic
variants of the polypeptide. In certain embodiments of the
invention, the native sequence TAT polypeptides disclosed herein
are mature or full-length native sequence polypeptides comprising
the full-length amino acids sequences shown in the accompanying
figures. Start and stop codons (if indicated) are shown in bold
font and underlined in the figures. Nucleic acid residues indicated
as "N" in the accompanying figures are any nucleic acid residue.
However, while the TAT polypeptides disclosed in the accompanying
figures are shown to begin with methionine residues designated
herein as amino acid position 1 in the figures, it is conceivable
and possible that other methionine residues located either upstream
or downstream from the amino acid position 1 in the figures may be
employed as the starting amino acid residue for the TAT
polypeptides.
[0434] The TAT polypeptide "extracellular domain" or "ECD" refers
to a form of the TAT polypeptide which is essentially free of the
transmembrane and cytoplasmic domains. Ordinarily, a TAT
polypeptide ECD will have less than 1% of such transmembrane and/or
cytoplasmic domains and preferably, will have less than 0.5% of
such domains. It will be understood that any transmembrane domains
identified for the TAT polypeptides of the present invention are
identified pursuant to criteria routinely employed in the art for
identifying that type of hydrophobic domain. The exact boundaries
of a transmembrane domain may vary but most likely by no more than
about 5 amino acids at either end of the domain as initially
identified herein. Optionally, therefore, an extracellular domain
of a TAT polypeptide may contain from about 5 or fewer amino acids
on either side of the transmembrane domain/extracellular domain
boundary as identified in the Examples or specification and such
polypeptides, with or without the associated signal peptide, and
nucleic acid encoding them, are contemplated by the present
invention.
[0435] The approximate location of the "signal peptides" of the
various TAT polypeptides disclosed herein may be shown in the
present specification and/or the accompanying figures. It is noted,
however, that the C-terminal boundary of a signal peptide may vary,
but most likely by no more than about 5 amino acids on either side
of the signal peptide C-terminal boundary as initially identified
herein, wherein the C-terminal boundary of the signal peptide may
be identified pursuant to criteria routinely employed in the art
for identifying that type of amino acid sequence element (e.g.,
Nielsen et al., Prot. Eng. 10:1-6 (1997) and von Heinje et al.,
Nucl. Acids. Res. 14:4683-4690 (1986)). Moreover, it is also
recognized that, in some cases, cleavage of a signal sequence from
a secreted polypeptide is not entirely uniform, resulting in more
than one secreted species. These mature polypeptides, where the
signal peptide is cleaved within no more than about 5 amino acids
on either side of the C-terminal boundary of the signal peptide as
identified herein, and the polynucleotides encoding them, are
contemplated by the present invention.
[0436] "TAT polypeptide variant" means a TAT polypeptide,
preferably an active TAT polypeptide, as defined herein having at
least about 80% amino acid sequence identity with a full-length
native sequence TAT polypeptide sequence as disclosed herein, a TAT
polypeptide sequence lacking the signal peptide as disclosed
herein, an extracellular domain of a TAT polypeptide, with or
without the signal peptide, as disclosed herein or any other
fragment of a full-length TAT polypeptide sequence as disclosed
herein (such as those encoded by a nucleic acid that represents
only a portion of the complete coding sequence for a full-length
TAT polypeptide). Such TAT polypeptide variants include, for
instance, TAT polypeptides wherein one or more amino acid residues
are added, or deleted, at the N- or C-terminus of the full-length
native amino acid sequence. Ordinarily, a TAT 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 full-length native sequence TAT
polypeptide sequence as disclosed herein, a TAT polypeptide
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a TAT polypeptide, with or without the
signal peptide, as disclosed herein or any other specifically
defined fragment of a full-length TAT polypeptide sequence as
disclosed herein. Ordinarily, TAT 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, TAT
variant polypeptides will have no more than one conservative amino
acid substitution as compared to the native TAT 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 TAT
polypeptide sequence.
[0437] "Percent (%) amino acid sequence identity" with respect to
the TAT 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 TAT
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.
[0438] 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 "TAT", wherein "TAT"
represents the amino acid sequence of a hypothetical TAT
polypeptide of interest, "Comparison Protein" represents the amino
acid sequence of a polypeptide against which the "TAT" 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.
[0439] "TAT variant polynucleotide" or "TAT variant nucleic acid
sequence" means a nucleic acid molecule which encodes a TAT
polypeptide, preferably an active TAT polypeptide, as defined
herein and which has at least about 80% nucleic acid sequence
identity with a nucleotide acid sequence encoding a full-length
native sequence TAT polypeptide sequence as disclosed herein, a
full-length native sequence TAT polypeptide sequence lacking the
signal peptide as disclosed herein, an extracellular domain of a
TAT polypeptide, with or without the signal peptide, as disclosed
herein or any other fragment of a full-length TAT polypeptide
sequence as disclosed herein (such as those encoded by a nucleic
acid that represents only a portion of the complete coding sequence
for a full-length TAT polypeptide). Ordinarily, a TAT 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 full-length native sequence TAT polypeptide sequence as
disclosed herein, a full-length native sequence TAT polypeptide
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a TAT polypeptide, with or without the
signal sequence, as disclosed herein or any other fragment of a
full-length TAT polypeptide sequence as disclosed herein. Variants
do not encompass the native nucleotide sequence.
[0440] Ordinarily, TAT 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.
[0441] "Percent (%) nucleic acid sequence identity" with respect to
TAT-encoding nucleic acid sequences identified herein is defined as
the percentage of nucleotides in a candidate sequence that are
identical with the nucleotides in the TAT 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.
[0442] 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 "TAT-DNA", wherein "TAT-DNA" represents a
hypothetical TAT-encoding nucleic acid sequence of interest,
"Comparison DNA" represents the nucleotide sequence of a nucleic
acid molecule against which the "TAT-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.
[0443] In other embodiments, TAT variant polynucleotides are
nucleic acid molecules that encode a TAT polypeptide and which are
capable of hybridizing, preferably under stringent hybridization
and wash conditions, to nucleotide sequences encoding a full-length
TAT polypeptide as disclosed herein. TAT variant polypeptides may
be those that are encoded by a TAT variant polynucleotide.
[0444] The term "full-length coding region" when used in reference
to a nucleic acid encoding a TAT polypeptide refers to the sequence
of nucleotides which encode the full-length TAT polypeptide of the
invention (which is often shown between start and stop codons,
inclusive thereof, in the accompanying figures). The term
"full-length coding region" when used in reference to an ATCC
deposited nucleic acid refers to the TAT polypeptide-encoding
portion of the cDNA that is inserted into the vector deposited with
the ATCC (which is often shown between start and stop codons,
inclusive thereof, in the accompanying figures).
[0445] "Isolated," when used to describe the various TAT
polypeptides disclosed herein, means polypeptide that has been
identified and separated and/or recovered from a component of its
natural environment.
[0446] Contaminant components of its natural environment are
materials that would typically interfere with diagnostic or
therapeutic uses for the polypeptide, and may include enzymes,
hormones, and other proteinaceous or non-proteinaceous solutes. In
preferred embodiments, the polypeptide will be purified (1) to a
degree sufficient to obtain at least 15 residues of N-terminal or
internal amino acid sequence by use of a spinning cup sequenator,
or (2) to homogeneity by SDS-PAGE under non-reducing or reducing
conditions using Coomassie blue or, preferably, silver stain.
Isolated polypeptide includes polypeptide in situ within
recombinant cells, since at least one component of the TAT
polypeptide natural environment will not be present. Ordinarily,
however, isolated polypeptide will be prepared by at least one
purification step.
[0447] An "isolated" TAT polypeptide-encoding nucleic acid or other
polypeptide-encoding nucleic acid is a nucleic acid molecule that
is identified and separated from at least one contaminant nucleic
acid molecule with which it is ordinarily associated in the natural
source of the polypeptide-encoding nucleic acid. An isolated
polypeptide-encoding nucleic acid molecule is other than in the
form or setting in which it is found in nature. Isolated
polypeptide-encoding nucleic acid molecules therefore are
distinguished from the specific polypeptide-encoding nucleic acid
molecule as it exists in natural cells. However, an isolated
polypeptide-encoding nucleic acid molecule includes
polypeptide-encoding nucleic acid molecules contained in cells that
ordinarily express the polypeptide where, for example, the nucleic
acid molecule is in a chromosomal location different from that of
natural cells.
[0448] 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 all operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0449] 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.
[0450] "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).
[0451] "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.
[0452] "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.
[0453] The term "epitope tagged" when used herein refers to a
chimeric polypeptide comprising a TAT polypeptide or anti-TAT
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).
[0454] "Active" or "activity" for the purposes herein refers to
form(s) of a TAT polypeptide which retain a biological and/or an
immunological activity of native or naturally-occurring TAT,
wherein "biological" activity refers to a biological function
(either inhibitory or stimulatory) caused by a native or
naturally-occurring TAT other than the ability to induce the
production of an antibody against an antigenic epitope possessed by
a native or naturally-occurring TAT 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 TAT.
[0455] 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 TAT polypeptide
disclosed herein. In a similar manner, the term "agonist" is used
in the broadest sense and includes any molecule that mimics a
biological activity of a native TAT polypeptide disclosed herein.
Suitable agonist or antagonist molecules specifically include
agonist or antagonist antibodies or antibody fragments, fragments
or amino acid sequence variants of native TAT polypeptides,
peptides, antisense oligonucleotides, small organic molecules, etc.
Methods for identifying agonists or antagonists of a TAT
polypeptide may comprise contacting a TAT polypeptide with a
candidate agonist or antagonist molecule and measuring a detectable
change in one or more biological activities normally associated
with the TAT polypeptide.
[0456] "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 TAT
polypeptide-expressing cancer if, after receiving a therapeutic
amount of an anti-TAT antibody, TAT binding oligopeptide or TAT
binding organic molecule according to the methods of the present
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 anti-TAT
antibody or TAT binding oligopeptide 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.
[0457] 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).
[0458] For bladder cancer, which is a more localized cancer,
methods to determine progress of disease include urinary cytologic
evaluation by cystoscopy, monitoring for presence of blood in the
urine, visualization of the urothelial tract by sonography or an
intravenous pyelogram, computed tomography (CT) and magnetic
resonance imaging (MRI). The presence of distant metastases can be
assessed by CT of the abdomen, chest x-rays, or radionuclide
imaging of the skeleton.
[0459] "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.
[0460] "Mammal" for purposes of the treatment of, alleviating the
symptoms of or diagnosis 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.
[0461] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0462] "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..
[0463] By "solid phase" or "solid support" is meant a non-aqueous
matrix to which an antibody, TAT binding oligopeptide or TAT
binding organic molecule of the present 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 teen also includes
a discontinuous solid phase of discrete particles, such as those
described in U.S. Pat. No. 4,275,149.
[0464] 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 TAT polypeptide, an antibody thereto
or a TAT 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.
[0465] A "small" molecule or "small" organic molecule is defined
herein to have a molecular weight below about 500 Daltons.
[0466] An "effective amount" of a polypeptide, antibody, TAT
binding oligopeptide, TAT binding organic molecule or an agonist or
antagonist thereof 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.
[0467] The term "therapeutically effective amount" refers to an
amount of an antibody, polypeptide, TAT binding oligopeptide, TAT
binding organic molecule or other drug effective to "treat" a
disease or disorder in a subject or mammal. In the case of cancer,
the therapeutically effective amount of the drug 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 drug may prevent growth and/or kill
existing cancer cells, it may be cytostatic and/or cytotoxic.
[0468] A "growth inhibitory amount" of an anti-TAT antibody, TAT
polypeptide, TAT binding oligopeptide or TAT binding organic
molecule 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 an anti-TAT antibody, TAT
polypeptide, TAT binding oligopeptide or TAT binding organic
molecule for purposes of inhibiting neoplastic cell growth may be
determined empirically and in a routine manner.
[0469] A "cytotoxic amount" of an anti-TAT antibody, TAT
polypeptide, TAT binding oligopeptide or TAT binding organic
molecule 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 an anti-TAT antibody, TAT polypeptide, TAT
binding oligopeptide or TAT binding organic molecule for purposes
of inhibiting neoplastic cell growth may be determined empirically
and in a routine manner.
[0470] The term "antibody" is used in the broadest sense and
specifically covers, for example, single anti-TAT monoclonal
antibodies (including agonist, antagonist, and neutralizing
antibodies), anti-TAT antibody compositions with polyepitopic
specificity, polyclonal antibodies, single chain anti-TAT
antibodies, and fragments of anti-TAT antibodies (see below) as
long as they exhibit the desired biological or immunological
activity. The term "immunoglobulin" (Ig) is used interchangeable
with antibody herein.
[0471] An "isolated antibody" is one which has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials which would interfere with diagnostic or therapeutic uses
for the antibody, and may include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In preferred
embodiments, be antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, preferably, silver stain. Isolated antibody
includes the antibody in situ within recombinant cells since at
least one component of the antibody's natural environment will not
be present. Ordinarily, however, isolated antibody will be prepared
by at least one purification step.
[0472] 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.
[0473] 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.
[0474] 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).
[0475] 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 (113) in the V.sub.H; Chothia and Lesk J. Mol. Biol.
196:901-917 (1987)).
[0476] 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.
[0477] 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.
[0478] 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.
[0479] "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.
[0480] 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.
[0481] The Fc fragment comprises the carboxy-terminal portions of
both H chains held together by disulfides.
[0482] 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.
[0483] "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.
[0484] "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.
[0485] 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).
[0486] "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).
[0487] 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.
[0488] A "TAT binding oligopeptide" is an oligopeptide that binds,
preferably specifically, to a TAT polypeptide as described herein.
TAT binding oligopeptides may be chemically synthesized using known
oligopeptide synthesis methodology or may be prepared and purified
using recombinant technology. TAT 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 TAT polypeptide as described
herein. TAT 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. F. 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).
[0489] A "TAT binding organic molecule" is an organic molecule
other than an oligopeptide or antibody as defined herein that
binds, preferably specifically, to a TAT polypeptide as described
herein. TAT binding organic molecules may be identified and
chemically synthesized using known methodology (see, e.g., PCT
Publication Nos. WO00/00823 and WO00/39585). TAT binding organic
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 molecules that are capable of binding,
preferably specifically, to a TAT 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 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).
[0490] An antibody, oligopeptide or other organic molecule "which
binds" an antigen of interest, e.g. a tumor-associated polypeptide
antigen target, is one that binds the antigen with sufficient
affinity such that the antibody, oligopeptide or other organic
molecule is useful as a diagnostic and/or 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 molecule to a "non-target" protein will be less than about
10% of the binding of the antibody, oligopeptide or other organic
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 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.-6M, 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.
[0491] An antibody, oligopeptide or other organic molecule that
"inhibits the growth of tumor cells expressing a TAT polypeptide"
or a "growth inhibitory" antibody, oligopeptide or other organic
molecule is one which results in measurable growth inhibition of
cancer cells expressing or overexpressing the appropriate TAT
polypeptide. The TAT 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. Preferred growth
inhibitory anti-TAT antibodies, oligopeptides or organic molecules
inhibit growth of TAT-expressing tumor cells by greater than 20%,
preferably from about 20% to about 50%, and even more 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 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. Growth inhibition of tumor cells in
vivo can be determined in various ways such as is described in the
Experimental Examples section below. The antibody is growth
inhibitory in vivo if administration of the anti-TAT 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,
preferably within about 5 to 30 days.
[0492] An antibody, oligopeptide or other organic molecule 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 a TAT polypeptide.
Preferably the cell is a tumor cell, e.g., a prostate, breast,
ovarian, stomach, endometrial, lung, kidney, colon, bladder cell.
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 antibody,
oligopeptide or other organic molecule which induces apoptosis is
one which results in about 2 to 50 fold, preferably about 5 to 50
fold, and most preferably about 10 to 50 fold, induction of annexin
binding relative to untreated cell in an annexin binding assay.
[0493] 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.
[0494] "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).
[0495] "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)).
[0496] "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.
[0497] "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.
[0498] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include, but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include squamous cell cancer (e.g., epithelial squamous cell
cancer), lung cancer including small-cell lung cancer, non-small
cell lung cancer, adenocarcinoma of the lung and squamous carcinoma
of the lung, cancer of the peritoneum, hepatocellular cancer,
gastric or stomach cancer including gastrointestinal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, cancer of the urinary tract,
hepatoma, breast cancer, colon cancer, rectal cancer, colorectal
cancer, endometrial or uterine carcinoma, salivary gland carcinoma,
kidney or renal cancer, prostate cancer, vulval cancer, thyroid
cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,
melanoma, multiple myeloma and B-cell lymphoma, brain, as well as
head and neck cancer, and associated metastases.
[0499] 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.
[0500] "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.
[0501] An antibody, oligopeptide or other organic molecule which
"induces cell death" is one which causes a viable cell to become
nonviable. The cell is one which expresses a TAT polypeptide,
preferably a cell that overexpresses a TAT polypeptide as compared
to a normal cell of the same tissue type. The TAT 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. Preferably, the cell is a cancer cell, e.g., a breast,
ovarian, stomach, endometrial, salivary gland, lung, kidney, colon,
thyroid, pancreatic or bladder 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 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 molecules are those which induce PI
uptake in the PI uptake assay in BT474 cells.
[0502] A "TAT-expressing cell" is a cell which expresses an
endogenous or transfected TAT polypeptide either on the cell
surface or in a secreted form. A "TAT-expressing cancer" is a
cancer comprising cells that have a TAT polypeptide present on the
cell surface or that produce and secrete a TAT polypeptide. A
"TAT-expressing cancer" optionally produces sufficient levels of
TAT polypeptide on the surface of cells thereof, such that an
anti-TAT antibody, oligopeptide or other organic molecule can bind
thereto and have a therapeutic effect with respect to the cancer.
In another embodiment, a "TAT-expressing cancer" optionally
produces and secretes sufficient levels of TAT polypeptide, such
that an anti-TAT antibody, oligopeptide or other organic molecule
antagonist can bind thereto and have a therapeutic effect with
respect to the cancer. With regard to the latter, the antagonist
may be an antisense oligonucleotide which reduces, inhibits or
prevents production and secretion of the secreted TAT polypeptide
by tumor cells. A cancer which "overexpresses" a TAT polypeptide is
one which has significantly higher levels of TAT polypeptide at the
cell surface thereof, or produces and secretes, compared to a
noncancerous cell of the same tissue type. Such overexpression may
be caused by gene amplification or by increased transcription or
translation. TAT polypeptide overexpression may be determined in a
diagnostic or prognostic assay by evaluating increased levels of
the TAT protein present on the surface of a cell, or secreted by
the cell (e.g., via an immunohistochemistry assay using anti-TAT
antibodies prepared against an isolated TAT polypeptide which may
be prepared using recombinant DNA technology from an isolated
nucleic acid encoding the TAT polypeptide; FACS analysis, etc.).
Alternatively, or additionally, one may measure levels of TAT
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 TAT-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 TAT polypeptide overexpression 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.
[0503] 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.
[0504] 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 molecule so as to
generate a "labeled" antibody, oligopeptide or other organic
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.
[0505] 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.sub.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, daunoribicin 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.
[0506] A "growth inhibitory agent" when used herein refers to a
compound or composition which inhibits growth of a cell, especially
a TAT-expressing cancer cell, either in vitro or in vivo. Thus, the
growth inhibitory agent may be one which significantly reduces the
percentage of TAT-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 (31
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.
[0507] "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.
[0508] 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;
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-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.
[0509] 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 TAT XXXXXXXXXXXXXXX (Length = 15 amino
acids) Comparison XXXXXYYYYYYY (Length = 12 amino acids) Protein %
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 TAT polypeptide) = 5 divided by 15 = 33.3%
TABLE-US-00002 TABLE 3 TAT XXXXXXXXXX (Length = 10 amino acids)
Comparison XXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein %
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 TAT polypeptide) = 5 divided by 10 = 50%
TABLE-US-00003 TABLE 4 TAT-DNA NNNNNNNNNNNNNN (Length = 14
nucleotides) Comparison NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
DNA % 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 TAT-DNA nucleic acid sequence) = 6 divided by 14 = 42.9%
TABLE-US-00004 TABLE 5 TAT-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 TAT-DNA nucleic acid sequence) = 4 divided by 12 = 33.3%
II. Compositions and Methods of the Invention
[0510] A. Anti-TAT Antibodies
[0511] In one embodiment, the present invention provides anti-TAT
antibodies which may find use herein as therapeutic and/or
diagnostic agents. Exemplary antibodies include polyclonal,
monoclonal, humanized, bispecific, and heteroconjugate
antibodies.
[0512] 1. Polyclonal Antibodies
[0513] 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.
[0514] 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 liter. 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.
[0515] 2. Monoclonal Antibodies
[0516] 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).
[0517] 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)).
[0518] 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.
[0519] 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)).
[0520] 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).
[0521] 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).
[0522] 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.
[0523] 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.
[0524] 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 ii 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).
[0525] 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 marine 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.
[0526] 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.
[0527] 3. Human and Humanized Antibodies
[0528] The anti-TAT 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)].
[0529] 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 coworkers [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.
[0530] 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 Proc. Natl. Acad. Sci. USA, 89:4285
(1992); Presta et al., J. Immunol. 151:2623 (1993)).
[0531] 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.
[0532] Various forms of a humanized anti-TAT 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.
[0533] 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 hill 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.
[0534] 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.
[0535] 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).
[0536] 4. Antibody Fragments
[0537] 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.
[0538] 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.
[0539] 5. Bispecific Antibodies
[0540] 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 TAT
protein as described herein. Other such antibodies may combine a
TAT binding site with a binding site for another protein.
Alternatively, an anti-TAT 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 TAT-expressing cell. Bispecific antibodies may also be used to
localize cytotoxic agents to cells which express TAT. These
antibodies possess a TAT-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).
[0541] 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.
[0542] 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).
[0543] 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 (C.sub.H1) 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.
[0544] 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).
[0545] 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.
[0546] 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.
[0547] 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.
[0548] 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.
[0549] 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).
[0550] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
[0551] 6. Heteroconjugate Antibodies
[0552] 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.
[0553] 7. Multivalent Antibodies
[0554] 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, an 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.
[0555] 8. Effector Function Engineering
[0556] 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
halflife 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.
[0557] 9. Immunoconjugates
[0558] The invention also pertains to immunoconjugates comprising
an antibody conjugated to a cytotoxic agent such as a
chemotherapeutic agent, 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).
[0559] 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 glutareldehyde),
bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine),
bis-(diazonium derivatives (such as
bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as
tolyene 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.
[0560] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, a trichothene, and
CC1065, and the derivatives of these toxins that have toxin
activity, are also contemplated herein.
Maytansine and Maytansinoids
[0561] In one preferred embodiment, an anti-TAT antibody (full
length or fragments) of the invention is conjugated to one or more
maytansinoid molecules.
[0562] 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
[0563] 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 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 maytansonid 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-TAT Polypeptide Antibody-Maytansinoid Conjugates
(Immunoconjugates)
[0564] Anti-TAT antibody-maytansinoid conjugates are prepared by
chemically linking an anti-TAT 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.
[0565] 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.
[0566] 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 glutareldehyde), 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.
[0567] 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
[0568] Another immunoconjugate of interest comprises an anti-TAT
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
[0569] Other antitumor agents that can be conjugated to the
anti-TAT 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).
[0570] 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.
[0571] 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).
[0572] 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-TAT 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, Pb.sup.212 and radioactive isotopes of Lu.
When the conjugate is used for diagnosis, 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-111, fluorine-19, carbon-13,
nitrogen-15, oxygen-17, gadolinium, manganese or iron.
[0573] 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-19 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.
[0574] 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 glutareldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such is bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 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.
[0575] Alternatively, a fusion protein comprising the anti-TAT
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.
[0576] 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).
[0577] 10. Immunoliposomes
[0578] The anti-TAT 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.
[0579] 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).
[0580] B. TAT Binding Oligopeptides
[0581] TAT binding oligopeptides of the present invention are
oligopeptides that bind, preferably specifically, to a TAT
polypeptide as described herein. TAT binding oligopeptides may be
chemically synthesized using known oligopeptide synthesis
methodology or may be prepared and purified using recombinant
technology. TAT 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 TAT polypeptide as described herein. TAT 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).
[0582] 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.
[0583] 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 T17 phage
display systems (Smith and Scott, Methods in Enzymology, 217:
228-257 (1993); U.S. Pat. No. 5,766,905) are also known.
[0584] 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 Staphylococcus 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.
[0585] 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.
[0586] C. TAT Binding Organic Molecules
[0587] TAT binding organic molecules are organic molecules other
than oligopeptides or antibodies as defined herein that bind,
preferably specifically, to a TAT polypeptide as described herein.
TAT binding organic molecules may be identified and chemically
synthesized using known methodology (see, e.g., PCT Publication
Nos. WO00/00823 and WO00/39585). TAT binding organic 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 molecules that are capable of binding, preferably
specifically, to a TAT 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 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). TAT binding
organic 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.
[0588] D. Screening for Anti-TAT Antibodies, TAT Binding
Oligopeptides and TAT Binding Organic Molecules with the Desired
Properties
[0589] Techniques for generating antibodies, oligopeptides and
organic molecules that bind to TAT polypeptides have been described
above. One may further select antibodies, oligopeptides or other
organic molecules with certain biological characteristics, as
desired.
[0590] The growth inhibitory effects of an anti-TAT antibody,
oligopeptide or other organic molecule of the invention may be
assessed by methods known in the art, e.g., using cells which
express a TAT polypeptide either endogenously or following
transfection with the TAT gene. For example, appropriate tumor cell
lines and TAT-transfected cells may treated with an anti-TAT
monoclonal antibody, oligopeptide or other organic 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
calorimetric 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-TAT antibody, TAT binding
oligopeptide or TAT binding organic 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. Preferably, the tumor cell is one that overexpresses a TAT
polypeptide. Preferably, the anti-TAT antibody, TAT binding
oligopeptide or TAT binding organic molecule will inhibit cell
proliferation of a TAT-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-TAT 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.
[0591] To select for an anti-TAT antibody, TAT binding oligopeptide
or TAT binding organic 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. TAT polypeptide-expressing tumor cells are
incubated with medium alone or medium containing the appropriate
anti-TAT antibody (e.g., at about 10 .mu.g/ml), TAT binding
oligopeptide or TAT binding organic 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-TAT antibodies,
TAT binding oligopeptides or TAT binding organic molecules that
induce statistically significant levels of cell death as determined
by PI uptake may be selected as cell death-inducing anti-TAT
antibodies, TAT binding oligopeptides or TAT binding organic
molecules.
[0592] To screen for antibodies, oligopeptides or other organic
molecules which bind to an epitope on a TAT 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 molecule binds the same site or epitope as a known
anti-TAT 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 TAT 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.
[0593] E. Antibody Dependent Enzyme Mediated Prodrug Therapy
(ADEPT)
[0594] 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.
[0595] 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.
[0596] 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.
[0597] The enzymes of this invention can be covalently bound to the
anti-TAT 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).
[0598] F. Full-Length TAT Polypeptides
[0599] The present invention also provides newly identified and
isolated nucleotide sequences encoding polypeptides referred to in
the present application as TAT polypeptides. In particular, cDNAs
(partial and full-length) encoding various TAT polypeptides have
been identified and isolated, as disclosed in further detail in the
Examples below.
[0600] As disclosed in the Examples below, various cDNA clones have
been deposited with the ATCC. The actual nucleotide sequences of
those clones can readily be determined by the skilled artisan by
sequencing of the deposited clone using routine methods in the art.
The predicted amino acid sequence can be determined from the
nucleotide sequence using routine skill. For the TAT polypeptides
and encoding nucleic acids described herein, in some cases,
Applicants have identified what is believed to be the reading frame
best identifiable with the sequence information available at the
time.
[0601] G. Anti-TAT Antibody and TAT Polypeptide Variants
[0602] In addition to the anti-TAT antibodies and full-length
native sequence TAT polypeptides described herein, it is
contemplated that anti-TAT antibody and TAT polypeptide variants
can be prepared. Anti-TAT antibody and TAT polypeptide variants can
be prepared by introducing appropriate nucleotide changes into the
encoding DNA, and/or by synthesis of the desired antibody or
polypeptide. Those skilled in the art will appreciate that amino
acid changes may alter post-translational processes of the anti-TAT
antibody or TAT polypeptide, such as changing the number or
position of glycosylation sites or altering the membrane anchoring
characteristics.
[0603] Variations in the anti-TAT antibodies and TAT polypeptides
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 or polypeptide 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-TAT antibody or TAT
polypeptide. 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-TAT antibody or TAT polypeptide 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 full-length or
mature native sequence.
[0604] Anti-TAT antibody and TAT 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-TAT antibody or TAT
polypeptide.
[0605] Anti-TAT antibody and TAT 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-TAT antibody and TAT polypeptide fragments share
at least one biological and/or immunological activity with the
native anti-TAT antibody or TAT polypeptide disclosed herein.
[0606] 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; lys; arg gln Asp (D) glu glu Cys (C)
ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His
(H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; leu
norleucine Leu (L) norleucine; ile; val; ile met; ala; phe Lys (K)
arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile;
ala; tyr leu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp
(W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu;
met; phe; leu ala; norleucine
[0607] Substantial modifications in function or immunological
identity of the anti-TAT antibody or TAT polypeptide are
accomplished by selecting substitutions that differ significantly
in their effect on maintaining (a) the structure of the polypeptide
backbone in the area of the substitution, for example, as a sheet
or helical conformation, (b) the charge or hydrophobicity of the
molecule at the target site, or (c) the bulk of the side chain.
Naturally occurring residues are divided into groups based on
common side-chain properties:
(1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral
hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn,
gln, his, lys, arg; (5) residues that influence chain orientation:
gly, pro; and (6) aromatic: trp, tyr, phe.
[0608] 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.
[0609] 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-TAT antibody or TAT polypeptide variant DNA.
[0610] 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
isoteric amino acid can be used.
[0611] Any cysteine residue not involved in maintaining the proper
conformation of the anti-TAT antibody or TAT 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-TAT antibody
or TAT polypeptide to improve its stability (particularly where the
antibody is an antibody fragment such as an Fv fragment).
[0612] 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 TAT
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.
[0613] Nucleic acid molecules encoding amino acid sequence variants
of the anti-TAT 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-TAT antibody.
H. Modifications of Anti-TAT Antibodies and TAT Polypeptides
[0614] Covalent modifications of anti-TAT antibodies and TAT
polypeptides are included within the scope of this invention. One
type of covalent modification includes reacting targeted amino acid
residues of an anti-TAT antibody or TAT 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-TAT antibody or
TAT polypeptide. Derivatization with bifunctional agents is useful,
for instance, for crosslinking anti-TAT antibody or TAT polypeptide
to a water-insoluble support matrix or surface for use in the
method for purifying anti-TAT 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.
