U.S. patent application number 10/712892 was filed with the patent office on 2005-06-09 for compositions and methods for the diagnosis and treatment of tumor.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Ashkenazi, Avi J., Frantz, Gretchen, Goddard, Audrey, Gonzalez, Lino, Gurney, Austin L., Polakis, Paul, Polson, Andrew, Wood, William I., Wu, Thomas D., Zhang, Zemin.
Application Number | 20050123925 10/712892 |
Document ID | / |
Family ID | 32329854 |
Filed Date | 2005-06-09 |
United States Patent
Application |
20050123925 |
Kind Code |
A1 |
Ashkenazi, Avi J. ; et
al. |
June 9, 2005 |
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: |
Ashkenazi, Avi J.; (San
Mateo, CA) ; Frantz, Gretchen; (San Francisco,
CA) ; Goddard, Audrey; (San Francisco, CA) ;
Gonzalez, Lino; (Hollister, CA) ; Gurney, Austin
L.; (San Francisco, CA) ; Polakis, Paul;
(Burlingame, CA) ; Polson, Andrew; (San Francisco,
CA) ; Wood, William I.; (Cupertino, CA) ; Wu,
Thomas D.; (San Francisco, CA) ; Zhang, Zemin;
(Foster City, CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
1 DNA Way
South San Francisco
CA
94080
|
Family ID: |
32329854 |
Appl. No.: |
10/712892 |
Filed: |
November 12, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60437344 |
Dec 31, 2002 |
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60431250 |
Dec 6, 2002 |
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60426847 |
Nov 15, 2002 |
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Current U.S.
Class: |
435/6.16 ;
435/7.23 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61P 35/02 20180101; A61P 35/00 20180101; A61P 43/00 20180101; C07K
16/30 20130101; A61P 35/04 20180101 |
Class at
Publication: |
435/006 ;
435/007.23 |
International
Class: |
C12Q 001/68; G01N
033/574 |
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 the polypeptide shown as SEQ ID NO:22 in a test
sample of tissue cells obtained from said mammal and in a control
sample of normal tissue cells of the same tissue type as the test
sample, wherein a higher level of expression of said gene in the
test sample, as compared to the control sample, is indicative of
the presence of a 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 said gene comprises employing an
oligonucleotide in an in situ hybridization or RT-PCR analysis.
23. The method of claim 21, wherein the step determining the level
of expression of said gene comprises employing an antibody in an
immunohistochemistry or Western blot analysis.
24. The method of claim 21, wherein said test and control samples
are obtained from breast tissue.
25. The method of claim 21, wherein said test and control samples
are obtained from brain tissue.
26. The method of claim 21, wherein said test and control samples
are obtained from bone tissue.
27. The method of claim 21, wherein said test and control samples
are obtained from colon tissue.
28. The method of claim 21, wherein said test and control samples
are obtained from kidney tissue.
29. The method of claim 21, wherein said test and control samples
are obtained from pancreatic tissue.
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: (1) 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 defmed 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 defmed
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 defmed 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%, 99% or animo acide 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
detectably 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. 22-42 (SEQ ID NOS:22-42);
[0041] (b) a DNA molecule encoding the amino acid sequence shown in
any one of FIGS. 22-42 (SEQ ID NOS:22-42), lacking its associated
signal peptide;
[0042] (c) a DNA molecule encoding an extracellular domain of the
polypeptide shown in any one of FIGS. 22-42 (SEQ ID NOS:22-42),
with its associated signal peptide;
[0043] (d) a DNA molecule encoding an extracellular domain of the
polypeptide shown in any one of FIGS. 22-42 (SEQ ID NOS:22-42),
lacking its associated signal peptide;
[0044] (e) the nucleotide sequence shown in any one of FIGS. 1-21
(SEQ ID NOS:1-21);
[0045] (f) the full-length coding region of the nucleotide sequence
shown in any one of FIGS. 1-21 (SEQ ID NOS:1-21); 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. 22-42 (SEQ ID NOS:22-42);
[0049] (b) a nucleotide sequence that encodes the amino acid
sequence shown in any one of FIGS. 22-42 (SEQ ID NOS:22-42),
lacking its associated signal peptide;
[0050] (c) a nucleotide sequence that encodes an extracellular
domain of the polypeptide shown in any one of FIGS. 22-42 (SEQ ID
NOS:22-42), with its associated signal peptide;
[0051] (d) a nucleotide sequence that encodes an extracellular
domain of the polypeptide shown in any one of FIGS. 22-42 (SEQ ID
NOS:22-42), lacking its associated signal peptide;
[0052] (e) the nucleotide sequence shown in any one of FIGS. 1-21
(SEQ ID NOS: 1-21);
[0053] (f) the full-length coding region of the nucleotide sequence
shown in any one of FIGS. 1-21 (SEQ ID NOS:1-21); 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. 22-42 (SEQ ID NOS:22-42);
[0057] (b) a nucleic acid that encodes the amino acid sequence
shown in any one of FIGS. 22-42 (SEQ ID NOS:22-42), lacking its
associated signal peptide;
[0058] (c) a nucleic acid that encodes an extracellular domain of
the polypeptide shown in any one of FIGS. 22-42 (SEQ ID NOS:22-42),
with its associated signal peptide;
[0059] (d) a nucleic acid that encodes an extracellular domain of
the polypeptide shown in any one of FIGS. 22-42 (SEQ ID NOS:22-42),
lacking its associated signal peptide;
[0060] (e) the nucleotide sequence shown in any one of FIGS. 1-21
(SEQ ID NOS: 1-21);
[0061] (f) the full-length coding region of the nucleotide sequence
shown in any one of FIGS. 1-21 (SEQ ID NOS:1-21); 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. 22-42 (SEQ ID
NOS:22-42);
[0072] (b) the polypeptide shown in any one of FIGS. 22-42 (SEQ ID
NOS:22-42), lacking its associated signal peptide;
[0073] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), with its associated signal
peptide;
[0074] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), lacking its associated
signal peptide;
[0075] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0076] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-21 (SEQ ID
NOS:1-21).
[0077] 12. An isolated polypeptide having:
[0078] (a) the amino acid sequence shown in any one of FIGS. 22-42
(SEQ ID NOS:22-42);
[0079] (b) the amino acid sequence shown in any one of FIGS. 22-42
(SEQ ID NOS:22-42), 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. 22-42 (SEQ ID NOS:22-42),
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. 22-42 (SEQ ID NOS:22-42),
lacking its associated signal peptide sequence;
[0082] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0083] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-21
(SEQ ID NOS:1-21).
[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. 22-42 (SEQ ID
NOS:22-42);
[0088] (b) the polypeptide shown in any one of FIGS. 22-42 (SEQ ID
NOS:22-42), lacking its associated signal peptide;
[0089] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), with its associated signal
peptide;
[0090] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), lacking its associated
signal peptide;
[0091] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0092] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-21 (SEQ ID
NOS:1-21).
[0093] 16. An isolated antibody that binds to a polypeptide
having:
[0094] (a) the amino acid sequence shown in any one of FIGS. 22-42
(SEQ ID NOS:22-42);
[0095] (b) the amino acid sequence shown in any one of FIGS. 22-42
(SEQ ID NOS:22-42), 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. 22-42 (SEQ ID NOS:22-42),
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. 22-42 (SEQ ID NOS:22-42),
lacking its associated signal peptide sequence;
[0098] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0099] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-21
(SEQ ID NOS: 1-21).
[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. 22-42 (SEQ ID
NOS:22-42);
[0120] (b) the polypeptide shown in any one of FIGS. 22-42 (SEQ ID
NOS:22-42), lacking its associated signal peptide;
[0121] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), with its associated signal
peptide;
[0122] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), lacking its associated
signal peptide;
[0123] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0124] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-21 (SEQ ID
NOS: 1-21).
[0125] 36. An isolated oligopeptide that binds to a polypeptide
having:
[0126] (a) the amino acid sequence shown in any one of FIGS. 22-42
(SEQ ID NOS:22-42);
[0127] (b) the amino acid sequence shown in any one of FIGS. 22-42
(SEQ ID NOS:22-42), 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. 22-42 (SEQ ID NOS:22-42),
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. 22-42 (SEQ ID NOS:22-42),
lacking its associated signal peptide sequence;
[0130] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0131] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-21
(SEQ ID NOS:1-21).
[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. 22-42 (SEQ ID
NOS:22-42);
[0142] (b) the polypeptide shown in any one of FIGS. 22-42 (SEQ ID
NOS:22-42), lacking its associated signal peptide;
[0143] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), with its associated signal
peptide;
[0144] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), lacking its associated
signal peptide;
[0145] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0146] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-21 (SEQ ID
NOS:1-21).
[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. 22-42
(SEQ ID NOS:22-42);
[0149] (b) the amino acid sequence shown in any one of FIGS. 22-42
(SEQ ID NOS:22-42), 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. 22-42 (SEQ ID NOS:2242), 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. 22-42 (SEQ ID NOS:22-42),
lacking its associated signal peptide sequence;
[0152] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0153] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-21
(SEQ ID NOS: 1-21).
[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. 22-42 (SEQ ID
NOS:22-42);
[0179] (b) the polypeptide shown in any one of FIGS. 22-42 (SEQ ID
NOS:22-42), lacking its associated signal peptide;
[0180] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), with its associated signal
peptide;
[0181] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), lacking its associated
signal peptide;
[0182] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0183] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-21 (SEQ ID
NOS: 1-21), 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. 22-42
(SEQ ID NOS:22-42);
[0202] (b) the amino acid sequence shown in any one of FIGS. 22-42
(SEQ ID NOS:22-42), 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. 22-42 (SEQ ID NOS:22-42),
with its associated signal peptide sequence;
[0204] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown in any one of FIGS. 22-42 (SEQ ID NOS:22-42),
lacking its associated signal peptide sequence;
[0205] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0206] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-21
(SEQ ID NOS: 1-21).
[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. 22-42 (SEQ ID
NOS:22-42);
[0209] (b) the polypeptide shown in any one of FIGS. 22-42 (SEQ ID
NOS:22-42), lacking its associated signal peptide;
[0210] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), with its associated signal
peptide;
[0211] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), lacking its associated
signal peptide;
[0212] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0213] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-21 (SEQ ID
NOS:1-21), 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. 22:42
(SEQ ID NOS:22-42);
[0230] (b) the amino acid sequence shown in any one of FIGS. 22-42
(SEQ ID NOS:22-42), 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. 22-42 (SEQ ID NOS:22-42),
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. 22-42 (SEQ ID NOS:22-42),
lacking its associated signal peptide sequence;
[0233] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0234] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-21
(SEQ ID NOS: 1-21).
[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. 22-42 (SEQ ID
NOS:22-42);
[0237] (b) the polypeptide shown in any one of FIGS. 22-42 (SEQ ID
NOS:22-42), lacking its associated signal peptide;
[0238] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), with its associated signal
peptide;
[0239] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), lacking its associated
signal peptide;
[0240] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0241] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-21 (SEQ ID
NOS:1-21), 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. 22-42
(SEQ ID NOS:22-42);
[0247] (b) the amino acid sequence shown in any one of FIGS. 22-42
(SEQ ID NOS:22-42), 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. 22-42 (SEQ ID NOS:22-42),
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. 22-42 (SEQ ID NOS:22-42),
lacking its associated signal peptide sequence;
[0250] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0251] 1(f) an amino acid sequence encoded by the full-length
coding region of the nucleotide sequence shown in any one of FIGS.
1-21 (SEQ ID NOS: 1-21).
[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. 22-42 (SEQ ID
NOS:22-42);
[0254] (b) the polypeptide shown in any one of FIGS. 22-42 (SEQ ID
NOS:22-42), lacking its associated signal peptide;
[0255] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), with its associated signal
peptide;
[0256] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), lacking its associated
signal peptide;
[0257] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0258] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-21 (SEQ ID
NOS:1-21), 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. 22-42
(SEQ ID NOS:22-42);
[0263] (b) the amino acid sequence shown in any one of FIGS. 22-42
(SEQ ID NOS:22-42), 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. 22-42 (SEQ ID NOS:22-42),
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. 22-42 (SEQ ID NOS:22-42),
lacking its associated signal peptide sequence;
[0266] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0267] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-21
(SEQ ID NOS:1-21).