[0615] 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.
[0616] Another type of covalent modification of the anti-TAT
antibody or TAT 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-TAT
antibody or TAT 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-TAT antibody
or TAT 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.
[0617] 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.
[0618] Addition of glycosylation sites to the anti-TAT antibody or
TAT 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-TAT antibody or TAT polypeptide (for
O-linked glycosylation sites). The anti-TAT antibody or TAT
polypeptide amino acid sequence may optionally be altered through
changes at the DNA level, particularly by mutating the DNA encoding
the anti-TAT antibody or TAT polypeptide at preselected bases such
that codons are generated that will translate into the desired
amino acids.
[0619] Another means of increasing the number of carbohydrate
moieties on the anti-TAT antibody or TAT 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).
[0620] Removal of carbohydrate moieties present on the anti-TAT
antibody or TAT 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).
[0621] Another type of covalent modification of anti-TAT antibody
or TAT 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. Nos. 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).
[0622] The anti-TAT antibody or TAT polypeptide of the present
invention may also be modified in a way to form chimeric molecules
comprising an anti-TAT antibody or TAT polypeptide fused to
another, heterologous polypeptide or amino acid sequence.
[0623] In one embodiment, such a chimeric molecule comprises a
fusion of the anti-TAT antibody or TAT 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-TAT antibody or TAT
polypeptide. The presence of such epitope-tagged forms of the
anti-TAT antibody or TAT polypeptide can be detected using an
antibody against the tag polypeptide. Also, provision of the
epitope tag enables the anti-TAT antibody or TAT 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)].
[0624] In an alternative embodiment, the chimeric molecule may
comprise a fusion of the anti-TAT antibody or TAT 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-TAT antibody or TAT 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.
[0625] 1. Preparation of Anti-TAT Antibodies and TAT
Polypeptides
[0626] The description below relates primarily to production of
anti-TAT antibodies and TAT polypeptides by culturing cells
transformed or transfected with a vector containing anti-TAT
antibody- and TAT 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-TAT antibodies and TAT
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-TAT antibody or TAT polypeptide may be chemically synthesized
separately and combined using chemical or enzymatic methods to
produce the desired anti-TAT antibody or TAT polypeptide.
[0627] 1. Isolation of DNA Encoding Anti-TAT Antibody or TAT
Polypeptide
[0628] DNA encoding anti-TAT antibody or TAT polypeptide may be
obtained from a cDNA library prepared from tissue believed to
possess the anti-TAT antibody or TAT polypeptide mRNA and to
express it at a detectable level. Accordingly, human anti-TAT
antibody or TAT polypeptide DNA can be conveniently obtained from a
cDNA library prepared from human tissue. The anti-TAT antibody- or
TAT polypeptide-encoding gene may also be obtained from a genomic
library or by known synthetic procedures (e.g., automated nucleic
acid synthesis).
[0629] 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-TAT antibody or TAT polypeptide is
to use PCR methodology [Sambrook et al., supra; Dieffenbach et al.,
PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory
Press, 1995)].
[0630] 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.
[0631] 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.
[0632] 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.
[0633] 2. Selection and Transformation of Host Cells
[0634] Host cells are transfected or transformed with expression or
cloning vectors described herein for anti-TAT antibody or TAT
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.
[0635] 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).
[0636] 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.
lichenformis (e.g., B. lichenformis 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 rbs7 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.
[0637] 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 region
(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.
[0638] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for anti-TAT antibody- or TAT polypeptide-encoding vectors.
Saccharomyces cerevisiae is a commonly used lower eukaryotic host
microorganism. Others include Schizo saccharomyces 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. thermotolerans, 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).
[0639] Suitable host cells for the expression of glycosylated
anti-TAT antibody or TAT 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.
[0640] 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).
[0641] Host cells are transformed with the above-described
expression or cloning vectors for anti-TAT antibody or TAT
polypeptide production and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences.
[0642] 3. Selection and Use of a Replicable Vector
[0643] The nucleic acid (e.g., cDNA or genomic DNA) encoding
anti-TAT antibody or TAT 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.
[0644] The TAT 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 par of the anti-TAT antibody- or TAT
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,
Ipp, 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.
[0645] 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.
[0646] 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.
[0647] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the anti-TAT antibody- or TAT 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)].
[0648] Expression and cloning vectors usually contain a promoter
operably linked to the anti-TAT antibody- or TAT
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-TAT antibody or
TAT polypeptide.
[0649] 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.
[0650] 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.
[0651] Anti-TAT antibody or TAT 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.
[0652] Transcription of a DNA encoding the anti-TAT antibody or TAT
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-TAT antibody or TAT polypeptide
coding sequence, but is preferably located at a site 5' from the
promoter.
[0653] 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-TAT
antibody or TAT polypeptide.
[0654] Still other methods, vectors, and host cells suitable for
adaptation to the synthesis of anti-TAT antibody or TAT 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.
[0655] 4. Culturing the Host Cells
[0656] The host cells used to produce the anti-TAT antibody or TAT
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), an
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. Nos. 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, pit, and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan.
[0657] 5. Detecting Gene Amplification/Expression
[0658] 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.
[0659] 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 TAT polypeptide or against a synthetic peptide
based on the DNA sequences provided herein or against exogenous
sequence fused to TAT DNA and encoding a specific antibody
epitope.
[0660] 6. Purification of Anti-TAT Antibody and TAT Polypeptide
[0661] Forms of anti-TAT antibody and TAT 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-TAT antibody and TAT
polypeptide can be disrupted by various physical or chemical means,
such as freeze-thaw cycling, sonication, mechanical disruption, or
cell lysing agents.
[0662] It may be desired to purify anti-TAT antibody and TAT
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-TAT antibody and TAT 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-TAT antibody or TAT
polypeptide produced.
[0663] 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.
[0664] 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.
[0665] 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).
[0666] J. Pharmaceutical Formulations
[0667] Therapeutic formulations of the anti-TAT antibodies, TAT
binding oligopeptides, TAT binding organic molecules and/or TAT
polypeptides used in accordance with the present invention are
prepared for storage by mixing the antibody, polypeptide,
oligopeptide or organic 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, mid 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.
[0668] 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-TAT antibody, TAT binding oligopeptide, or TAT binding organic
molecule, it may be desirable to include in the one formulation, an
additional antibody, e.g., a second anti-TAT antibody which binds a
different epitope on the TAT 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.
[0669] 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).
[0670] 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.
[0671] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0672] K. Diagnosis and Treatment with Anti-TAT Antibodies, TAT
Binding Oligopeptides and TAT Binding Organic Molecules
[0673] To determine TAT expression in the cancer, various
diagnostic assays are available. In one embodiment, TAT polypeptide
overexpression may be analyzed by immunohistochemistry (1HC).
Parrafin embedded tissue sections from a tumor biopsy may be
subjected to the 1HC assay and accorded a TAT protein staining
intensity criteria as follows:
[0674] Score 0--no staining is observed or membrane staining is
observed in less than 10% of tumor cells.
[0675] 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.
[0676] Score 2+--a weak to moderate complete membrane staining is
observed in more than 10% of the tumor cells.
[0677] Score 3+--a moderate to strong complete membrane staining is
observed in more than 10% of the tumor cells.
[0678] Those tumors with 0 or 1+ scores for TAT polypeptide
expression may be characterized as not overexpressing TAT, whereas
those tumors with 2+ or 3+ scores may be characterized as
overexpressing TAT.
[0679] 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 TAT overexpression in
the tumor.
[0680] TAT overexpression or amplification may be evaluated using
an in vivo diagnostic assay, e.g., by administering a molecule
(such as an antibody, oligopeptide or organic 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.
[0681] As described above, the anti-TAT antibodies, oligopeptides
and organic molecules of the invention have various non-therapeutic
applications. The anti-TAT antibodies, oligopeptides and organic
molecules of the present invention can be useful for diagnosis and
staging of TAT polypeptide-expressing cancers (e.g., in
radioimaging). The antibodies, oligopeptides and organic molecules
are also useful for purification or immunoprecipitation of TAT
polypeptide from cells, for detection and quantitation of TAT
polypeptide in vitro, e.g., in an ELISA or a Western blot, to kill
and eliminate TAT-expressing cells from a population of mixed cells
as a step in the purification of other cells.
[0682] 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-TAT antibody, oligopeptide or organic 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-TAT antibodies, oligopeptides and organic
molecules of the invention are useful to alleviate TAT-expressing
cancers upon initial diagnosis of the disease or during relapse.
For therapeutic applications, the anti-TAT antibody, oligopeptide
or organic 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-TAT antibody,
oligopeptide or organic 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-TAT antibody,
oligopeptide or organic 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-TAT
antibody, oligopeptide or organic molecule will be administered
with a therapeutically effective dose of the chemotherapeutic
agent. In another embodiment, the anti-TAT antibody, oligopeptide
or organic 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.
[0683] In one particular embodiment, a conjugate comprising an
anti-TAT antibody, oligopeptide or organic molecule conjugated with
a cytotoxic agent is administered to the patient. Preferably, the
immunoconjugate bound to the TAT 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.
[0684] The anti-TAT antibodies, oligopeptides, organic 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 molecule is
preferred.
[0685] Other therapeutic regimens may be combined with the
administration of the anti-TAT antibody, oligopeptide or organic
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.
[0686] It may also be desirable to combine administration of the
anti-TAT antibody or antibodies, oligopeptides or organic
molecules, with administration of an antibody directed against
another tumor antigen associated with the particular cancer.
[0687] In another embodiment, the therapeutic treatment methods of
the present invention involves the combined administration of an
anti-TAT antibody (or antibodies), oligopeptides or organic
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 (losing schedules for such
chemotherapy are also described in Chemotherapy Service Ed., M. C.
Perry, Williams & Wilkins, Baltimore, Md. (1992).
[0688] The antibody, oligopeptide or organic molecule may be
combined with an anti-hormonal compound; e.g., an anti-estrogen
compound such as tamoxifen; an anti-progesterone such as
onapristone (se, 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, alter
the cancer becomes androgen independent, the anti-TAT antibody,
oligopeptide or organic molecule (and optionally other agents as
described herein) may be administered to the patient.
[0689] 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 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-TAT antibody, oligopeptide or organic molecule.
[0690] 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 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 molecule is administered for
preventive or therapeutic purposes, previous therapy, the patient's
clinical history and response to the antibody, oligopeptide or
organic molecule, and the discretion of the attending physician.
The antibody, oligopeptide or organic molecule is suitably
administered to the patient at one time or over a series of
treatments. Preferably, the antibody, oligopeptide or organic
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-TAT 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.
[0691] 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.
[0692] There are two major approaches to getting the nucleic acid
(optionally contained in a vector) into the patient's cells; in
vivo and ex viva. 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 viva delivery of the gene is a
retroviral vector.
[0693] The currently preferred in viva 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.
[0694] The anti-TAT 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.
[0695] 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-TAT antibodies of the invention are also contemplated,
specifically including the in viva tumor targeting and any cell
proliferation inhibition or cytotoxic characteristics.
[0696] Methods of producing the above antibodies are described in
detail herein.
[0697] The present anti-TAT antibodies, oligopeptides and organic
molecules are useful for treating a TAT-expressing cancer or
alleviating one or more symptoms of the cancer in a mammal. Such a
cancer includes prostate cancer, cancer of the urinary tract, lung
cancer, breast cancer, colon cancer and ovarian cancer, more
specifically, prostate adenocarcinoma, renal cell carcinomas,
colorectal adenocarcinomas, lung adenocarcinomas, lung squamous
cell carcinomas, and pleural mesothelioma. The cancers encompass
metastatic cancers of any of the preceding. The antibody,
oligopeptide or organic molecule is able to bind to at least a
portion of the cancer cells that express TAT polypeptide in the
mammal. In a preferred embodiment, the antibody, oligopeptide or
organic molecule is effective to destroy or kill TAT-expressing
tumor cells or inhibit the growth of such tumor cells, in vitro or
in vivo, upon binding to TAT polypeptide on the cell. Such an
antibody includes a naked anti-TAT 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-TAT antibody by,
e.g., conjugating the antibody with a cytotoxic agent, to form an
immunoconjugate as described herein. The cytotoxic agent or a
growth inhibitory agent is preferably a small molecule. Toxins such
as calicheamicin or a maytansinoid and analogs or derivatives
thereof, are preferable.
[0698] The invention provides a composition comprising an anti-TAT
antibody, oligopeptide or organic 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-TAT antibodies present as
an immunoconjugate or as the naked antibody. In a further
embodiment, the compositions can comprise these antibodies,
oligopeptides or organic 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-TAT antibody, oligopeptide or
organic molecule of the invention, and a carrier. In one
embodiment, the formulation is a therapeutic formulation comprising
a pharmaceutically acceptable carrier.
[0699] Another aspect of the invention is isolated nucleic acids
encoding the anti-TAT 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.
[0700] The invention also provides methods useful for treating a
TAT polypeptide-expressing cancer or alleviating one or more
symptoms of the cancer in a mammal, comprising administering a
therapeutically effective amount of an anti-TAT antibody,
oligopeptide or organic molecule to the mammal. The antibody,
oligopeptide or organic 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 TAT polypeptide-expressing cell.
[0701] The invention also provides kits and articles of manufacture
comprising at least one anti-TAT antibody, oligopeptide or organic
molecule. Kits containing anti-TAT antibodies, oligopeptides or
organic molecules find use, e.g., for TAT cell killing assays, for
purification or immunoprecipitation of TAT polypeptide from cells.
For example, for isolation and purification of TAT, the kit can
contain an anti-TAT antibody, oligopeptide or organic molecule
coupled to beads (e.g., sepharose beads). Kits can be provided
which contain the antibodies, oligopeptides or organic molecules
for detection and quantitation of TAT in vitro, e.g., in an ELISA
or a Western blot. Such antibody, oligopeptide or organic molecule
useful for detection may be provided with a label such as a
fluorescent or radiolabel.
[0702] L. Articles of Manufacture and Kits
[0703] Another embodiment of the invention is an article of
manufacture containing materials useful for the treatment of
anti-TAT expressing cancer. The article of manufacture comprises a
container mid 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-TAT antibody, oligopeptide or
organic 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 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.
[0704] Kits are also provided that are useful for various purposes,
e.g., for TAT-expressing cell killing assays, for purification or
immunoprecipitation of TAT polypeptide from cells. For isolation
and purification of TAT polypeptide, the kit can contain an
anti-TAT antibody, oligopeptide or organic molecule coupled to
beads (e.g., sepharose beads). Kits can be provided which contain
the antibodies, oligopeptides or organic molecules for detection
and quantitation of TAT 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-TAT antibody, oligopeptide or organic
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
diagnostic use.
[0705] M. Uses for TAT Polypeptides and TAT-Polypeptide Encoding
Nucleic Acids
[0706] Nucleotide sequences (or their complement) encoding TAT
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. TAT-encoding nucleic acid will also be useful for the
preparation of TAT polypeptides by the recombinant techniques
described herein, wherein those TAT polypeptides may find use, for
example, in the preparation of anti-TAT antibodies as described
herein.
[0707] The full-length native sequence TAT gene, or portions
thereof, may be used as hybridization probes for a cDNA library to
isolate the full-length TAT cDNA or to isolate still other cDNAs
(for instance, those encoding naturally-occurring variants of TAT
or TAT from other species) which have a desired sequence identity
to the native TAT 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 TAT. By way of example, a screening method will comprise
isolating the coding region of the TAT 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 TAT 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.
[0708] Other useful fragments of the TAT-encoding nucleic acids
include antisense or sense oligonucleotides comprising a
singe-stranded nucleic acid sequence (either RNA or DNA) capable of
binding to target TAT mRNA (sense) or TAT DNA (antisense)
sequences. Antisense or sense oligonucleotides, according to the
present invention, comprise a fragment of the coding region of TAT
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).
[0709] 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
TAT proteins, wherein those TAT proteins 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.
[0710] 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 Bun mRNA or corresponding nucleotides on the gene.
[0711] Specific examples of preferred antisense compounds useful
for inhibiting expression of TAT proteins 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 a basic (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.
[0712] 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.
[0713] 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 all 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.
[0714] 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(C.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.
[0715] 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; O-alkenyl, S-alkenyl, 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.2 CH.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).
[0716] 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--), group
bridging the 2' oxygen atom and the 4' carbon atom wherein n is 1
or 2. 1.NAs and preparation thereof are described in WO 98/39352
and WO 99/14226.
[0717] Other preferred modifications include
2'-methoxy(2'-O--CH.sub.3),
2'-aminopropoxy(2'-OCH.sub.2CH.sub.2CH.sub.2 NH.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.
[0718] 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.
[0719] Another modification of antisense oligonucleotides
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.
[0720] 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 loin 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.
[0721] 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.
[0722] 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.
[0723] 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).
[0724] 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.
[0725] 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.
[0726] 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.
[0727] The probes may also be employed in PCR techniques to
generate a pool of sequences for identification of closely related
TAT coding sequences.
[0728] Nucleotide sequences encoding a TAT can also be used to
construct hybridization probes for mapping the gene which encodes
that TAT 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.
[0729] When the coding sequences for TAT encode a protein which
binds to another protein (example, where the TAT is a receptor),
the TAT can be used in assays to identify the other proteins or
molecules involved in the binding interaction. 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 or
agonists of the binding interaction. Also, the receptor TAT can be
used to isolate correlative ligand(s). Screening assays can be
designed to find lead compounds that mimic the biological activity
of a native TAT or a receptor for TAT. 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.
[0730] Nucleic acids which encode TAT 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 TAT
can be used to clone genomic DNA encoding TAT in accordance with
established techniques and the genomic sequences used to generate
transgenic animals that contain cells which express DNA encoding
TAT. 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 TAT
transgene incorporation with tissue-specific enhancers. Transgenic
animals that include a copy of a transgene encoding TAT 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 TAT.
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.
[0731] Alternatively, non-human homologues of TAT can be used to
construct a TAT "knock out" animal which has a defective or altered
gene encoding TAT as a result of homologous recombination between
the endogenous gene encoding TAT and altered genomic DNA encoding
TAT introduced into an embryonic stein cell of the animal. For
example, cDNA encoding TAT can be used to clone genomic DNA
encoding TAT in accordance with established techniques. A portion
of the genomic DNA encoding TAT 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 TAT polypeptide.
[0732] Nucleic acid encoding the TAT 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.
[0733] 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
halflife. 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).
[0734] The nucleic acid molecules encoding the TAT 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 TAT nucleic acid molecule of the present invention
can be used as a chromosome marker.
[0735] The TAT polypeptides and nucleic acid molecules of the
present invention may also be used diagnostically for tissue
typing, wherein the TAT polypeptides of the present invention 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. TAT nucleic acid molecules will find use for
generating probes for PCR, Northern analysis, Southern analysis and
Western analysis.
[0736] This invention encompasses methods of screening compounds to
identify those that mimic the TAT polypeptide (agonists) or prevent
the effect of the TAT polypeptide (antagonists). Screening assays
for antagonist drug candidates are designed to identify compounds
that bind or complex with the TAT 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 TAT 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.
[0737] 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.
[0738] All assays for antagonists are common in that they call for
contacting the drug candidate with a TAT polypeptide encoded by a
nucleic acid identified herein under conditions and for a time
sufficient to allow these two components to interact.
[0739] 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 TAT polypeptide encoded by the gene
identified herein 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 TAT polypeptide
and drying. Alternatively, an immobilized antibody, e.g., a
monoclonal antibody, specific for the TAT 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.
[0740] If the candidate compound interacts with but does not bind
to a particular TAT polypeptide encoded by a gene identified
herein, 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")
tikes 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.
[0741] Compounds that interfere with the interaction of a gene
encoding a TAT 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.
[0742] To assay for antagonists, the TAT 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 TAT polypeptide indicates that the
compound is an antagonist to the TAT polypeptide. Alternatively,
antagonists may be detected by combining the TAT polypeptide and a
potential antagonist with membrane-bound TAT polypeptide receptors
or recombinant receptors under appropriate conditions for a
competitive inhibition assay. The TAT polypeptide can be labeled,
such as by radioactivity, such that the number of TAT 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 TAT
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 TAT polypeptide. Transfected cells that are
grown on glass slides are exposed to labeled TAT polypeptide. The
TAT 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.
[0743] As an alternative approach for receptor identification,
labeled TAT 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.
[0744] In another assay for antagonists, mammalian cells or a
membrane preparation expressing the receptor would be incubated
with labeled TAT polypeptide in the presence of the candidate
compound. The ability of the compound to enhance or block this
interaction could then be measured.
[0745] More specific examples of potential antagonists include an
oligonucleotide that binds to the fusions of immunoglobulin with
TAT 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 TAT polypeptide that recognizes the receptor but
imparts no effect, thereby competitively inhibiting the action of
the TAT polypeptide.
[0746] Another potential TAT polypeptide 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 TAT polypeptides
herein, is 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 TAT polypeptide. The antisense RNA
oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into the TAT polypeptide
(antisense-Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides
as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton,
Fla., 19.times.8). 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 TAT 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.
[0747] 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 TAT polypeptide, thereby
blocking the normal biological activity of the TAT 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.
[0748] 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).
[0749] 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.
[0750] 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.
[0751] Isolated TAT polypeptide-encoding nucleic acid can be used
herein for recombinantly producing TAT polypeptide using techniques
well known in the art and as described herein. In turn, the
produced TAT polypeptides can be employed for generating anti-TAT
antibodies using techniques well known in the art and as described
herein.
[0752] Antibodies specifically binding a TAT 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.
[0753] If the TAT 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).
[0754] 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.
[0755] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0756] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirely.
EXAMPLES
[0757] Commercially available reagents referred to in the examples
were used according to manufacturer's instructions unless otherwise
indicated. The source of those cells identified in the following
examples, and throughout the specification, by ATCC accession
numbers is the American Type Culture Collection, Manassas, Va.
Example 1
Tissue Expression Profiling Using GeneExpress.RTM.
[0758] A proprietary database containing gene expression
information (GeneExpress.RTM., Gene Logic Inc., Gaithersburg, Md.)
was analyzed in an attempt to identify polypeptides (and their
encoding nucleic acids) whose expression is significantly
upregulated in a particular tumor tissue(s) of interest as compared
to other tumor(s) and/or normal tissues. Specifically, analysis of
the GeneExpress.RTM.database was conducted using either software
available through Gene Logic Inc., Gaithersburg, Md., for use with
the GeneExpress.RTM. database or with proprietary software written
and developed at Genentech, Inc. for use with the GeneExpress.RTM.
database. The rating of positive hits in the analysis is based upon
several criteria including, for example, tissue specificity, tumor
specificity and expression level in normal essential and/or normal
proliferating tissues. The following is a list of molecules whose
tissue expression profile as determined from an analysis of the
GeneExpress.RTM. database evidences high tissue expression and
significant upregulation of expression in a specific tumor or
tumors as compared to other tumor(s) and/or normal tissues and
optionally relatively low expression in normal essential and/or
normal proliferating tissues. As such, the molecules listed below
are excellent polypeptide targets for the diagnosis and therapy of
cancer in mammals.
TABLE-US-00006 upregulation of Molecule expression in: as compared
to: DNA225785 (TAT295) breast tumor normal breast tissue DNA225785
(TAT295) colon tumor normal colon tissue DNA225785 (TAT295)
lymphoid tumor normal lymphoid tissue DNA88158 (TAT298) ovarian
tumor normal ovarian tissue DNA88158 (TAT298) uterine tumor normal
uterine tissue DNA88158 (TAT298) breast tumor normal breast tissue
DNA88158 (TAT298) kidney tumor normal kidney tissue DNA225800
(TAT347) ovarian tumor normal ovarian tissue DNA225800 (TAT347)
uterine tumor normal uterine tissue DNA225800 (TAT347) breast tumor
normal breast tissue DNA225800 (TAT347) kidney tumor normal kidney
tissue DNA299884 (TAT304) lung tumor normal lung tissue DNA299884
(TAT304) colon tumor normal colon tissue DNA299884 (TAT304) rectum
tumor normal rectum tissue DNA299884 (TAT304) ovarian tumor normal
ovarian tissue DNA299884 (TAT304) breast tumor normal breast tissue
DNA299884 (TAT304) uterine tumor normal uterine tissue DNA293550
(TAT293) breast tumor normal breast tissue DNA293550 (TAT293) colon
tumor normal colon tissue DNA293550 (TAT293) rectum tumor normal
rectum tissue DNA293550 (TAT293) uterine tumor normal uterine
tissue DNA293550 (TAT293) lung tumor normal lung tissue DNA293550
(TAT293) ovarian tumor normal ovarian tissue DNA293550 (TAT293)
pancreatic tumor normal pancreatic tissue DNA293550 (TAT293)
prostate tumor normal prostate tissue DNA226313 (TAT296) skin tumor
normal skin tissue DNA226313 (TAT296) melanoma tumor normal
melanocyte tissue DNA225865 (TAT300) breast tumor normal breast
tissue DNA225865 (TAT300) colon tumor normal colon tissue DNA225865
(TAT300) rectum tumor normal rectum tissue DNA225865 (TAT300) lung
tumor normal lung tissue DNA225865 (TAT300) ovarian tumor normal
ovarian tissue DNA225865 (TAT300) pancreatic tumor normal
pancreatic tissue DNA225865 (TAT300) skin tumor normal skin tissue
DNA225865 (TAT300) soft tissue tumor normal soft tissue DNA226403
(TAT302) breast tumor normal breast tissue DNA226403 (TAT302)
prostate tumor normal prostate tissue DNA73736 (TAT303) uterine
tumor normal uterine tissue DNA73736 (TAT303) lung tumor normal
lung tissue DNA73736 (TAT303) ovarian tumor normal ovarian tissue
DNA73736 (TAT303) testicular tumor normal testicular tissue
DNA299885 (TAT305) skin tumor normal skin tissue
Example 2
Quantitative Analysis of TAT mRNA Expression
[0759] In this assay, a 5' nuclease assay (for example,
TaqMan.RTM.) and real-time quantitative PCR (for example, ABI Prizm
7700 Sequence Detection System.RTM. (Perkin Elmer, Applied
Biosystems Division, Foster City, Calif.)), were used to find genes
that are significantly overexpressed in a cancerous tumor or tumors
as compared to other cancerous tumors or normal non-cancerous
tissue. The 5' nuclease assay reaction is a fluorescent PCR-based
technique which makes use of the 5' exonuclease activity of Taq DNA
polymerase enzyme to monitor gene expression in real time. Two
oligonucleotide primers (whose sequences are based upon the gene or
EST sequence of interest) are used to generate an amplicon typical
of a PCR reaction. A third oligonucleotide, or probe, is designed
to detect nucleotide sequence located between the two PCR primers.
The probe is non-extendible by Taq DNA polymerase enzyme, and is
labeled with a reporter fluorescent dye and a quencher fluorescent
dye. Any laser-induced emission from the reporter dye is quenched
by the quenching dye when the two dyes are located close together
as they are on the probe. During the PCR amplification reaction,
the Taq DNA polymerase enzyme cleaves the probe in a
template-dependent manner. The resultant probe fragments
disassociate in solution, and signal from the released reporter dye
is free from the quenching effect of the second fluorophore. One
molecule of reporter dye is liberated for each new molecule
synthesized, and detection of the unquenched reporter dye provides
the basis for quantitative interpretation of the data.
[0760] The 5' nuclease procedure is run on a real-time quantitative
PCR device such as the ABI Prism 7700.TM. Sequence Detection. The
system consists of a thermocycler, laser, charge-coupled device
(CCD) camera and computer. The system amplifies samples in a
96-well format on a thermocycler. During amplification,
laser-induced fluorescent signal is collected in real-time through
fiber optics cables for all 96 wells, and detected at the CCD. The
system includes software for running the instrument and for
analyzing the data.
[0761] The starting material for the screen was mRNA isolated from
a variety of different cancerous tissues. The mRNA is quantitated
precisely, e.g., fluorometrically. As a negative control, RNA was
isolated from various normal tissues of the same tissue type as the
cancerous tissues being tested.
[0762] 5' nuclease assay data are initially expressed as Ct, or the
threshold cycle. This is defined as the cycle at which the reporter
signal accumulates above the background level of fluorescence. The
.DELTA.Ct values are used as quantitative measurement of the
relative number of starting copies of a particular target sequence
in a nucleic acid sample when comparing cancer mRNA results to
normal human mRNA results. As one Ct unit corresponds to 1 PCR
cycle or approximately a 2-fold relative increase relative to
normal, two units corresponds to a 4-fold relative increase, 3
units corresponds to an 8-fold relative increase and so on, one can
quantitatively measure the relative fold increase in mRNA
expression between two or more different tissues. Using this
technique, the molecules listed below have been identified as being
significantly overexpressed (i.e., at least 2-fold) in a particular
tumor(s) as compared to their normal non-cancerous counterpart
tissue(s) (from both the same and different tissue donors) and
thus, represent excellent polypeptide targets for the diagnosis and
therapy of cancer in mammals.
TABLE-US-00007 upregulation of Molecule expression in: as compared
to: DNA88158 (TAT298) ovarian tumor normal ovarian tissue DNA225800
(TAT347) ovarian tumor normal ovarian tissue DNA293550 (TAT293)
breast tumor normal breast tissue DNA103386 (TAT299) lung tumor
normal lung tissue DNA273438 (TAT351) lung tumor normal lung tissue
DNA225865 (TAT300) colon tumor normal colon tissue DNA226403
(TAT302) breast tumor normal breast tissue DNA73736 (TAT303)
ovarian tumor normal ovarian tissue
Example 3
In Situ Hybridization
[0763] In situ hybridization is a powerful and versatile technique
for the detection and localization of nucleic acid sequences within
cell or tissue preparations. It may be useful, for example, to
identify sites of gene expression, analyze the tissue distribution
of transcription, identify and localize viral infection, follow
changes in specific mRNA synthesis and aid in chromosome
mapping.
[0764] In situ hybridization was performed following an optimized
version of the protocol by Lu and Gillett, Cell Vision 1:169-176
(1994), using PCR-generated .sup.33P-labeled riboprobes. Briefly,
formalin-fixed, paraffin-embedded human tissues were sectioned,
deparaffinized, deproteinated in proteinase K (20 g/ml) for 15
minutes at 37.degree. C., and further processed for in situ
hybridization as described by Lu and Gillett, supra. A [.sup.33-P]
UTP-labeled antisense riboprobe was generated from a PCR product
and hybridized at 55.degree. C. overnight. The slides were dipped
in Kodak NTB2 nuclear track emulsion and exposed for 4 weeks.