[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. 22-42 (SEQ ID
NOS:22-42);
[0270] (b) the polypeptide shown in any one of FIGS. 22-42 (SEQ ID
NOS:22-42), lacking its associated signal peptide;
[0271] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), with its associated signal
peptide;
[0272] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), lacking its associated
signal peptide;
[0273] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0274] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-21 (SEQ ID
NOS:1-21), 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. 22-42
(SEQ ID NOS:22-42);
[0279] (b) the amino acid sequence shown in any one of FIGS. 22-42
(SEQ ID NOS:22-42), 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. 22-42 (SEQ ID NOS:22-42),
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. 22-42 (SEQ ID NOS:22-42),
lacking its associated signal peptide sequence;
[0282] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0283] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-21
(SEQ ID NOS:1-21).
[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. 22-42 (SEQ ID
NOS:22-42);
[0286] (b) the polypeptide shown in any one of FIGS. 22-42 (SEQ ID
NOS:22-42), lacking its associated signal peptide;
[0287] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), with its associated signal
peptide;
[0288] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), lacking its associated
signal peptide;
[0289] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0290] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-21 (SEQ ID
NOS: 1-21), 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. 22-42 (SEQ ID
NOS:22-42);
[0295] (b) the polypeptide shown in any one of FIGS. 22-42 (SEQ ID
NOS:22-42), lacking its associated signal peptide;
[0296] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), with its associated signal
peptide;
[0297] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), lacking its associated
signal peptide;
[0298] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0299] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-21 (SEQ ID
NOS: 1-21), 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. 22-42 (SEQ ID
NOS:22-42);
[0345] (b) the polypeptide shown in any one of FIGS. 22-42 (SEQ ID
NOS:22-42), lacking its associated signal peptide;
[0346] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), with its associated signal
peptide;
[0347] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), lacking its associated
signal peptide;
[0348] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0349] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-21 (SEQ ID
NOS: 1-21), 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. 22-42
(SEQ ID NOS:22-42);
[0367] (b) the amino acid sequence shown in any one of FIGS. 22-42
(SEQ ID NOS:22-42), 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. 22-42 (SEQ ID NOS:22-42),
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. 22-42 (SEQ ID NOS:22-42),
lacking its associated signal peptide sequence;
[0370] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0371] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-21
(SEQ ID NOS:1-21).
[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. 22-42 (SEQ ID
NOS:22-42);
[0374] (b) the polypeptide shown in any one of FIGS. 22-42 (SEQ ID
NOS:2242), lacking its associated signal peptide;
[0375] (c) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), with its associated signal
peptide;
[0376] (d) an extracellular domain of the polypeptide shown in any
one of FIGS. 22-42 (SEQ ID NOS:22-42), lacking its associated
signal peptide;
[0377] (e) a polypeptide encoded by the nucleotide sequence shown
in any one of FIGS. 1-21 (SEQ ID NOS:1-21); or
[0378] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown in any one of FIGS. 1-21 (SEQ ID
NOS:1-21), 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. 22-42
(SEQ ID NOS:22-42);
[0394] (b) the amino acid sequence shown in any one of FIGS. 22-42
(SEQ ID NOS:22-42), 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. 22-42 (SEQ ID NOS:22-42),
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. 22-42 (SEQ ID NOS:22-42),
lacking its associated signal peptide sequence;
[0397] (e) an amino acid sequence encoded by the nucleotide
sequence shown in any one of FIGS. 1-21 (SEQ ID NOS: 1-21); or
[0398] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown in any one of FIGS. 1-21
(SEQ ID NOS: 1-21).
[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 TAT400
cDNA, wherein SEQ ID NO:1 is a clone designated herein as
"DNA225795".
[0401] FIG. 2 shows a nucleotide sequence (SEQ ID NO:2) of a TAT401
cDNA, wherein SEQ ID NO:2 is a clone designated herein as
"DNA62814".
[0402] FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a TAT402
cDNA, wherein SEQ ID NO:3 is a clone designated herein as
"DNA334601".
[0403] FIG. 4 shows a nucleotide sequence (SEQ ID NO:4) of a TAT403
cDNA, wherein SEQ ID NO:4 is a clone designated herein as
"DNA255030".
[0404] FIG. 5 shows a nucleotide sequence (SEQ ID NO:5) of a TAT404
cDNA, wherein SEQ ID NO:5 is a clone designated herein as
"DNA257428".
[0405] FIG. 6 shows a nucleotide sequence (SEQ ID NO:6) of a TAT405
cDNA, wherein SEQ ID NO:6 is a clone designated herein as
"DNA194132".
[0406] FIG. 7 shows a nucleotide sequence (SEQ ID NO:7) of a TAT406
cDNA, wherein SEQ ID NO:7 is a clone designated herein as
"DNA255706".
[0407] FIG. 8 shows a nucleotide sequence (SEQ ID NO:8) of a TAT407
cDNA, wherein SEQ ID NO:8 is a clone designated herein as
"DNA329863".
[0408] FIG. 9 shows a nucleotide sequence (SEQ ID NO:9) of a TAT408
cDNA, wherein SEQ ID NO:9 is a clone designated herein as
"DNA22780".
[0409] FIG. 10 shows a nucleotide sequence (SEQ ID NO:10) of a
TAT206 cDNA, wherein SEQ ID NO:10 is a clone designated herein as
"DNA56041".
[0410] FIGS. 11A-B show a nucleotide sequence (SEQ ID NO:11) of a
TAT409 cDNA, wherein SEQ ID NO:11 is a clone designated herein as
"DNA220758".
[0411] FIGS. 12A-B show a nucleotide sequence (SEQ ID NO:12) of a
TAT410 cDNA, wherein SEQ ID NO:12 is a clone designated herein as
"DNA225549".
[0412] FIGS. 13A-B show a nucleotide sequence (SEQ ID NO:13) of a
TAT411 cDNA, wherein SEQ ID NO:13 is a clone designated herein as
"DNA225701".
[0413] FIG. 14 shows a nucleotide sequence (SEQ ID NO:14) of a
TAT412 cDNA, wherein SEQ ID NO:14 is a clone designated herein as
"DNA226115".
[0414] FIG. 15 shows a nucleotide sequence (SEQ ID NO:15) of a
TAT413 cDNA, wherein SEQ ID NO:15 is a clone designated herein as
"DNA227150".
[0415] FIG. 16 shows a nucleotide sequence (SEQ ID NO:16) of a
TAT414 cDNA, wherein SEQ ID NO:16 is a clone designated herein as
"DNA247426".
[0416] FIG. 17 shows a nucleotide sequence (SEQ ID NO:17) of a
TAT415 cDNA, wherein SEQ ID NO:17 is a clone designated herein as
"DNA84914".
[0417] FIGS. 18A-B show a nucleotide sequence (SEQ ID NO:18) of a
TAT416 cDNA, wherein SEQ ID NO:18 is a clone designated herein as
"DNA226751".
[0418] FIG. 19 shows a nucleotide sequence (SEQ ID NO:19) of a
TAT417 cDNA, wherein SEQ ID NO:19 is a clone designated herein as
"DNA151312".
[0419] FIGS. 20A-B show a nucleotide sequence (SEQ ID NO:20) of a
TAT418 cDNA, wherein SEQ ID NO:20 is a clone designated herein as
"DNA225703".
[0420] FIG. 21 shows a nucleotide sequence (SEQ ID NO:21) of a
TAT419 cDNA, wherein SEQ ID NO:21 is a clone designated herein as
"DNA96945".
[0421] FIG. 22 shows the amino acid sequence (SEQ ID NO:22) derived
from the coding sequence of SEQ ID NO:1 shown in FIG. 1.
[0422] FIG. 23 shows the amino acid sequence (SEQ ID NO:23) derived
from the coding sequence of SEQ ID NO:2 shown in FIG. 2.
[0423] FIG. 24 shows the amino acid sequence (SEQ ID NO:24) derived
from the coding sequence of SEQ ID NO:3 shown in FIG. 3.
[0424] FIG. 25 shows the amino acid sequence (SEQ ID NO:25) derived
from the coding sequence of SEQ ID NO:4 shown in FIG. 4.
[0425] FIG. 26 shows the amino acid sequence (SEQ ID NO:26) derived
from the coding sequence of SEQ ID NO:5 shown in FIG. 5.
[0426] FIG. 27 shows the amino acid sequence (SEQ ID NO:27) derived
from the coding sequence of SEQ ID NO:6 shown in FIG. 6.
[0427] FIG. 28 shows the amino acid sequence (SEQ ID NO:28) derived
from the coding sequence of SEQ ID NO:7 shown in FIG. 7.
[0428] FIG. 29 shows the amino acid sequence (SEQ ID NO:29) derived
from the coding sequence of SEQ ID NO:8 shown in FIG. 8.
[0429] FIG. 30 shows the amino acid sequence (SEQ ID NO:30) derived
from the coding sequence of SEQ ID NO:9 shown in FIG. 9.
[0430] FIG. 31 shows the amino acid sequence (SEQ ID NO:31) derived
from the coding sequence of SEQ ID NO:10 shown in FIG. 10.
[0431] FIG. 32 shows the amino acid sequence (SEQ ID NO:32) derived
from the coding sequence of SEQ ID NO:11 shown in FIGS. 11A-B.
[0432] FIG. 33 shows the amino acid sequence (SEQ ID NO:33) derived
from the coding sequence of SEQ ID NO:12 shown in FIGS. 12A-B.
[0433] FIG. 34 shows the amino acid sequence (SEQ ID NO:34) derived
from the coding sequence of SEQ ID NO:13 shown in FIGS. 13A-B.
[0434] FIG. 35 shows the amino acid sequence (SEQ ID NO:35) derived
from the coding sequence of SEQ ID NO:14 shown in FIG. 14.
[0435] FIG. 36 shows the amino acid sequence (SEQ ID NO:36) derived
from the coding sequence of SEQ ID NO:15 shown in FIG. 15.
[0436] FIG. 37 shows the amino acid sequence (SEQ ID NO:37) derived
from the coding sequence of SEQ ID NO:16 shown in FIG. 16.
[0437] FIG. 38 shows the amino acid sequence (SEQ ID NO:38) derived
from the coding sequence of SEQ ID NO:17 shown in FIG. 17.
[0438] FIG. 39 shows the amino acid sequence (SEQ ID NO:39) derived
from the coding sequence of SEQ ID NO:18 shown in FIGS. 18A-B.
[0439] FIG. 40 shows the amino acid sequence (SEQ ID NO:40) derived
from the coding sequence of SEQ ID NO:19 shown in FIG. 19.
[0440] FIG. 41 shows the amino acid sequence (SEQ ID NO:41) derived
from the coding sequence of SEQ ID NO:20 shown in FIGS. 20A-B.
[0441] FIG. 42 shows the amino acid sequence (SEQ ID NO:42) derived
from the coding sequence of SEQ ID NO:21 shown in FIG. 21.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0442] I. Definitions
[0443] 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.
[0444] 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" or "X" 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 I in the
figures may be employed as the starting amino acid residue for the
TAT polypeptides.
[0445] 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.
[0446] 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.
[0447] "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.
[0448] "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.
[0449] 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
[0450] 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.
[0451] "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%, 8485%, 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.
[0452] 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.
[0453] "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.
[0454] 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
[0455] 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.
[0456] 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.
[0457] 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).
[0458] "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. 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.
[0459] 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.
[0460] The term "control sequences" refers to DNA sequences
necessary for the expression of an operably linked coding sequence
in a particular host organism. The control sequences that are
suitable for prokaryotes, for example, include a promoter,
optionally an operator sequence, and a ribosome binding site.
Eukaryotic cells are known to utilize promoters, polyadenylation
signals, and enhancers.
[0461] 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.
[0462] "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).
[0463] "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 x SSC containing EDTA at 55.degree. C.
[0464] "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.
[0465] 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).
[0466] "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.
[0467] 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.
[0468] "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.
[0469] 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).
[0470] 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.
[0471] "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.
[0472] "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.
[0473] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0474] "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..
[0475] 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 term also includes
a discontinuous solid phase of discrete particles, such as those
described in U.S. Pat. No. 4,275,149.
[0476] 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.
[0477] A "small" molecule or "small" organic molecule is defined
herein to have a molecular weight below about 500 Daltons.
[0478] 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.
[0479] 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.
[0480] 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.
[0481] 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.
[0482] 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.
[0483] 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, the antibody will be purified (1) to greater than 95%
by weight of antibody as determined by the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by 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.
[0484] 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.
[0485] 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.
[0486] 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 P-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).
[0487] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g. around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3)
in the V.sub.L, and around about 1-35 (H1), 50-65 (H2) and 95-102
(H3) in the V.sub.H; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2)
and 91-96 (L3) in the V.sub.L, and 26-32 (H1), 53-55 (H2) and
96-101 (H3) in the V.sub.H; Chothia and Lesk J. Mol. Biol.
196:901-917 (1987)).
[0488] 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.
[0489] 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.
[0490] 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.
[0491] "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.
[0492] 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.
[0493] The Fc fragment comprises the carboxy-terminal portions of
both H chains held together by disulfides. The effector functions
of antibodies are determined by sequences in the Fc region, which
region is also the part recognized by Fc receptors (FcR) found on
certain types of cells.
[0494] "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.
[0495] "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.
[0496] 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).
[0497] "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).
[0498] 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.
[0499] 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. 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).
[0500] 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).
[0501] 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.-6 M, alternatively at least about 10.sup.-7 M,
alternatively at least about 10.sup.-8 M, alternatively at least
about 10.sup.-9 M, alternatively at least about 10.sup.-10M,
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.
[0502] 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.
[0503] 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 fold, induction of annexin
binding relative to untreated cell in an annexin binding assay.
[0504] 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.
[0505] "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. Nos. 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).
[0506] "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)).
[0507] "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.
[0508] "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.
[0509] 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.
[0510] 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.
[0511] "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.
[0512] 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.
[0513] 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 ot other organic molecule can bind
thereto and have a therapeutic effect with respect to the cancer.
In another embodimnent, a "TAT-expressing cancer" optionally
produces and secretes sufficient levels of TAT polypeptide, such
that an anti-TAT antibody, oligopeptide ot 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.
[0514] 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.
[0515] 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.
[0516] 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.86, Re.sup.188, Sm.sup.153, Bi.sup.212, P32 and
radioactive isotopes of Lu), chemotherapeutic methotrexate,
adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide),
doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or
other intercalating agents, enzymes and fragments thereof such as
nucleolytic enzymes, antibiotics, and toxins such as small molecule
toxins or enzymatically active toxins of bacterial, fungal, plant
or animal origin, including fragments and/or variants thereof, and
the various antitumor or anticancer agents disclosed below. Other
cytotoxic agents are described below. A tumoricidal agent causes
destruction of tumor cells.
[0517] 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 G1
arrest and M-phase arrest. Classical M-phase blockers include the
vincas (vincristine and vinblastine), taxanes, and topoisomerase II
inhibitors such as doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin. Those agents that arrest G1 also spill
over into S-phase arrest, for example, DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,
methotrexate, 5-fluorouracil, and ara-C. Further information can be
found in The Molecular Basis of Cancer, Mendelsohn and Israel,
eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and
antineoplastic drugs" by Murakami et al. (W B 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.
[0518] "Doxorubicin" is an anthracycline antibiotic. The full
chemical name of doxorubicin is
(8S-cis)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyx-
o-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacety-
l)-1-methoxy-5,1 2-naphthacenedione.
[0519] 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-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.
[0520] 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.
1TABLE 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%
[0521]
2TABLE 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%
[0522]
3TABLE 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%
[0523]
4TABLE 5 TAT-DNA NNNNNNNNNNNN (Length = 12 nucleotides) Comparison
NNNNLLLVV (Length = 9 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) = 4 divided by 12 = 33.3%
[0524] II. Compositions and Methods of the Invention
[0525] A. Anti-TAT Antibodies
[0526] 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.
1. Polyclonal Antibodies
[0527] 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.
[0528] 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 {fraction (1/10)} the original
amount of peptide or conjugate in Freund's complete adjuvant by
subcutaneous injection at multiple sites. Seven to 14 days later,
the animals are bled and the serum is assayed for antibody titer.
Animals are boosted until the titer plateaus. Conjugates also can
be made in recombinant cell culture as protein fusions. Also,
aggregating agents such as alum are suitably used to enhance the
immune response.
2. Monoclonal Antibodies
[0529] 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).
[0530] 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 immunizedn 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)).
[0531] 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.
[0532] 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)).
[0533] 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).
[0534] 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).
[0535] 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.
[0536] 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.
[0537] DNA encoding the monoclonal antibodies is readily isolated
and sequenced using conventional procedures (e.g., by using
oligonucleotide probes that are capable of binding specifically to
genes encoding the heavy and light chains of murine antibodies).
The hybridoma cells serve as a preferred source of such DNA. Once
isolated, the DNA may be placed into expression vectors, which are
then transfected into host cells such as E. coli cells, simian COS
cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do
not otherwise produce antibody protein, to obtain the synthesis of
monoclonal antibodies in the recombinant host cells. Review
articles on recombinant expression in bacteria of DNA encoding the
antibody include Skerra et al., Curr. Opinion in Immunol.,
5:256-262 (1993) and Pluckthun, Immunol. Revs. 130:151-188
(1992).
[0538] In a further embodiment, monoclonal antibodies or antibody
fragments can be isolated from antibody phage libraries generated
using the techniques described in McCafferty et al., Nature,
348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and
Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the
isolation of murine and human antibodies, respectively, using phage
libraries. Subsequent publications describe the production of high
affinity (nM range) human antibodies by chain shuffling (Marks et
al., Bio/Technology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0539] 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.
3. Human and Humanized Antibodies
[0540] 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)].
[0541] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers [Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0542] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity and HAMA response (human anti-mouse antibody)
when the antibody is intended for human therapeutic use. According
to the so-called "best-fit" method, the sequence of the variable
domain of a rodent antibody is screened against the entire library
of known human variable domain sequences. The human V domain
sequence which is closest to that of the rodent is identified and
the human framework region (FR) within it accepted for the
humanized antibody (Sims et al., J. Immunol. 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework region derived from the consensus sequence
of all human antibodies of a particular subgroup of light or heavy
chains. The same framework may be used for several different
humanized antibodies (Carter et al. Proc. Natl. Acad. Sci. USA,
89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).
[0543] 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.
[0544] 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.
[0545] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array into such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno. 7:33
(1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of
GenPharm); U.S. Pat. No. 5,545,807; and WO 97/17852.
[0546] 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.
[0547] 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).
4. Antibody Fragments
[0548] 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.
[0549] 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') 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.
5. Bispecific Antibodies
[0550] 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).
[0551] 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 a antibody is shown in WO98/02463. U.S.
Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3
antibody.
[0552] 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).
[0553] 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, CH.sup.2, and CH.sup.3
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.
[0554] 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).
[0555] 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.
[0556] 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.
[0557] 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.
[0558] 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.
[0559] 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).
[0560] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
6. Heteroconjugate Antibodies
[0561] 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.
7. Multivalent Antibodies
[0562] A multivalent antibody may be internalized (and/or
catabolized) faster than a bivalent antibody by a cell expressing
an antigen to which the antibodies bind. The antibodies of the
present invention can be multivalent antibodies (which are other
than of the IgM class) with three or more antigen binding sites
(e.g. tetravalent antibodies), which can be readily produced by
recombinant expression of nucleic acid encoding the polypeptide
chains of the antibody. The multivalent antibody can comprise a
dimerization domain and three or more antigen binding sites. The
preferred dimerization domain comprises (or consists of) an Fc
region or a hinge region. In this scenario, the antibody will
comprise an Fc region and three or more antigen binding sites
amino-terminal to the Fc region. The preferred multivalent antibody
herein comprises (or consists of) three to about eight, but
preferably four, antigen binding sites. The multivalent antibody
comprises at least one polypeptide chain (and preferably two
polypeptide chains), wherein the polypeptide chain(s) comprise two
or more variable domains. For instance, the polypeptide chain(s)
may comprise VD1-(X1).sub.n-VD2-(X2).sub.n-Fc, wherein VD1 is a
first variable domain, VD2 is a second variable domain, Fc is one
polypeptide chain of an Fc region, X1 and X2 represent an amino
acid or polypeptide, and n is 0 or 1. For instance, the polypeptide
chain(s) may comprise: VH--CH1-flexible linker-VH--CH1-Fc region
chain; or VH-CH1-VH--CH1-Fc region chain. The multivalent antibody
herein preferably further comprises at least two (and preferably
four) light chain variable domain polypeptides. The multivalent
antibody herein may, for instance, comprise from about two to about
eight light chain variable domain polypeptides. The light chain
variable domain polypeptides contemplated here comprise a light
chain variable domain and, optionally, further comprise a CL
domain.
8. Effector Function Engineering
[0563] It may be desirable to modify the antibody of the invention
with respect to effector function, e.g., so as to enhance
antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B.
J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.,
Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can
be engineered which has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design 3:219-230 (1989). To increase the
serum half life of the antibody, one may incorporate a salvage
receptor binding epitope into the antibody (especially an antibody
fragment) as described in U.S. Pat. No. 5,739,277, for example. As
used herein, the term "salvage receptor binding epitope" refers to
an epitope of the Fc region of an IgG molecule (e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, or IgG.sub.4) that is responsible for
increasing the in vivo serum half-life of the IgG molecule.
9. Immunoconjugate
[0564] 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).
[0565] 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.
[0566] 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.
[0567] Maytansine and Maytansinoids
[0568] In one preferred embodiment, an anti-TAT antibody (full
length or fragments) of the invention is conjugated to one or more
maytansinoid molecules.
[0569] 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.
[0570] Maytansinoid-Antibody Coniugates
[0571] 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.
[0572] The cytotoxicity of the TA. 1-maytansonoid conjugate was
tested in vitro on the human breast cancer cell line SK-BR-3, which
expresses 3.times.10.sup.5 HER-2 surface antigens per cell. The
drug conjugate achieved a degree of cytotoxicity similar to the
free 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.
[0573] Anti-TAT Polypeptide Antibody-Maytansinoid Coniugates
(Immunoconjugate)
[0574] 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.
[0575] 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 disufide 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.
[0576] 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.
[0577] 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 hyrdoxymethyl, 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.
[0578] Calicheamicin
[0579] 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..sub.1.sup.I (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.
[0580] Other Cytotoxic Agents
[0581] 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).
[0582] 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.
[0583] 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).
[0584] 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-I111, fluorine-19, carbon-13,
nitrogen-15, oxygen-17, gadolinium, manganese or iron.
[0585] 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.
[0586] 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 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. 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.
[0587] 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.
[0588] 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).
10. Immunoliposomes
[0589] 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. USA77:4030 (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.
[0590] 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 defmed 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).
[0591] B. TAT Binding Oligonevtides
[0592] 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).
[0593] 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.
[0594] Although most phage display methods have used filamentous
phage, lambdoid phage display systems (WO 95/34683; U.S. Pat. No.
5,627,024), T4 phage display systems (Ren et al., Gene, 215: 439
(1998); Zhu et al., Cancer Research, 58(15): 3209-3214 (1998);
Jiang et al., Infection & Immunity, 65(11): 4770-4777 (1997)
Ren et al., Gene, 195(2):303-311 (1997); Ren, Protein Sci., 5: 1833
(1996); Efimov et al., Virus Genes, 10: 173 (1995)) and T7 phage
display systems (Smith and Scott, Methods in Enzymology, 217:
228-257 (1993); U.S. Pat. No. 5,766,905) are also known.
[0595] Many other improvements and variations of the basic phage
display concept have now been developed. These improvements enhance
the ability of display systems to screen peptide libraries for
binding to selected target molecules and to display functional
proteins with the potential of screening these proteins for desired
properties. Combinatorial reaction devices for phage display
reactions have been developed (WO 98/14277) and phage display
libraries have been used to analyze and control bimolecular
interactions (WO 98/20169; WO 98/20159) and properties of
constrained helical peptides (WO 98/20036). WO 97/35196 describes a
method of isolating an affinity ligand in which a phage display
library is contacted with one solution in which the ligand will
bind to a target molecule and a second solution in which the
affinity ligand will not bind to the target molecule, to
selectively isolate binding ligands. WO 97/46251 describes a method
of biopanning a random phage display library with an affinity
purified antibody and then isolating binding phage, followed by a
micropanning process using microplate wells to isolate high
affinity binding phage. The use of Staphlylococcus aureus protein A
as an affinity tag has also been reported (Li et al. (1998) Mol
Biotech., 9:187). WO 15 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.