.sup.33P-Riboprobe Synthesis
[0765] 6.0 .mu.l (125 mCi) of .sup.33P-UTP (Amersham BF 1002,
SA<2000 Ci/mmol) were speed vac dried. To each tube containing
dried .sup.33P-UTP, the following ingredients were added:
[0766] 2.0 .mu.l 5.times. transcription buffer
[0767] 1.0 .mu.l DTT (100 mM)
[0768] 2.0 .mu.l NTP mix (2.5 mM: 10.mu.; each of 10 mM GTP, CTP
& ATP+10 .mu.l H.sub.2O)
[0769] 1.0 .mu.l UTP (50 .mu.M)
[0770] 1.0 .mu.l Rnasin
[0771] 1.0 .mu.l DNA template (1 .mu.g)
[0772] 1.0 .mu.l H.sub.2O
[0773] 1.0 .mu.l RNA polymerase (for PCR products T3-AS, T7=S,
usually)
[0774] The tubes were incubated at 37.degree. C. for one hour.
.mu.l RQ1 DNase were added, followed by incubation at 37.degree. C.
for 15 minutes. 90 .mu.l TE (10 mM Tris pH 7.6/1 mM EDTA pH 8.0)
were added, and the mixture was pipetted onto DE81 paper. The
remaining solution was loaded in a Microcon-50 ultrafiltration
unit, and spun using program 10 (6 minutes). The filtration unit
was inverted over a second tube and spun using program 2 (3
minutes). After the final recovery spin, 100 .mu.l TE were added. 1
.mu.l of the final product was pipetted on DE81 paper and counted
in 6 ml of Biofluor II.
[0775] The probe was run on a TBE/urea gel. 1-3 .mu.l of the probe
or 5 .mu.l of RNA Mrk III were added to 3 .mu.l of loading buffer.
After heating on a 95.degree. C. heat block for three minutes, the
probe was immediately placed on ice. The wells of gel were flushed,
the sample loaded, and run at 180-250 volts for 45 minutes. The gel
was wrapped in saran wrap and exposed to XAR film with an
intensifying screen in -70.degree. C. freezer one hour to
overnight.
.sup.33P-Hybridization
[0776] A. Pretreatment of Frozen Sections
[0777] The slides were removed from the freezer, placed on
aluminium trays and thawed at room temperature for 5 minutes. The
trays were placed in 55.degree. C. incubator for five minutes to
reduce condensation. The slides were fixed for 10 minutes in 4%
paraformaldehyde on ice in the fume hood, and washed in
0.5.times.SSC for 5 minutes, at room temperature (25 ml
20.times.SSC+975 ml SQ H.sub.2O). After deproteination in 0.5
.mu.g/ml proteinase K for 10 minutes at 37.degree. C. (12.5 .mu.l
of 10 mg/ml stock in 250 ml prewarmed RNase-free RNAse buffer), the
sections were washed in 0.5.times.SSC for 10 minutes at room
temperature. The sections were dehydrated in 70%, 95%, 100%
ethanol, 2 minutes each.
[0778] B. Pretreatment of Paraffin-Embedded Sections
[0779] The slides were deparaffinized, placed in SQ H.sub.2O, and
rinsed twice in 2.times.SSC at room temperature, for 5 minutes each
time. The sections were deproteinated in 20 .mu.g/ml proteinase K
(500 .mu.l of 10 mg/ml in 250 ml RNase-free RNase buffer;
37.degree. C., 15 minutes)-human embryo, or 8.times. proteinase K
(100 .mu.l in 250 ml Rnase buffer, 37.degree. C., 30
minutes)-formalin tissues. Subsequent rinsing in 0.5.times.SSC and
dehydration were performed as described above.
C. Prehybridization
[0780] The slides were laid out in a plastic box lined with Box
buffer (4.times.SSC, 50% formamide)-saturated filter paper.
[0781] D. Hybridization
[0782] 1.0.times.10.sup.6 cpm probe and 1.0 .mu.l tRNA (50 mg/ml
stock) per slide were heated at 95.degree. C. for 3 minutes. The
slides were cooled on ice, and 48 .mu.l hybridization buffer were
added per slide. After vortexing, 50 .mu.l .sup.33P mix were added
to 50 .mu.l prehybridization on slide. The slides were incubated
overnight at 55.degree. C.
[0783] E. Washes
[0784] Washing was done 2.times.10 minutes with 2.times.SSC, EDTA
at room temperature (400 ml 20.times.SSC+16 ml 0.25M EDTA,
V.sub.f=4 L), followed by RNaseA treatment at 37.degree. C. for 30
minutes (500 .mu.L of 10 mg/ml in 250 ml Rnase buffer=20 .mu.g/ml),
The slides were washed 2.times.10 minutes with 2.times.SSC, EDTA at
room temperature. The stringency wash conditions were as follows: 2
hours at 55.degree. C., 0.1.times.SSC, EDTA (20 ml 20.times.SSC+16
ml EDTA, V.sub.f=4 L).
[0785] F. Oligonucleotides
[0786] In situ analysis was performed on a variety of DNA sequences
disclosed herein. The oligonucleotides employed for these analyses
were obtained so as to be complementary to the nucleic acids (or
the complements thereof) as shown in the accompanying figures.
[0787] G. Results
[0788] In situ analysis was performed on a variety of DNA sequences
disclosed herein. The results from these analyses are as
follows.
(1) DNA226313 (TAT296)
[0789] All normal tissues tested are negative for expression. 11/21
malignant melanoma tissue samples are positive for expression and
of the positive cases 6 display an extremely strong signal.
(2) DNA 103386 (TAT299)
[0790] A strong signal is observed in the malignant cells of a
testicular neoplasm (seminoma) while normal testicular tissue is
negative for expression.
(3) DNA273438 (TAT351)
[0791] A strong signal is observed in the malignant cells of a
testicular neoplasm (seminoma) while normal testicular tissue is
negative for expression.
(4) DNA73736 (TAT303)
[0792] Normal ovarian and endometrial tissues are negative for
expression. On the other hand, 6 of 11 ovarian and 2 of 8
endometrial adenocarcinomas are positive for expression.
Additionally, in another experiment, 5 of 11 clear cell carcinomas,
18 of 26 serous cystadenocarcinomas and 15 of 21 endometrioid
adenocarcinomas are positive for expression.
(5) DNA299885 (TAT305)
[0793] Normal skin tissue is negative for expression while 5 of 5
malignant melanoma samples tested are positive for expression.
Example 4
Verification and Analysis of Differential TAT Polypeptide
Expression by GEPIS
[0794] TAT polypetides which may have been identified as a tumor
antigen as described in one or more of the above Examples were
analyzed and verified as follows. An expressed sequence tag (EST)
DNA database (LIFESEQ.RTM., Incyte Pharmaceuticals, Palo Alto,
Calif.) was searched and interesting EST sequences were identified
by GEPIS. Gene expression profiling in silico (GEPIS) is a
bioinformatics tool developed at Genentech, Inc. that characterizes
genes of interest for new cancer therapeutic targets. GEPIS takes
advantage of large amounts of EST sequence and library information
to determine gene expression profiles. GEPIS is capable of
determining the expression profile of a gene based upon its
proportional correlation with the number of its occurrences in EST
databases, and it works by integrating the LIFESEQ.RTM. EST
relational database and Genentech proprietary information in a
stringent and statistically meaningful way. In this example, GEPIS
is used to identify and cross-validate novel tumor antigens,
although GEPIS can be configured to perform either very specific
analyses or broad screening tasks. For the initial screen, GEPIS is
used to identify EST sequences from the LIFESEQ.RTM. database that
correlate to expression in a particular tissue or tissues of
interest (often a tumor tissue of interest). The EST sequences
identified in this initial screen (or consensus sequences obtained
from aligning multiple related and overlapping EST sequences
obtained from the initial screen) were then subjected to a screen
intended to identify the presence of at least one transmembrane
domain in the encoded protein. Finally, GEPIS was employed to
generate a complete tissue expression profile for the various
sequences of interest. Using this type of screening bioinformatics,
various TAT polypeptides (and their encoding nucleic acid
molecules) were identified as being significantly overexpressed in
a particular type of cancer or certain cancers as compared to other
cancers and/or normal non-cancerous tissues. The rating of GEPIS
hits is based upon several criteria including, for example, tissue
specificity, tumor specificity and expression level in normal
essential and/or normal proliferating tissues. The following is a
list of molecules whose tissue expression profile as determined by
GEPIS evidences high tissue expression and significant upregulation
of expression in a specific tumor or tumors as compared to other
tumor(s) and/or normal tissues and optionally relatively low
expression in normal essential and/or normal proliferating tissues.
As such, the molecules listed below are excellent polypeptide
targets for the diagnosis and therapy of cancer in mammals.
TABLE-US-00008 upregulation of Molecule expression in: as compared
to: DNA225785 (TAT295) colon tumor normal colon tissue DNA88158
(TAT298) uterine tumor normal uterine tissue DNA225800 (TAT347)
uterine tumor normal uterine tissue DNA293550 (TAT293) prostate
tumor normal prostate tissue DNA293550 (TAT293) breast tumor normal
breast tissue DNA293550 (TAT293) colon tumor normal colon tissue
DNA293550 (TAT293) uterine tumor normal uterine tissue DNA293550
(TAT293) pancreatic tumor normal pancreatic tissue DNA293550
(TAT293) ovarian tumor normal ovarian tissue DNA293550 (TAT293)
bladder tumor normal bladder tissue DNA293550 (TAT293) brain tumor
normal brain tissue DNA103386 (TAT299) testicular tumor normal
testicular tissue DNA273438 (TAT351) testicular tumor normal
testicular tissue DNA225865 (TAT300) uterine tumor normal uterine
tissue DNA225865 (TAT300) kidney tumor normal kidney tissue
DNA225865 (TAT300) bone tumor normal bone tissue DNA225865 (TAT300)
esophagus tumor normal esophagus tissue DNA225865 (TAT300) bladder
tumor normal bladder tissue DNA226403 (TAT302) prostate tumor
normal prostate tissue DNA73736 (TAT303) testicular tumor normal
testicular tissue DNA73736 (TAT303) ovarian tumor normal ovarian
tissue DNA73736 (TAT303) prostate tumor normal prostate tissue
Example 5
Identification of TAT Polypeptides that are Specifically Expressed
by or Overexpressed in Lymphoid Tissues
[0795] Using the GeneExpress.RTM.- and GEPIS-associated tissue
expression analyses described in certain Examples above, various
TAT polypeptides have been identified as being either specifically
expressed on the surface of both normal and malignant B cells or
overexpressed in cancerous lymphoid tissue(s) as compared to its
normal tissue counterpart. By "specifically expressed" on a certain
tissue or cell type, it is meant that the TAT polypeptide is
expressed by that certain tissue or cell type and is not
significantly expressed by any other tissue or cell type. TAT
polypeptides that are specifically expressed on the surface of both
normal and malignant B cells or overexpressed in cancerous lymphoid
tissue(s) as compared to its normal tissue counterpart are
excellent polypeptide targets for the treatment of B cell and other
lymphoid-associated cancers including, for example, high,
intermediate and low grade lymphomas (including B cell lymphomas
such as, for example, mucosa-associated-lymphoid tissue B cell
lymphoma and non-Hodgkin's lymphoma, mantle cell lymphoma,
Burkitt's lymphoma, marginal zone lymphoma, diffuse large cell
lymphoma, follicular lymphoma, and Hodgkin's lymphoma) and
leukemias (including chronic lymphocytic leukemia), multiple
myeloma, and other hematological and/or B cell-associated cancers.
In this regard, we note that RITUXAN.RTM. (Genentech Inc., South
San Francisco, Calif.) is an antibody that binds to the CD20
antigen that is specifically expressed on the surface of both
normal and malignant B cells and which has been used successfully
to treat non-Hodgkin's lymphoma in humans. The following are TAT
polypeptides that have been herein identified as being specifically
expressed on the surface of both normal and malignant B-cells or
overexpressed in cancerous lymphoid tissue(s) as compared to their
normal tissue counterpart and, therefore, will serve as excellent
polypeptide targets for the treatment of B-cell and other
lymphoid-associated cancers: TAT294 (DNA150813), TAT297 (DNA
103365), TAT301 (DNA196665) and TAT352 (DNA287183).
Example 6
Use of TAT as a Hybridization Probe
[0796] The following method describes use of a nucleotide sequence
encoding TAT as a hybridization probe for, i.e., diagnosis of the
presence of a tumor in a mammal.
[0797] DNA comprising the coding sequence of full-length or mature
TAT as disclosed herein can also be employed as a probe to screen
for homologous DNAs (such as those encoding naturally-occurring
variants of TAT) in human tissue cDNA libraries or human tissue
genomic libraries.
[0798] Hybridization and washing of filters containing either
library DNAs is performed under the following high stringency
conditions. Hybridization of radiolabeled TAT-derived probe to the
filters is performed in a solution of 50% formamide, 5.times.SSC,
0.1% SDS, 0.1% sodium pyrophosphate, 50 mM sodium phosphate, pH
6.8, 2.times.Denhardt's solution, and 10% dextran sulfate at
42.degree. C. for 20 hours. Washing of the filters is performed in
an aqueous solution of 0.1.times.SSC and 0.1% SDS at 42.degree.
C.
[0799] DNAs having a desired sequence identity with the DNA
encoding full-length native sequence TAT can then be identified
using standard techniques known in the art.
Example 7
Expression of TAT in E. coli
[0800] This example illustrates preparation of an unglycosylated
form of TAT by recombinant expression in E. coli.
[0801] The DNA sequence encoding TAT is initially amplified using
selected PCR primers. The primers should contain restriction enzyme
sites which correspond to the restriction enzyme sites on the
selected expression vector. A variety of expression vectors may be
employed. An example of a suitable vector is pBR322 (derived from
E. coli; see Bolivar et al., Gene, 2:95 (1977)) which contains
genes for ampicillin and tetracycline resistance. The vector is
digested with restriction enzyme and dephosphorylated. The PCR
amplified sequences are then ligated into the vector. The vector
will preferably include sequences which encode for an antibiotic
resistance gene, a trp promoter, a polyhis leader (including the
first six STII codons, polyhis sequence, and enterokinase cleavage
site), the TAT coding region, lambda transcriptional terminator,
and an argU gene.
[0802] The ligation mixture is then used to transform a selected E.
coli strain using the methods described in Sambrook et al., supra.
Transformants are identified by their ability to grow on LB plates
and antibiotic resistant colonies are then selected. Plasmid DNA
can be isolated and confirmed by restriction analysis and DNA
sequencing.
[0803] Selected clones can be grown overnight in liquid culture
medium such as LB broth supplemented with antibiotics. The
overnight culture may subsequently be used to inoculate a larger
scale culture. The cells are then grown to a desired optical
density, during which the expression promoter is turned on.
[0804] After culturing the cells for several more hours, the cells
can be harvested by centrifugation. The cell pellet obtained by the
centrifugation can be solubilized using various agents known in the
art, and the solubilized TAT protein can then be purified using a
metal chelating column under conditions that allow tight binding of
the protein.
[0805] TAT may be expressed in E. coli in a poly-His tagged form,
using the following procedure. The DNA encoding TAT is initially
amplified using selected PCR primers. The primers will contain
restriction enzyme sites which correspond to the restriction enzyme
sites on the selected expression vector, and other useful sequences
providing for efficient and reliable translation initiation, rapid
purification on a metal chelation column, and proteolytic removal
with enterokinase. The PCR-amplified, poly-His tagged sequences are
then ligated into an expression vector, which is used to transform
an E. coli host based on strain 52 (W3110 fuhA(tonA) Ion galE
rpoHts(htpRts) clpP(lacIq). Transformants are first grown in LB
containing 50 mg/ml carbenicillin at 30.degree. C. with shaking
until an O.D. 600 of 3-5 is reached. Cultures are then diluted
50-100 fold into CRAP media (prepared by mixing 3.57 g
(NH.sub.4).sub.2SO.sub.4, 0.71 g sodium citrate.2H.sub.2O, 1.07 g
KCl, 5.36 g Difco yeast extract, 5.36 g Sheffield hycase SF in 500
mL water, as well as 110 mM MPOS, pH 7.3, 0.55% (w/v) glucose and 7
mM MgSO.sub.4) and grown for approximately 20-30 hours at
30.degree. C. with shaking. Samples are removed to verify
expression by SDS-PAGE analysis, and the bulk culture is
centrifuged to pellet the cells. Cell pellets are frozen until
purification and refolding.
[0806] E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets)
is resuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH
8 buffer. Solid sodium sulfite and sodium tetrathionate is added to
make final concentrations of 0.1M and 0.02 M, respectively, and the
solution is stirred overnight at 4.degree. C. This step results in
a denatured protein with all cysteine residues blocked by
sulfitolization. The solution is centrifuged at 40,000 rpm in a
Beckman Ultracentifuge for 30 min. The supernatant is diluted with
3-5 volumes of metal chelate column buffer (6 M guanidine, 20 mM
Tris, pH 7.4) and filtered through 0.22 micron filters to clarify.
The clarified extract is loaded onto a 5 ml Qiagen Ni-NTA metal
chelate column equilibrated in the metal chelate column buffer. The
column is washed with additional buffer containing 50 mM imidazole
(Calbiochem, Utrol grade), pH 7.4. The protein is eluted with
buffer containing 250 mM imidazole. Fractions containing the
desired protein are pooled and stored at 4.degree. C. Protein
concentration is estimated by its absorbance at 280 nm using the
calculated extinction coefficient based on its amino acid
sequence.
[0807] The proteins are refolded by diluting the sample slowly into
freshly prepared refolding buffer consisting of: 20 mM Tris, pH
8.6, 0.3 M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM
EDTA. Refolding volumes are chosen so that the final protein
concentration is between 50 to 100 micrograms/ml. The refolding
solution is stirred gently at 4.degree. C. for 12-36 hours. The
refolding reaction is quenched by the addition of TFA to a final
concentration of 0.4% (pH of approximately 3). Before further
purification of the protein, the solution is filtered through a
0.22 micron filter and acetonitrile is added to 2-10% final
concentration. The refolded protein is chromatographed on a Poros
R1/H reversed phase column using a mobile buffer of 0.1% TFA with
elution with a gradient of acetonitrile from 10 to 80%. Aliquots of
fractions with A280 absorbance are analyzed on SDS polyacrylamide
gels and fractions containing homogeneous refolded protein are
pooled. Generally, the properly refolded species of most proteins
are eluted at the lowest concentrations of acetonitrile since those
species are the most compact with their hydrophobic interiors
shielded from interaction with the reversed phase resin. Aggregated
species are usually eluted at higher acetonitrile concentrations.
In addition to resolving misfolded forms of proteins from the
desired form, the reversed phase step also removes endotoxin from
the samples.
[0808] Fractions containing the desired folded TAT polypeptide are
pooled and the acetonitrile removed using a gentle stream of
nitrogen directed at the solution. Proteins are formulated into 20
mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by
dialysis or by gel filtration using G25 Superfine (Pharmacia)
resins equilibrated in the formulation buffer and sterile
filtered.
[0809] Certain of the TAT polypeptides disclosed herein have been
successfully expressed and purified using this technique(s).
Example 8
Expression of TAT in Mammalian Cells
[0810] This example illustrates preparation of a potentially
glycosylated form of TAT by recombinant expression in mammalian
cells.
[0811] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989),
is employed as the expression vector. Optionally, the TAT DNA is
ligated into pRK5 with selected restriction enzymes to allow
insertion of the TAT DNA using ligation methods such as described
in Sambrook et al., supra. The resulting vector is called
pRK5-TAT.
[0812] In one embodiment, the selected host cells may be 293 cells.
Human 293 cells (ATCC CCL 1573) are grown to confluence in tissue
culture plates in medium such as DMEM supplemented with fetal calf
serum and optionally, nutrient components and/or antibiotics. About
10 .mu.g pRK5-TAT DNA is mixed with about 1 .mu.g DNA encoding the
VA RNA gene [Thimmappaya et al., Cell, 31:543 (1982)] and dissolved
in 500 .mu.l of 1 mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl.sub.2. To
this mixture is added, dropwise, 500 .mu.l of 50 mM HEPES (pH
7.35), 280 mM NaCl, 1.5 mM NaPO.sub.4, and a precipitate is allowed
to form for 10 minutes at 25.degree. C. The precipitate is
suspended and added to the 293 cells and allowed to settle for
about four hours at 37.degree. C. The culture medium is aspirated
off and 2 ml of 20% glycerol in PBS is added for 30 seconds. The
293 cells are then washed with serum free medium, fresh medium is
added and the cells are incubated for about 5 days.
[0813] Approximately 24 hours after the transfections, the culture
medium is removed and replaced with culture medium (alone) or
culture medium containing 200 .mu.Ci/ml .sup.35S-cysteine and 200
.mu.Ci/ml .sup.35S-methionine. After a 12 hour incubation, the
conditioned medium is collected, concentrated on a spin filter, and
loaded onto a 15% SDS gel. The processed gel may be dried and
exposed to film for a selected period of time to reveal the
presence of TAT polypeptide. The cultures containing transfected
cells may undergo further incubation (in serum free medium) and the
medium is tested in selected bioassays.
[0814] In an alternative technique, TAT may be introduced into 293
cells transiently using the dextran sulfate method described by
Somparyrac et al., Proc. Natl. Acad. Sci., 12:7575 (1981). 293
cells are grown to maximal density in a spinner flask and 700 .mu.g
pRK5-TAT DNA is added. The cells are first concentrated from the
spinner flask by centrifugation and washed with PBS. The
DNA-dextran precipitate is incubated on the cell pellet for four
hours. The cells are treated with 20% glycerol for 90 seconds,
washed with tissue culture medium, and re-introduced into the
spinner flask containing tissue culture medium, 5 .mu.g/ml bovine
insulin and 0.1 .mu.g/ml bovine transferrin. After about four days,
the conditioned media is centrifuged and filtered to remove cells
and debris. The sample containing expressed TAT can then be
concentrated and purified by any selected method, such as dialysis
and/or column chromatography.
[0815] In another embodiment, TAT can be expressed in CHO cells.
The pRK5-TAT can be transfected into CHO cells using known reagents
such as CaPO.sub.4 or DEAE-dextran. As described above, the cell
cultures can be incubated, and the medium replaced with culture
medium (alone) or medium containing a radiolabel such as
.sup.35S-methionine. After determining the presence of TAT
polypeptide, the culture medium may be replaced with serum free
medium. Preferably, the cultures are incubated for about 6 days,
and then the conditioned medium is harvested. The medium containing
the expressed TAT can then be concentrated and purified by any
selected method.
[0816] Epitope-tagged TAT may also be expressed in host CHO cells.
The TAT may be subcloned out of the pRK5 vector. The subclone
insert can undergo PCR to fuse in frame with a selected epitope tag
such as it poly-his tag into a Baculovirus expression vector. The
poly-his tagged TAT insert can then be subcloned into a SV40 driven
vector containing a selection marker such as DHFR for selection of
stable clones. Finally, the CHO cells can be transfected (as
described above) with the SV40 driven vector Labeling may be
performed, as described above, to verify expression. The culture
medium containing the expressed poly-His tagged TAT can then be
concentrated and purified by any selected method, such as by
Ni.sup.2+-chelate affinity chromatography.
[0817] TAT may also be expressed in CHO and/or COS cells by a
transient expression procedure or in CHO cells by another stable
expression procedure.
[0818] Stable expression in CHO cells is performed using the
following procedure. The proteins are expressed as an IgG construct
(immunoadhesin), in which the coding sequences for the soluble
forms (e.g. extracellular domains) of the respective proteins are
fused to an IgG1 constant region sequence containing the hinge, CH2
and CH2 domains and/or is a poly-His tagged form.
[0819] Following PCR amplification, the respective DNAs are
subcloned in a CHO expression vector using standard techniques as
described in Ausubel et al., Current Protocols of Molecular
Biology, Unit 3.16, John Wiley and Sons (1997). CHO expression
vectors are constructed to have compatible restriction sites 5' and
3' of the DNA of interest to allow the convenient shuttling of
cDNA's. The vector used expression in CHO cells is as described in
Lucas et al., Nucl. Acids Res. 24:9 (1774-1779 (1996), and uses the
SV40 early promoter/enhancer to drive expression of the cDNA of
interest and dihydrofolate reductase (DHFR). DHFR expression
permits selection for stable maintenance of the plasmid following
transfection.
[0820] Twelve micrograms of the desired plasmid DNA is introduced
into approximately 10 million CHO cells using commercially
available transfection reagents Superfect.RTM. (Quiagen),
Dosper.RTM. or Fugene.RTM. (Boehringer Mannheim). The cells are
grown as described in Lucas et al., supra. Approximately
3.times.10.sup.7 cells are frozen in an ampule for further growth
and production as described below.
[0821] The ampules containing the plasmid DNA are thawed by
placement into water bath and mixed by vortexing. The contents are
pipetted into a centrifuge tube containing 10 mLs of media and
centrifuged at 1000 rpm for 5 minutes. The supernatant is aspirated
and the cells are resuspended in 10 mL of selective media (0.2
.mu.m filtered PS20 with 5% 0.2 .mu.m diafiltered fetal bovine
serum). The cells are then aliquoted into a 100 mL spinner
containing 90 mL of selective media. After 1-2 days, the cells are
transferred into a 250 mL spinner filled with 150 mL selective
growth medium and incubated at 37.degree. C. After another 2-3
days, 250 mL, 500 mL and 2000 mL spinners are seeded with
3.times.10.sup.5 cells/mL. The cell media is exchanged with fresh
media by centrifugation and resuspension in production medium.
Although any suitable CHO media may be employed, a production
medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992
may actually be used. A 3 L production spinner is seeded at
1.2.times.10.sup.6 cells/mL. On day 0, the cell number pH is
determined. On day 1, the spinner is sampled and sparging with
filtered air is commenced. On day 2, the spinner is sampled, the
temperature shifted to 33.degree. C., and 30 mL of 500 g/L glucose
and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane
emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout
the production, the pH is adjusted as necessary to keep it at
around 7.2. After 10 days, or until the viability dropped below
70%, the cell culture is harvested by centrifugation and filtering
through a 0.22 .mu.m filter. The filtrate was either stored at
4.degree. C. or immediately loaded onto columns for
purification.
[0822] For the poly-His tagged constructs, the proteins are
purified using a Ni-NTA column (Qiagen). Before purification,
imidazole is added to the conditioned media to a concentration of 5
mM. The conditioned media is pumped onto a 6 ml Ni-NTA column
equilibrated in 20 mM Hepes. pH 7.4, buffer containing 0.3 M NaCl
and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4.degree. C.
After loading, the column is washed with additional equilibration
buffer and the protein eluted with equilibration buffer containing
0.25 M imidazole. The highly purified protein is subsequently
desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl
and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia)
column and stored at -80.degree. C.
[0823] Immunoadhesin (Fc-containing) constructs are purified from
the conditioned media as follows. The conditioned medium is pumped
onto a 5 ml Protein A column (Pharmacia) which had been
equilibrated in 20 mM Na phosphate buffer, pH 6.8. After loading,
the column is washed extensively with equilibration buffer before
elution with 100 mM citric acid, pH 3.5. The eluted protein is
immediately neutralized by collecting 1 ml fractions into tubes
containing 275 .mu.L of 1 M Tris buffer, pH 9. The highly purified
protein is subsequently desalted into storage buffer as described
above for the poly-His tagged proteins. The homogeneity is assessed
by SDS polyacrylamide gels and by N-terminal amino acid sequencing
by Edman degradation.
[0824] Certain of the TAT polypeptides disclosed herein have been
successfully expressed and purified using this technique(s).
Example 9
Expression of TAT in Yeast
[0825] The following method describes recombinant expression of TAT
in yeast.
[0826] First, yeast expression vectors are constructed for
intracellular production or secretion of TAT from the ADH2/GAPDH
promoter. DNA encoding TAT and the promoter is inserted into
suitable restriction enzyme sites in the selected plasmid to direct
intracellular expression of TAT. For secretion, DNA encoding TAT
can be cloned into the selected plasmid, together with DNA encoding
the ADH2/GAPDH promoter, a native TAT signal peptide or other
mammalian signal peptide, or, for example, a yeast alpha-factor or
invertase secretory signal/leader sequence, and linker sequences
(if needed) for expression of TAT.
[0827] Yeast cells, such as yeast strain AB110, can then be
transformed with the expression plasmids described above and
cultured in selected fermentation media. The transformed yeast
supernatants can be analyzed by precipitation with 10%
trichloroacetic acid and separation by SDS-PAGE, followed by
staining of the gels with Coomassie Blue stain.
[0828] Recombinant TAT can subsequently be isolated and purified by
removing the yeast cells from the fermentation medium by
centrifugation and then concentrating the medium using selected
cartridge filters. The concentrate containing TAT may further be
purified using selected column chromatography resins.
[0829] Certain of the TAT polypeptides disclosed herein have been
successfully expressed and purified using this technique(s).
Example 10
Expression of TAT in Baculovirus-Infected Insect Cells
[0830] The following method describes recombinant expression of TAT
in Baculovirus-infected insect cells.
[0831] The sequence coding for TAT is fused upstream of an epitope
tag contained within a baculovirus expression vector. Such epitope
tags include poly-his tags and immunoglobulin tags (like Fc regions
of IgG). A variety of plasmids may be employed, including plasmids
derived from commercially available plasmids such as pVL1393
(Novagen). Briefly, the sequence encoding TAT or the desired
portion of the coding sequence of TA T such as the sequence
encoding an extracellular domain of a transmembrane protein or the
sequence encoding the mature protein if the protein is
extracellular is amplified by PCR with primers complementary to the
5' and 3' regions. The 5' primer may incorporate flanking
(selected) restriction enzyme sites. The product is then digested
with those selected restriction enzymes and subcloned into the
expression vector.
[0832] Recombinant baculovirus is generated by co-transfecting the
above plasmid and BaculoGold.TM. virus DNA (Pharmingen) into
Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using
lipofectin (commercially available from GIBCO-BRL). After 4-5 days
of incubation at 28.degree. C., the released viruses are harvested
and used for further amplifications. Viral infection and protein
expression are performed as described by O'Reilley et al.,
Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford
University Press (1994).
[0833] Expressed poly-his tagged TAT can then be purified, for
example, by Ni.sup.2+-chelate affinity chromatography as follows.
Extracts are prepared from recombinant virus-infected Sf9 cells as
described by Rupert et al., Nature, 362:175-179 (1993). Briefly,
Sf9 cells are washed, resuspended in sonication buffer (25 mL
Hepes, pH 7.9; 12.5 mM MgCl.sub.2; 0.1 mM EDTA; 10% glycerol; 01.1%
NP-40; 0.4 M KCl), and sonicated twice for 20 seconds on ice. The
sonicates are cleared by centrifugation, and the supernatant is
diluted 50-fold in loading buffer (50 mM phosphate, 300 mM NaCl,
10% glycerol, pH 7.8) and filtered through a 0.45 .mu.m filter. A
Ni.sup.2+-NTA agarose column (commercially available from Qiagen)
is prepared with a bed volume of 5 mL, washed with 25 mL of water
and equilibrated with 25 mL of loading buffer. The filtered cell
extract is loaded onto the column at 0.5 mL per minute. The column
is washed to baseline n with loading buffer, at which point
fraction collection is started. Next, the column is washed with a
secondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol,
pH 6.0), which elutes nonspecifically bound protein. After reaching
A.sub.280 baseline again, the column is developed with a 0 to 500
mM Imidazole gradient in the secondary wash buffer. One mL
fractions are collected and analyzed by SDS-PAGE and silver
staining or Western blot with Ni.sup.2+-NTA-conjugated to alkaline
phosphatase (Qiagen). Fractions containing the eluted
His.sub.10-tagged TAT are pooled and dialyzed against loading
buffer.
[0834] Alternatively, purification of the IgG tagged (or Fc tagged)
TAT can be performed using known chromatography techniques,
including for instance, Protein A or protein G column
chromatography.
[0835] Certain of the TAT polypeptides disclosed herein have been
successfully expressed and purified using this technique(s).
Example 11
Preparation of Antibodies that Bind Tat
[0836] This example illustrates preparation of monoclonal
antibodies which can specifically bind TAT.
[0837] Techniques for producing the monoclonal antibodies are known
in the art and are described, for instance, in Goding, supra.
Immunogens that may be employed include purified TAT, fusion
proteins containing TAT, and cells expressing recombinant TAT on
the cell surface. Selection of the immunogen can be made by the
skilled artisan without undue experimentation.
[0838] Mice, such as Balb/c, are immunized with the TAT immunogen
emulsified in complete Freund's adjuvant and injected
subcutaneously or intraperitoneally in an amount from 1-100
micrograms. Alternatively, the immunogen is emulsified in MPL-TDM
adjuvant (Ribi Immunochemical Research, Hamilton, MIT) and injected
into the animal's hind foot pads. The immunized mice are then
boosted 10 to 12 days later with additional immunogen emulsified in
the selected adjuvant. Thereafter, for several weeks, the mice may
also be boosted with additional immunization injections. Serum
samples may be periodically obtained from the mice by retro-orbital
bleeding for testing in ELISA assays to detect anti-TAT
antibodies.
[0839] After a suitable antibody titer has been detected, the
animals "positive" for antibodies can be injected with a final
intravenous injection of TAT. Three to four days later, the mice
are sacrificed and the spleen cells are harvested. The spleen cells
are then fused (using 35% polyethylene glycol) to a selected murine
myeloma cell line such as P3X63AgU.1, available from ATCC, No. CRL
1597. The fusions generate hybridoma cells which can then be plated
in 96 well tissue culture plates containing HAT (hypoxanthine,
aminopterin, and thymidine) medium to inhibit proliferation of
non-fused cells, myeloma hybrids, and spleen cell hybrids.
[0840] The hybridoma cells will be screened in an ELISA for
reactivity against TAT. Determination of "positive" hybridoma cells
secreting the desired monoclonal antibodies against TAT is within
the skill in the art.
[0841] The positive hybridoma cells can be injected
intraperitoneally into syngeneic Balb/c mice to produce ascites
containing the anti-TAT monoclonal antibodies. Alternatively, the
hybridoma cells can be grown in tissue culture flasks or roller
bottles. Purification of the monoclonal antibodies produced in the
ascites can be accomplished using ammonium sulfate precipitation,
followed by gel exclusion chromatography. Alternatively, affinity
chromatography based upon binding of antibody to protein A or
protein G can be employed.
Example 12
Purification of TAT Polypeptides Using Specific Antibodies
[0842] Native or recombinant TAT polypeptides may be purified by a
variety of standard techniques in the art of protein purification.
For example, pro-TAT polypeptide, mature TAT polypeptide, or
pre-TAT polypeptide is purified by immunoaffinity chromatography
using antibodies specific for the TAT polypeptide of interest. In
general, an immunoaffinity column is constructed by covalently
coupling the anti-TAT polypeptide antibody to an activated
chromatographic resin.
[0843] Polyclonal immunoglobulins are prepared from immune sera
either by precipitation with ammonium sulfate or by purification on
immobilized Protein A (Pharmacia LKB Biotechnology, Piscataway,
N.J.). Likewise, monoclonal antibodies are prepared from mouse
ascites fluid by ammonium sulfate precipitation or chromatography
on immobilized Protein A. Partially purified immunoglobulin is
covalently attached to a chromatographic resin such as
CnBr-activated SEPHAROSE.TM. (Pharmacia LKB Biotechnology). The
antibody is coupled to the resin, the resin is blocked, and the
derivative resin is washed according to the manufacturer's
instructions.
[0844] Such an immunoaffinity column is utilized in the
purification of TAT polypeptide by preparing a fraction from cells
containing TAT polypeptide in a soluble form. This preparation is
derived by solubilization of the whole cell or of a subcellular
fraction obtained via differential centrifugation by the addition
of detergent or by other methods well known in the art.
Alternatively, soluble TAT polypeptide containing a signal sequence
may be secreted in useful quantity into the medium in which the
cells are grown.
[0845] A soluble TAT polypeptide-containing preparation is passed
over the immunoaffinity column, and the column is washed under
conditions that allow the preferential absorbance of TAT
polypeptide (e.g., high ionic strength buffers in the presence of
detergent). Then, the column is eluted under conditions that
disrupt antibody/TAT polypeptide binding (e.g., a low pH buffer
such as approximately pH 2-3, or a high concentration of a
chaotrope such as urea or thiocyanate ion), and TAT polypeptide is
collected.
Example 13
In Vitro Tumor Cell Killing Assay
[0846] Mammalian cells expressing the TAT polypeptide of interest
may be obtained using standard expression vector and cloning
techniques. Alternatively, many tumor cell lines expressing TAT
polypeptides of interest are publicly available, for example,
through the ATCC and can be routinely identified using standard
ELISA or FACS analysis. Anti-TAT polypeptide monoclonal antibodies
(and toxin conjugated derivatives thereof) may then be employed in
assays to determine the ability of the antibody to kill TAT
polypeptide expressing cells in vitro.
[0847] For example, cells expressing the TAT polypeptide of
interest are obtained as described above and plated into 96 well
dishes. In one analysis, the antibody/toxin conjugate (or naked
antibody) is included throughout the cell incubation for a period
of 4 days. In a second independent analysis, the cells are
incubated for 1 hour with the antibody/toxin conjugate (or naked
antibody) and then washed and incubated in the absence of
antibody/toxin conjugate for a period of 4 days. Cell viability is
then measured using the CellTiter-Glo Luminescent Cell Viability
Assay from Promega (Cat# G7571). Untreated cells serve as a
negative control.
Example 14
In Vivo Tumor Cell Killing Assay
[0848] To test the efficacy of conjugated or unconjugated anti-TAT
polypeptide monoclonal antibodies, anti-TAT antibody is injected
intraperitoneally into nude mice 24 hours prior to receiving tumor
promoting cells subcutaneously in the flank. Antibody injections
continue twice per week for the remainder of the study. Tumor
volume is then measured twice per week.
[0849] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the construct deposited, since the deposited embodiment is intended
as a single illustration of certain aspects of the invention and
any constructs that are functionally equivalent are within the
scope of this invention. The deposit of material herein does not
constitute an admission that the written description herein
contained is inadequate to enable the practice of any aspect of the
invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Sequence CWU 1
1
3212442DNAHomo sapien 1gagcgagagg ccgggggtgc cgagccgggc ggggagagct
gggccgggag 50agcagaacag ggaggctaga gcgcagcggg aaccggcccg gagccggagc
100cggagcccca caggcaccta ctaaaccgcc cagccgatcg gcccccacag
150agtggcccgc gggcctccgg ccgggcccag tcccctcccg ggccctccat
200ggcccgggcc gctgccctcc tgccgtcgag atcgccgccg acgccgctgc
250tgtggccgct gctgctgctg ctgctcctgg aaaccggagc ccaggatgtg
300cgagttcaag tgctacccga ggtgcgaggc cagctcgggg gcaccgtgga
350gctgccgtgc cacctgctgc cacctgttcc tggactgtac atctccctgg
400tgacctggca gcgcccagat gcacctgcga accaccagaa tgtggccgcc
450ttccacccta agatgggtcc cagcttcccc agcccgaagc ctggcagcga
500gcggctgtcc ttcgtctctg ccaagcagag cactgggcaa gacacagagg
550cagagctcca ggacgccacg ctggccctcc acgggctcac ggtggaggac
600gagggcaact acacttgcga gtttgccacc ttccccaagg ggtccgtccg
650agggatgacc tggctcagag tcatagccaa gcccaagaac caagctgagg
700cccagaaggt cacgttcagc caggacccta cgacagtggc cctctgcatc
750tccaaagagg gccgcccacc tgcccggatc tcctggctct catccctgga
800ctgggaagcc aaagagactc aggtgtcagg gaccctggcc ggaactgtca
850ctgtcaccag ccgcttcacc ttggtgccct cgggccgagc agatggtgtc
900acggtcacct gcaaagtgga gcatgagagc ttcgaggaac cagccctgat
950acctgtgacc ctctctgtac gctaccctcc tgaagtgtcc atctccggct
1000atgatgacaa ctggtacctc ggccgtactg atgccaccct gagctgtgac
1050gtccgcagca acccagagcc cacgggctat gactggagca cgacctcagg
1100caccttcccg acctccgcag tggcccaggg ctcccagctg gtcatccacg
1150cagtggacag tctgttcaat accaccttcg tctgcacagt caccaatgcc
1200gtgggcatgg gccgcgctga gcaggtcatc tttgtccgag agacccccaa
1250cacagcaggc gcaggggcca caggcggcat catcgggggc atcatcgccg
1300ccatcattgc tactgctgtg gctgccacgg gcatccttat ctgccggcag
1350cagcggaagg agcagacgct gcagggggca gaggaggacg aagacctgga
1400gggacctccc tcctacaagc caccaacccc aaaagcgaag ctggaggcac
1450aggagatgcc ctcccagctc ttcactctgg gggcctcgga gcacagccca
1500ctcaagaccc cctactttga tgctggcgcc tcatgcactg agcaggaaat
1550gcctcgatac catgagctgc ccaccttgga agaacggtca ggacccttgc
1600accctggagc cacaagcctg gggtccccca tcccggtgcc tccagggcca
1650cctgctgtgg aagacgtttc cctggatcta gaggatgagg agggggagga
1700ggaggaagag tatctggaca agatcaaccc catctatgat gctctgtcct
1750atagcagccc ctctgattcc taccagggca aaggctttgt catgtcccgg
1800gccatgtatg tgtgagctgc catgcgcctg gcgtctcaca tctcacctgt
1850tgatccctta gctttcttgc caaggatcta gtgccccctg acctctggcc
1900aggccactgt cagttaacac atatgcattc catttgtgat gtctaccttg
1950gtggctccac tatgacccct aacccatgag cccagagaaa ttcaccgtga
2000taatggaatc ctggcaacct tatctcatga ggcaggaggt ggggaaggtg
2050cttctgcaca acctctgatc ccaaggactc ctctcccaga ctgtgacctt
2100agaccatacc tctcaccccc caatgcctcg actcccccaa aatcacaaag
2150aagaccctag acctataatt tgtcttcagg tagtaaattc ctgcctacca
2200agcaagcagc cccagcctag ggtcagacag ggtgagcctc atacagactg
2250tgccttgatg gccccagcct tgggagaaga atttactgtt aacctggaag
2300actactgaat cattttaccc ttgcccagtg gaataggacc taaacatccc
2350ccttccgggg aaagtgggtc atctgaattg ggggtagcaa ttgatactgt
2400tttgtaaact acatttccta caaaatatga atttatactt tg 24422993DNAHomo
sapien 2atgatcaccc tgaacaatca agatcaacct gtcactttta acagctcaca
50tccagatgaa tacaaaattg cagcccttgt cttctatagc tgtatcttca
100taattggatt atttgttaac atcactgcat tatgggtttt cagttgtacc
150accaagaaga gaaccacggt aaccatctat atgatgaatg tggcattagt
200ggacttgata tttataatga ctttaccctt tcgaatgttt tattatgcaa
250aagatgcatg gccatttgga gagtacttct gccagattat tggagctctc
300acagtgtttt acccaagcat tgctttatgg cttcttgcct ttattagtgc
350tgacagatac atggccattg tacagccgaa gtacgccaaa gaacttaaaa
400acacgtgcaa agccgtgctg gcgtgtgtgg gagtctggat aatgaccctg
450accacgacca cccctctgct actgctctat aaagacccag ataaagactc
500cactcccgcc acctgcctca agatttctga catcatctat ctaaaagctg
550tgaacgtgct gaacctcact cgactgacat tttttttctt gattcctttg
600ttcatcatga ttgggtgcta cttggtcatt attcataatc tccttcacgg
650caggacgtct aagctgaaac ccaaagtcaa ggagaagtcc ataaggatca
700tcatcacgct gctggtgcag gtgctcgtct gctttatgcc cttccacatc
750tgtttcgctt tcctgatgct gggaacgggg gagaacagtt acaatccctg
800gggagccttt accaccttcc tcatgaacct cagcacgtgt ctggatgtga
850ttctctacta catcgtttca aaacaatttc aggctcgagt cattagtgtc
900atgctatacc gtaattacct tcgaagcctg cgcagaaaaa gtttccgatc
950tggtagtcta aggtcactaa gcaatataaa cagtgaaatg tta 99331107DNAHomo
sapien 3tgctgcaact caaactaacc aacccactgg gagaagatgc ctgggggtcc
50aggagtcctc caagctctgc ctgccaccat cttcctcctc ttcctgctgt
100ctgctgtcta cctgggccct gggtgccagg ccctgtggat gcacaaggtc
150ccagcatcat tgatggtgag cctgggggaa gacgcccact tccaatgccc
200gcacaatagc agcaacaacg ccaacgtcac ctggtggcgc gtcctccatg
250gcaactacac gtggccccct gagttcttgg gcccgggcga ggaccccaat
300ggtacgctga tcatccagaa tgtgaacaag agccatgggg gcatatacgt
350gtgccgggtc caggagggca acgagtcata ccagcagtcc tgcggcacct
400acctccgcgt gcgccagccg ccccccaggc ccttcctgga catgggggag
450ggcaccaaga accgaatcat cacagccgag gggatcatcc tcctgttctg
500cgcggtggtg cctgggacgc tgctgctgtt caggaaacga tggcagaacg
550agaagctcgg gttggatgcc ggggatgaat atgaagatga aaacctttat
600gaaggcctga acctggacga ctgctccatg tatgaggaca tctcccgggg
650cctccagggc acctaccagg atgtgggcag cctcaacata ggagatgtcc
700agctggagaa gccgtgacac ccctactcct gccaggctgc ccccgcctgc
750tgtgcaccca gctccagtgt ctcagctcac ttccctggga cattctcctt
800tcagcccttc tgggggcttc cttagtcata ttcccccagt ggggggtggg
850agggtaacct cactcttctc caggccaggc ctccttggac tcccctgggg
900gtgtcccact cttcttccct ctaaactgcc ccacctccta acctaatccc
950cacgccccgc tgcctttccc aggctcccct cacccagcgg gtaatgagcc
1000cttaatcgct gcctctaggg gagctgattg tagcagcctc gttagtgtca
1050ccccctcctc cctgatctgt cagggccact tagtgataat aaattcttcc
1100caactgc 110741524DNAHomo sapien 4agcagacaga ggactctcat
taaggaaggt gtcctgtgcc ctgaccctac 50aagatgccaa gagaagatgc tcacttcatc
tatggttacc ccaagaaggg 100gcacggccac tcttacacca cggctgaaga
ggccgctggg atcggcatcc 150tgacagtgat cctgggagtc ttactgctca
tcggctgttg gtattgtaga 200agacgaaatg gatacagagc cttgatggat
aaaagtcttc atgttggcac 250tcaatgtgcc ttaacaagaa gatgcccaca
agaagggttt gatcatcggg 300acagcaaagt gtctcttcaa gagaaaaact
gtgaacctgt ggttcccaat 350gctccacctg cttatgagaa actctctgca
gaacagtcac caccacctta 400ttcaccttaa gagccagcga gacacctgag
acatgctgaa attatttctc 450tcacactttt gcttgaattt aatacagaca
tctaatgttc tcctttggaa 500tggtgtagga aaaatgcaag ccatctctaa
taataagtca gtgttaaaat 550tttagtaggt ccgctagcag tactaatcat
gtgaggaaat gatgagaaat 600attaaattgg gaaaactcca tcaataaatg
ttgcaatgca tgatactatc 650tgtgccagag gtaatgttag taaatccatg
gtgttatttt ctgagagaca 700gaattcaagt gggtattctg gggccatcca
atttctcttt acttgaaatt 750tggctaataa caaactagtc aggttttcga
accttgaccg acatgaactg 800tacacagaat tgttccagta ctatggagtg
ctcacaaagg atacttttac 850aggttaagac aaagggttga ctggcctatt
tatctgatca agaacatgtc 900agcaatgtct ctttgtgctc taaaattcta
ttatactaca ataatatatt 950gtaaagatcc tatagctctt tttttttgag
atggagtttc gcttttgttg 1000cccaggctgg agtgcaatgg cgcgatcttg
gctcaccata acctccgcct 1050cccaggttca agcaattctc ctgccttagc
ctcctgagta gctgggatta 1100caggcgtgcg ccactatgcc tgactaattt
tgtagtttta gtagagacgg 1150ggtttctcca tgttggtcag gctggtctca
aactcctgac ctcaggtgat 1200ctgcccgcct cagcctccca aagtgctgga
attacaggcg tgagccacca 1250cgcctggctg gatcctatat cttaggtaag
acatataacg cagtctaatt 1300acatttcact tcaaggctca atgctattct
aactaatgac aagtattttc 1350tactaaacca gaaattggta gaaggattta
aataagtaaa agctactatg 1400tactgcctta gtgctgatgc ctgtgtactg
ccttaaatgt acctatggca 1450atttagctct cttgggttcc caaatccctc
tcacaagaat gtgcagaaga 1500aatcataaag gatcagagat tctg
152451125DNAHomo sapien 5gtctccccca ctgtcagcac ctcttctgtg
tggtgagtgg accgcttacc 50ccactaggtg aagatgtcag cccaggagag ctgcctcagc
ctcatcaagt 100acttcctctt cgttttcaac ctcttcttct tcgtcctcgg
cagcctgatc 150ttctgcttcg gcatctggat cctcatcgac aagaccagct
tcgtgtcctt 200tgtgggcttg gccttcgtgc ctctgcagat ctggtccaaa
gtcctggcca 250tctcaggaat cttcaccatg ggcatcgccc tcctgggttg
tgtgggggcc 300ctcaaggagc tccgctgcct cctgggcctg tattttggga
tgctgctgct 350cctgtttgcc acacagatca ccctgggaat cctcatctcc
actcagcggg 400cccagctgga gcgaagcttg cgggacgtcg tagagaaaac
catccaaaag 450tacggcacca accccgagga gaccgcggcc gaggagagct
gggactatgt 500gcagttccag ctgcgctgct gcggctggca ctacccgcag
gactggttcc 550aagtcctcat cctgagaggt aacgggtcgg aggcgcaccg
cgtgccctgc 600tcctgctaca acttgtcggc gaccaacgac tccacaatcc
tagataaggt 650gatcttgccc cagctcagca ggcttggaca cctggcgcgg
tccagacaca 700gtgcagacat ctgcgctgtc cctgcagaga gccacatcta
ccgcgagggc 750tgcgcgcagg gcctccagaa gtggctgcac aacaacctta
tttccatagt 800gggcatttgc ctgggcgtcg gcctactcga gctcgggttc
atgacgctct 850cgatattcct gtgcagaaac ctggaccacg tctacaaccg
gctcgctcga 900taccgttagg ccccgccctc cccaaagtcc cgccccgccc
ccgtcacgtg 950cgctgggcac ttccctgctg cctgtaaata tttgtttaat
ccccagttcg 1000cctggagccc tccgccttca cattcccctg gggacccacg
tggctgcgtg 1050cccctgctgc tgtcacctct cccacgggac ctggggcttt
cgtccacagc 1100ttcctgtccc catctgtcgg cctac 112564523DNAHomo sapien
6ccgcggtgcc gccgggaaag atggtcgtgg cgctgcggta cgtgtggcct
50ctcctcctct gcagcccctg cctgcttatc cagatccccg aggaatatga
100aggacaccat gtgatggagc cacctgtcat cacggaacag tctccacggc
150gcctggttgt cttccccaca gatgacatca gcctcaagtg tgaggccagt
200ggcaagcccg aagtgcagtt ccgctggacg agggatggtg tccacttcaa
250acccaaggaa gagctgggtg tgaccgtgta ccagtcgccc cactctggct
300ccttcaccat cacgggcaac aacagcaact ttgctcagag gttccagggc
350atctaccgct gctttgccag caataagctg ggcaccgcca tgtcccatga
400gatccggctc atggccgagg gtgcccccaa gtggccaaag gagacagtga
450agcccgtgga ggtggaggaa ggggagtcag tggttctgcc ttgcaaccct
500cccccaagtg cagagcctct ccggatctac tggatgaaca gcaagatctt
550gcacatcaag caggacgagc gggtgacgat gggccagaac ggcaacctct
600actttgccaa tgtgctcacc tccgacaacc actcagacta catctgccac
650gcccacttcc caggcaccag gaccatcatt cagaaggaac ccattgacct
700ccgggtcaag gccaccaaca gcatgattga caggaagccg cgcctgctct
750tccccaccaa ctccagcagc cacctggtgg ccttgcaggg gcagccattg
800gtcctggagt gcatcgccga gggctttccc acgcccacca tcaaatggct
850gcgccccagt ggccccatgc cagctgaccg tgtcacctac cagaaccaca
900acaagaccct gcagctgctg aaagtgggcg aggaggatga tggcgagtac
950cgctgcctgg ccgagaactc actgggcagt gcccggcatg cgtactatgt
1000caccgtggag gctgccccgt actggctgca caagccccag agccatctat
1050atgggccagg agagactgcc cgcctggact gccaagtcca gggcaggccc
1100caaccagagg tcacctggag aatcaacggg atccctgtgg aggagctggc
1150caaagaccag aagtaccgga ttcagcgtgg cgccctgatc ctgagcaacg
1200tgcagcccag tgacacaatg gtgacccaat gtgaggcccg caaccggcac
1250gggctcttgc tggccaatgc ctacatctac gttgtccagc tgccagccaa
1300gatcctgact gcggacaatc agacgtacat ggctgtccag ggcagcactg
1350cctaccttct gtgcaaggcc ttcggagcgc ctgtgcccag tgttcagtgg
1400ctggacgagg atgggacaac agtgcttcag gacgaacgct tcttccccta
1450tgccaatggg accctgggca ttcgagacct ccaggccaat gacaccggac
1500gctacttctg cctggctgcc aatgaccaaa acaatgttac catcatggct
1550aacctgaagg ttaaagatgc aactcagatc actcaggggc cccgcagcac
1600aatcgagaag aaaggttcca gggtgacctt cacgtgccag gcctcctttg
1650acccctcctt gcagcccagc atcacctggc gtggggacgg tcgagacctc
1700caggagcttg gggacagtga caagtacttc atagaggatg ggcgcctggt
1750catccacagc ctggactaca gcgaccaggg caactacagc tgcgtggcca
1800gtaccgaact ggatgtggtg gagagtaggg cacagctctt ggtggtgggg
1850agccctgggc cggtgccacg gctggtgctg tccgacctgc acctgctgac
1900gcagagccag gtgcgcgtgt cctggagtcc tgcagaagac cacaatgccc
1950ccattgagaa atatgacatt gaatttgagg acaaggaaat ggcgcctgaa
2000aaatggtaca gtctgggcaa ggttccaggg aaccagacct ctaccaccct
2050caagctgtcg ccctatgtcc actacacctt tagggttact gccataaaca