[0596] 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.
[0597] C. TAT Binding Organic Molecules
[0598] 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.
[0599] D. Screening for Anti-TAT Antibodies. TAT Binding
Oligopeptides and TAT Binding Organic Molecules with the Desired
Properties
[0600] 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.
[0601] 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
colorimetric assay. Another method of measuring proliferation would
be by comparing .sup.3H-thymidine uptake by the cells treated in
the presence or absence an anti-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 cellsin 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.
[0602] 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.
[0603] 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 initailly
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.
[0604] E. Antibody Dependent Enzyme Mediated Prodrug Theragp
(ADEPT)
[0605] 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.
[0606] 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.
[0607] 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.
[0608] 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).
[0609] F. Full-Length TAT Polyieptides
[0610] 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.
[0611] 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.
[0612] G. Anti-TAT Antibody and TAT Polypeptide Variants
[0613] 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.
[0614] 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.
[0615] 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.
[0616] 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.
[0617] 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.
5 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; ala;
norleucine leu
[0618] 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:
[0619] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[0620] (2) neutral hydrophilic: cys, ser, thr;
[0621] (3) acidic: asp, glu;
[0622] (4) basic: asn, gln, his, lys, arg;
[0623] (5) residues that influence chain orientation: gly, pro;
and
[0624] (6) aromatic: trp, tyr, phe.
[0625] 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.
[0626] 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.
[0627] 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.
[0628] 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).
[0629] 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.
[0630] 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.
[0631] H. Modifications of Anti-TAT Antibodies and TAT
Polypeptides
[0632] 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,
l-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.
[0633] 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.
[0634] 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.
[0635] 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.
[0636] 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.
[0637] 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).
[0638] 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).
[0639] 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).
[0640] 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.
[0641] 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 Biololy, 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)].
[0642] 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 IgGI molecule. For the production of immunoglobulin fusions
see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0643] I. Preparation of Anti-TAT Antibodies and TAT
Polypeptides
[0644] 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.
1. Isolation of DNA Encoding Anti-TAT Antibody or TAT
Polypeptide
[0645] 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).
[0646] 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)].
[0647] 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 minimnized. 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.
[0648] 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.
[0649] 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.
2. Selection and Transformation of Host Cells
[0650] 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.
[0651] 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.
[0652] 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).
[0653] Suitable host cells for cloning or expressing the DNA in the
vectors herein include prokaryote, yeast, or higher eukaryote
cells. Suitable prokaryotes include but are not limited to
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as E. coli. Various E. coli
strains are publicly-available, such as E. coli K12 strain MM294
(ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli strain W3110
(ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic
host cells include Enterobacteriaceae such as Escherichia, e.g., E.
coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. These examples are illustrative rather than
limiting.
[0654] 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 W3 110 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 W3
110 strain 40B4, which is strain 37D6 with a no 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.
[0655] Full length antibody, antibody fragments, and antibody
fusion proteins can be produced in bacteria, in particular when
glycosylation and Fc effector function are not needed, such as when
the therapeutic antibody is conjugated to a cytotoxic agent (e.g.,
a toxin) and the immunoconjugate by itself shows effectiveness in
tumor cell destruction. Full length antibodies have greater half
life in circulation. Production in E. coli is faster and more cost
efficient. For expression of antibody fragments and polypeptides in
bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et. al.), U.S.
Pat. No. 5,789,199 (Joly et al.), and U.S. Pat. No. 5,840,523
(Simmons et al.) which describes translation initiation regio (TIR)
and signal sequences for optimizing expression and secretion, these
patents incorporated herein by reference. After expression, the
antibody is isolated from the E. coli cell paste in a soluble
fraction and can be purified through, e.g., a protein A or G column
depending on the isotype. Final purification can be carried out
similar to the process for purifying antibody expressed e.g, in CHO
cells.
[0656] 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 Schizosaccharomyces pombe (Beach and
Nurse, Nature, 290: 140 [1981]; EP 139,383 published 2 May 1985);
Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,
Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis
(MW98-8C, CBS683, CBS4574; Louvencourtetal., 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. thennotolerans, 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).
[0657] 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.
[0658] 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 (WI38, 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).
[0659] 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.
3. Selection and Use of a Replicable Vector
[0660] 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.
[0661] 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 part 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,
lpp, or heat-stable enterotoxin II leaders. For yeast secretion the
signal sequence may be, e.g., the yeast invertase leader, alpha
factor leader (including Saccharomyces and Kluyveromyces
.alpha.-factor leaders, the latter described in U.S. Pat. No.
5,010,182), or acid phosphatase leader, the C. albicans
glucoamylase leader (EP 362,179 published 4 Apr. 1990), or the
signal described in WO 90/13646 published 15 Nov. 1990. In
mammalian cell expression, mammalian signal sequences may be used
to direct secretion of the protein, such as signal sequences from
secreted polypeptides of the same or related species, as well as
viral secretory leaders.
[0662] 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.
[0663] 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.
[0664] 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, 25 77:4216 (1980). A suitable
selection gene for use in yeast is thetrpl 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)].
[0665] 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.
[0666] 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.
[0667] 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.
[0668] 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.
[0669] 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.
[0670] 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.
[0671] 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.
4. Culturing the Host Cells
[0672] 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), and
Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for
culturing the host cells. In addition, any of the media described
in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.
Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;
4,927,762;
[0673] 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S.
Pat. Re. No. 30,985 may be used as culture media for the host
cells. Any of these media may be supplemented as necessary with
hormones and/or other growth factors (such as insulin, transferrin,
or epidermal growth factor), salts (such as sodium chloride,
calcium, magnesium, and phosphate), buffers (such as HEPES),
nucleotides (such as adenosine and thymidine), antibiotics (such as
GENTAMYCIN.TM. drug), trace elements (defined as inorganic
compounds usually present at final concentrations in the micromolar
range), and glucose or an equivalent energy source. Any other
necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. The
culture conditions, such as temperature, pH, and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan.
5. Detecting Gene Amplification/Expression
[0674] 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.
[0675] 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.
6. Purification of Anti-TAT Antibody and TAT Polypeptide
[0676] 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.
[0677] 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.
[0678] 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.
[0679] 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,3 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.
[0680] 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).
[0681] J. Pharmaceutical Formulations
[0682] 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, and other organic acids; antioxidants including
ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
tonicifiers such as trehalose and sodium chloride; sugars such as
sucrose, mannitol, trehalose or sorbitol; surfactant such as
polysorbate; salt-forming counter-ions such as sodium; metal
complexes (e.g., Zn-protein complexes); and/or non-ionic
surfactants such as TWEEN.RTM., PLURONICS.RTM. or polyethylene
glycol (PEG). The antibody preferably comprises the antibody at a
concentration of between 5-200 mg/ml, preferably between 10-100
mg/ml.
[0683] 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.
[0684] 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).
[0685] 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.
[0686] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0687] K. Diagnosis and Treatment with Anti-TAT Antibodies, TAT
Binding Oligopeptides and TAT Bindinig Organic Molecules
[0688] To determine TAT expression in the cancer, various
diagnostic assays are available. In one embodiment, TAT polypeptide
overexpression may be analyzed by immunohistochemistry (IHC).
Parrafin embedded tissue sections from a tumor biopsy may be
subjected to the IHC assay and accorded a TAT protein staining
intensity criteria as follows:
[0689] Score 0--no staining is observed or membrane staining is
observed in less than 10% of tumor cells.
[0690] 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.
[0691] Score 2+--a weak to moderate complete membrane staining is
observed in more than 10% of the tumor cells.
[0692] Score 3+--a moderate to strong complete membrane staining is
observed in more than 10% of the tumor cells.
[0693] 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.
[0694] Alternatively, or additionally, FISH assays such as the
INFORM.RTM. (sold by Ventana, Arizona) 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.
[0695] 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.
[0696] 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.
[0697] 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 conjuction 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.
[0698] 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.
[0699] 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.
[0700] 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.
[0701] 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.
[0702] 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 dosing schedules for such
chemotherapy are also described in Chemotherapy Service Ed., M. C.
Perry, Williams & Wilkins, Baltimore, Md. (1992).
[0703] 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 (see, EP 616 812); or an anti-androgen such as
flutamide, in dosages known for such molecules. Where the cancer to
be treated is androgen independent cancer, the patient may
previously have been subjected to anti-androgen therapy and, after
the cancer becomes androgen independent, the anti-TAT antibody,
oligopeptide or organic molecule (and optionally other agents as
described herein) may be administered to the patient.
[0704] 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.
[0705] 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 defmed 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.
1-15mg/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.
[0706] 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.
[0707] There are two major approaches to getting the nucleic acid
(optionally contained in a vector) into the patient's cells; in
vivo and ex vivo. For in vivo delivery the nucleic acid is injected
directly into the patient, usually at the site where the antibody
is required. For ex vivo treatment, the patient's cells are
removed, the nucleic acid is introduced into these isolated cells
and the modified cells are administered to the patient either
directly or, for example, encapsulated within porous membranes
which are implanted into the patient (see, e.g., U.S. Pat. Nos.
4,892,538 and 5,283,187). There are a variety of techniques
available for introducing nucleic acids into viable cells. The
techniques vary depending upon whether the nucleic acid is
transferred into cultured cells in vitro, or in vivo in the cells
of the intended host. Techniques suitable for the transfer of
nucleic acid into mammalian cells in vitro include the use of
liposomes, electroporation, microinjection, cell fusion,
DEAE-dextran, the calcium phosphate precipitation method, etc. A
commonly used vector forex vivo delivery of the gene is a
retroviral vector.
[0708] The currently preferred in vivo nucleic acid transfer
techniques include transfection with viral vectors (such as
adenovirus, Herpes simplex I virus, or adeno-associated virus) and
lipid-based systems (useful lipids for lipid-mediated transfer of
the gene are DOTMA, DOPE and DC-Chol, for example). For review of
the currently known gene marking and gene therapy protocols see
Anderson et al., Science 256:808-813 (1992). See also WO 93/25673
and the references cited therein.
[0709] 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.
[0710] 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 vivo tumor targeting and any cell
proliferation inhibition or cytotoxic characteristics.
[0711] Methods of producing the above antibodies are described in
detail herein.
[0712] 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.
[0713] 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.
[0714] 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.
[0715] 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.
[0716] 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.
[0717] L. Articles of Manufacture and Kits
[0718] 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 and a label or package insert on or associated with the
container. Suitable containers include, for example, bottles,
vials, syringes, etc. The containers may be formed from a variety
of materials such as glass or plastic. The container holds a
composition which is effective for treating the cancer condition
and may have a sterile access port (for example the container may
be an intravenous solution bag or a vial having a stopper
pierceable by a hypodermic injection needle). At least one active
agent in the composition is an anti-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.
[0719] 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.
[0720] M. Uses for TAT Polypeptides and TAT-Polypeptide Encoding
Nucleic Acids
[0721] 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.
[0722] 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.
[0723] 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 oligonucieotides, 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. (BioTechnigues 6:958, 1988).
[0724] 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.
[0725] Preferred intragenic sites for antisense binding include the
region incorporating the translation initiation/start codon
(5'-AUG/5'-ATG) or termination/stop codon (5'-UAA, 5'-UAG and
5-UGA/5'-TAA, 5'-TAG and 5'-TGA) of the open reading frame (ORF) of
the gene. These regions refer to a portion of the mRNA or gene that
encompasses from about 25 to about 50 contiguous nucleotides in
either direction (i.e., 5' or 3') from a translation initiation or
termination codon. Other preferred regions for antisense binding
include: introns; exons; intron-exon junctions; the open reading
frame (ORF) or "coding region," which is the region between the
translation initiation codon and the translation termination codon;
the 5' cap of an mRNA which comprises an N7-methylated guanosine
residue joined to the 5'-most residue of the mRNA via a 5'-5'
triphosphate linkage and includes 5' cap structure itself as well
as the first 50 nucleotides adjacent to the cap; the 5'
untranslated region (5' UTR), the portion of an mRNA in the
5'direction from the translation initiation codon, and thus
including nucleotides between the 5' cap site and the translation
initiation codon of an mRNA or corresponding nucleotides on the
gene; and the 3' untranslated region (3' UTR), the portion of an
mRNA in the 3' direction from the translation termination codon,
and thus including nucleotides between the translation termination
codon and 3' end of an mRNA or corresponding nucleotides on the
gene.