2100aatatggccc cggggagccc agcccggtct ctgagactgt ggtcacacct
2150gaggcagccc cagagaagaa ccctgtggat gtgaaggggg aaggaaatga
2200gaccaccaat atggtcatca cgtggaagcc gctccggtgg atggactgga
2250acgcccccca ggttcagtac cgcgtgcagt ggcgccctca ggggacacga
2300gggccctggc aggagcagat tgtcagcgac cccttcctgg tggtgtccaa
2350cacgtccacc ttcgtgccct atgagatcaa agtccaggcc gtcaacagcc
2400agggcaaggg accagagccc caggtcacta tcggctactc tggagaggac
2450tacccccagg caatccctga gctggaaggc attgaaatcc tcaactcaag
2500tgccgtgctg gtcaagtggc ggccggtgga cctggcccag gtcaagggcc
2550acctccgcgg atacaatgtg acgtactgga gggagggcag tcagaggaag
2600cacagcaaga gacatatcca caaagaccat gtggtggtgc ccgccaacac
2650caccagtgtc atcctcagtg gcttgcggcc ctatagctcc taccacctgg
2700aggtgcaggc ctttaacggg cgaggatcgg ggcccgccag cgagttcacc
2750ttcagcaccc cagagggagt gcctggccac cccgaggcgt tgcacctgga
2800gtgccagtcg aacaccagcc tgctgctgcg ctggcagccc ccactcagcc
2850acaacggcgt gctcaccggc tacgtgctct cctaccaccc cctggatgag
2900gggggcaagg ggcaactgtc cttcaacctt cgggaccccg aacttcggac
2950acacaacctg accgatctca gcccccacct gcggtaccgc ttccagcttc
3000aggccaccac caaagagggc cctggtgaag ccatcgtacg ggaaggaggc
3050actatggcct tgtctgggat ctcagatttt ggcaacatct cagccacagc
3100gggtgaaaac tacagtgtcg tctcctgggt ccccaaggag ggccagtgca
3150acttcaggtt ccatatcttg ttcaaagcct tgggagaaga gaagggtggg
3200gcttcccttt cgccacagta tgtcagctac aaccagagct cctacacgca
3250gtgggacctg cagcctgaca ctgactacga gatccacttg tttaaggaga
3300ggatgttccg gcaccaaatg gctgtgaaga ccaatggcac aggccgcgtg
3350aggctccctc ctgctggctt cgccactgag ggctggttca tcggctttgt
3400gagtgccatc atcctcctgc tcctcgtcct gctcatcctc tgcttcatca
3450agcgcagcaa gggcggcaaa tactcagtga aggataagga ggacacccag
3500gtggactctg aggcccgacc gatgaaagat gagaccttcg gcgagtacag
3550gtccctggag agtgacaacg aggagaaggc ctttggcagc agccagccat
3600cgctcaacgg ggacatcaag cccctgggca gtgacgacag cctggccgat
3650tatgggggca gcgtggatgt tcagttcaac gaggatggtt cgttcattgg
3700ccagtacagt ggcaagaagg agaaggaggc ggcagggggc aatgacagct
3750caggggccac ttcccccatc aaccctgccg tggccctaga atagtggagt
3800acggacagga gatgctgtgc cccctggcct tgggatccag gcccctccct
3850ctccagcagg cccatgggag gctggagttg gggcagagga gaacttgctg
3900cctcggatcc ccttcctacc acccggtccc cactttattg ccaaaaccca
3950gctgcacccc ttcctgggca cacgctgctc tgccccagct tgggcagatc
4000tcccacatgc caggggcctt tgggtgctgt tttgccagcc catttgggca
4050gagaggctgt ggtttggggg agaagaagta ggggtggccc gaaaggtctc
4100cgaaatgctg tctttcttgc tccctgactg ggggcagaca tggtggggtc
4150tcctcaggac cagggttggc accttccccc tcccccagcc accactccag
4200cagcctggct gggactggga acagaactcg tgtccccacc atctgctgtc
4250ttttctttgc catctctgct ccaaccggga tggcagccgg gcaaactggc
4300cgcgggggca ggggaggcca tctggagagc ccagagtccc cccactccca
4350gcatcgcact ctggcagcac cgcctcttcc cgccgcccag cccaccccat
4400ggccggcttt caggagctcc atacacacgc tgccttcggt acccaccaca
4450caacatccaa gtggcctccg tcactacctg gctgcggggc gggcacacct
4500cctcccactg cccactggcc ggc 452374525DNAHomo sapien 7gcgcggtgcc
gccgggaaag atggtcgtgg cgctgcggta cgtgtggcct 50ctcctcctct gcagcccctg
cctgcttatc cagatccccg aggaatatga 100aggacaccat gtgatggagc
cacctgtcat cacggaacag tctccacggc 150gcctggttgt cttccccaca
gatgacatca gcctcaagtg tgaggccagt 200ggcaagcccg aagtgcagtt
ccgctggacg agggatggtg tccacttcaa 250acccaaggaa gagctgggtg
tgaccgtgta ccagtcgccc cactctggct 300ccttcaccat cacgggcaac
aacagcaact ttgctcagag gttccagggc 350atctaccgct gctttgccag
caataagctg ggcaccgcca tgtcccatga 400gatccggctc atggccgagg
gtgcccccaa gtggccaaag gagacagtga 450agcccgtgga ggtggaggaa
ggggagtcag tggttctgcc ttgcaaccct 500cccccaagtg cagagcctct
ccggatctac tggatgaaca gcaagatctt 550gcacatcaag caggacgagc
gggtgacgat gggccagaac
ggcaacctct 600actttgccaa tgtgctcacc tccgacaacc actcagacta
catctgccac 650gcccacttcc caggcaccag gaccatcatt cagaaggaac
ccattgacct 700ccgggtcaag gccaccaaca gcatgattga caggaagccg
cgcctgctct 750tccccaccaa ctccagcagc cacctggtgg ccttgcaggg
gcagccattg 800gtcctggagt gcatcgccga gggctttccc acgcccacca
tcaaatggct 850gcgccccagt ggccccatgc cagccgaccg tgtcacctac
cagaaccaca 900acaagaccct gcagctgctg aaagtgggcg aggaggatga
tggcgagtac 950cgctgcctgg ccgagaactc actgggcagt gcccggcatg
cgtactatgt 1000caccgtggag gctgccccgt actggctgca caagccccag
agccatctat 1050atgggccagg agagactgcc cgcctggact gccaagtcca
gggcaggccc 1100caaccagagg tcacctggag aatcaacggg atccctgtgg
aggagctggc 1150caaagaccag aagtaccgga ttcagcgtgg cgccctgatc
ctgagcaacg 1200tgcagcccag tgacacaatg gtgacccaat gtgaggcccg
caaccggcac 1250gggctcttgc tggccaatgc ctacatctac gttgtccagc
tgccagccaa 1300gatcctgact gcggacaatc agacgtacat ggctgtccag
ggcagcactg 1350cctaccttct gtgcaaggcc ttcggagcgc ctgtgcccag
tgttcagtgg 1400ctggacgagg atgggacaac agtgcttcag gacgaacgct
tcttccccta 1450tgccaatggg accctgggca ttcgagacct ccaggccaat
gacaccggac 1500gctacttctg cctggctgcc aatgaccaaa acaatgttac
catcatggct 1550aacctgaagg ttaaagatgc aactcagatc actcaggggc
cccgcagcac 1600aatcgagaag aaaggttcca gggtgacctt cacgtgccag
gcctcctttg 1650acccctcctt gcagcccagc atcacctggc gtggggacgg
tcgagacctc 1700caggagcttg gggacagtga caagtacttc atagaggatg
ggcgcctggt 1750catccacagc ctggactaca gcgaccaggg caactacagc
tgcgtggcca 1800gtaccgaact ggatgtggtg gagagtaggg cacagctctt
ggtggtgggg 1850agccctgggc cggtgccacg gctggtgctg tccgacctgc
acctgctgac 1900gcagagccag gtgcgcgtgt cctggagtcc tgcagaagac
cacaatgccc 1950ccattgagaa atatgacatt gaatttgagg acaaggaaat
ggcgcctgaa 2000aaatggtaca gtctgggcaa ggttccaggg aaccagacct
ctaccaccct 2050caagctgtcg ccctatgtcc actacacctt tagggttact
gccataaaca 2100aatatggccc cggggagccc agcccggtct ctgagactgt
ggtcacacct 2150gaggcagccc cagagaagaa ccctgtggat gtgaaggggg
aaggaaatga 2200gaccaccaat atggtcatca cgtggaagcc gctccggtgg
atggactgga 2250acgcccccca ggttcagtac cgcgtgcagt ggcgccctca
ggggacacga 2300gggccctggc aggagcagat tgtcagcgac cccttcctgg
tggtgtccaa 2350cacgtccacc ttcgtgccct atgagatcaa agtccaggcc
gtcaacagcc 2400agggcaaggg accagagccc caggtcacta tcggctactc
tggagaggac 2450tacccccagg caatccctga gctggaaggc attgaaatcc
tcaactcaag 2500tgccgtgctg gtcaagtggc ggccggtgga cctggcccag
gtcaagggcc 2550acctccgcgg atacaatgtg acgtactgga gggagggcag
tcagaggaag 2600cacagcaaga gacatatcca caaagaccat gtggtggtgc
ccgccaacac 2650caccagtgtc atcctcagtg gcttgcggcc ctatagctcc
taccacctgg 2700aggtgcaggc ctttaacggg cgaggatcgg ggcccgccag
cgagttcacc 2750ttcagcaccc cagagggagt gcctggccac cccgaggcgt
tgcacctgga 2800gtgccagtcg aacaccagcc tgctgctgcg ctggcagccc
ccactcagcc 2850acaacggcgt gctcaccggc tacgtgctct cctaccaccc
cctggatgag 2900gggggcaagg ggcaactgtc cttcaacctt cgggaccccg
aacttcggac 2950acacaacctg accgatctca gcccccacct gcggtaccgc
ttccagcttc 3000aggccaccac caaagagggc cctggtgaag ccatcgtacg
ggaaggaggc 3050actatggcct tgtctgggat ctcagatttt ggcaacatct
cagccacagc 3100gggtgaaaac tacagtgtcg tctcctgggt ccccaaggag
ggccagtgca 3150acttcaggtt ccatatcttg ttcaaagcct tgggagaaga
gaagggtggg 3200gcttcccttt cgccacagta tgtcagctac aaccagagct
cctacacgca 3250gtgggacctg cagcctgaca ctgactacga gatccacttg
tttaaggaga 3300ggatgttccg gcaccaaatg gctgtgaaga ccaatggcac
aggccgcgtg 3350aggctccctc ctgctggctt cgccactgag ggctggttca
tcggctttgt 3400gagtgccatc atcctcctgc tcctcgtcct gctcatcctc
tgcttcatca 3450agcgcagcaa gggcggcaaa tactcagtga aggataagga
ggacacccag 3500gtggactctg aggcccgacc gatgaaagat gagaccttcg
gcgagtacag 3550gtccctggag agtgacaacg aggagaaggc ctttggcagc
agccagccat 3600cgctcaacgg ggacatcaag cccctgggca gtgacgacag
cctggccgat 3650tatgggggca gcgtggatgt tcagttcaac gaggatggtt
cgttcattgg 3700ccagtacagt ggcaagaagg agaaggaggc ggcagggggc
aatgacagct 3750caggggccac ttcccccatc aaccctgccg tggccctaga
atagtggagt 3800ccaggacagg agatgctgtg cccctggcct tgggatccag
gcccctccct 3850ctccagcagg cccatgggag gctggagttg gggcagagga
gaacttgctg 3900cctcggatcc ccttcctacc acccggtccc cactttattg
ccaaaaccca 3950gctgcacccc ttcctgggca cacgctgctc tgccccagct
tgggcagatc 4000tcccacatgc caggggcctt tgggtgctgt tttgccagcc
catttgggca 4050gagaggctgt ggtttggggg agaagaagta ggggtggccc
gaaagggtct 4100ccgaaatgct gtctttcttg ctccctgact gggggcagac
atggtggggt 4150ctcctcagga ccagggttgg caccttcccc ctcccccagc
cactccccag 4200ccagcctggc tgggactggg aacagaactc ggtgtcccca
ccatctgctg 4250tcttttcttt gccatctctg ctccaaccgg gatgggagcc
gggcaaactg 4300gccgcggggg caggggaggc catctggaga gcccagagtc
cccccactcc 4350cagcatcgca ctctggcagc accgcctctt cccgccgccc
agcccacccc 4400atggccggct ttcaggagct ccatacacac gctgccttcg
gtacccacca 4450cacaacatcc aagtggcctc cgtcactacc tggctgcggg
gcgggcacac 4500ctcctcccac tgcccactgg ccggc 45258768DNAHomo sapien
8gatcccaccc ttccggcccc cccacggtcg cgctcctcca ggctgggcct
50gtggccgcgg tgctttttaa ttttccccca gctcagaatc ttgctgctcg
100gcccccagga gagcaacaac tcaacgggaa cgatgtggaa ggtgtcagct
150ctgctcttcg ttttgggaag cgcgtcgctc tgggtcctgg cagaaggagc
200cagcacaggc cagccagaag atgacactga gactacaggt ttggaaggcg
250gcgttgccat gccaggtgcc gaagatgatg tggtgactcc aggaaccagc
300gaagaccgct ataagtctgg cttgacaact ctggtggcaa caagtgtcaa
350cagtgtaaca ggcattcgca tcgaggatct gccaacttca gaaagcacag
400tccacgcgca agaacaaagt ccaagcgcca cagcctcaaa cgtggccacc
450agtcactcca cggagaaagt ggatggagac acacagacaa cagttgagaa
500agatggtttg tcaacagtga ccctggttgg aatcatagtt ggggtcttac
550tagccatcgg tttcattggt ggaatcatcg ttgtggttat gcgaaaaatg
600tcgggaaggt actcgcccta aagagctgaa gggttacgcc ctgctgccaa
650cgtgcttaaa aaaagaccgt ttctgacttc tgtggccctg ttcccttgag
700cttcgtgggg agaagattga cccgtgggaa catttgcctg ggcccattca
750gaatccacgg tgaccttt 76892163DNAHomo sapienUnsure2118,
2136Unknown base 9tttgctgtcc ggctgcctak ggtctgggaa gctcgggcac
cctccctctc 50cggggctcct gctcccaccc ctccggcccc cccaccgtcg cgctcctcca
100ggctgggcct gtggccgcgg tgctttttaa ttttccccca gctcagaatc
150ttgctgctcg gcccccagga gagcaacaac tcaacgggaa cgatgtggaa
200ggtgtcagct ctgctcttcg ttttgggaag cgcgtcgctc tgggtcctgg
250cagaaggagc cagcacaggc cagccagaag atgacactga gactacaggt
300ttggaaggcg gcgttgccat gccaggtgcc gaagatgatg tggtgactcc
350aggaaccagc gaagaccgct ataagtctgg cttgacaact ctggtggcaa
400caagtgtcaa cagtgtaaca ggcattcgca tcgaggatct gccaacttca
450gaaagcacag tccacgcgca agaacaaagt ccaagcgcca cagcctcaaa
500cgtggccacc agtcactcca cggagaaagt ggatggagac acacagacaa
550cagttgagaa agatggtttg tcaacagtga ccctggttgg aatcatagtt
600ggggtcttac tagccatcgg yttcattggt ggaatcatcg ttgtggttat
650gcgaaaaatg tcgggaaggc cctaaagagc tgaagggtta cgccctgctg
700ccaacgtgct taaaaaaaga ccgtttctga ctctgtgccc tgtccctgag
750ctcgtgggag aagatgaccc gtggaacact tgcctggccc actcagaatc
800cacggtgacc tctccgcttg ccaaaataac cgaagaaaga ccgttcacca
850gacttggctc ctctaaacat ttgctgttca aacatgtttt tgaatataca
900ttctataaaa gattatttga aagacaaaat tcatagaaaa tggagcaaaa
950ctgtataaac tgatttgtaa ctaacactgg accattggat cgatattaya
1000tgctgtaacc atgtgtctcc gtctgaccat tcttgttatt gttaaaatgc
1050agaggaatct ggaaatattt atatccacgg agtccttgga tccagtgcta
1100cgtcagtaaa tagcaccagc attttgcaat tgctgatctg ctgaaatgta
1150cacattctgg tctagtttgg tctatctttt aaagcctgat ctggtgtgaa
1200taatcaacta ggaaatctaa acttggataa cacgtggtga acaactgcct
1250ttagctggtc cagattaatc atttcaaaga catccatttt agatcacaag
1300caggaagtcg atagtctcaa aggcactttg tttctcccaa gtaggccacc
1350aggcagcctc tagagttgct ttacccaaat ccttctccag ccatgacttg
1400gtgactctaa gcttgctccc acctgccccc tccacttccc tcagatgatg
1450aggagccagg gctaaggggg cagccttctc tcttcccagt gatgcacatc
1500cttcacattg gctgctttgt tctggaatat ggatatctca gcctggatgc
1550cgaggaagct gctggatgct taatggtgct agaggctcaa gtgtgtttga
1600aaccaagagc cagttgtccc ccatgcagaa agaaatcctg tgtgagcctc
1650tggtatgaga aataaaatct gccagtttta taacattcac tttctgcctc
1700tgaggaaaga tacagggaac aaaaatcaat ttgtacagtc ttaatattaa
1750aagcagcttg actaaatacc tgatttaaaa atagaagaca tccccagtcc
1800tcatgacata ccgcaaatat ctgtggggtc ctgttgaaaa gaacaaaata
1850aaggagccca aggggtcatt ctgtctcagc accatccagc ctggcacttc
1900tcttcccata tatccattgg attttttttt tttttttcct aaacaaagtt
1950tttacactga gcagatgctc tgtcatgatg gcggttgtgc aattctggta
2000tcctctaaat ttgtaagcat tcataaaaaa aaaaaaaaaa aactcgaggg
2050ggggcccggw cccaattgcc ctatagggag tcgtattaca attcactgsc
2100cgcgttttac aacgtcgnga ctgggaaaac cctggngtta cccaacttaa
2150tcgccttgca gaa 2163103558DNAHomo sapien 10cagaccccag ttcgccgact
aagcagaaga aagatcaaaa accggaaaag 50aggagaagag caaacaggca ctttgaggaa
caatcccctt taactccaag 100ccgacagcgg tctaggaatt caagttcagt
gcctaccgaa gacaaaggcg 150ccccgaggga gtggcggtgc gaccccaggg
cgtgggcccg gccgcggagc 200ccacactgcc cggctgaccc ggtggtctcg
gaccatgtct cccgccccaa 250gacccccccg ttgtctcctg ctccccctgc
tcacgctcgg caccgcgctc 300gcctccctcg gctcggccca aagcagcagc
ttcagccccg aagcctggct 350acagcaatat ggctacctgc ctcccgggga
cctacgtacc cacacacagc 400gctcacccca gtcactctca gcggccatcg
ctgccatgca gaagttttac 450ggcttgcaag taacaggcaa agctgatgca
gacaccatga aggccatgag 500gcgcccccga tgtggtgttc cagacaagtt
tggggctgag atcaaggcca 550atgttcgaag gaagcgctac gccatccagg
gtctcaaatg gcaacataat 600gaaatcactt tctgcatcca gaattacacc
cccaaggtgg gcgagtatgc 650cacatacgag gccattcgca aggcgttccg
cgtgtgggag agtgccacac 700cactgcgctt ccgcgaggtg ccctatgcct
acatccgtga gggccatgag 750aagcaggccg acatcatgat cttctttgcc
gagggcttcc atggcgacag 800cacgcccttc gatggtgagg gcggcttcct
ggcccatgcc tacttcccag 850gccccaacat tggaggagac acccactttg
actctgccga gccttggact 900gtcaggaatg aggatctgaa tggaaatgac
atcttcctgg tggctgtgca 950cgagctgggc catgccctgg ggctcgagca
ttccagtgac ccctcggcca 1000tcatggcacc cttttaccag tggatggaca
cggagaattt tgtgctgccc 1050gatgatgacc gccggggcat ccagcaactt
tatgggggtg agtcagggtt 1100ccccaccaag atgccccctc aacccaggac
tacctcccgg ccttctgttc 1150ctgataaacc caaaaacccc acctatgggc
ccaacatctg tgacgggaac 1200tttgacaccg tggccatgct ccgaggggag
atgtttgtct tcaaggagcg 1250ctggttctgg cgggtgagga ataaccaagt
gatggatgga tacccaatgc 1300ccattggcca gttctggcgg ggcctgcctg
cgtccatcaa cactgcctac 1350gagaggaagg atggcaaatt cgtcttcttc
aaaggagaca agcattgggt 1400gtttgatgag gcgtccctgg aacctggcta
ccccaagcac attaaggagc 1450tgggccgagg gctgcctacc gacaagattg
atgctgctct cttctggatg 1500cccaatggaa agacctactt cttccgtgga
aacaagtact accgtttcaa 1550cgaagagctc agggcagtgg atagcgagta
ccccaagaac atcaaagtct 1600gggaagggat ccctgagtct cccagagggt
cattcatggg cagcgatgaa 1650gtcttcactt acttctacaa ggggaacaaa
tactggaaat tcaacaacca 1700gaagctgaag gtagaaccgg gctaccccaa
gtcagccctg agggactgga 1750tgggctgccc atcgggaggc cggccggatg
aggggactga ggaggagacg 1800gaggtgatca tcattgaggt ggacgaggag
ggcggcgggg cggtgagcgc 1850ggctgccgtg gtgctgcccg tgctgctgct
gctcctggtg ctggcggtgg 1900gccttgcagt cttcttcttc agacgccatg
ggacccccag gcgactgctc 1950tactgccagc gttccctgct ggacaaggtc
tgacgcccac cgccggcccg 2000cccactccta ccacaaggac tttgcctctg
aaggccagtg gcagcaggtg 2050gtggtgggtg ggctgctccc atcgtcccga
gccccctccc cgcagcctcc 2100ttgcttctct ctgtcccctg gctggcctcc
ttcaccctga ccgcctccct 2150ccctcctgcc ccggcattgc atcttcccta
gataggtccc ctgagggctg 2200agtgggaggg cggccctttc cagcctctgc
ccctcagggg aaccctgtag 2250ctttgtgtct gtccagcccc atctgaatgt
gttgggggct ctgcacttga 2300aggcaggacc ctcagacctc gctggtaaag
gtcaaatggg gtcatctgct 2350ccttttccat cccctgacat accttaacct
ctgaactctg acctcaggag 2400gctctgggca ctccagccct gaaagcccca
ggtgtaccca attggcagcc 2450tctcactact ctttctggct aaaaggaatc
taatcttgtt gagggtagag 2500accctgagac agtgtgaggg ggtggggact
gccaagccac cctaagacct 2550tgggaggaaa actcagagag ggtcttcgtt
gctcagtcag tcaagttcct 2600cggagatctg cctctgcctc acctacccca
gggaacttcc aaggaaggag 2650cctgagccac tggggactaa gtgggcagaa
gaaacccttg gcagccctgt 2700gcctctcgaa tgttagcctt ggatggggct
ttcacagtta gaagagctga 2750aaccaggggt gcagctgtca ggtagggtgg
ggccggtggg agaggcccgg 2800gtcagagccc tgggggtgag cctgaaggcc
acagagaaag aaccttgccc 2850aaactcaggc agctggggct gaggcccaaa
ggcagaacag ccagaggggg 2900caggagggga ccaaaaagga aaatgaggac
gtgcagcagc attggaaggc 2950tggggccggg caggccaggc caagccaagc
agggggccac agggtgggct 3000gtggagctct caggaagggc cctgaggaag
gcacacttgc tcctgttggt 3050ccctgtcctt gctgcccagg cagcgtggag
gggaagggta gggcagccag 3100agaaaggagc agagaaggca cacaaacgag
gaatgagggg cttcacgaga 3150ggccacaggg cctggctggc cacgctgtcc
cggcctgctc accatctcag 3200tgaggggcag gagctggggc tcgcttaggc
tgggtccacg cttccctggt 3250gccagcaccc ctcaagcctg tctcaccagt
ggcctgccct ctcgctcccc 3300cacccagccc acccattgaa gtctccttgg
gccaccaaag gtggtggcca 3350tggtaccggg gacttgggag agtgagaccc
agtggaggga gcaagaggag 3400agggatgtcg ggggggtggg gcacggggta
ggggaaatgg ggtgaacggt 3450gctggcagtt cggctagatt tctgtcttgt
ttgttttttt gttttgttta 3500atgtatattt ttattataat tattatatat
gaattccaaa aaaaaaaaaa 3550aaaaaaaa 3558111872DNAHomo sapien
11tcaggctgcc tgatctgccc agctttccag ctttcctctg gattccggcc
50tctggtcatc cctccccacc ctctctccaa ggccctctcc tggtctccct
100tcttctagaa ccccttcctc cacctccctc tctgcagaac ttctccttta
150ccccccaccc cccaccactg ccccctttcc ttttctgacc tccttttgga
200gggctcagcg ctgcccagac cataggagag atgtgggagg ctcagttcct
250gggcttgctg tttctgcagc cgctttgggt ggctccagtg aagcctctcc
300agccaggggc tgaggtcccg gtggtgtggg cccaggaggg ggctcctgcc
350cagctcccct gcagccccac aatccccctc caggatctca gccttctgcg
400aagagcaggg gtcacttggc agcatcagcc agacagtggc ccgcccgctg
450ccgcccccgg ccatcccctg gcccccggcc ctcacccggc ggcgccctcc
500tcctgggggc ccaggccccg ccgctacacg gtgctgagcg tgggtcccgg
550aggcctgcgc agcgggaggc tgcccctgca gccccgcgtc cagctggatg
600agcgcggccg gcagcgcggg gacttctcgc tatggctgcg cccagcccgg
650cgcgcggacg ccggcgagta ccgcgccgcg gtgcacctca gggaccgcgc
700cctctcctgc cgcctccgtc tgcgcctggg ccaggcctcg atgactgcca
750gccccccagg atctctcaga gcctccgact gggtcatttt gaactgctcc
800ttcagccgcc ctgaccgccc agcctctgtg cattggttcc ggaaccgggg
850ccagggccga gtccctgtcc gggagtcccc ccatcaccac ttagcggaaa
900gcttcctctt cctgccccaa gtcagcccca tggactctgg gccctggggc
950tgcatcctca cctacagaga tggcttcaac gtctccatca tgtataacct
1000cactgttctg ggtctggagc ccccaactcc cttgacagtg tacgctggag
1050caggttccag ggtggggctg ccctgccgcc tgcctgctgg tgtggggacc
1100cggtctttcc tcactgccaa gtggactcct cctgggggag gccctgacct
1150cctggtgact ggagacaatg gcgactttac ccttcgacta gaggatgtga
1200gccaggccca ggctgggacc tacacctgcc atatccatct gcaggaacag
1250cagctcaatg ccactgtcac attggcaatc atcacagtga ctcccaaatc
1300ctttgggtca cctggatccc tggggaagct gctttgtgag gtgactccag
1350tatctggaca agaacgcttt gtgtggagct ctctggacac cccatcccag
1400aggagtttct caggaccttg gctggaggca caggaggccc agctcctttc
1450ccagccttgg caatgccagc tgtaccaggg ggagaggctt cttggagcag
1500cagtgtactt cacagagctg tctagcccag gtgcccaacg ctctgggaga
1550gccccaggtg ccctcccagc aggccacctc ctgctgtttc tcacccttgg
1600tgtcctttct ctgctccttt tggtgactgg agcctttggc tttcaccttt
1650ggagaagaca gtggcgacca agacgatttt ctgccttaga gcaagggatt
1700caccctcgcc aggctcagag caagatagag gagctggagc aagaaccgga
1750gccggagccg gagccggaac cggagcccga gcccgagccc gagccggagc
1800agctctgacc tggagctgag gcagccagca gatctcagca gcccagtcca
1850aataaacgtc ctgtctagca gc
1872121991DNAHomo sapien 12acaggggtga aggcccagag accagcagaa
cggcatccca gccacgacgg 50ccactttgct ctgtctgctg tccgccacgg ccctgctctg
ttccctggga 100cacccccgcc cccacctcct caggctgcct gatctgccca
gctttccagc 150tttcctctgg attccggcct ctggtcatcc ctccccaccc
tctctccaag 200gccctctcct ggtctccctt cttctagaac cccttcctcc
acctccctct 250ctgcagaact tctcctttac cccccacccc ccaccactgc
cccctttcct 300tttctgacct ccttttggag ggctcagcgc tgcccagacc
ataggagaga 350tgtgggaggc tcagttcctg ggcttgctgt ttctgcagcc
gctttgggtg 400gctccagtga agcctctcca gccaggggct gaggtcccgg
tggtgtgggc 450ccaggagggg gctcctgccc agctcccctg cagccccaca
atccccctcc 500aggatctcag ccttctgcga agagcagggg tcacttggca
gcatcagcca 550gacagtggcc cgcccgctgc cgcccccggc catcccctgg
cccccggccc 600tcacccggcg gcgccctcct cctgggggcc caggccccgc
cgctacacgg 650tgctgagcgt gggtcccgga ggcctgcgca gcgggaggct
gcccctgcag 700ccccgcgtcc agctggatga gcgcggccgg cagcgcgggg
acttctcgct 750atggctgcgc ccagcccggc gcgcggacgc cggcgagtac
cgcgccgcgg 800tgcacctcag ggaccgcgcc ctctcctgcc gcctccgtct
gcgcctgggc 850caggcctcga tgactgccag ccccccagga tctctcagag
cctccgactg 900ggtcattttg aactgctcct tcagccgccc tgaccgccca
gcctctgtgc 950attggttccg gaaccggggc cagggccgag tccctgtccg
ggagtccccc 1000catcaccact tagcggaaag cttcctcttc ctgccccaag
tcagccccat 1050ggactctggg ccctggggct gcatcctcac ctacagagat
ggcttcaacg 1100tctccatcat gtataacctc actgttctgg gtctggagcc
cccaactccc 1150ttgacagtgt acgctggagc aggttccagg gtggggctgc
cctgccgcct 1200gcctgctggt gtggggaccc ggtctttcct cactgccaag
tggactcctc 1250ctgggggagg ccctgacctc ctggtgactg gagacaatgg
cgactttacc 1300cttcgactag aggatgtgag ccaggcccag gctgggacct
acacctgcca 1350tatccatctg caggaacagc agctcaatgc cactgtcaca
ttggcaatca 1400tcacagtgac tcccaaatcc tttgggtcac ctggatccct
ggggaagctg 1450ctttgtgagg tgactccagt atctggacaa gaacgctttg
tgtggagctc 1500tctggacacc ccatcccaga ggagtttctc aggaccttgg
ctggaggcac 1550aggaggccca gctcctttcc cagccttggc aatgccagct
gtaccagggg 1600gagaggcttc ttggagcagc agtgtacttc acagagctgt
ctagcccagg 1650tgcccaacgc tctgggagag ccccaggtgc cctcccagca
ggccacctcc 1700tgctgtttct cacccttggt gtcctttctc tgctcctttt
ggtgactgga 1750gcctttggct ttcacctttg gagaagacag tggcgaccaa
gacgattttc 1800tgccttagag caagggattc accctccgca ggctcagagc
aagatagagg 1850agctggagca agaaccggag ccggagccgg agccggaacc
ggagcccgag 1900cccgagcccg agccggagca gctctgacct ggagctgagg
cagccagcag 1950atctcagcag cccagtccaa ataaacgtcc tgtctagcag c
1991137193DNAHomo sapien 13agaataaggg cagggaccgc ggctcctatc
tcttggtgat ccccttcccc 50attccgcccc cgcctcaacg cccagcacag tgccctgcac
acagtagtcg 100ctcaataaat gttcgtggat gatgatgatg atgatgatga
aaaaaatgca 150gcatcaacgg cagcagcaag cggaccacgc gaacgaggca
aactatgcaa 200gaggcaccag acttcctctt tctggtgaag gaccaacttc
tcagccgaat 250agctccaagc aaactgtcct gtcttggcaa gctgcaatcg
atgctgctag 300acaggccaag gctgcccaaa ctatgagcac ctctgcaccc
ccacctgtag 350gatctctctc ccaaagaaaa cgtcagcaat acgccaagag
caaaaaacag 400ggtaactcgt ccaacagccg acctgcccgc gcccttttct
gtttatcact 450caataacccc atccgaagag cctgcattag tatagtggaa
tggaaaccat 500ttgacatatt tatattattg gctatttttg ccaattgtgt
ggccttagct 550atttacatcc cattccctga agatgattct aattcaacaa
atcataactt 600ggaaaaagta gaatatgcct tcctgattat ttttacagtc
gagacatttt 650tgaagattat agcgtatgga ttattgctac atcctaatgc
ttatgttagg 700aatggatgga atttactgga ttttgttata gtaatagtag
gattgtttag 750tgtaattttg gaacaattaa ccaaagaaac agaaggcggg
aaccactcaa 800gcggcaaatc tggaggcttt gatgtcaaag ccctccgtgc
ctttcgagtg 850ttgcgaccac ttcgactagt gtcaggggtg cccagtttac
aagttgtcct 900gaactccatt ataaaagcca tggttcccct ccttcacata
gcccttttgg 950tattatttgt aatcataatc tatgctatta taggattgga
actttttatt 1000ggaaaaatgc acaaaacatg tttttttgct gactcagata
tcgtagctga 1050agaggaccca gctccatgtg cgttctcagg gaatggacgc
cagtgtactg 1100ccaatggcac ggaatgtagg agtggctggg ttggcccgaa
cggaggcatc 1150accaactttg ataactttgc ctttgccatg cttactgtgt
ttcagtgcat 1200caccatggag ggctggacag acgtgctcta ctgggtaaat
gatgcgatag 1250gatgggaatg gccatgggtg tattttgtta gtctgatcat
ccttggctca 1300tttttcgtcc ttaacctggt tcttggtgtc cttagtggag
aattctcaaa 1350ggaaagagag aaggcaaaag cacggggaga tttccagaag
ctccgggaga 1400agcagcagct ggaggaggat ctaaagggct acttggattg
gatcacccaa 1450gctgaggaca tcgatccgga gaatgaggaa gaaggaggag
aggaaggcaa 1500acgaaatact agcatgccca ccagcgagac tgagtctgtg
aacacagaga 1550acgtcagcgg tgaaggcgag aaccgaggct gctgtggaag
tctctggtgc 1600tggtggagac ggagaggcgc ggccaaggcg gggccctctg
ggtgtcggcg 1650gtggggtcaa gccatctcaa aatccaaact cagccgacgc
tggcgtcgct 1700ggaaccgatt caatcgcaga agatgtaggg ccgccgtgaa
gtctgtcacg 1750ttttactggc tggttatcgt cctggtgttt ctgaacacct
taaccatttc 1800ctctgagcac tacaatcagc cagattggtt gacacagatt
caagatattg 1850ccaacaaagt cctcttggct ctgttcacct gcgagatgct
ggtaaaaatg 1900tacagcttgg gcctccaagc atatttcgtc tctcttttca
accggtttga 1950ttgcttcgtg gtgtgtggtg gaatcactga gacgatcctg
gtggaactgg 2000aaatcatgtc tcccctgggg atctctgtgt ttcggtgtgt
gcgcctctta 2050agaatcttca aagtgaccag gcactggact tccctgagca
acttagtggc 2100atccttatta aactccatga agtccatcgc ttcgctgttg
cttctgcttt 2150ttctcttcat tatcatcttt tccttgcttg ggatgcagct
gtttggcggc 2200aagtttaatt ttgatgaaac gcaaaccaag cggagcacct
ttgacaattt 2250ccctcaagca cttctcacag tgttccagat cctgacaggc
gaagactgga 2300atgctgtgat gtacgatggc atcatggctt acgggggccc
atcctcttca 2350ggaatgatcg tctgcatcta cttcatcatc ctcttcattt
gtggtaacta 2400tattctactg aatgtcttct tggccatcgc tgtagacaat
ttggctgatg 2450ctgaaagtct gaacactgct cagaaagaag aagcggaaga
aaaggagagg 2500aaaaagattg ccagaaaaga gagcctagaa aataaaaaga
acaacaaacc 2550agaagtcaac cagatagcca acagtgacaa caaggttaca
attgatgact 2600atagagaaga ggatgaagac aaggacccct atccgccttg
cgatgtgcca 2650gtaggggaag aggaagagga agaggaggag gatgaacctg
aggttcctgc 2700cggaccccgt cctcgaagga tctcggagtt gaacatgaag
gaaaaaattg 2750cccccatccc tgaagggagc gctttcttca ttcttagcaa
gaccaacccg 2800atccgcgtag gctgccacaa gctcatcaac caccacatct
tcaccaacct 2850catccttgtc ttcatcatgc tgagcagcgc tgccctggcc
gcagaggacc 2900ccatccgcag ccactccttc cggaacacga tactgggtta
ctttgactat 2950gccttcacag ccatctttac tgttgagatc ctgttgaaga
tgacaacttt 3000tggagctttc ctccacaaag gggccttctg caggaactac
ttcaatttgc 3050tggatatgct ggtggttggg gtgtctctgg tgtcatttgg
gattcaatcc 3100agtgccatct ccgttgtgaa gattctgagg gtcttaaggg
tcctgcgtcc 3150cctcagggcc atcaacagag caaaaggact taagcacgtg
gtccagtgcg 3200tcttcgtggc catccggacc atcggcaaca tcatgatcgt
cactaccctc 3250ctgcagttca tgtttgcctg tatcggggtc cagttgttca
aggggaagtt 3300ctatcgctgt acggatgaag ccaaaagtaa ccctgaagaa
tgcaggggac 3350ttttcatcct ctacaaggat ggggatgttg acagtcctgt
ggtccgtgaa 3400cggatctggc aaaacagtga tttcaacttc gacaacgtcc
tctctgctat 3450gatggcgctc ttcacagtct ccacgtttga gggctggcct
gcgttgctgt 3500ataaagccat cgactcgaat ggagagaaca tcggcccaat
ctacaaccac 3550cgcgtggaga tctccatctt cttcatcatc tacatcatca
ttgtagcttt 3600cttcatgatg aacatctttg tgggctttgt catcgttaca
tttcaggaac 3650aaggagaaaa agagtataag aactgtgagc tggacaaaaa
tcagcgtcag 3700tgtgttgaat acgccttgaa agcacgtccc ttgcggagat
acatccccaa 3750aaacccctac cagtacaagt tctggtacgt ggtgaactct
tcgcctttcg 3800aatacatgat gtttgtcctc atcatgctca acacactctg
cttggccatg 3850cagcactacg agcagtccaa gatgttcaat gatgccatgg
acattctgaa 3900catggtcttc accggggtgt tcaccgtcga gatggttttg
aaagtcatcg 3950catttaagcc taaggggtat tttagtgacg cctggaacac
gtttgactcc 4000ctcatcgtaa tcggcagcat tatagacgtg gccctcagcg
aagcggaccc 4050aactgaaagt gaaaatgtcc ctgtcccaac tgctacacct
gggaactctg 4100aagagagcaa tagaatctcc atcacctttt tccgtctttt
ccgagtgatg 4150cgattggtga agcttctcag caggggggaa ggcatccgga
cattgctgtg 4200gacttttatt aagtcctttc aggcgctccc gtatgtggcc
ctcctcatag 4250ccatgctgtt cttcatctat gcggtcattg gcatgcagat
gtttgggaaa 4300gttgccatga gagataacaa ccagatcaat aggaacaata
acttccagac 4350gtttccccag gcggtgctgc tgctcttcag gtgtgcaaca
ggtgaggcct 4400ggcaggagat catgctggcc tgtctcccag ggaagctctg
tgaccctgag 4450tcagattaca accccgggga ggagtataca tgtgggagca
actttgccat 4500tgtctatttc atcagttttt acatgctctg tgcatttctg
atcatcaatc 4550tgtttgtggc tgtcatcatg gataatttcg actatctgac
ccgggactgg 4600tctattttgg ggcctcacca tttagatgaa ttcaaaagaa
tatggtcaga 4650atatgaccct gaggcaaagg gaaggataaa acaccttgat
gtggtcactc 4700tgcttcgacg catccagcct cccctggggt ttgggaagtt
atgtccacac 4750agggtagcgt gcaagagatt agttgccatg aacatgcctc
tcaacagtga 4800cgggacagtc atgtttaatg caaccctgtt tgctttggtt
cgaacggctc 4850ttaagatcaa gaccgaaggg aacctggagc aagctaatga
agaacttcgg 4900gctgtgataa agaaaatttg gaagaaaacc agcatgaaat
tacttgacca 4950agttgtccct ccagctggtg atgatgaggt aaccgtgggg
aagttctatg 5000ccactttcct gatacaggac tactttagga aattcaagaa
acggaaagaa 5050caaggactgg tgggaaagta ccctgcgaag aacaccacaa
ttgccctaca 5100ggcgggatta aggacactgc atgacattgg gccagaaatc
cggcgtgcta 5150tatcgtgtga tttgcaagat gacgagcctg aggaaacaaa
acgagaagaa 5200gaagatgatg tgttcaaaag aaatggtgcc ctgcttggaa
accatgtcaa 5250tcatgttaat agtgatagga gagattccct tcagcagacc
aataccaccc 5300accgtcccct gcatgtccaa aggccttcaa ttccacctgc
aagtgatact 5350gagaaaccgc tgtttcctcc agcaggaaat tcggtgtgtc
ataaccatca 5400taaccataat tccataggaa agcaagttcc cacctcaaca
aatgccaatc 5450tcaataatgc caatatgtcc aaagctgccc atggaaagcg
gcccagcatt 5500gggaaccttg agcatgtgtc tgaaaatggg catcattctt
cccacaagca 5550tgaccgggag cctcagagaa ggtccagtgt gaaaagaacc
cgctattatg 5600aaacttacat taggtccgac tcaggagatg aacagctccc
aactatttgc 5650cgggaagacc cagagataca tggctatttc agggaccccc
actgcttggg 5700ggagcaggag tatttcagta gtgaggaatg ctacgaggat
gacagctcgc 5750ccacctggag caggcaaaac tatggctact acagcagata
cccaggcaga 5800aacatcgact ctgagaggcc ccgaggctac catcatcccc
aaggattctt 5850ggaggacgat gactcgcccg tttgctatga ttcacggaga
tctccaagga 5900gacgcctact acctcccacc ccagcatccc accggagatc
ctccttcaac 5950tttgagtgcc tgcgccggca gagcagccag gaagaggtcc
cgtcgtctcc 6000catcttcccc catcgcacgg ccctgcctct gcatctaatg
cagcaacaga 6050tcatggcagt tgccggccta gattcaagta aagcccagaa
gtactcaccg 6100agtcactcga cccggtcgtg ggccacccct ccagcaaccc
ctccctaccg 6150ggactggaca ccgtgctaca cccccctgat ccaagtggag
cagtcagagg 6200ccctggacca ggtgaacggc agcctgccgt ccctgcaccg
cagctcctgg 6250tacacagacg agcccgacat ctcctaccgg actttcacac
cagccagcct 6300gactgtcccc agcagcttcc ggaacaaaaa cagcgacaag
cagaggagtg 6350cggacagctt ggtggaggca gtcctgatat ccgaaggctt
gggacgctat 6400gcaagggacc caaaatttgt gtcagcaaca aaacacgaaa
tcgctgatgc 6450ctgtgacctc accatcgacg agatggagag tgcagccagc
accctgctta 6500atgggaacgt gcgtccccga gccaacgggg atgtgggccc
cctctcacac 6550cggcaggact atgagctaca ggactttggt cctggctaca
gcgacgaaga 6600gccagaccct gggagggatg aggaggacct ggcggatgaa
atgatatgca 6650tcaccacctt gtagccccca gcgaggggca gactggctct
ggcctcaggt 6700ggggcgcagg agagccaggg gaaaagtgcc tcatagttag
gaaagtttag 6750gcactagttg ggagtaatat tcaattaatt agacttttgt
ataagagatg 6800tcatgcctca agaaagccat aaacctggta ggaacaggtc
ccaagcggtt 6850gagcctggca gagtaccatg cgctcggccc cagctgcagg
aaacagcagg 6900ccccgccctc tcacagagga tgggtgagga ggccagacct
gccctgcccc 6950attgtccaga tgggcactgc tgtggagtct gcttctccca
tgtaccaggg 7000caccaggccc acccaactga aggcatggcg gcggggtgca
ggggaaagtt 7050aaaggtgatg acgatcatca cacctcgtgt cgttacctca
gccatcggtc 7100tagcatatca gtcactgggc ccaacatatc catttttaaa
ccctttcccc 7150caaatacact gcgtcctggt tcctgtttag ctgttctgaa ata
7193141315DNAHomo sapien 14tcgccatggc ctctgccgga atgcagatcc
tgggagtcgt cctgacactg 50ctgggctggg tgaatggcct ggtctcctgt gccctgccca
tgtggaaggt 100gaccgctttc atcggcaaca gcatcgtggt ggcccaggtg
gtgtgggagg 150gcctgtggat gtcctgcgtg gtgcagagca ccggccagat
gcagtgcaag 200gtgtacgact cactgctggc gctgccacag gacctgcagg
ctgcacgtgc 250cctctgtgtc atcgccctcc ttgtggccct gttcggcttg
ctggtctacc 300ttgctggggc caagtgtacc acctgtgtgg aggagaagga
ttccaaggcc 350cgcctggtgc tcacctctgg gattgtcttt gtcatctcag
gggtcctgac 400gctaatcccc gtgtgctgga cggcgcatgc catcatccgg
gacttctata 450accccctggt ggctgaggcc caaaagcggg agctgggggc
ctccctctac 500ttgggctggg cggcctcagg ccttttgttg ctgggtgggg
ggttgctgtg 550ctgcacttgc ccctcggggg ggtcccaggg ccccagccat
tacatggccc 600gctactcaac atctgcccct gccatctctc gggggccctc
tgagtaccct 650accaagaatt acgtctgacg tggaggggaa tgggggctcc
gctggcgcta 700gagccatcca gaagtggcag tgcccaacag ctttgggatg
ggttcgtacc 750ttttgtttct gcctcctgct atttttcttt tgactgagga
tatttaaaat 800tcatttgaaa actgagccaa ggtgttgact cagactctca
cttaggctct 850gctgtttctc acccttggat gatggagcca aagaggggat
gctttgagat 900tctggatctt gacatgccca tcttagaagc cagtcaagct
atggaactaa 950tgcggaggct gcttgctgtg ctggctttgc aacaagacag
actgtcccca 1000agagttcctg ctgctgctgg gggctgggct tccctagatg
tcactggaca 1050gctgcccccc atcctactca ggtctctgga gctcctctct
tcacccctgg 1100aaaaacaaat catctgttaa caaaggactg cccacctccg
gaacttctga 1150cctctgtttc ctccgtcctg ataagacgtc caccccccag
ggccaggtcc 1200cagctatgta gacccccgcc cccacctcca acactgcacc
cttctgccct 1250gcccccctcg tctcaccccc tttacactca catttttatc
aaataaagca 1300tgttttgtta gtgca 1315152000DNAHomo sapien
15cctgtgaaca ctatagcgct gagagagaca gtctgaaagc agaggaagac
50atcgatcagt aacaccaaga gacaccaaag ttgaaagttt tgttttcttt
100ccctctgttt tatttttccc ccgtgtgtcc ctactatggt cagaaagcct
150gttgtgtcca ccatctccaa aggaggttac ctgcagggaa atgttaacgg
200gaggctgcct tccctgggca acaaggagcc acctgggcag gagaaagtgc
250agctgaagag gaaagtcact ttactgaggg gagtctccat tatcattggc
300accatcattg gagcaggaat cttcatctct cctaagggcg tgctccagaa
350cacgggcagc gtgggcatgt ctctgaccat ctggacggtg tgtggggtcc
400tgtcactatt tggagctttg tcttatgctg aattgggaac aactataaag
450aaatctggag gtcattacac atatattttg gaagtctttg gtccattacc
500agcttttgta cgagtctggg tggaactcct cataatacgc cctgcagcta
550ctgctgtgat atccctggca tttggacgct acattctgga accatttttt
600attcaatgtg aaatccctga acttgcgatc aagctcatta cagctgtggg
650cataactgta gtgatggtcc taaatagcat gagtgtcagc tggagcgccc
700ggatccagat tttcttaacc ttttgcaagc tcacagcaat tctgataatt
750atagtccctg gagttatgca gctaattaaa ggtcaaacgc agaactttaa
800agacgccttt tcaggaagag attcaagtat tacgcggttg ccactggctt
850tttattatgg aatgtatgca tatgctggct ggttttacct caactttgtt
900actgaagaag tagaaaaccc tgaaaaaacc attccccttg caatatgtat
950atccatggcc attgtcacca ttggctatgt gctgacaaat gtggcctact
1000ttacgaccat taatgctgag gagctgctgc tttcaaatgc agtggcagtg
1050accttttctg agcggctact gggaaatttc tcattagcag ttccgatctt
1100tgttgccctc tcctgctttg gctccatgaa cggtggtgtg tttgctgtct
1150ccaggttatt ctatgttgcg tctcgagagg gtcaccttcc agaaatcctc
1200tccatgattc atgtccgcaa gcacactcct ctaccagctg ttattgtttt
1250gcaccctttg acaatgataa tgctcttctc tggagacctc gacagtcttt
1300tgaatttcct cagttttgcc aggtggcttt ttattgggct ggcagttgct
1350gggctgattt atcttcgata caaatgccca gatatgcatc gtcctttcaa
1400ggtgccactg ttcatcccag ctttgttttc cttcacatgc ctcttcatgg
1450ttgccctttc cctctattcg gacccattta gtacagggat tggcttcgtc
1500atcactctga ctggagtccc tgcgtattat ctctttatta tatgggacaa
1550gaaacccagg tggtttagaa taatgtcagg gttcctagca ctgatgcctg
1600cacaagcatg tgatatgtga aataaaatgg attcttctat agctaaatga
1650gttccctctg gggagagttc tggtactgca atcacaatgc cagatggtgt
1700ttatgggcta tttgtgtaag taagtggtaa gatgctatga agtaagtgtg
1750tttgttttca tcttatggaa actcttgatg catgtgcttt tgtatggaat
1800aaattttggt gcaatatgat gtcattcaac tttgcattga attgaatttt
1850ggttgtattt atatgtatta tacctgtcac gcttctagtt gcttcaacca
1900ttttataacc atttttgtac atattttact tgaaaatatt
ttaaatggaa 1950atttaaataa acatttgata gtttacataa taaaaaaaaa
aaaaaaaaaa 2000161601DNAHomo sapien 16cacagaggag ccagcgaacc
tctcccggcg cctgttctgg gggctttctg 50ttccagcgtc aaggatggag gagggggaga
ggagcccctt actgtcccag 100gaaactgcag gccagaagcc cctctctgtg
cacaggccac ccacctcagg 150ctgcctaggt ccagtgccca gggaggacca
ggcggaggcc tggggctgca 200gctgctgtcc cccggagacc aagcaccagg
ccttgagtgg cactcccaag 250aaaggaccag ccccttccct ctccccaggg
agcagctgcg tcaagtatct 300gatcttcctc tccaacttcc ccttctccct
gctggggctg ctggccctgg 350ccatcgggct ctggggcctg gctgtcaagg
ggtctctggg aagtgatctg 400ggggggcccc tgcccacaga ccccatgctg
gggctggcac tgggagggct 450ggtggtcagc gcagcgagcc tggctggctg
cctgggcgcc ctctgcgaga 500acacctgcct gttacgtggc ttctccgggg
gcatccttgc cttcctggtg 550cttgaggccg tggcgggggc cctggtggtg
gccctctggg gcccgctgca 600agacagcctg gagcacaccc tgcgtgtggc
catcgcccac taccaggacg 650acccagacct gcgcttcctc ctcgaccaag
tccagctcgg gctgaggtgc 700tgcggagctg cctcctacca ggactggcag
cagaacctgt actttaactg 750cagctccccc ggggtgcagg cctgcagcct
tcccgcctcc tgctgcatcg 800acccccgcga agatggagcc tctgtcaacg
accagtgcgg cttcggggtc 850ctgcgcctgg atgcggacgc agctcagaga
gtggtgtacc tggagggctg 900cggcccgccg ctccggcggt ggctgcgcgc
gaacctggct gcctcgggcg 950gctacgcaat cgcggtggtg ctgctgcagg
gggcggagct cctgctggcc 1000gcccggctac tcggggccct cgctgcccgc
agtggggcgg cgtacggccc 1050cggagcgcac ggggaggacc gcgctggccc
ccagagcccc agccccggcg 1100ccccgcccgc tgccaaaccc gcccggggct
gagcgcacgc cccgaggtcc 1150gagaccgcca cgcacaggga tacagggggc
gcctccgccc ggctaaaaag 1200cgctgcctgc gccgccgccg ccgcctgatt
tcgctcgggc ttcgggtgac 1250ttcgccgcag gacctaccca gctcgctcac
ttcgctcgct ccgcgtcccc 1300catgccagcc cccaacgcag ggcgcccggc
gaagccacgg gactggcggg 1350aggagcacgc ggggccggag gaaatcctgg
agctgaccct cacctccgag 1400cccccactcc caccccagcc gcacagttcc
cacctcctgg cacctccctc 1450ccctggggcc gccacccctt ctgggctcgt
gatggtggag ctaaggtcca 1500ggcctctccc tcccgagtgc atttttgggg
agatagtaaa tgttttattc 1550gggtgtatca ttcatacagt aaagacacca
atcttcaaaa aaaaaaaaaa 1600a 160117538PRTHomo sapien 17Met Ala Arg
Ala Ala Ala Leu Leu Pro Ser Arg Ser Pro Pro Thr 1 5 10 15Pro Leu
Leu Trp Pro Leu Leu Leu Leu Leu Leu Leu Glu Thr Gly 20 25 30Ala Gln
Asp Val Arg Val Gln Val Leu Pro Glu Val Arg Gly Gln 35 40 45Leu Gly
Gly Thr Val Glu Leu Pro Cys His Leu Leu Pro Pro Val 50 55 60Pro Gly
Leu Tyr Ile Ser Leu Val Thr Trp Gln Arg Pro Asp Ala 65 70 75Pro Ala
Asn His Gln Asn Val Ala Ala Phe His Pro Lys Met Gly 80 85 90Pro Ser
Phe Pro Ser Pro Lys Pro Gly Ser Glu Arg Leu Ser Phe 95 100 105Val
Ser Ala Lys Gln Ser Thr Gly Gln Asp Thr Glu Ala Glu Leu 110 115
120Gln Asp Ala Thr Leu Ala Leu His Gly Leu Thr Val Glu Asp Glu 125
130 135Gly Asn Tyr Thr Cys Glu Phe Ala Thr Phe Pro Lys Gly Ser Val
140 145 150Arg Gly Met Thr Trp Leu Arg Val Ile Ala Lys Pro Lys Asn
Gln 155 160 165Ala Glu Ala Gln Lys Val Thr Phe Ser Gln Asp Pro Thr
Thr Val 170 175 180Ala Leu Cys Ile Ser Lys Glu Gly Arg Pro Pro Ala
Arg Ile Ser 185 190 195Trp Leu Ser Ser Leu Asp Trp Glu Ala Lys Glu
Thr Gln Val Ser 200 205 210Gly Thr Leu Ala Gly Thr Val Thr Val Thr
Ser Arg Phe Thr Leu 215 220 225Val Pro Ser Gly Arg Ala Asp Gly Val
Thr Val Thr Cys Lys Val 230 235 240Glu His Glu Ser Phe Glu Glu Pro
Ala Leu Ile Pro Val Thr Leu 245 250 255Ser Val Arg Tyr Pro Pro Glu
Val Ser Ile Ser Gly Tyr Asp Asp 260 265 270Asn Trp Tyr Leu Gly Arg
Thr Asp Ala Thr Leu Ser Cys Asp Val 275 280 285Arg Ser Asn Pro Glu
Pro Thr Gly Tyr Asp Trp Ser Thr Thr Ser 290 295 300Gly Thr Phe Pro
Thr Ser Ala Val Ala Gln Gly Ser Gln Leu Val 305 310 315Ile His Ala
Val Asp Ser Leu Phe Asn Thr Thr Phe Val Cys Thr 320 325 330Val Thr
Asn Ala Val Gly Met Gly Arg Ala Glu Gln Val Ile Phe 335 340 345Val
Arg Glu Thr Pro Asn Thr Ala Gly Ala Gly Ala Thr Gly Gly 350 355
360Ile Ile Gly Gly Ile Ile Ala Ala Ile Ile Ala Thr Ala Val Ala 365
370 375Ala Thr Gly Ile Leu Ile Cys Arg Gln Gln Arg Lys Glu Gln Thr
380 385 390Leu Gln Gly Ala Glu Glu Asp Glu Asp Leu Glu Gly Pro Pro
Ser 395 400 405Tyr Lys Pro Pro Thr Pro Lys Ala Lys Leu Glu Ala Gln
Glu Met 410 415 420Pro Ser Gln Leu Phe Thr Leu Gly Ala Ser Glu His
Ser Pro Leu 425 430 435Lys Thr Pro Tyr Phe Asp Ala Gly Ala Ser Cys
Thr Glu Gln Glu 440 445 450Met Pro Arg Tyr His Glu Leu Pro Thr Leu
Glu Glu Arg Ser Gly 455 460 465Pro Leu His Pro Gly Ala Thr Ser Leu
Gly Ser Pro Ile Pro Val 470 475 480Pro Pro Gly Pro Pro Ala Val Glu
Asp Val Ser Leu Asp Leu Glu 485 490 495Asp Glu Glu Gly Glu Glu Glu
Glu Glu Tyr Leu Asp Lys Ile Asn 500 505 510Pro Ile Tyr Asp Ala Leu
Ser Tyr Ser Ser Pro Ser Asp Ser Tyr 515 520 525Gln Gly Lys Gly Phe
Val Met Ser Arg Ala Met Tyr Val 530 53518331PRTHomo sapien 18Met
Ile Thr Leu Asn Asn Gln Asp Gln Pro Val Thr Phe Asn Ser 1 5 10
15Ser His Pro Asp Glu Tyr Lys Ile Ala Ala Leu Val Phe Tyr Ser 20 25
30Cys Ile Phe Ile Ile Gly Leu Phe Val Asn Ile Thr Ala Leu Trp 35 40
45Val Phe Ser Cys Thr Thr Lys Lys Arg Thr Thr Val Thr Ile Tyr 50 55
60Met Met Asn Val Ala Leu Val Asp Leu Ile Phe Ile Met Thr Leu 65 70
75Pro Phe Arg Met Phe Tyr Tyr Ala Lys Asp Ala Trp Pro Phe Gly 80 85
90Glu Tyr Phe Cys Gln Ile Ile Gly Ala Leu Thr Val Phe Tyr Pro 95
100 105Ser Ile Ala Leu Trp Leu Leu Ala Phe Ile Ser Ala Asp Arg Tyr
110 115 120Met Ala Ile Val Gln Pro Lys Tyr Ala Lys Glu Leu Lys Asn
Thr 125 130 135Cys Lys Ala Val Leu Ala Cys Val Gly Val Trp Ile Met
Thr Leu 140 145 150Thr Thr Thr Thr Pro Leu Leu Leu Leu Tyr Lys Asp
Pro Asp Lys 155 160 165Asp Ser Thr Pro Ala Thr Cys Leu Lys Ile Ser
Asp Ile Ile Tyr 170 175 180Leu Lys Ala Val Asn Val Leu Asn Leu Thr
Arg Leu Thr Phe Phe 185 190 195Phe Leu Ile Pro Leu Phe Ile Met Ile
Gly Cys Tyr Leu Val Ile 200 205 210Ile His Asn Leu Leu His Gly Arg
Thr Ser Lys Leu Lys Pro Lys 215 220 225Val Lys Glu Lys Ser Ile Arg
Ile Ile Ile Thr Leu Leu Val Gln 230 235 240Val Leu Val Cys Phe Met
Pro Phe His Ile Cys Phe Ala Phe Leu 245 250 255Met Leu Gly Thr Gly
Glu Asn Ser Tyr Asn Pro Trp Gly Ala Phe 260 265 270Thr Thr Phe Leu
Met Asn Leu Ser Thr Cys Leu Asp Val Ile Leu 275 280 285Tyr Tyr Ile
Val Ser Lys Gln Phe Gln Ala Arg Val Ile Ser Val 290 295 300Met Leu
Tyr Arg Asn Tyr Leu Arg Ser Leu Arg Arg Lys Ser Phe 305 310 315Arg
Ser Gly Ser Leu Arg Ser Leu Ser Asn Ile Asn Ser Glu Met 320 325
330Leu19226PRTHomo sapien 19Met Pro Gly Gly Pro Gly Val Leu Gln Ala
Leu Pro Ala Thr Ile 1 5 10 15Phe Leu Leu Phe Leu Leu Ser Ala Val
Tyr Leu Gly Pro Gly Cys 20 25 30Gln Ala Leu Trp Met His Lys Val Pro
Ala Ser Leu Met Val Ser 35 40 45Leu Gly Glu Asp Ala His Phe Gln Cys
Pro His Asn Ser Ser Asn 50 55 60Asn Ala Asn Val Thr Trp Trp Arg Val
Leu His Gly Asn Tyr Thr 65 70 75Trp Pro Pro Glu Phe Leu Gly Pro Gly
Glu Asp Pro Asn Gly Thr 80 85 90Leu Ile Ile Gln Asn Val Asn Lys Ser
His Gly Gly Ile Tyr Val 95 100 105Cys Arg Val Gln Glu Gly Asn Glu