[0726] 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
invverted polarity comprise a single 3' to 3' linkage at the
3'-most intemucleotide linkage i.e. a single inverted nucleoside
residue which may be abasic (the nucleobase is missing or has a
hydroxyl group in place thereof). Various salts, mixed salts and
free acid forms are also included. Representative United States
patents that teach the preparation of phosphorus-containing
linkages include, but are not limited to, U.S. Pat. Nos.:
3,687,808; 4;469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897;
5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676;
5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126;
5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361;
5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and
5,625,050, each of which is herein incorporated by reference.
[0727] 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.
[0728] In other preferred antisense oligonucleotides, both the
sugar and the internucleoside linkage, i.e., the backbone, of the
nucleotide units are replaced with novel groups. The base units are
maintained for hybridization with an appropriate nucleic acid
target compound. One such oligomeric compound, an oligonucleotide
mimetic that has been shown to have excellent hybridization
properties, is referred to as a peptide nucleic acid (PNA). In PNA
compounds, the sugar-backbone of an oligonucleotide is replaced
with an amide containing backbone, in particular an
aminoethylglycine backbone. The nucleobases are retained and are
bound directly or indirectly to aza nitrogen atoms of the amide
portion of the backbone. Representative United States patents that
teach the preparation of PNA compounds include, but are not limited
to, U.S. Pat. Nos.: 5,539,082; 5,714,331; and 5,719,262, each of
which is herein incorporated by reference. Further teaching of PNA
compounds can be found in Nielsen et al., Science, 1991, 254,
1497-1500.
[0729] Preferred antisense oligonucleotides incorporate
phosphorothioate backbones and/or heteroatom backbones, and in
particular --CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub- .3)--CH.sub.2-- and
--O--N(CH.sub.3)--CH.sub.2the 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.
[0730] 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-alkeynyl, or N-alkenyl; O-alkynyl, S-alkynyl
or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and
alkynyl may be substituted or unsubstituted C.sub.1 to C.sub.10
alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Particularly
preferred are O[(CH.sub.2).sub.nO].sub.mCH.sub.3,
O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m
are from 1 to about 10. Other preferred antisense oligonucleotides
comprise one of the following at the 2' position: C.sub.1 to
C.sub.10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl,
alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl,
Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2CH.sub.3,
ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl,
heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted
silyl, an RNA cleaving group, a reporter group, an intercalator, a
group for improving the pharmacokinetic properties of an
oligonucleotide, or a group for improving the pharmacodynamic
properties of an oligonucleotide, and other substituents having
similar properties. A preferred modification includes
2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta,
1995, 78, 486-504) i.e., an alkoxyalkoxy group. A further preferred
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples hereinbelow, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).
[0731] A further prefered 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. LNAs and preparation thereof are described in WO 98/39352 and
WO 99/14226.
[0732] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy
(2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), 2'-allyl
(2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl
(2'-O--CH.sub.2--CH.dbd.CH.sub- .2) and 2'-fluoro (2'-F). The
2'-modification may be in the arabino (up) position or ribo (down)
position. A preferred 2'-arabino modification is 2'-F.
[0733] 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.
[0734] 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.dbd.C--CH.sub.3 or --CH.sub.2--C.dbd.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 a 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]ben-
zothiazin-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(3 H)-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 purin 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.
[0735] 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-S-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.
[0736] 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 an region with at least 4 contiguous
2'-H sugars to confer RNase H activity. Such compounds have also
been referred to in the art as hybrids or gapmers. Preferred
gapmers have a region of 2' modified sugars (preferably
2'-O--(CH.sub.2).sub.2--O- --CH.sub.3) at the 3'-terminal and at
the 5' terminal separated by at least one region having 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.
[0737] 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.
[0738] 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.
[0739] 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 DCTSA, DCT5B and DCT5C (see WO 90/13641).
[0740] 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.
[0741] 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.
[0742] 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.
[0743] The probes may also be employed in PCR techniques to
generate a pool of sequences for identification of closely related
TAT coding sequences.
[0744] 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.
[0745] 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.
[0746] 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.
[0747] 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 stem 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.
[0748] 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 rnRNA. 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.
[0749] There are a variety of techniques available for introducing
nucleic acids into viable cells. The techniques vary depending upon
whether the nucleic acid is transferred into cultured cells in
vitro, or in vivo in the cells of the intended host. Techniques
suitable for the transfer of nucleic acid into mammalian cells in
vitro include the use of liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate
precipitation method, etc. The currently preferred in vivo gene
transfer techniques include transfection with viral (typically
retroviral) vectors and viral coat protein-liposome mediated
transfection (Dzau et al., Trends in Biotechnology 11, 205-210
[1993]). In some situations it is desirable to provide the nucleic
acid source with an agent that targets the target cells, such as an
antibody specific for a cell surface membrane protein or the target
cell, a ligand for a receptor on the target cell, etc. Where
liposomes are employed, proteins which bind to a cell surface
membrane protein associated with endocytosis may be used for
targeting and/or to facilitate uptake, e.g. capsid proteins or
fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, proteins that
target intracellular localization and enhance intracellular
half-life. The technique of receptor-mediated endocytosis is
described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432
(1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414
(1990). For review of gene marking and gene therapy protocols see
Anderson et al., Science 256, 808-813 (1992).
[0750] 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.
[0751] 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.
[0752] 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.
[0753] 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.
[0754] 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.
[0755] 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.
[0756] 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")
takes advantage of this property, and employs two hybrid proteins,
one in which the target protein is fused to the DNA-binding domain
of GAL4, and another, in which candidate activating proteins are
fused to the activation domain. The expression of a GALL-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.
[0757] 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.
[0758] 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.
[0759] As an alternative approach for receptor identification,
labeled TAT polypeptide can be photoaffmity-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.
[0760] 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.
[0761] 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.
[0762] 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 mnRNA 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., 35
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); Oligodeoxvnucleotides
as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton,
Fla., 1988). The oligonucleotides described above can also be
delivered to cells such that the antisense RNA or DNA may be
expressed in vivo to inhibit production of the 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.
[0763] 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.
[0764] 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).
[0765] 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.
[0766] 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.
[0767] 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.
[0768] 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.
[0769] 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).
[0770] 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.
[0771] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0772] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0773] 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.
[0774] 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. Molecule upregulation of expression in: as
compared to:
6 upregulation of Molecule expression in: as compared to: DNA225795
brain tumor normal brain tissue (TAT400) DNA225795 breast tumor
normal breast tissue (TAT400) DNA225795 bone tumor normal bone
tissue (TAT400) DNA225795 colon tumor normal colon tissue (TAT400)
DNA225795 kidney tumor normal kidney tissue (TAT400) DNA225795
pancreatic tumor normal pancreatic tissue (TAT400) DNA225795 soft
tissue tumor normal soft tissue (TAT400) DNA62814 lung tumor normal
lung tissue (TAT401) DNA62814 thyroid tumor normal thyroid tissue
(TAT401) DNA62814 bladder tumor normal bladder tissue (TAT401)
DNA62814 uterine tumor normal uterine tissue (TAT401) DNA62814
ovarian tumor normal ovarian tissue (TAT401) DNA62814 colon tumor
normal colon tissue (TAT401) DNA62814 brain tumor normal brain
tissue (TAT401) DNA62814 pancreatic tumor normal pancreatic tissue
(TAT401) DNA334601 breast tumor normal breast tissue (TAT402)
DNA334601 uterine tumor normal uterine tissue (TAT402) DNA334601
ovarian tumor normal ovarian tissue (TAT402) DNA334601 colon tumor
normal colon tissue (TAT402) DNA255030 prostate tumor normal
prostate tissue (TAT403) DNA255030 uterine tumor normal uterine
tissue (TAT403) DNA255030 breast tumor normal breast tissue
(TAT403) DNA257428 breast tumor normal breast tissue (TAT404)
DNA257428 ovarian tumor normal ovarian tissue (TAT404) DNA257428
kidney tumor normal kidney tissue (TAT404) DNA257428 uterine tumor
normal uterine tissue (TAT404) DNA257428 testis tumor normal testis
tissue (TAT404) DNA257428 adrenal tumor normal adrenal tissue
(TAT404) DNA194132 breast tumor normal breast tissue (TAT405)
DNA194132 lung tumor normal lung tissue (TAT405) DNA194132 ovarian
tumor normal ovarian tissue (TAT405) DNA22780 kidney tumor normal
kidney tissue (TAT408) DNA220758 colon tumor normal colon tissue
(TAT409) DNA220758 uterine tumor normal uterine tissue (TAT409)
DNA220758 prostate tumor normal prostate tissue (TAT409) DNA220758
breast tumor normal breast tissue (TAT409) DNA220758 pancreatic
tumor normal pancreatic tissue (TAT409) DNA220758 bladder tumor
normal bladder tissue (TAT409) DNA220758 kidney tumor normal kidney
tissue (TAT409) DNA220758 lung tumor normal lung tissue (TAT409)
DNA220758 fallopian tube tumor normal fallopian tube tissue
(TAT409) DNA220758 head and neck tumor normal head and neck tissue
(TAT409) DNA220758 ovarian tumor normal ovarian tissue (TAT409)
DNA220758 esophageal tumor normal esophageal tissue (TAT409)
DNA220758 stomach tumor normal stomach tissue (TAT409) DNA220758
urinary system tumor normal urinary system tissue (TAT409)
DNA225549 prostate tumor normal prostate tissue (TAT410) DNA225549
uterine tumor normal uterine tissue (TAT410) DNA225549 breast tumor
normal breast tissue (TAT410) DNA225549 pancreatic tumor normal
pancreatic tissue (TAT410) DNA225549 thyroid tumor normal thyroid
tissue (TAT410) DNA225701 ovarian tumor normal ovarian tissue
(TAT411) DNA225701 breast tumor normal breast tissue (TAT411)
DNA225701 urinary tumor normal urinary tissue (TAT411) DNA226115
lung tumor normal lung tissue (TAT412) DNA226115 colon tumor normal
colon tissue (TAT412) DNA226115 bladder tumor normal bladder tissue
(TAT412) DNA226115 breast tumor normal breast tissue (TAT412)
DNA226115 prostate tumor normal prostate tissue TAT412) DNA226115
kidney tumor normal kidney tissue (TAT412) DNA226115 pancreatic
tumor normal pancreatic tissue (TAT412) DNA226115 liver tumor
normal liver tissue (TAT412) DNA226115 uterine tumor normal uterine
tissue (TAT412) DNA227150 breast tumor normal breast tissue
(TAT413) DNA227150 brain tumor normal brain tissue (TAT413)
DNA227150 lung tumor normal lung tissue (TAT413) DNA227150 uterine
tumor normal uterine tissue (TAT413) DNA227150 ovarian tumor normal
ovarian tissue (TAT413) DNA227150 fallopian tube tumor normal
fallopian tube tissue (TAT413) DNA227150 kidney tumor normal kidney
tissue (TAT413) DNA227150 testis tumor normal testis tissue
(TAT413) DNA227150 pancreatic tumor normal pancreatic tissue
(TAT413) DNA247426 breast tumor normal breast tissue (TAT414)
DNA84914 lung tumor normal lung tissue (TAT415) DNA84914 breast
tumor normal breast tissue (TAT415) DNA84914 uterine tumor normal
uterine tissue (TAT415) DNA84914 lymphoid tumor normal lymphoid
tissue (TAT415) DNA226751 skin tumor normal skin tissue (TAT416)
DNA226751 melanoma tumor normal associated tissue (TAT416)
DNA151312 lung tumor normal lung tissue (TAT417) DNA151312 non
small cell normal lung tissue (TAT417) lung tumor DNA151312 uterine
tumor normal uterine tissue (TAT417) DNA151312 breast tumor normal
breast tissue (TAT417) DNA151312 kidney tumor normal kidney tissue
(TAT417) DNA151312 adrenal tumor normal adrenal tissue (TAT417)
DNA151312 lymphoid tumor normal lymphoid tissue (TAT417) DNA225703
prostate tumor normal prostate tissue (TAT418) DNA96945 skin tumor
normal skin tissue (TAT419) DNA96945 melanoma tumor normal
associated tissue (TAT419) DNA96945 kidney tumor normal kidney
tissue (TAT419) DNA96945 ovarian tumor normal ovarian tissue
(TAT419) DNA96945 stomach tumor normal stomach tissue (TAT419)
DNA96945 pancreatic tumor normal pancreatic tissue (TAT419)
Example 2
Quantitative Analysis of TAT mRNA Expression
[0775] 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.
[0776] The 5' nuclease procedure is run on a real-time quantitative
PCR device such as the ABI Prism 7700TM 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.
[0777] 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.
[0778] 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.
7 upregulation of Molecule expression in: as compared to: DNA225795
brain tumor normal brain tissue (TAT400) DNA225795 breast tumor
normal breast tissue (TAT400) DNA225795 bone tumor normal bone
tissue (TAT400) DNA225795 colon tumor normal colon tissue (TAT400)
DNA225795 kidney tumor normal kidney tissue (TAT400) DNA225795
pancreatic tumor normal pancreatic tissue (TAT400) DNA225795 soft
tissue tumor normal soft tissue (TAT400) DNA22780 kidney tumor
normal kidney tissue (TAT408) DNA220758 ovarian tumor normal
ovarian tissue (TAT409) DNA227150 breast tumor normal breast tissue
(TAT413)
Example 3
In Situ Hybridization
[0779] 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.
[0780] 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.
[0781] .sup.33P-Riboprobe Synthesis
[0782] 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:
[0783] 2.0 .mu.l 5.times. transcription buffer
[0784] 1.0 .mu.l DTT (100 mM)
[0785] 2.0 .mu.l NTP mix (2.5 mM: 10 .mu.; each of 10 mM GTP, CTP
& ATP+10 .mu.l H.sub.2O)
[0786] 1.0 .mu.l UTP (50 .mu.M)
[0787] 1.0 .mu.l Rnasin
[0788] 1.0 .mu.l DNA template (1 .mu.g)
[0789] 1.0 .mu.l H.sub.2O
[0790] 1.0 .mu.l RNA polymerase (for PCR products T3=AS, T7=S,
usually)
[0791] The tubes were incubated at 37.degree. C. for one hour. 1.0
.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.
[0792] 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.
[0793] .sup.33P-Hybridization
[0794] A. Pretreatment of frozen Sections
[0795] 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.
[0796] B. Pretreatment of Paraffm-Embedded Sections
[0797] 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.
[0798] C. Prehybridization
[0799] The slides were laid out in a plastic box lined with Box
buffer (4.times.SSC, 50% formamide)--saturated filter paper.
[0800] D. Hybridization 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
minute 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.
[0801] E. Washes
[0802] 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 x 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).
[0803] F. Oligonucleotides
[0804] 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.
[0805] G. Results
[0806] In situ analysis was performed on a variety of DNA sequences
disclosed herein. The results from these analyses are as
follows.
[0807] (1) DNA225795 (TAT400)
[0808] 1/1 breast carcinomas shows a positive signal in tumor cells
and stroma. 1/1 NSCLC shows a very strong signal in tumor stroma,
but is negative in malignant epithelium. 2/2 colorectal carcinomas
and 1/1 chondrosarcoma is strongly positive. 2/2 leiomyosarcomas
are also positive for expression.
Example 4
Verification and Analysis of Differential TAT Polypeptide
Expression by GEPIS
[0809] TAT polypeptides 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.
Molecule upregulation of expression in: as compared to:
8 upregulation of Molecule expression in: as compared to: DNA225795
brain tumor normal brain tissue (TAT400) DNA225795 breast tumor
normal breast tissue (TAT400) DNA225795 bone tumor normal bone
tissue (TAT400) DNA225795 colon tumor normal colon tissue (TAT400)
DNA225795 kidney tumor normal kidney tissue (TAT400) DNA225795
pancreatic tumor normal pancreatic tissue (TAT400) DNA225795 soft
tissue tumor normal soft tissue (TAT400) DNA62814 lung tumor normal
lung tissue (TAT401) DNA62814 thyroid tumor normal thyroid tissue
(TAT401) DNA62814 bladder tumor normal bladder tissue (TAT401)
DNA62814 uterine tumor normal uterine tissue (TAT401) DNA62814
ovarian tumor normal ovarian tissue (TAT401) DNA62814 colon tumor
normal colon tissue (TAT401) DNA62814 brain tumor normal brain
tissue (TAT401) DNA62814 pancreatic tumor normal pancreatic tissue
(TAT401) DNA334601 breast tumor normal breast tissue (TAT402)
DNA334601 uterine tumor normal uterine tissue (TAT402) DNA334601
ovarian tumor normal ovarian tissue (TAT402) DNA334601 colon tumor
normal colon tissue (TAT402) DNA255030 prostate tumor normal
prostate tissue (TAT403) DNA255030 uterine tumor normal uterine
tissue (TAT403) DNA255030 breast tumor normal breast tissue
(TAT403) DNA257428 breast tumor normal breast tissue (TAT404)
DNA257428 ovarian tumor normal ovarian tissue (TAT404) DNA257428
kidney tumor normal kidney tissue (TAT404) DNA257428 uterine tumor
normal uterine tissue (TAT404) DNA257428 testis tumor normal testis
tissue (TAT404) DNA257428 adrenal tumor normal adrenal tissue
(TAT404) DNA194132 breast tumor normal breast tissue (TAT405)
DNA194132 lung tumor normal lung tissue (TAT405) DNA194132 ovarian
tumor normal ovarian tissue (TAT405) DNA22780 kidney tumor normal
kidney tissue (TAT408) DNA220758 colon tumor normal colon tissue
(TAT409) DNA220758 uterine tumor normal uterine tissue (TAT409)
DNA220758 prostate tumor normal prostate tissue (TAT409) DNA220758
breast tumor normal breast tissue (TAT409) DNA220758 pancreatic
tumor normal pancreatic tissue (TAT409) DNA220758 bladder tumor
normal bladder tissue (TAT409) DNA220758 kidney tumor normal kidney
tissue (TAT409) DNA220758 lung tumor normal lung tissue (TAT409)
DNA220758 fallopian tube tumor normal fallopian tube tissue
(TAT409) DNA220758 head and neck tumor normal head and neck tissue
(TAT409) DNA220758 ovarian tumor normal ovarian tissue (TAT409)
DNA220758 esophageal tumor normal esophageal tissue (TAT409)
DNA220758 stomach tumor normal stomach tissue (TAT409) DNA220758
urinary system tumor normal urinary system tissue (TAT409)
DNA225549 prostate tumor normal prostate tissue (TAT410) DNA225549
uterine tumor normal uterine tissue (TAT410) DNA225549 breast tumor
normal breast tissue (TAT410) DNA225549 pancreatic tumor normal
pancreatic tissue (TAT410) DNA225549 thyroid tumor normal thyroid
tissue (TAT410) DNA225701 ovarian tumor normal ovarian tissue
(TAT411) DNA225701 breast tumor normal breast tissue (TAT411)
DNA225701 urinary tumor normal urinary tissue (TAT411) DNA226115
lung tumor normal lung tissue (TAT412) DNA226115 colon tumor normal
colon tissue (TAT412) DNA226115 bladder tumor normal bladder tissue
(TAT412) DNA226115 breast tumor normal breast tissue (TAT412)
DNA226115 prostate tumor normal prostate tissue (TAT412) DNA226115
kidney tumor normal kidney tissue (TAT412) DNA226115 pancreatic
tumor normal pancreatic tissue (TAT412) DNA226115 liver tumor
normal liver tissue (TAT412) DNA226115 uterine tumor normal uterine
tissue (TAT412) DNA227150 breast tumor normal breast tissue
(TAT413) DNA227150 brain tumor normal brain tissue (TAT413)
DNA227150 lung tumor normal lung tissue (TAT413) DNA227150 uterine
tumor normal uterine tissue (TAT413) DNA227150 ovarian tumor normal
ovarian tissue (TAT413) DNA227150 fallopian tube tumor normal
fallopian tube tissue (TAT413) DNA227150 kidney tumor normal kidney
tissue (TAT413) DNA227150 testis tumor normal testis tissue
(TAT413) DNA227150 pancreatic tumor normal pancreatic tissue
(TAT413) DNA247426 breast tumor normal breast tissue (TAT414)
DNA84914 lung tumor normal lung tissue (TAT415) DNA84914 breast
tumor normal breast tissue (TAT415) DNA84914 uterine tumor normal
uterine tissue (TAT415) DNA84914 lymphoid tumor normal lymphoid
tissue (TAT415) DNA226751 skin tumor normal skin tissue (TAT416)
DNA226751 melanoma tumor normal associated tissue (TAT416)
DNA151312 lung tumor normal lung tissue (TAT417) DNA151312 non
small cell normal lung tissue (TAT417) lung tumor DNA151312 uterine
tumor normal uterine tissue (TAT417) DNA151312 breast tumor normal
breast tissue (TAT417) DNA151312 kidney tumor normal kidney tissue
(TAT417) DNA151312 adrenal tumor normal adrenal tissue (TAT417)
DNA151312 lymphoid tumor normal lymphoid tissue (TAT417) DNA225703
prostate tumor normal prostate tissue (TAT418) DNA96945 skin tumor
normal skin tissue (TAT419) DNA96945 melanoma tumor normal
associated tissue (TAT419) DNA96945 kidney tumor normal kidney
tissue (TAT419) DNA96945 ovarian tumor normal ovarian tissue
(TAT419) DNA96945 stomach tumor normal stomach tissue (TAT419)
DNA96945 pancreatic tumor normal pancreatic tissue (TAT419)
Example 5
Identification of TAT Polypeptides that are Specifically Expressed
by B Cells
[0810] Using the GeneExpress.RTM.- and GEPIS-associated tissue
expression analyses described in Examples 1 and 4 above, various
TAT polypeptides have been identified as being specifically
expressed on the surface of both normal and malignant B cells. 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 are excellent
polypeptide targets for the treatment of B cell-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 RITUXANO
(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 and, therefore, will serve as excellent
polypeptide targets for the treatment of B-cell-associated cancers:
TAT406 (DNA255706), TAT407 (DNA329863) and TAT206 (DNA56041).
Example 6
Use of TAT as a Hybridization Probe
[0811] 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.
[0812] 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.
[0813] 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.
[0814] 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
[0815] This example illustrates preparation of an unglycosylated
form of TAT by recombinant expression in E. coli.
[0816] 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.
[0817] 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.
[0818] 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.
[0819] 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.
[0820] 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) lon 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.2H2O, 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.
[0821] 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.
[0822] 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 fmal
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.
[0823] 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 20 0.14 M sodium chloride and 4% mannitol by
dialysis or by gel filtration using G25 Superfme (Pharmacia) resins
equilibrated in the formulation buffer and sterile filtered.
[0824] 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
[0825] This example illustrates preparation of a potentially
glycosylated form of TAT by recombinant expression in mammalian
cells.
[0826] 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.
[0827] 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.
[0828] 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-methio 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.
[0829] 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.
[0830] 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.
[0831] 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 a 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.
[0832] 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.
[0833] 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.
[0834] 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.
[0835] 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.
[0836] 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 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 3L production spinner is seeded at
1.2.times.10.sup.6 cells/mL. On day 0, the cell number pH ie
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.
[0837] 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 Superfme (Pharmacia)
column and stored at -80.degree. C.
[0838] 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.
[0839] Certain of the TAT polypeptides disclosed herein have been
successfully expressed and purified using this technique(s).
Example 9
Expression of TAT in Yeast
[0840] The following method describes recombinant expression of TAT
in yeast.
[0841] 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.
[0842] 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.
[0843] 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.
[0844] 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
[0845] The following method describes recombinant expression of TAT
in Baculovirus-infected insect cells.
[0846] 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 TAT 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.