Ser Tyr Gln Gln Ser Cys Gly 110 115 120Thr Tyr Leu Arg Val Arg Gln
Pro Pro Pro Arg Pro Phe Leu Asp 125 130 135Met Gly Glu Gly Thr Lys
Asn Arg Ile Ile Thr Ala Glu Gly Ile 140 145 150Ile Leu Leu Phe Cys
Ala Val Val Pro Gly Thr Leu Leu Leu Phe 155 160 165Arg Lys Arg Trp
Gln Asn Glu Lys Leu Gly Leu Asp Ala Gly Asp 170 175 180Glu Tyr Glu
Asp Glu Asn Leu Tyr Glu Gly Leu Asn Leu Asp Asp 185 190 195Cys Ser
Met Tyr Glu Asp Ile Ser Arg Gly Leu Gln Gly Thr Tyr 200 205 210Gln
Asp Val Gly Ser Leu Asn Ile Gly Asp Val Gln Leu Glu Lys 215 220
225Pro20118PRTHomo sapien 20Met Pro Arg Glu Asp Ala His Phe Ile Tyr
Gly Tyr Pro Lys Lys 1 5 10 15Gly His Gly His Ser Tyr Thr Thr Ala
Glu Glu Ala Ala Gly Ile 20 25 30Gly Ile Leu Thr Val Ile Leu Gly Val
Leu Leu Leu Ile Gly Cys 35 40 45Trp Tyr Cys Arg Arg Arg Asn Gly Tyr
Arg Ala Leu Met Asp Lys 50 55 60Ser Leu His Val Gly Thr Gln Cys Ala
Leu Thr Arg Arg Cys Pro 65 70 75Gln Glu Gly Phe Asp His Arg Asp Ser
Lys Val Ser Leu Gln Glu 80 85 90Lys Asn Cys Glu Pro Val Val Pro Asn
Ala Pro Pro Ala Tyr Glu 95 100 105Lys Leu Ser Ala Glu Gln Ser Pro
Pro Pro Tyr Ser Pro 110 11521281PRTHomo sapien 21Met Ser Ala Gln
Glu Ser Cys Leu Ser Leu Ile Lys Tyr Phe Leu 1 5 10 15Phe Val Phe
Asn Leu Phe Phe Phe Val Leu Gly Ser Leu Ile Phe 20 25 30Cys Phe Gly
Ile Trp Ile Leu Ile Asp Lys Thr Ser Phe Val Ser 35 40 45Phe Val Gly
Leu Ala Phe Val Pro Leu Gln Ile Trp Ser Lys Val 50 55 60Leu Ala Ile
Ser Gly Ile Phe Thr Met Gly Ile Ala Leu Leu Gly 65 70 75Cys Val Gly
Ala Leu Lys Glu Leu Arg Cys Leu Leu Gly Leu Tyr 80 85 90Phe Gly Met
Leu Leu Leu Leu Phe Ala Thr Gln Ile Thr Leu Gly 95 100 105Ile Leu
Ile Ser Thr Gln Arg Ala Gln Leu Glu Arg Ser Leu Arg 110 115 120Asp
Val Val Glu Lys Thr Ile Gln Lys Tyr Gly Thr Asn Pro Glu 125 130
135Glu Thr Ala Ala Glu Glu Ser Trp Asp Tyr Val Gln Phe Gln Leu 140
145 150Arg Cys Cys Gly Trp His Tyr Pro Gln Asp Trp Phe Gln Val Leu
155 160 165Ile Leu Arg Gly Asn Gly Ser Glu Ala His Arg Val Pro Cys
Ser 170 175 180Cys Tyr Asn Leu Ser Ala Thr Asn Asp Ser Thr Ile Leu
Asp Lys 185 190 195Val Ile Leu Pro Gln Leu Ser Arg Leu Gly His Leu
Ala Arg Ser 200 205 210Arg His Ser Ala Asp Ile Cys Ala Val Pro Ala
Glu Ser His Ile 215 220 225Tyr Arg Glu Gly Cys Ala Gln Gly Leu Gln
Lys Trp Leu His Asn 230 235 240Asn Leu Ile Ser Ile Val Gly Ile Cys
Leu Gly Val Gly Leu Leu 245 250 255Glu Leu Gly Phe Met Thr Leu Ser
Ile Phe Leu Cys Arg Asn Leu 260 265 270Asp His Val Tyr Asn Arg Leu
Ala Arg Tyr Arg 275 280221257PRTHomo sapien 22Met Val Val Ala Leu
Arg Tyr Val Trp Pro Leu Leu Leu Cys Ser 1 5 10 15Pro Cys Leu Leu
Ile Gln Ile Pro Glu Glu Tyr Glu Gly His His 20 25 30Val Met Glu Pro
Pro Val Ile Thr Glu Gln Ser Pro Arg Arg Leu 35 40 45Val Val Phe Pro
Thr Asp Asp Ile Ser Leu Lys Cys Glu Ala Ser 50 55 60Gly Lys Pro Glu
Val Gln Phe Arg Trp Thr Arg Asp Gly Val His 65 70 75Phe Lys Pro Lys
Glu Glu Leu Gly Val Thr Val Tyr Gln Ser Pro 80 85 90His Ser Gly Ser
Phe Thr Ile Thr Gly Asn Asn Ser Asn Phe Ala 95 100 105Gln Arg Phe
Gln Gly Ile Tyr Arg Cys Phe Ala Ser Asn Lys Leu 110 115 120Gly Thr
Ala Met Ser His Glu Ile Arg Leu Met Ala Glu Gly Ala 125 130 135Pro
Lys Trp Pro Lys Glu Thr Val Lys Pro Val Glu Val Glu Glu 140 145
150Gly Glu Ser Val Val Leu Pro Cys Asn Pro Pro Pro Ser Ala Glu 155
160 165Pro Leu Arg Ile Tyr Trp Met Asn Ser Lys Ile Leu His Ile Lys
170 175 180Gln Asp Glu Arg Val Thr Met Gly Gln Asn Gly Asn Leu Tyr
Phe 185 190 195Ala Asn Val Leu Thr Ser Asp Asn His Ser Asp Tyr Ile
Cys His 200 205 210Ala His Phe Pro Gly Thr Arg Thr Ile Ile Gln Lys
Glu Pro Ile 215 220 225Asp Leu Arg Val Lys Ala Thr Asn Ser Met Ile
Asp Arg Lys Pro 230 235 240Arg Leu Leu Phe Pro Thr Asn Ser Ser Ser
His Leu Val Ala Leu 245 250 255Gln Gly Gln Pro Leu Val Leu Glu Cys
Ile Ala Glu Gly Phe Pro 260 265 270Thr Pro Thr Ile Lys Trp Leu Arg
Pro Ser Gly Pro Met Pro Ala 275 280 285Asp Arg Val Thr Tyr Gln Asn
His Asn Lys Thr Leu Gln Leu Leu 290 295 300Lys Val Gly Glu Glu Asp
Asp Gly Glu Tyr Arg Cys Leu Ala Glu 305 310 315Asn Ser Leu Gly Ser
Ala Arg His Ala Tyr Tyr Val Thr Val Glu 320 325 330Ala Ala Pro Tyr
Trp Leu His Lys Pro Gln Ser His Leu Tyr Gly 335 340 345Pro Gly Glu
Thr Ala Arg Leu Asp Cys Gln Val Gln Gly Arg Pro 350 355 360Gln Pro
Glu Val Thr Trp Arg Ile Asn Gly Ile Pro Val Glu Glu 365 370 375Leu
Ala Lys Asp Gln Lys Tyr Arg Ile Gln Arg Gly Ala Leu Ile 380 385
390Leu Ser Asn Val Gln Pro Ser Asp Thr Met Val Thr Gln Cys Glu 395
400 405Ala Arg Asn Arg His Gly Leu Leu Leu Ala Asn Ala Tyr Ile Tyr
410 415 420Val Val Gln Leu Pro Ala Lys Ile Leu Thr Ala Asp Asn Gln
Thr 425 430 435Tyr Met Ala Val Gln Gly Ser Thr Ala Tyr Leu Leu Cys
Lys Ala 440 445 450Phe Gly Ala Pro Val Pro Ser Val Gln Trp Leu Asp
Glu Asp Gly 455 460 465Thr Thr Val Leu Gln Asp Glu Arg Phe Phe Pro
Tyr Ala Asn Gly 470 475 480Thr Leu Gly Ile Arg Asp Leu Gln Ala Asn
Asp Thr Gly Arg Tyr 485 490
495Phe Cys Leu Ala Ala Asn Asp Gln Asn Asn Val Thr Ile Met Ala 500
505 510Asn Leu Lys Val Lys Asp Ala Thr Gln Ile Thr Gln Gly Pro Arg
515 520 525Ser Thr Ile Glu Lys Lys Gly Ser Arg Val Thr Phe Thr Cys
Gln 530 535 540Ala Ser Phe Asp Pro Ser Leu Gln Pro Ser Ile Thr Trp
Arg Gly 545 550 555Asp Gly Arg Asp Leu Gln Glu Leu Gly Asp Ser Asp
Lys Tyr Phe 560 565 570Ile Glu Asp Gly Arg Leu Val Ile His Ser Leu
Asp Tyr Ser Asp 575 580 585Gln Gly Asn Tyr Ser Cys Val Ala Ser Thr
Glu Leu Asp Val Val 590 595 600Glu Ser Arg Ala Gln Leu Leu Val Val
Gly Ser Pro Gly Pro Val 605 610 615Pro Arg Leu Val Leu Ser Asp Leu
His Leu Leu Thr Gln Ser Gln 620 625 630Val Arg Val Ser Trp Ser Pro
Ala Glu Asp His Asn Ala Pro Ile 635 640 645Glu Lys Tyr Asp Ile Glu
Phe Glu Asp Lys Glu Met Ala Pro Glu 650 655 660Lys Trp Tyr Ser Leu
Gly Lys Val Pro Gly Asn Gln Thr Ser Thr 665 670 675Thr Leu Lys Leu
Ser Pro Tyr Val His Tyr Thr Phe Arg Val Thr 680 685 690Ala Ile Asn
Lys Tyr Gly Pro Gly Glu Pro Ser Pro Val Ser Glu 695 700 705Thr Val
Val Thr Pro Glu Ala Ala Pro Glu Lys Asn Pro Val Asp 710 715 720Val
Lys Gly Glu Gly Asn Glu Thr Thr Asn Met Val Ile Thr Trp 725 730
735Lys Pro Leu Arg Trp Met Asp Trp Asn Ala Pro Gln Val Gln Tyr 740
745 750Arg Val Gln Trp Arg Pro Gln Gly Thr Arg Gly Pro Trp Gln Glu
755 760 765Gln Ile Val Ser Asp Pro Phe Leu Val Val Ser Asn Thr Ser
Thr 770 775 780Phe Val Pro Tyr Glu Ile Lys Val Gln Ala Val Asn Ser
Gln Gly 785 790 795Lys Gly Pro Glu Pro Gln Val Thr Ile Gly Tyr Ser
Gly Glu Asp 800 805 810Tyr Pro Gln Ala Ile Pro Glu Leu Glu Gly Ile
Glu Ile Leu Asn 815 820 825Ser Ser Ala Val Leu Val Lys Trp Arg Pro
Val Asp Leu Ala Gln 830 835 840Val Lys Gly His Leu Arg Gly Tyr Asn
Val Thr Tyr Trp Arg Glu 845 850 855Gly Ser Gln Arg Lys His Ser Lys
Arg His Ile His Lys Asp His 860 865 870Val Val Val Pro Ala Asn Thr
Thr Ser Val Ile Leu Ser Gly Leu 875 880 885Arg Pro Tyr Ser Ser Tyr
His Leu Glu Val Gln Ala Phe Asn Gly 890 895 900Arg Gly Ser Gly Pro
Ala Ser Glu Phe Thr Phe Ser Thr Pro Glu 905 910 915Gly Val Pro Gly
His Pro Glu Ala Leu His Leu Glu Cys Gln Ser 920 925 930Asn Thr Ser
Leu Leu Leu Arg Trp Gln Pro Pro Leu Ser His Asn 935 940 945Gly Val
Leu Thr Gly Tyr Val Leu Ser Tyr His Pro Leu Asp Glu 950 955 960Gly
Gly Lys Gly Gln Leu Ser Phe Asn Leu Arg Asp Pro Glu Leu 965 970
975Arg Thr His Asn Leu Thr Asp Leu Ser Pro His Leu Arg Tyr Arg 980
985 990Phe Gln Leu Gln Ala Thr Thr Lys Glu Gly Pro Gly Glu Ala Ile
995 1000 1005Val Arg Glu Gly Gly Thr Met Ala Leu Ser Gly Ile Ser
Asp Phe 1010 1015 1020Gly Asn Ile Ser Ala Thr Ala Gly Glu Asn Tyr
Ser Val Val Ser 1025 1030 1035Trp Val Pro Lys Glu Gly Gln Cys Asn
Phe Arg Phe His Ile Leu 1040 1045 1050Phe Lys Ala Leu Gly Glu Glu
Lys Gly Gly Ala Ser Leu Ser Pro 1055 1060 1065Gln Tyr Val Ser Tyr
Asn Gln Ser Ser Tyr Thr Gln Trp Asp Leu 1070 1075 1080Gln Pro Asp
Thr Asp Tyr Glu Ile His Leu Phe Lys Glu Arg Met 1085 1090 1095Phe
Arg His Gln Met Ala Val Lys Thr Asn Gly Thr Gly Arg Val 1100 1105
1110Arg Leu Pro Pro Ala Gly Phe Ala Thr Glu Gly Trp Phe Ile Gly
1115 1120 1125Phe Val Ser Ala Ile Ile Leu Leu Leu Leu Val Leu Leu
Ile Leu 1130 1135 1140Cys Phe Ile Lys Arg Ser Lys Gly Gly Lys Tyr
Ser Val Lys Asp 1145 1150 1155Lys Glu Asp Thr Gln Val Asp Ser Glu
Ala Arg Pro Met Lys Asp 1160 1165 1170Glu Thr Phe Gly Glu Tyr Arg
Ser Leu Glu Ser Asp Asn Glu Glu 1175 1180 1185Lys Ala Phe Gly Ser
Ser Gln Pro Ser Leu Asn Gly Asp Ile Lys 1190 1195 1200Pro Leu Gly
Ser Asp Asp Ser Leu Ala Asp Tyr Gly Gly Ser Val 1205 1210 1215Asp
Val Gln Phe Asn Glu Asp Gly Ser Phe Ile Gly Gln Tyr Ser 1220 1225
1230Gly Lys Lys Glu Lys Glu Ala Ala Gly Gly Asn Asp Ser Ser Gly
1235 1240 1245Ala Thr Ser Pro Ile Asn Pro Ala Val Ala Leu Glu 1250
1255231257PRTHomo sapien 23Met Val Val Ala Leu Arg Tyr Val Trp Pro
Leu Leu Leu Cys Ser 1 5 10 15Pro Cys Leu Leu Ile Gln Ile Pro Glu
Glu Tyr Glu Gly His His 20 25 30Val Met Glu Pro Pro Val Ile Thr Glu
Gln Ser Pro Arg Arg Leu 35 40 45Val Val Phe Pro Thr Asp Asp Ile Ser
Leu Lys Cys Glu Ala Ser 50 55 60Gly Lys Pro Glu Val Gln Phe Arg Trp
Thr Arg Asp Gly Val His 65 70 75Phe Lys Pro Lys Glu Glu Leu Gly Val
Thr Val Tyr Gln Ser Pro 80 85 90His Ser Gly Ser Phe Thr Ile Thr Gly
Asn Asn Ser Asn Phe Ala 95 100 105Gln Arg Phe Gln Gly Ile Tyr Arg
Cys Phe Ala Ser Asn Lys Leu 110 115 120Gly Thr Ala Met Ser His Glu
Ile Arg Leu Met Ala Glu Gly Ala 125 130 135Pro Lys Trp Pro Lys Glu
Thr Val Lys Pro Val Glu Val Glu Glu 140 145 150Gly Glu Ser Val Val
Leu Pro Cys Asn Pro Pro Pro Ser Ala Glu 155 160 165Pro Leu Arg Ile
Tyr Trp Met Asn Ser Lys Ile Leu His Ile Lys 170 175 180Gln Asp Glu
Arg Val Thr Met Gly Gln Asn Gly Asn Leu Tyr Phe 185 190 195Ala Asn
Val Leu Thr Ser Asp Asn His Ser Asp Tyr Ile Cys His 200 205 210Ala
His Phe Pro Gly Thr Arg Thr Ile Ile Gln Lys Glu Pro Ile 215 220
225Asp Leu Arg Val Lys Ala Thr Asn Ser Met Ile Asp Arg Lys Pro 230
235 240Arg Leu Leu Phe Pro Thr Asn Ser Ser Ser His Leu Val Ala Leu
245 250 255Gln Gly Gln Pro Leu Val Leu Glu Cys Ile Ala Glu Gly Phe
Pro 260 265 270Thr Pro Thr Ile Lys Trp Leu Arg Pro Ser Gly Pro Met
Pro Ala 275 280 285Asp Arg Val Thr Tyr Gln Asn His Asn Lys Thr Leu
Gln Leu Leu 290 295 300Lys Val Gly Glu Glu Asp Asp Gly Glu Tyr Arg
Cys Leu Ala Glu 305 310 315Asn Ser Leu Gly Ser Ala Arg His Ala Tyr
Tyr Val Thr Val Glu 320 325 330Ala Ala Pro Tyr Trp Leu His Lys Pro
Gln Ser His Leu Tyr Gly 335 340 345Pro Gly Glu Thr Ala Arg Leu Asp
Cys Gln Val Gln Gly Arg Pro 350 355 360Gln Pro Glu Val Thr Trp Arg
Ile Asn Gly Ile Pro Val Glu Glu 365 370 375Leu Ala Lys Asp Gln Lys
Tyr Arg Ile Gln Arg Gly Ala Leu Ile 380 385 390Leu Ser Asn Val Gln
Pro Ser Asp Thr Met Val Thr Gln Cys Glu 395 400 405Ala Arg Asn Arg
His Gly Leu Leu Leu Ala Asn Ala Tyr Ile Tyr 410 415 420Val Val Gln
Leu Pro Ala Lys Ile Leu Thr Ala Asp Asn Gln Thr 425 430 435Tyr Met
Ala Val Gln Gly Ser Thr Ala Tyr Leu Leu Cys Lys Ala 440 445 450Phe
Gly Ala Pro Val Pro Ser Val Gln Trp Leu Asp Glu Asp Gly 455 460
465Thr Thr Val Leu Gln Asp Glu Arg Phe Phe Pro Tyr Ala Asn Gly 470
475 480Thr Leu Gly Ile Arg Asp Leu Gln Ala Asn Asp Thr Gly Arg Tyr
485 490 495Phe Cys Leu Ala Ala Asn Asp Gln Asn Asn Val Thr Ile Met
Ala 500 505 510Asn Leu Lys Val Lys Asp Ala Thr Gln Ile Thr Gln Gly
Pro Arg 515 520 525Ser Thr Ile Glu Lys Lys Gly Ser Arg Val Thr Phe
Thr Cys Gln 530 535 540Ala Ser Phe Asp Pro Ser Leu Gln Pro Ser Ile
Thr Trp Arg Gly 545 550 555Asp Gly Arg Asp Leu Gln Glu Leu Gly Asp
Ser Asp Lys Tyr Phe 560 565 570Ile Glu Asp Gly Arg Leu Val Ile His
Ser Leu Asp Tyr Ser Asp 575 580 585Gln Gly Asn Tyr Ser Cys Val Ala
Ser Thr Glu Leu Asp Val Val 590 595 600Glu Ser Arg Ala Gln Leu Leu
Val Val Gly Ser Pro Gly Pro Val 605 610 615Pro Arg Leu Val Leu Ser
Asp Leu His Leu Leu Thr Gln Ser Gln 620 625 630Val Arg Val Ser Trp
Ser Pro Ala Glu Asp His Asn Ala Pro Ile 635 640 645Glu Lys Tyr Asp
Ile Glu Phe Glu Asp Lys Glu Met Ala Pro Glu 650 655 660Lys Trp Tyr
Ser Leu Gly Lys Val Pro Gly Asn Gln Thr Ser Thr 665 670 675Thr Leu
Lys Leu Ser Pro Tyr Val His Tyr Thr Phe Arg Val Thr 680 685 690Ala
Ile Asn Lys Tyr Gly Pro Gly Glu Pro Ser Pro Val Ser Glu 695 700
705Thr Val Val Thr Pro Glu Ala Ala Pro Glu Lys Asn Pro Val Asp 710
715 720Val Lys Gly Glu Gly Asn Glu Thr Thr Asn Met Val Ile Thr Trp
725 730 735Lys Pro Leu Arg Trp Met Asp Trp Asn Ala Pro Gln Val Gln
Tyr 740 745 750Arg Val Gln Trp Arg Pro Gln Gly Thr Arg Gly Pro Trp
Gln Glu 755 760 765Gln Ile Val Ser Asp Pro Phe Leu Val Val Ser Asn
Thr Ser Thr 770 775 780Phe Val Pro Tyr Glu Ile Lys Val Gln Ala Val
Asn Ser Gln Gly 785 790 795Lys Gly Pro Glu Pro Gln Val Thr Ile Gly
Tyr Ser Gly Glu Asp 800 805 810Tyr Pro Gln Ala Ile Pro Glu Leu Glu
Gly Ile Glu Ile Leu Asn 815 820 825Ser Ser Ala Val Leu Val Lys Trp
Arg Pro Val Asp Leu Ala Gln 830 835 840Val Lys Gly His Leu Arg Gly
Tyr Asn Val Thr Tyr Trp Arg Glu 845 850 855Gly Ser Gln Arg Lys His
Ser Lys Arg His Ile His Lys Asp His 860 865 870Val Val Val Pro Ala
Asn Thr Thr Ser Val Ile Leu Ser Gly Leu 875 880 885Arg Pro Tyr Ser
Ser Tyr His Leu Glu Val Gln Ala Phe Asn Gly 890 895 900Arg Gly Ser
Gly Pro Ala Ser Glu Phe Thr Phe Ser Thr Pro Glu 905 910 915Gly Val
Pro Gly His Pro Glu Ala Leu His Leu Glu Cys Gln Ser 920 925 930Asn
Thr Ser Leu Leu Leu Arg Trp Gln Pro Pro Leu Ser His Asn 935 940
945Gly Val Leu Thr Gly Tyr Val Leu Ser Tyr His Pro Leu Asp Glu 950
955 960Gly Gly Lys Gly Gln Leu Ser Phe Asn Leu Arg Asp Pro Glu Leu
965 970 975Arg Thr His Asn Leu Thr Asp Leu Ser Pro His Leu Arg Tyr
Arg 980 985 990Phe Gln Leu Gln Ala Thr Thr Lys Glu Gly Pro Gly Glu
Ala Ile 995 1000 1005Val Arg Glu Gly Gly Thr Met Ala Leu Ser Gly
Ile Ser Asp Phe 1010 1015 1020Gly Asn Ile Ser Ala Thr Ala Gly Glu
Asn Tyr Ser Val Val Ser 1025 1030 1035Trp Val Pro Lys Glu Gly Gln
Cys Asn Phe Arg Phe His Ile Leu 1040 1045 1050Phe Lys Ala Leu Gly
Glu Glu Lys Gly Gly Ala Ser Leu Ser Pro 1055 1060 1065Gln Tyr Val
Ser Tyr Asn Gln Ser Ser Tyr Thr Gln Trp Asp Leu 1070 1075 1080Gln
Pro Asp Thr Asp Tyr Glu Ile His Leu Phe Lys Glu Arg Met 1085 1090
1095Phe Arg His Gln Met Ala Val Lys Thr Asn Gly Thr Gly Arg Val
1100 1105 1110Arg Leu Pro Pro Ala Gly Phe Ala Thr Glu Gly Trp Phe
Ile Gly 1115 1120 1125Phe Val Ser Ala Ile Ile Leu Leu Leu Leu Val
Leu Leu Ile Leu 1130 1135 1140Cys Phe Ile Lys Arg Ser Lys Gly Gly
Lys Tyr Ser Val Lys Asp 1145 1150 1155Lys Glu Asp Thr Gln Val Asp
Ser Glu Ala Arg Pro Met Lys Asp 1160 1165 1170Glu Thr Phe Gly Glu
Tyr Arg Ser Leu Glu Ser Asp Asn Glu Glu 1175 1180 1185Lys Ala Phe
Gly Ser Ser Gln Pro Ser Leu Asn Gly Asp Ile Lys 1190 1195 1200Pro
Leu Gly Ser Asp Asp Ser Leu Ala Asp Tyr Gly Gly Ser Val 1205 1210
1215Asp Val Gln Phe Asn Glu Asp Gly Ser Phe Ile Gly Gln Tyr Ser
1220 1225 1230Gly Lys Lys Glu Lys Glu Ala Ala Gly Gly Asn Asp Ser
Ser Gly 1235 1240 1245Ala Thr Ser Pro Ile Asn Pro Ala Val Ala Leu
Glu 1250 125524162PRTHomo sapien 24Met Trp Lys Val Ser Ala Leu Leu
Phe Val Leu Gly Ser Ala Ser 1 5 10 15Leu Trp Val Leu Ala Glu Gly
Ala Ser Thr Gly Gln Pro Glu Asp 20 25 30Asp Thr Glu Thr Thr Gly Leu
Glu Gly Gly Val Ala Met Pro Gly 35 40 45Ala Glu Asp Asp Val Val Thr
Pro Gly Thr Ser Glu Asp Arg Tyr 50 55 60Lys Ser Gly Leu Thr Thr Leu
Val Ala Thr Ser Val Asn Ser Val 65 70 75Thr Gly Ile Arg Ile Glu Asp
Leu Pro Thr Ser Glu Ser Thr Val 80 85 90His Ala Gln Glu Gln Ser Pro
Ser Ala Thr Ala Ser Asn Val Ala 95 100 105Thr Ser His Ser Thr Glu
Lys Val Asp Gly Asp Thr Gln Thr Thr 110 115 120Val Glu Lys Asp Gly
Leu Ser Thr Val Thr Leu Val Gly Ile Ile 125 130 135Val Gly Val Leu
Leu Ala Ile Gly Phe Ile Gly Gly Ile Ile Val 140 145 150Val Val Met
Arg Lys Met Ser Gly Arg Tyr Ser Pro 155 16025160PRTHomo sapien
25Met Trp Lys Val Ser Ala Leu Leu Phe Val Leu Gly Ser Ala Ser 1 5
10 15Leu Trp Val Leu Ala Glu Gly Ala Ser Thr Gly Gln Pro Glu Asp 20
25 30Asp Thr Glu Thr Thr Gly Leu Glu Gly Gly Val Ala Met Pro Gly 35
40 45Ala Glu Asp Asp Val Val Thr Pro Gly Thr Ser Glu Asp Arg Tyr 50
55 60Lys Ser Gly Leu Thr Thr Leu Val Ala Thr Ser Val Asn Ser Val 65
70 75Thr Gly Ile Arg Ile Glu Asp Leu Pro Thr Ser Glu Ser Thr Val 80
85 90His Ala Gln Glu Gln Ser Pro Ser Ala Thr Ala Ser Asn Val Ala 95
100 105Thr Ser His Ser Thr Glu Lys Val Asp Gly Asp Thr Gln Thr Thr
110 115 120Val Glu Lys Asp Gly Leu Ser Thr Val Thr Leu Val Gly Ile
Ile 125
130 135Val Gly Val Leu Leu Ala Ile Gly Phe Ile Gly Gly Ile Ile Val
140 145 150Val Val Met Arg Lys Met Ser Gly Arg Pro 155
16026582PRTHomo sapien 26Met Ser Pro Ala Pro Arg Pro Pro Arg Cys
Leu Leu Leu Pro Leu 1 5 10 15Leu Thr Leu Gly Thr Ala Leu Ala Ser
Leu Gly Ser Ala Gln Ser 20 25 30Ser Ser Phe Ser Pro Glu Ala Trp Leu
Gln Gln Tyr Gly Tyr Leu 35 40 45Pro Pro Gly Asp Leu Arg Thr His Thr
Gln Arg Ser Pro Gln Ser 50 55 60Leu Ser Ala Ala Ile Ala Ala Met Gln
Lys Phe Tyr Gly Leu Gln 65 70 75Val Thr Gly Lys Ala Asp Ala Asp Thr
Met Lys Ala Met Arg Arg 80 85 90Pro Arg Cys Gly Val Pro Asp Lys Phe
Gly Ala Glu Ile Lys Ala 95 100 105Asn Val Arg Arg Lys Arg Tyr Ala
Ile Gln Gly Leu Lys Trp Gln 110 115 120His Asn Glu Ile Thr Phe Cys
Ile Gln Asn Tyr Thr Pro Lys Val 125 130 135Gly Glu Tyr Ala Thr Tyr
Glu Ala Ile Arg Lys Ala Phe Arg Val 140 145 150Trp Glu Ser Ala Thr
Pro Leu Arg Phe Arg Glu Val Pro Tyr Ala 155 160 165Tyr Ile Arg Glu
Gly His Glu Lys Gln Ala Asp Ile Met Ile Phe 170 175 180Phe Ala Glu
Gly Phe His Gly Asp Ser Thr Pro Phe Asp Gly Glu 185 190 195Gly Gly
Phe Leu Ala His Ala Tyr Phe Pro Gly Pro Asn Ile Gly 200 205 210Gly
Asp Thr His Phe Asp Ser Ala Glu Pro Trp Thr Val Arg Asn 215 220
225Glu Asp Leu Asn Gly Asn Asp Ile Phe Leu Val Ala Val His Glu 230
235 240Leu Gly His Ala Leu Gly Leu Glu His Ser Ser Asp Pro Ser Ala
245 250 255Ile Met Ala Pro Phe Tyr Gln Trp Met Asp Thr Glu Asn Phe
Val 260 265 270Leu Pro Asp Asp Asp Arg Arg Gly Ile Gln Gln Leu Tyr
Gly Gly 275 280 285Glu Ser Gly Phe Pro Thr Lys Met Pro Pro Gln Pro
Arg Thr Thr 290 295 300Ser Arg Pro Ser Val Pro Asp Lys Pro Lys Asn
Pro Thr Tyr Gly 305 310 315Pro Asn Ile Cys Asp Gly Asn Phe Asp Thr
Val Ala Met Leu Arg 320 325 330Gly Glu Met Phe Val Phe Lys Glu Arg
Trp Phe Trp Arg Val Arg 335 340 345Asn Asn Gln Val Met Asp Gly Tyr
Pro Met Pro Ile Gly Gln Phe 350 355 360Trp Arg Gly Leu Pro Ala Ser
Ile Asn Thr Ala Tyr Glu Arg Lys 365 370 375Asp Gly Lys Phe Val Phe
Phe Lys Gly Asp Lys His Trp Val Phe 380 385 390Asp Glu Ala Ser Leu
Glu Pro Gly Tyr Pro Lys His Ile Lys Glu 395 400 405Leu Gly Arg Gly
Leu Pro Thr Asp Lys Ile Asp Ala Ala Leu Phe 410 415 420Trp Met Pro
Asn Gly Lys Thr Tyr Phe Phe Arg Gly Asn Lys Tyr 425 430 435Tyr Arg
Phe Asn Glu Glu Leu Arg Ala Val Asp Ser Glu Tyr Pro 440 445 450Lys
Asn Ile Lys Val Trp Glu Gly Ile Pro Glu Ser Pro Arg Gly 455 460
465Ser Phe Met Gly Ser Asp Glu Val Phe Thr Tyr Phe Tyr Lys Gly 470
475 480Asn Lys Tyr Trp Lys Phe Asn Asn Gln Lys Leu Lys Val Glu Pro
485 490 495Gly Tyr Pro Lys Ser Ala Leu Arg Asp Trp Met Gly Cys Pro
Ser 500 505 510Gly Gly Arg Pro Asp Glu Gly Thr Glu Glu Glu Thr Glu
Val Ile 515 520 525Ile Ile Glu Val Asp Glu Glu Gly Gly Gly Ala Val
Ser Ala Ala 530 535 540Ala Val Val Leu Pro Val Leu Leu Leu Leu Leu
Val Leu Ala Val 545 550 555Gly Leu Ala Val Phe Phe Phe Arg Arg His
Gly Thr Pro Arg Arg 560 565 570Leu Leu Tyr Cys Gln Arg Ser Leu Leu
Asp Lys Val 575 58027525PRTHomo sapien 27Met Trp Glu Ala Gln Phe
Leu Gly Leu Leu Phe Leu Gln Pro Leu 1 5 10 15Trp Val Ala Pro Val
Lys Pro Leu Gln Pro Gly Ala Glu Val Pro 20 25 30Val Val Trp Ala Gln
Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser 35 40 45Pro Thr Ile Pro Leu
Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly 50 55 60Val Thr Trp Gln His
Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala 65 70 75Pro Gly His Pro Leu
Ala Pro Gly Pro His Pro Ala Ala Pro Ser 80 85 90Ser Trp Gly Pro Arg
Pro Arg Arg Tyr Thr Val Leu Ser Val Gly 95 100 105Pro Gly Gly Leu
Arg Ser Gly Arg Leu Pro Leu Gln Pro Arg Val 110 115 120Gln Leu Asp
Glu Arg Gly Arg Gln Arg Gly Asp Phe Ser Leu Trp 125 130 135Leu Arg
Pro Ala Arg Arg Ala Asp Ala Gly Glu Tyr Arg Ala Ala 140 145 150Val
His Leu Arg Asp Arg Ala Leu Ser Cys Arg Leu Arg Leu Arg 155 160
165Leu Gly Gln Ala Ser Met Thr Ala Ser Pro Pro Gly Ser Leu Arg 170
175 180Ala Ser Asp Trp Val Ile Leu Asn Cys Ser Phe Ser Arg Pro Asp
185 190 195Arg Pro Ala Ser Val His Trp Phe Arg Asn Arg Gly Gln Gly