[0847] Recombinant baculovirus is generated by co-transfecting the
above plasmid and BaculoGold" 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 Laboratorv Manual, Oxford: Oxford
University Press (1994).
[0848] 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; 0.1%
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 10 is loaded onto the column at 0.5 mL per minute. The
column is washed to baseline A.sub.280 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.
[0849] 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.
[0850] 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
[0851] This example illustrates preparation of monoclonal
antibodies which can specifically bind TAT.
[0852] 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.
[0853] 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, Mont.) 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.
[0854] After a suitable antibody titer has been detected, the
animals "positive" for antibodies can be injected with a fmal
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 (bypoxanthine,
aminopterin, and thymidine) medium to inhibit proliferation of
non-fused cells, myeloma hybrids, and spleen cell hybrids.
[0855] 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.
[0856] 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.
[0857] Antibodies directed against certain of the TAT polypeptides
disclosed herein have been successfully produced using this
technique(s). More specifically, functional monoclonal antibodies
that are capable of recognizing and binding to TAT protein (as
measured by standard ELISA, FACS sorting analysis and/or
immunohistochemistry analysis) have been successfully generated
against the following TAT proteins as disclosed herein: TAT400
(DNA225795), TAT401 (DNA62814), TAT406 (DNA255706), TAT407
(DNA329863), TAT206 (DNA56041), TAT412 (DNA226115), TAT413
(DNA227150), TAT416 (DNA226751) and TAT419 (DNA96945).
Example 12
Purification of TAT Polypeptides Using Specific Antibodies
[0858] 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.
[0859] 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.
[0860] Such an immunoaffmity 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.
[0861] A soluble TAT polypeptide-containing preparation is passed
over the immunoaffrnity 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
[0862] 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.
[0863] 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
[0864] 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.
[0865] 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
42 1 2625 DNA Homo sapiens 1 cggcagccct gacgtgatga gctcaaccag
cagagacatt ccatcccaag 50 agaggtctgc gtgacgcgtc cgggaggcca
ccctcagcaa gaccaccgta 100 cagttggtgg aaggggtgac agctgcattc
tcctgtgcct accacgtaac 150 caaaaatgaa ggagaactac tgtttacaag
ccgccctggt gtgcctgggc 200 atgctgtgcc acagccatgc ctttgcccca
gagcggcggg ggcacctgcg 250 gccctccttc catgggcacc atgagaaggg
caaggagggg caggtgctac 300 agcgctccaa gcgtggctgg gtctggaacc
agttcttcgt gatagaggag 350 tacaccgggc ctgaccccgt gcttgtgggc
aggcttcatt cagatattga 400 ctctggtgat gggaacatta aatacattct
ctcaggggaa ggagctggaa 450 ccatttttgt gattgatgac aaatcaggga
acattcatgc caccaagacg 500 ttggatcgag aagagagagc ccagtacacg
ttgatggctc aggcggtgga 550 cagggacacc aatcggccac tggagccacc
gtcggaattc attgtcaagg 600 tccaggacat taatgacaac cctccggagt
tcctgcacga gacctatcat 650 gccaacgtgc ctgagaggtc caatgtggga
acgtcagtaa tccaggtgac 700 agcttcagat gcagatgacc ccacttatgg
aaatagcgcc aagttagtgt 750 acagtatcct cgaaggacaa ccctattttt
cggtggaagc acagacaggt 800 atcatcagaa cagccctacc caacatggac
agggaggcca aggaggagta 850 ccacgtggtg atccaggcca aggacatggg
tggacatatg ggcggactct 900 cagggacaac caaagtgacg atcacactga
ccgatgtcaa tgacaaccca 950 ccaaagtttc cgcagaggct ataccagatg
tctgtgtcag aagcagccgt 1000 ccctggggag gaagtaggaa gagtgaaagc
taaagatcca gacattggag 1050 aaaatggctt agtcacatac aatattgttg
atggagatgg tatggaatcg 1100 tttgaaatca caacggacta tgaaacacag
gagggggtga taaagctgaa 1150 aaagcctgta gattttgaaa ccgaaagagc
ctatagcttg aaggtagagg 1200 cagccaacgt gcacatcgac ccgaagttta
tcagcaatgg ccctttcaag 1250 gacactgtga ccgtcaagat ctcagtagaa
gatgctgatg agccccctat 1300 gttcttggcc ccaagttaca tccacgaagt
ccaagaaaat gcagctgctg 1350 gcaccgtggt tgggagagtg catgccaaag
accctgatgc tgccaacagc 1400 ccgataaggt attccatcga tcgtcacact
gacctcgaca gatttttcac 1450 tattaatcca gaggatggtt ttattaaaac
tacaaaacct ctggatagag 1500 aggaaacagc ctggctcaac atcactgtct
ttgcagcaga aatccacaat 1550 cggcatcagg aagcccaagt cccagtggcc
attagggtcc ttgatgtcaa 1600 cgataatgct cccaagtttg ctgcccctta
tgaaggtttc atctgtgaga 1650 gtgatcagac caagccactt tccaaccagc
caattgttac aattagtgca 1700 gatgacaagg atgacacggc caatggacca
agatttatct tcagcctacc 1750 ccctgaaatc attcacaatc caaatttcac
agtcagagac aaccgagata 1800 acacagcagg cgtgtacgcc cggcgtggag
ggttcagtcg gcagaagcag 1850 gacttgtacc ttctgcccat agtgatcagc
gatggcggca tcccgcccat 1900 gagtagcacc aacaccctca ccatcaaagt
ctgcgggtgc gacgtgaacg 1950 gggcactgct ctcctgcaac gcagaggcct
acattctgaa cgccggcctg 2000 agcacaggcg ccctgatcgc catcctcgcc
tgcatcgtca ttctcctggt 2050 cattgtagta ttgtttgtga ccctgagaag
gcaaaagaaa gaaccactca 2100 ttgtctttga ggaagaagat gtccgtgaga
acatcattac ttatgatgat 2150 gaagggggtg gggaagaaga cacagaagcc
tttgatattg ccaccctcca 2200 gaatcctgat ggtatcaatg gatttatccc
ccgcaaagac atcaaacctg 2250 agtatcagta catgcctaga cctgggctcc
ggccagcgcc caacagcgtg 2300 gatgtcgatg acttcatcaa cacgagaata
caggaggcag acaatgaccc 2350 cacggctcct ccttatgact ccattcaaat
ctacggttat gaaggcaggg 2400 gctcagtggc cgggtccctg agctccctag
agtcggccac cacagattca 2450 gacttggact atgattatct acagaactgg
ggacctcgtt ttaagaaact 2500 agcagatttg tatggttcca aagacacttt
tgatgacgat tcttaacaat 2550 aacgatacaa atttggcctt aagaactgtg
tctggcgttc tcaagaatct 2600 agaagatgtg taacaggtat ttttt 2625 2 1371
DNA Homo sapiens 2 aactcaaact cctctctctg ggaaaacgcg gtgcttgctc
ctcccggagt 50 ggccttggca gggtgttgga gccctcggtc tgccccgtcc
ggtctctggg 100 gccaaggctg ggtttccctc atgtatggca agagctctac
tcgtgcggtg 150 cttcttctcc ttggcataca gctcacagct ctttggccta
tagcagctgt 200 ggaaatttat acctcccggg tgctggaggc tgttaatggg
acagatgctc 250 ggttaaaatg cactttctcc agctttgccc ctgtgggtga
tgctctaaca 300 gtgacctgga attttcgtcc tctagacggg ggacctgagc
agtttgtatt 350 ctactaccac atagatccct tccaacccat gagtgggcgg
tttaaggacc 400 gggtgtcttg ggatgggaat cctgagcggt acgatgcctc
catccttctc 450 tggaaactgc agttcgacga caatgggaca tacacctgcc
aggtgaagaa 500 cccacctgat gttgatgggg tgatagggga gatccggctc
agcgtcgtgc 550 acactgtacg cttctctgag atccacttcc tggctctggc
cattggctct 600 gcctgtgcac tgatgatcat aatagtaatt gtagtggtcc
tcttccagca 650 ttaccggaaa aagcgatggg ccgaaagagc tcataaagtg
gtggagataa 700 aatcaaaaga agaggaaagg ctcaaccaag agaaaaaggt
ctctgtttat 750 ttagaagaca cagactaaca attttagatg gaagctgaga
tgatttccaa 800 gaacaagaac cctagtattt cttgaagtta atggaaactt
ttctttggct 850 tttccagttg tgacccgttt tccaaccagt tctgcagcat
attagattct 900 agacaagcaa cacccctctg gagccagcac agtgctcctc
catatcacca 950 gtcatacaca gcctcattat taaggtctta tttaatttca
gagtgtaaat 1000 tttttcaagt gctcattagg ttttataaac aagaagctac
atttttgccc 1050 ttaagacact acttacagtg ttatgacttg tatacacata
tattggtatc 1100 aaaggggata aaagccaatt tgtctgttac atttcctttc
acgtatttct 1150 tttagcagca cttctgctac taaagttaat gtgtttactc
tctttccttc 1200 ccacattctc aattaaaagg tgagctaagc ctcctcggtg
tttctgatta 1250 acagtaaatc ctaaattcaa actgttaaat gacattttta
tttttatgtc 1300 tctccttaac tatgagacac atcttgtttt actgaatttc
tttcaatatt 1350 ccaggtgata gatttttgtc g 1371 3 1274 DNA Homo
sapiens 3 ggcacgaggc gattcagggg agggagcaac tggagcctca ggccctccag 50
agtagtctgc ctgaccaccc tggagcccac agaagcccag gacgtctccc 100
gcgaggcctc cccgtgtgtg gctgaggatg gctgagcagc agggccggga 150
gcttgaggct gagtgccccg tctgctggaa ccccttcaac aacacgttcc 200
atacccccaa aatgctggat tgctgccact ccttctgcgt ggaatgtctg 250
gcccacctca gccttgtgac tccagcccgg cgccgcctgc tgtgcccact 300
ctgtcgccag cccacagtgc tggcctcagg gcagcctgtc actgacttgc 350
ccacggacac tgccatgctc accctgctcc gcctggagcc ccaccatgtc 400
atcctggaag gccatcagct gtgcctcaag gaccagccca agagccgcta 450
cttcctgcgc cagcctcgag tctacacgct ggaccttggc ccccagcctg 500
ggggccagac tgggccgccc ccagacacgg cctctgccac cgtgtctacg 550
cccatcctca tccccagcca ccactctttg agggagtgtt tccgcaaccc 600
tcagttccgc atctttgcct acctgatggc cgtcatcctc agtgtcactc 650
tgttgctcat attctccatc ttttggacca agcagttcct ttggggtgtg 700
gggtgagtgc tgttcccaga caagaaacca aacctttttc ggttgctgct 750
gggtatggtg actacggagc ctcatttggt attgtcttcc tttgtagtgt 800
tgtttatttt acaatccagg gattgttcag gccatgtgtt tgcttctggg 850
aacaatttaa aaaaaaaaca aaaaaacgaa aagcttgaag gactgggaga 900
tgtggagcga cctccgggtg tgagtgtggc gtcatggaag ggcagagaag 950