Arg 200 205 210Val Pro Val Arg Glu Ser Pro His His His Leu Ala Glu
Ser Phe 215 220 225Leu Phe Leu Pro Gln Val Ser Pro Met Asp Ser Gly
Pro Trp Gly 230 235 240Cys Ile Leu Thr Tyr Arg Asp Gly Phe Asn Val
Ser Ile Met Tyr 245 250 255Asn Leu Thr Val Leu Gly Leu Glu Pro Pro
Thr Pro Leu Thr Val 260 265 270Tyr Ala Gly Ala Gly Ser Arg Val Gly
Leu Pro Cys Arg Leu Pro 275 280 285Ala Gly Val Gly Thr Arg Ser Phe
Leu Thr Ala Lys Trp Thr Pro 290 295 300Pro Gly Gly Gly Pro Asp Leu
Leu Val Thr Gly Asp Asn Gly Asp 305 310 315Phe Thr Leu Arg Leu Glu
Asp Val Ser Gln Ala Gln Ala Gly Thr 320 325 330Tyr Thr Cys His Ile
His Leu Gln Glu Gln Gln Leu Asn Ala Thr 335 340 345Val Thr Leu Ala
Ile Ile Thr Val Thr Pro Lys Ser Phe Gly Ser 350 355 360Pro Gly Ser
Leu Gly Lys Leu Leu Cys Glu Val Thr Pro Val Ser 365 370 375Gly Gln
Glu Arg Phe Val Trp Ser Ser Leu Asp Thr Pro Ser Gln 380 385 390Arg
Ser Phe Ser Gly Pro Trp Leu Glu Ala Gln Glu Ala Gln Leu 395 400
405Leu Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln Gly Glu Arg Leu 410
415 420Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser Ser Pro Gly Ala
425 430 435Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu Pro Ala Gly His
Leu 440 445 450Leu Leu Phe Leu Thr Leu Gly Val Leu Ser Leu Leu Leu
Leu Val 455 460 465Thr Gly Ala Phe Gly Phe His Leu Trp Arg Arg Gln
Trp Arg Pro 470 475 480Arg Arg Phe Ser Ala Leu Glu Gln Gly Ile His
Pro Arg Gln Ala 485 490 495Gln Ser Lys Ile Glu Glu Leu Glu Gln Glu
Pro Glu Pro Glu Pro 500 505 510Glu Pro Glu Pro Glu Pro Glu Pro Glu
Pro Glu Pro Glu Gln Leu 515 520 52528525PRTHomo sapien 28Met Trp
Glu Ala Gln Phe Leu Gly Leu Leu Phe Leu Gln Pro Leu 1 5 10 15Trp
Val Ala Pro Val Lys Pro Leu Gln Pro Gly Ala Glu Val Pro 20 25 30Val
Val Trp Ala Gln Glu Gly Ala Pro Ala Gln Leu Pro Cys Ser 35 40 45Pro
Thr Ile Pro Leu Gln Asp Leu Ser Leu Leu Arg Arg Ala Gly 50 55 60Val
Thr Trp Gln His Gln Pro Asp Ser Gly Pro Pro Ala Ala Ala 65 70 75Pro
Gly His Pro Leu Ala Pro Gly Pro His Pro Ala Ala Pro Ser 80 85 90Ser
Trp Gly Pro Arg Pro Arg Arg Tyr Thr Val Leu Ser Val Gly 95 100
105Pro Gly Gly Leu Arg Ser Gly Arg Leu Pro Leu Gln Pro Arg Val 110
115 120Gln Leu Asp Glu Arg Gly Arg Gln Arg Gly Asp Phe Ser Leu Trp
125 130 135Leu Arg Pro Ala Arg Arg Ala Asp Ala Gly Glu Tyr Arg Ala
Ala 140 145 150Val His Leu Arg Asp Arg Ala Leu Ser Cys Arg Leu Arg
Leu Arg 155 160 165Leu Gly Gln Ala Ser Met Thr Ala Ser Pro Pro Gly
Ser Leu Arg 170 175 180Ala Ser Asp Trp Val Ile Leu Asn Cys Ser Phe
Ser Arg Pro Asp 185 190 195Arg Pro Ala Ser Val His Trp Phe Arg Asn
Arg Gly Gln Gly Arg 200 205 210Val Pro Val Arg Glu Ser Pro His His
His Leu Ala Glu Ser Phe 215 220 225Leu Phe Leu Pro Gln Val Ser Pro
Met Asp Ser Gly Pro Trp Gly 230 235 240Cys Ile Leu Thr Tyr Arg Asp
Gly Phe Asn Val Ser Ile Met Tyr 245 250 255Asn Leu Thr Val Leu Gly
Leu Glu Pro Pro Thr Pro Leu Thr Val 260 265 270Tyr Ala Gly Ala Gly
Ser Arg Val Gly Leu Pro Cys Arg Leu Pro 275 280 285Ala Gly Val Gly
Thr Arg Ser Phe Leu Thr Ala Lys Trp Thr Pro 290 295 300Pro Gly Gly
Gly Pro Asp Leu Leu Val Thr Gly Asp Asn Gly Asp 305 310 315Phe Thr
Leu Arg Leu Glu Asp Val Ser Gln Ala Gln Ala Gly Thr 320 325 330Tyr
Thr Cys His Ile His Leu Gln Glu Gln Gln Leu Asn Ala Thr 335 340
345Val Thr Leu Ala Ile Ile Thr Val Thr Pro Lys Ser Phe Gly Ser 350
355 360Pro Gly Ser Leu Gly Lys Leu Leu Cys Glu Val Thr Pro Val Ser
365 370 375Gly Gln Glu Arg Phe Val Trp Ser Ser Leu Asp Thr Pro Ser
Gln 380 385 390Arg Ser Phe Ser Gly Pro Trp Leu Glu Ala Gln Glu Ala
Gln Leu 395 400 405Leu Ser Gln Pro Trp Gln Cys Gln Leu Tyr Gln Gly
Glu Arg Leu 410 415 420Leu Gly Ala Ala Val Tyr Phe Thr Glu Leu Ser
Ser Pro Gly Ala 425 430 435Gln Arg Ser Gly Arg Ala Pro Gly Ala Leu
Pro Ala Gly His Leu 440 445 450Leu Leu Phe Leu Thr Leu Gly Val Leu
Ser Leu Leu Leu Leu Val 455 460 465Thr Gly Ala Phe Gly Phe His Leu
Trp Arg Arg Gln Trp Arg Pro 470 475 480Arg Arg Phe Ser Ala Leu Glu
Gln Gly Ile His Pro Pro Gln Ala 485 490 495Gln Ser Lys Ile Glu Glu
Leu Glu Gln Glu Pro Glu Pro Glu Pro 500 505 510Glu Pro Glu Pro Glu
Pro Glu Pro Glu Pro Glu Pro Glu Gln Leu 515 520 525292181PRTHomo
sapien 29Met Met Met Met Met Met Met Lys Lys Met Gln His Gln Arg
Gln 1 5 10 15Gln Gln Ala Asp His Ala Asn Glu Ala Asn Tyr Ala Arg
Gly Thr 20 25 30Arg Leu Pro Leu Ser Gly Glu Gly Pro Thr Ser Gln Pro
Asn Ser 35 40 45Ser Lys Gln Thr Val Leu Ser Trp Gln Ala Ala Ile Asp
Ala Ala 50 55 60Arg Gln Ala Lys Ala Ala Gln Thr Met Ser Thr Ser Ala
Pro Pro 65 70 75Pro Val Gly Ser Leu Ser Gln Arg Lys Arg Gln Gln Tyr
Ala Lys 80 85 90Ser Lys Lys Gln Gly Asn Ser Ser Asn Ser Arg Pro Ala
Arg Ala 95 100 105Leu Phe Cys Leu Ser Leu Asn Asn Pro Ile Arg Arg
Ala Cys Ile 110 115 120Ser Ile Val Glu Trp Lys Pro Phe Asp Ile Phe
Ile Leu Leu Ala 125 130 135Ile Phe Ala Asn Cys Val Ala Leu Ala Ile
Tyr Ile Pro Phe Pro 140 145 150Glu Asp Asp Ser Asn Ser Thr Asn His
Asn Leu Glu Lys Val Glu 155 160 165Tyr Ala Phe Leu Ile Ile Phe Thr
Val Glu Thr Phe Leu Lys Ile 170 175 180Ile Ala Tyr Gly Leu Leu Leu
His Pro Asn Ala Tyr Val Arg Asn 185 190 195Gly Trp Asn Leu Leu Asp
Phe Val Ile Val Ile Val Gly Leu Phe 200 205 210Ser Val Ile Leu Glu
Gln Leu Thr Lys Glu Thr Glu Gly Gly Asn 215 220 225His Ser Ser Gly
Lys Ser Gly Gly Phe Asp Val Lys Ala Leu Arg 230 235 240Ala Phe Arg
Val Leu Arg Pro Leu Arg Leu Val Ser Gly Val Pro 245 250 255Ser Leu
Gln Val Val Leu Asn Ser Ile Ile Lys Ala Met Val Pro 260 265 270Leu
Leu His Ile Ala Leu Leu Val Leu Phe Val Ile Ile Ile Tyr 275 280
285Ala Ile Ile Gly Leu Glu Leu Phe Ile Gly Lys Met His Lys Thr 290
295 300Cys Phe Phe Ala Asp Ser Asp Ile Val Ala Glu Glu Asp Pro Ala
305 310 315Pro Cys Ala Phe Ser Gly Asn Gly Arg Gln Cys Thr Ala Asn
Gly 320 325 330Thr Glu Cys Arg Ser Gly Trp Val Gly Pro Asn Gly Gly
Ile Thr 335 340 345Asn Phe Asp Asn Phe Ala Phe Ala Met Leu Thr Val
Phe Gln Cys 350 355 360Ile Thr Met Glu Gly Trp Thr Asp Val Leu Tyr
Trp Val Asn Asp 365 370 375Ala Ile Gly Trp Glu Trp Pro Trp Val Tyr
Phe Val Ser Leu Ile 380 385 390Ile Leu Gly Ser Phe Phe Val Leu Asn
Leu Val Leu Gly Val Leu 395 400 405Ser Gly Glu Phe Ser Lys Glu Arg
Glu Lys Ala Lys Ala Arg Gly 410 415 420Asp Phe Gln Lys Leu Arg Glu
Lys Gln Gln Leu Glu Glu Asp Leu 425 430 435Lys Gly Tyr Leu Asp Trp
Ile Thr Gln Ala Glu Asp Ile Asp Pro 440 445 450Glu Asn Glu Glu Glu
Gly Gly Glu Glu Gly Lys Arg Asn Thr Ser 455 460 465Met Pro Thr Ser
Glu Thr Glu Ser Val Asn Thr Glu Asn Val Ser 470 475 480Gly Glu Gly
Glu Asn Arg Gly Cys Cys Gly Ser Leu Trp Cys Trp 485 490 495Trp Arg
Arg Arg Gly Ala Ala Lys Ala Gly Pro Ser Gly Cys Arg 500 505 510Arg
Trp Gly Gln Ala Ile Ser Lys Ser Lys Leu Ser Arg Arg Trp 515 520
525Arg Arg Trp Asn Arg Phe Asn Arg Arg Arg Cys Arg Ala Ala Val 530
535 540Lys Ser Val Thr Phe Tyr Trp Leu Val Ile Val Leu Val Phe Leu
545 550 555Asn Thr Leu Thr Ile Ser Ser Glu His Tyr Asn Gln Pro Asp
Trp 560 565 570Leu Thr Gln Ile Gln Asp Ile Ala Asn Lys Val Leu Leu
Ala Leu 575 580 585Phe Thr Cys Glu Met Leu Val Lys Met Tyr Ser Leu
Gly Leu Gln 590 595 600Ala Tyr Phe Val Ser Leu Phe Asn Arg Phe Asp
Cys Phe Val Val 605 610 615Cys Gly Gly Ile Thr Glu Thr Ile Leu Val
Glu Leu Glu Ile Met 620 625 630Ser Pro Leu Gly Ile Ser Val Phe Arg
Cys Val Arg Leu Leu Arg 635 640 645Ile Phe Lys Val Thr Arg His Trp
Thr Ser Leu Ser
Asn Leu Val 650 655 660Ala Ser Leu Leu Asn Ser Met Lys Ser Ile Ala
Ser Leu Leu Leu 665 670 675Leu Leu Phe Leu Phe Ile Ile Ile Phe Ser
Leu Leu Gly Met Gln 680 685 690Leu Phe Gly Gly Lys Phe Asn Phe Asp
Glu Thr Gln Thr Lys Arg 695 700 705Ser Thr Phe Asp Asn Phe Pro Gln
Ala Leu Leu Thr Val Phe Gln 710 715 720Ile Leu Thr Gly Glu Asp Trp
Asn Ala Val Met Tyr Asp Gly Ile 725 730 735Met Ala Tyr Gly Gly Pro
Ser Ser Ser Gly Met Ile Val Cys Ile 740 745 750Tyr Phe Ile Ile Leu
Phe Ile Cys Gly Asn Tyr Ile Leu Leu Asn 755 760 765Val Phe Leu Ala
Ile Ala Val Asp Asn Leu Ala Asp Ala Glu Ser 770 775 780Leu Asn Thr
Ala Gln Lys Glu Glu Ala Glu Glu Lys Glu Arg Lys 785 790 795Lys Ile
Ala Arg Lys Glu Ser Leu Glu Asn Lys Lys Asn Asn Lys 800 805 810Pro
Glu Val Asn Gln Ile Ala Asn Ser Asp Asn Lys Val Thr Ile 815 820
825Asp Asp Tyr Arg Glu Glu Asp Glu Asp Lys Asp Pro Tyr Pro Pro 830
835 840Cys Asp Val Pro Val Gly Glu Glu Glu Glu Glu Glu Glu Glu Asp
845 850 855Glu Pro Glu Val Pro Ala Gly Pro Arg Pro Arg Arg Ile Ser
Glu 860 865 870Leu Asn Met Lys Glu Lys Ile Ala Pro Ile Pro Glu Gly
Ser Ala 875 880 885Phe Phe Ile Leu Ser Lys Thr Asn Pro Ile Arg Val
Gly Cys His 890 895 900Lys Leu Ile Asn His His Ile Phe Thr Asn Leu
Ile Leu Val Phe 905 910 915Ile Met Leu Ser Ser Ala Ala Leu Ala Ala
Glu Asp Pro Ile Arg 920 925 930Ser His Ser Phe Arg Asn Thr Ile Leu
Gly Tyr Phe Asp Tyr Ala 935 940 945Phe Thr Ala Ile Phe Thr Val Glu
Ile Leu Leu Lys Met Thr Thr 950 955 960Phe Gly Ala Phe Leu His Lys
Gly Ala Phe Cys Arg Asn Tyr Phe 965 970 975Asn Leu Leu Asp Met Leu
Val Val Gly Val Ser Leu Val Ser Phe 980 985 990Gly Ile Gln Ser Ser
Ala Ile Ser Val Val Lys Ile Leu Arg Val 995 1000 1005Leu Arg Val
Leu Arg Pro Leu Arg Ala Ile Asn Arg Ala Lys Gly 1010 1015 1020Leu
Lys His Val Val Gln Cys Val Phe Val Ala Ile Arg Thr Ile 1025 1030
1035Gly Asn Ile Met Ile Val Thr Thr Leu Leu Gln Phe Met Phe Ala
1040 1045 1050Cys Ile Gly Val Gln Leu Phe Lys Gly Lys Phe Tyr Arg
Cys Thr 1055 1060 1065Asp Glu Ala Lys Ser Asn Pro Glu Glu Cys Arg
Gly Leu Phe Ile 1070 1075 1080Leu Tyr Lys Asp Gly Asp Val Asp Ser
Pro Val Val Arg Glu Arg 1085 1090 1095Ile Trp Gln Asn Ser Asp Phe
Asn Phe Asp Asn Val Leu Ser Ala 1100 1105 1110Met Met Ala Leu Phe
Thr Val Ser Thr Phe Glu Gly Trp Pro Ala 1115 1120 1125Leu Leu Tyr
Lys Ala Ile Asp Ser Asn Gly Glu Asn Ile Gly Pro 1130 1135 1140Ile
Tyr Asn His Arg Val Glu Ile Ser Ile Phe Phe Ile Ile Tyr 1145 1150
1155Ile Ile Ile Val Ala Phe Phe Met Met Asn Ile Phe Val Gly Phe
1160 1165 1170Val Ile Val Thr Phe Gln Glu Gln Gly Glu Lys Glu Tyr
Lys Asn 1175 1180 1185Cys Glu Leu Asp Lys Asn Gln Arg Gln Cys Val
Glu Tyr Ala Leu 1190 1195 1200Lys Ala Arg Pro Leu Arg Arg Tyr Ile
Pro Lys Asn Pro Tyr Gln 1205 1210 1215Tyr Lys Phe Trp Tyr Val Val
Asn Ser Ser Pro Phe Glu Tyr Met 1220 1225 1230Met Phe Val Leu Ile
Met Leu Asn Thr Leu Cys Leu Ala Met Gln 1235 1240 1245His Tyr Glu
Gln Ser Lys Met Phe Asn Asp Ala Met Asp Ile Leu 1250 1255 1260Asn
Met Val Phe Thr Gly Val Phe Thr Val Glu Met Val Leu Lys 1265 1270
1275Val Ile Ala Phe Lys Pro Lys Gly Tyr Phe Ser Asp Ala Trp Asn
1280 1285 1290Thr Phe Asp Ser Leu Ile Val Ile Gly Ser Ile Ile Asp
Val Ala 1295 1300 1305Leu Ser Glu Ala Asp Pro Thr Glu Ser Glu Asn
Val Pro Val Pro 1310 1315 1320Thr Ala Thr Pro Gly Asn Ser Glu Glu
Ser Asn Arg Ile Ser Ile 1325 1330 1335Thr Phe Phe Arg Leu Phe Arg
Val Met Arg Leu Val Lys Leu Leu 1340 1345 1350Ser Arg Gly Glu Gly
Ile Arg Thr Leu Leu Trp Thr Phe Ile Lys 1355 1360 1365Ser Phe Gln
Ala Leu Pro Tyr Val Ala Leu Leu Ile Ala Met Leu 1370 1375 1380Phe
Phe Ile Tyr Ala Val Ile Gly Met Gln Met Phe Gly Lys Val 1385 1390
1395Ala Met Arg Asp Asn Asn Gln Ile Asn Arg Asn Asn Asn Phe Gln
1400 1405 1410Thr Phe Pro Gln Ala Val Leu Leu Leu Phe Arg Cys Ala
Thr Gly 1415 1420 1425Glu Ala Trp Gln Glu Ile Met Leu Ala Cys Leu
Pro Gly Lys Leu 1430 1435 1440Cys Asp Pro Glu Ser Asp Tyr Asn Pro
Gly Glu Glu Tyr Thr Cys 1445 1450 1455Gly Ser Asn Phe Ala Ile Val
Tyr Phe Ile Ser Phe Tyr Met Leu 1460 1465 1470Cys Ala Phe Leu Ile
Ile Asn Leu Phe Val Ala Val Ile Met Asp 1475 1480 1485Asn Phe Asp
Tyr Leu Thr Arg Asp Trp Ser Ile Leu Gly Pro His 1490 1495 1500His
Leu Asp Glu Phe Lys Arg Ile Trp Ser Glu Tyr Asp Pro Glu 1505 1510
1515Ala Lys Gly Arg Ile Lys His Leu Asp Val Val Thr Leu Leu Arg
1520 1525 1530Arg Ile Gln Pro Pro Leu Gly Phe Gly Lys Leu Cys Pro
His Arg 1535 1540 1545Val Ala Cys Lys Arg Leu Val Ala Met Asn Met
Pro Leu Asn Ser 1550 1555 1560Asp Gly Thr Val Met Phe Asn Ala Thr
Leu Phe Ala Leu Val Arg 1565 1570 1575Thr Ala Leu Lys Ile Lys Thr
Glu Gly Asn Leu Glu Gln Ala Asn 1580 1585 1590Glu Glu Leu Arg Ala
Val Ile Lys Lys Ile Trp Lys Lys Thr Ser 1595 1600 1605Met Lys Leu
Leu Asp Gln Val Val Pro Pro Ala Gly Asp Asp Glu 1610 1615 1620Val
Thr Val Gly Lys Phe Tyr Ala Thr Phe Leu Ile Gln Asp Tyr 1625 1630
1635Phe Arg Lys Phe Lys Lys Arg Lys Glu Gln Gly Leu Val Gly Lys
1640 1645 1650Tyr Pro Ala Lys Asn Thr Thr Ile Ala Leu Gln Ala Gly
Leu Arg 1655 1660 1665Thr Leu His Asp Ile Gly Pro Glu Ile Arg Arg
Ala Ile Ser Cys 1670 1675 1680Asp Leu Gln Asp Asp Glu Pro Glu Glu
Thr Lys Arg Glu Glu Glu 1685 1690 1695Asp Asp Val Phe Lys Arg Asn
Gly Ala Leu Leu Gly Asn His Val 1700 1705 1710Asn His Val Asn Ser
Asp Arg Arg Asp Ser Leu Gln Gln Thr Asn 1715 1720 1725Thr Thr His
Arg Pro Leu His Val Gln Arg Pro Ser Ile Pro Pro 1730 1735 1740Ala
Ser Asp Thr Glu Lys Pro Leu Phe Pro Pro Ala Gly Asn Ser 1745 1750
1755Val Cys His Asn His His Asn His Asn Ser Ile Gly Lys Gln Val
1760 1765 1770Pro Thr Ser Thr Asn Ala Asn Leu Asn Asn Ala Asn Met
Ser Lys 1775 1780 1785Ala Ala His Gly Lys Arg Pro Ser Ile Gly Asn
Leu Glu His Val 1790 1795 1800Ser Glu Asn Gly His His Ser Ser His
Lys His Asp Arg Glu Pro 1805 1810 1815Gln Arg Arg Ser Ser Val Lys
Arg Thr Arg Tyr Tyr Glu Thr Tyr 1820 1825 1830Ile Arg Ser Asp Ser
Gly Asp Glu Gln Leu Pro Thr Ile Cys Arg 1835 1840 1845Glu Asp Pro
Glu Ile His Gly Tyr Phe Arg Asp Pro His Cys Leu 1850 1855 1860Gly
Glu Gln Glu Tyr Phe Ser Ser Glu Glu Cys Tyr Glu Asp Asp 1865 1870
1875Ser Ser Pro Thr Trp Ser Arg Gln Asn Tyr Gly Tyr Tyr Ser Arg
1880 1885 1890Tyr Pro Gly Arg Asn Ile Asp Ser Glu Arg Pro Arg Gly
Tyr His 1895 1900 1905His Pro Gln Gly Phe Leu Glu Asp Asp Asp Ser
Pro Val Cys Tyr 1910 1915 1920Asp Ser Arg Arg Ser Pro Arg Arg Arg
Leu Leu Pro Pro Thr Pro 1925 1930 1935Ala Ser His Arg Arg Ser Ser
Phe Asn Phe Glu Cys Leu Arg Arg 1940 1945 1950Gln Ser Ser Gln Glu
Glu Val Pro Ser Ser Pro Ile Phe Pro His 1955 1960 1965Arg Thr Ala
Leu Pro Leu His Leu Met Gln Gln Gln Ile Met Ala 1970 1975 1980Val
Ala Gly Leu Asp Ser Ser Lys Ala Gln Lys Tyr Ser Pro Ser 1985 1990
1995His Ser Thr Arg Ser Trp Ala Thr Pro Pro Ala Thr Pro Pro Tyr
2000 2005 2010Arg Asp Trp Thr Pro Cys Tyr Thr Pro Leu Ile Gln Val
Glu Gln 2015 2020 2025Ser Glu Ala Leu Asp Gln Val Asn Gly Ser Leu
Pro Ser Leu His 2030 2035 2040Arg Ser Ser Trp Tyr Thr Asp Glu Pro
Asp Ile Ser Tyr Arg Thr 2045 2050 2055Phe Thr Pro Ala Ser Leu Thr
Val Pro Ser Ser Phe Arg Asn Lys 2060 2065 2070Asn Ser Asp Lys Gln
Arg Ser Ala Asp Ser Leu Val Glu Ala Val 2075 2080 2085Leu Ile Ser
Glu Gly Leu Gly Arg Tyr Ala Arg Asp Pro Lys Phe 2090 2095 2100Val
Ser Ala Thr Lys His Glu Ile Ala Asp Ala Cys Asp Leu Thr 2105 2110
2115Ile Asp Glu Met Glu Ser Ala Ala Ser Thr Leu Leu Asn Gly Asn
2120 2125 2130Val Arg Pro Arg Ala Asn Gly Asp Val Gly Pro Leu Ser
His Arg 2135 2140 2145Gln Asp Tyr Glu Leu Gln Asp Phe Gly Pro Gly
Tyr Ser Asp Glu 2150 2155 2160Glu Pro Asp Pro Gly Arg Asp Glu Glu
Asp Leu Ala Asp Glu Met 2165 2170 2175Ile Cys Ile Thr Thr Leu
218030220PRTHomo sapien 30Met Ala Ser Ala Gly Met Gln Ile Leu Gly
Val Val Leu Thr Leu 1 5 10 15Leu Gly Trp Val Asn Gly Leu Val Ser
Cys Ala Leu Pro Met Trp 20 25 30Lys Val Thr Ala Phe Ile Gly Asn Ser
Ile Val Val Ala Gln Val 35 40 45Val Trp Glu Gly Leu Trp Met Ser Cys
Val Val Gln Ser Thr Gly 50 55 60Gln Met Gln Cys Lys Val Tyr Asp Ser
Leu Leu Ala Leu Pro Gln 65 70 75Asp Leu Gln Ala Ala Arg Ala Leu Cys
Val Ile Ala Leu Leu Val 80 85 90Ala Leu Phe Gly Leu Leu Val Tyr Leu
Ala Gly Ala Lys Cys Thr 95 100 105Thr Cys Val Glu Glu Lys Asp Ser
Lys Ala Arg Leu Val Leu Thr 110 115 120Ser Gly Ile Val Phe Val Ile
Ser Gly Val Leu Thr Leu Ile Pro 125 130 135Val Cys Trp Thr Ala His
Ala Ile Ile Arg Asp Phe Tyr Asn Pro 140 145 150Leu Val Ala Glu Ala
Gln Lys Arg Glu Leu Gly Ala Ser Leu Tyr 155 160 165Leu Gly Trp Ala
Ala Ser Gly Leu Leu Leu Leu Gly Gly Gly Leu 170 175 180Leu Cys Cys
Thr Cys Pro Ser Gly Gly Ser Gln Gly Pro Ser His 185 190 195Tyr Met
Ala Arg Tyr Ser Thr Ser Ala Pro Ala Ile Ser Arg Gly 200 205 210Pro
Ser Glu Tyr Pro Thr Lys Asn Tyr Val 215 22031494PRTHomo sapien
31Met Val Arg Lys Pro Val Val Ser Thr Ile Ser Lys Gly Gly Tyr 1 5
10 15Leu Gln Gly Asn Val Asn Gly Arg Leu Pro Ser Leu Gly Asn Lys 20
25 30Glu Pro Pro Gly Gln Glu Lys Val Gln Leu Lys Arg Lys Val Thr 35
40 45Leu Leu Arg Gly Val Ser Ile Ile Ile Gly Thr Ile Ile Gly Ala 50
55 60Gly Ile Phe Ile Ser Pro Lys Gly Val Leu Gln Asn Thr Gly Ser 65
70 75Val Gly Met Ser Leu Thr Ile Trp Thr Val Cys Gly Val Leu Ser 80
85 90Leu Phe Gly Ala Leu Ser Tyr Ala Glu Leu Gly Thr Thr Ile Lys 95
100 105Lys Ser Gly Gly His Tyr Thr Tyr Ile Leu Glu Val Phe Gly Pro
110 115 120Leu Pro Ala Phe Val Arg Val Trp Val Glu Leu Leu Ile Ile
Arg 125 130 135Pro Ala Ala Thr Ala Val Ile Ser Leu Ala Phe Gly Arg
Tyr Ile 140 145 150Leu Glu Pro Phe Phe Ile Gln Cys Glu Ile Pro Glu
Leu Ala Ile 155 160 165Lys Leu Ile Thr Ala Val Gly Ile Thr Val Val
Met Val Leu Asn 170 175 180Ser Met Ser Val Ser Trp Ser Ala Arg Ile
Gln Ile Phe Leu Thr 185 190 195Phe Cys Lys Leu Thr Ala Ile Leu Ile
Ile Ile Val Pro Gly Val 200 205 210Met Gln Leu Ile Lys Gly Gln Thr
Gln Asn Phe Lys Asp Ala Phe 215 220 225Ser Gly Arg Asp Ser Ser Ile
Thr Arg Leu Pro Leu Ala Phe Tyr 230 235 240Tyr Gly Met Tyr Ala Tyr
Ala Gly Trp Phe Tyr Leu Asn Phe Val 245 250 255Thr Glu Glu Val Glu
Asn Pro Glu Lys Thr Ile Pro Leu Ala Ile 260 265 270Cys Ile Ser Met
Ala Ile Val Thr Ile Gly Tyr Val Leu Thr Asn 275 280 285Val Ala Tyr
Phe Thr Thr Ile Asn Ala Glu Glu Leu Leu Leu Ser 290 295 300Asn Ala
Val Ala Val Thr Phe Ser Glu Arg Leu Leu Gly Asn Phe 305 310 315Ser
Leu Ala Val Pro Ile Phe Val Ala Leu Ser Cys Phe Gly Ser 320 325
330Met Asn Gly Gly Val Phe Ala Val Ser Arg Leu Phe Tyr Val Ala 335
340 345Ser Arg Glu Gly His Leu Pro Glu Ile Leu Ser Met Ile His Val
350 355 360Arg Lys His Thr Pro Leu Pro Ala Val Ile Val Leu His Pro
Leu 365 370 375Thr Met Ile Met Leu Phe Ser Gly Asp Leu Asp Ser Leu
Leu Asn 380 385 390Phe Leu Ser Phe Ala Arg Trp Leu Phe Ile Gly Leu
Ala Val Ala 395 400 405Gly Leu Ile Tyr Leu Arg Tyr Lys Cys Pro Asp
Met His Arg Pro 410 415 420Phe Lys Val Pro Leu Phe Ile Pro Ala Leu
Phe Ser Phe Thr Cys 425 430 435Leu Phe Met Val Ala Leu Ser Leu Tyr
Ser Asp Pro Phe Ser Thr 440 445 450Gly Ile Gly Phe Val Ile Thr Leu
Thr Gly Val Pro Ala Tyr Tyr 455 460 465Leu Phe Ile Ile Trp Asp Lys
Lys Pro Arg Trp Phe Arg Ile Met 470 475 480Ser Gly Phe Leu Ala Leu
Met Pro Ala Gln Ala Cys Asp Met 485 49032355PRTHomo sapien 32Met
Glu Glu Gly Glu Arg Ser Pro Leu Leu Ser Gln Glu Thr Ala 1 5 10
15Gly Gln Lys Pro Leu Ser Val His Arg Pro Pro Thr Ser Gly Cys 20 25
30Leu Gly Pro Val Pro Arg Glu Asp Gln Ala Glu Ala Trp Gly Cys 35 40
45Ser Cys Cys Pro Pro Glu Thr Lys His Gln Ala Leu Ser Gly Thr 50 55
60Pro Lys Lys Gly Pro Ala Pro Ser Leu Ser Pro Gly Ser Ser Cys
65 70 75Val Lys Tyr Leu Ile Phe Leu Ser Asn Phe Pro Phe Ser Leu Leu
80 85 90Gly Leu Leu Ala Leu Ala Ile Gly Leu Trp Gly Leu Ala Val Lys
95 100 105Gly Ser Leu Gly Ser Asp Leu Gly Gly Pro Leu Pro Thr Asp
Pro 110 115 120Met Leu Gly Leu Ala Leu Gly Gly Leu Val Val Ser Ala
Ala Ser 125 130 135Leu Ala Gly Cys Leu Gly Ala Leu Cys Glu Asn Thr
Cys Leu Leu 140 145 150Arg Gly Phe Ser Gly Gly Ile Leu Ala Phe Leu
Val Leu Glu Ala 155 160 165Val Ala Gly Ala Leu Val Val Ala Leu Trp
Gly Pro Leu Gln Asp 170 175 180Ser Leu Glu His Thr Leu Arg Val Ala
Ile Ala His Tyr Gln Asp 185 190 195Asp Pro Asp Leu Arg Phe Leu Leu
Asp Gln Val Gln Leu Gly Leu 200 205 210Arg Cys Cys Gly Ala Ala Ser
Tyr Gln Asp Trp Gln Gln Asn Leu 215 220 225Tyr Phe Asn Cys Ser Ser
Pro Gly Val Gln Ala Cys Ser Leu Pro 230 235 240Ala Ser Cys Cys Ile
Asp Pro Arg Glu Asp Gly Ala Ser Val Asn 245 250 255Asp Gln Cys Gly
Phe Gly Val Leu Arg Leu Asp Ala Asp Ala Ala 260 265 270Gln Arg Val
Val Tyr Leu Glu Gly Cys Gly Pro Pro Leu Arg Arg 275 280 285Trp Leu
Arg Ala Asn Leu Ala Ala Ser Gly Gly Tyr Ala Ile Ala 290 295 300Val
Val Leu Leu Gln Gly Ala Glu Leu Leu Leu Ala Ala Arg Leu 305 310
315Leu Gly Ala Leu Ala Ala Arg Ser Gly Ala Ala Tyr Gly Pro Gly 320
325 330Ala His Gly Glu Asp Arg Ala Gly Pro Gln Ser Pro Ser Pro Gly
335 340 345Ala Pro Pro Ala Ala Lys Pro Ala Arg Gly 350 355
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