cggttctgac cacagagctc cacagcaagt tgtgccaaag ggctgcacag 1000
tggtatccag gaacctgact agcccaaata gcaagttgca tttctcactg 1050
gagctgcttc aaaatcagtg catatttttt tgagttgctc ttttactatg 1100
ggttgctaaa aaaaaaaaaa aaaaaattgg gaagtgagct tcaattctgt 1150
gggtaaatgt gtgtttgttt ctctttgaat gtcttgccac tggttgcagt 1200
aaaagtgttc tgtattcatt aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1250
aaaaaaaaaa aaaaaaaaaa aaaa 1274 4 2032 DNA Homo sapiens 4
gaagcgcgct cccggggagg tgttgcagcc atggctacgg cagccggcgc 50
gacctacttt cagcgaggca gtctgttctg gttcacagtc atcaccctca 100
gctttggcta ctacacatgg gttgtcttct ggcctcagag tatcccttat 150
cagaaccttg ggcccctggg ccccttcact cagtacttgg tggaccacca 200
tcacaccctc ctttgcaatg ggtattggct tgcctggctg attcatgtgg 250
gagagtcctt gtatgccata gcattgtgca agcataaagg catcacaagt 300
ggtcgggctc agctactctg gttcctacag actttcttct ttgggatagc 350
gtctctcacc atcttgattg cttacaaacg gaagcgccaa aaacaaactt 400
gaagttgtct gaaagcttgc tctacacttt tacattcatc ctcacccttt 450
tttttgtggg gtagaggagg tgcagtaatt tactcagtga tctttctact 500
ttctagaaac tgtccttcaa agctctttaa gaccccctcg ttagtcagtt 550
tcttctctta tatgctctgg ttgagcttga atagaccagt tgttacttaa 600
gaaagaaaca gagaaagatt ttagcttttc aatcctattt ggcagaggac 650
ttcagctacc ttcttacagt ctttggctgt gttggtaccc tcgtgtgctc 700
tgagctaagc cacatactaa actgactttt tggtttgtat acccttgctc 750
ccgccttctg atgaaaacac cttaccctca caaccaccat ctttcctctc 800
ctttccaaag ctctttccac cttgctgcac taagataaag tgacacttcc 850
actatatgtc aattccacac acatttatta ggtacctgtg aggtaggatc 900
ctatcctctc aaacttccat ttctcatgct acagagaaag ataaggaaga 950
tgagcaagtg cctggaatgg ggcaggctga gcagtcacac aggcatagag 1000
gcacgctgag aacctggagg ggagactgca gagtgccttc cctgatgctg 1050
cagccggaag tgatccttcc ctccacctgg cccctgggac actgtgctct 1100
gcagtgtgca gggcctgatg gcactgctag attgctcctt cagctcaggg 1150
ccacagctta aacagcttta cctttcccct cagcacctgt cccactatct 1200
tgcacacagg tgctctaacc atgtttattg aacaaaggag ggaaactgat 1250
ttcactttca cttgttcatt atcattccaa tttttatgtg aaaatggcac 1300
aacccatttg gggtaccctc accccaaaat aaaagcccaa gtctaccttt 1350
gactggtacc accttttttg tggtttcgtt ggtgagaaac ctttatcttt 1400
ttcatacctt tctattctca atcacttctc caaaagtgtg tctttccagc 1450
tctgatttat tcaaaacaca agcatttctg tttagagatt ctagcccatg 1500
ggttatctgg ctagttatta cctctcctgt tcacttagtt atactttatt 1550
attgctcaca ggctggggag gcagaatgac tctgtcacca ctaggagcca 1600
ttagggcttc ttccctggag gactgcctgc ttgctttctg gggacactag 1650
ccctcatttc ccttctgtgg tacagtgggg caaattattt gtattaagca 1700
aacatttatg ggaaacaacc cgctcccgaa aacggagccc ccaagtaaag 1750
cacaaccctg aaagattatg aactatgaat tgtctctagt agagataaat 1800
ttctgcaaac atatctcagt cttccctctg tttctctggt gattaagaag 1850
ttcctttttg gtaaggaaaa ggatttttaa ccatagagtt aggcatcatg 1900
gaaattcaaa ccagatttct taatacctgg tcttcctcaa agagaaataa 1950
taacagtaat agtggtgctg ggaacaatat ggcagattat tgaatgaaat 2000
tgattaactt gaataaaatg ctgtgaattt tc 2032 5 1572 DNA Homo sapiens 5
ggcacgaggc cgcagcggac tgccctttcc caagatggcg tcgaagatag 50
gttcgagacg gtggatgttg cagctgatca tgcagttggg ttcggtgctg 100
ctcacacgct gccccttttg gggctgcttc agccagctca tgctgtacgc 150
tgagagggct gaggcacgcc ggaagcccga catcccagtg ccttacctgt 200
atttcgacat gggggcagcc gtgctgtgcg ctagtttcat gtcctttggc 250
gtgaagcggc gctggttcgc gctgggggcc gcactccaat tggccattag 300
cacctacgcc gcctacatcg ggggctacgt ccactacggg gactggctga 350
aggtccgtat gtactcgcgc acagttgcca tcatcggcgg ctttcttgtg 400
ttggccagcg gtgctgggga gctgtaccgc cggaaacctc gcagccgctc 450
cctgcagtcc accggccagg tgttcctggg tatctacctc atctgtgtgg 500
cctactcact gcagcacagc aaggaggacc ggctggcgta tctgaaccat 550
ctcccaggag gggagctgat gatccagctg ttcttcgtgc tgtatggcat 600
cctggccctg gcctttctgt caggctacta cgtgaccctc gctgcccaga 650
tcctggctgt actgctgccc cctgtcatgc tgctcattga tggcaatgtt 700
gcttactggc acaacacgcg gcgtgttgag ttctggaacc agatgaagct 750
ccttggagag agtgtgggca tcttcggaac tgctgtcatc ctggccactg 800
atggctgagt tttatggcaa gaggctgaga tgggcacagg gagccactga 850
gggtcaccct gccttcctcc ttgctggccc agctgctgtt tatttatgct 900
ttttggtctg tttgtttgat cttttgcttt tttaaaattg ttttttgcag 950
ttaagaggca gctcatttgt ccaaatttct gggctcagcg cttgggaggg 1000
caggagccct ggcactaatg ctgtacaggt ttttttcctg ttaggagagc 1050
tgaggccagc tgcccactga gtctcctgtc cctgagaagg gagtatggca 1100
gggctgggat gcggctactg agagtgggag agtgggagac agaggaagga 1150
agatggagat tggaagtgag caaatgtgaa aaattcctct ttgaacctgg 1200
cagatgcagc taggctctgc agtgctgttt ggagactgtg agagggagtg 1250
tgtgtgttga cacatgtgga tcaggcccag gaagggcaca ggggctgagc 1300
actacagaag tcacatgggt tctcagggta tgccaggggc agaaacagta 1350
ccggctctct gtcactcacc ttgagagtag agcagaccct gttctgctct 1400
gggctgtgaa ggggtggagc aggcagtggc cagctttgcc cttcctgctg 1450
tctctgtttc tagctccatg gttggcctgg tgggggtgga gttccctccc 1500
aaacaccaga ccacacagtc ctccaaaaat aaacatttta tatagacaaa 1550
aaaaaaaaaa aaaaaaaaaa aa 1572 6 1451 DNA Homo sapiens Unsure 1141
Unknown base 6 ggccgcggct cgcctttggc ccttcttatc aggatgaaaa
cgcttctgtt 50 tggtgtctgg gccctgctgg ccttgatcct ttgcccaggg
gtcccggaag 100 agttgtttga ggtttctatt tggccaagtc aggccctggt
ggagtttgga 150 cagtccctag tgtgcaactg cagcactact tgcccagacc
caggacccag 200 tggaattgag accttcttaa agaaaactca ggtggacaaa
gggcctcagt 250 ggaaagagtt tcttctggag gatgtcacag agaattccat
cctgcagtgc 300 ttcttctctt gtgcagggat tcaaaaggac acaagccttg
gcatcactgt 350 gtatcagcca ccagagcaag tgatcctgga gctgcagcct
gcctgggtgg 400 ccgtggacga agccttcaca gtgaagtgtc atgtacccag
tgtagcaccc 450 ttggagagtc tcacccttgc ccttctccag ggtaaccaag
aactgcatag 500 aaagaacttt acgagcttgg ctgtggcctc ccaaagagct
gaagtcatca 550 tcagtgtcag agcccaaaag gagaatgaca gatgcaattc
ttcctgccat 600 gcagaactgg acttgagttt gcaaggtggg aggctctttc
aaggcagctc 650 acccatcaga atagtccgga tctttgaatt ctctcagagt
ccccacatct 700 gggtctcttc ccttttggag gctgggatgg cggagactgt
gagctgcgag 750 gtggctaggg tgtttccagc caaagaagtt atgttccaca
tgttcctgga 800 agaccaagag ctgagctcct ccctttcctg ggagggggac
acagcatggg 850 ccaatgctac cattcggacc atggaggctg gtgatcagga
actgtcttgc 900 tttgcatctc tgggtgcaat ggaacagaag acaagaaagc
tagtgcatag 950 ctacagcttc cctccaccaa tcctggagct aaaagaatca
tacccattgg 1000 cagggaccga cattaatgtg acctgctcag ggcatgtatt
aacatcaccc 1050 agccctactc ttcggcttca gggagcccca gacctccctg
ctggggagcc 1100 tgcctggctt ctacttactg ccagggagga agatgatggc
ngaaatttct 1150 cctgcgaggc ctctttggtg gtgcagggtc agcggttgat
gaaaaccact 1200 gtgatccagc tccatatcct aaagccacag ttagaggaat
ccagttgccc 1250 tggcaaacag acctggctgg aagggatgga acacacgctc
gcctgcgtcc 1300 caaagggaaa cccagctcca gccttggtgt gtacctggaa
tggggtggtc 1350 tttgaccttg aagtgccaca gaaggcaacc tagaaccaca
ctggaaccta 1400 ccgctacaca gccactaacc agctgggctc tgtcagcaaa
gacattgctg 1450 t 1451 7 2652 DNA Homo sapiens 7 ccacgcgtcc
gttctgaggt gcattctttt tttgatgaga ggcatctcta 50 ggtaccatcc
ctgacctggt cctcatgctg ccgaggctgt tgctgttgat 100 ctgtgctcca
ctctgtgaac ctgccgagct gtttttgata gccagcccct 150 cccatcccac
agaggggagc ccagtgaccc tgacgtgtaa gatgcccttt 200 ctacagagtt
cagatgccca gttccagttc tgctttttca gagacacccg 250 ggccttgggc
ccaggctgga gcagctcccc caagctccag atcgctgcca 300 tgtggaaaga
agacacaggg tcatactggt gcgaggcaca gacaatggcg 350 tccaaagtct
tgaggagcag gagatcccag ataaatgtgc acatcccggt 400 gtctcgccca
atcctcatgc tcagggctcc cagggcccag gctgcagtgg 450 aggatgtgct
ggagcttcac tgtgaggccc tgagaggctc tcctccaatc 500 ctgtactggt
tttatcacga ggatatcacc ctggggagca ggtcggcccc 550 ctctggagga
ggagcctcct tcaacctttc cctgactgaa gaacattctg 600 gaaactactc
ctgtgaggcc aacaatggcc tgggggccca gcgcagtgag 650 gcggtgacac
tcaacttcac agtgcctact ggggccagaa gcaatcatct 700 tacctcagga
gtcattgagg ggctgctcag cacccttggt ccagccaccg 750 tggccttatt
attttgctac ggcctcaaaa gaaaaatagg aagacgttca 800 gccagggatc
cactcaggag ccttccagcc ttaccccaag agttcaccta 850 cctcaactca
cctaccccag ggcagctaca gcctatatat gaaaatgtga 900 atgttgtaag
tggggatgag gtttattcac tggcgtacta taaccagccg 950 gagcaggaat
cagtagcagc agaaaccctg gggacacata tggaggacaa 1000 ggtttcctta
gacatctatt ccaggctgag gaaagcaaac attacagatg 1050 tggactatga
agatgctatg taaggttatg gaagattctg ctctttgaaa 1100 accatccatg
accccaagcc tcaggcctga tatgttcttc agagatcctg 1150 gggcattagc
tttccagtat acctcttctg gatgccattc tccatggcac 1200 tattccttca
tctactgtga agtgaagttg gcgcagccct gaagaaacta 1250 cctaggagaa
ctaatagaca caggagtgac agggactttg ttatcagaac 1300 cagattcctg
ccggctcctt tgaaaacagg tcatattgtg ctcttctgtt 1350 tacaagagga
aacaagatgg aataaaagaa attgggatct tgggttggag 1400 ggacagtgaa
gcttagagca catgaactca aggttagtga ctctgcagga 1450 cttcacagag
agagctgtgc ccatcattca gtccaagtgc tttctctgcc 1500 cagacagcac
agaactccag ccccgctact tacatggatc atcgagtttc 1550 cacctaaaat
atgattctat ttattttgag tcactgttac caaattagaa 1600 ctaaaacaaa
gttacataaa aagttattgt gactccactt aattttagtg 1650 acgtattttt
gtatatatag gccaacctat accacatcca aaattatgta 1700 tctattacag
cccctagaag ctttataaat acagtgtgtc ttcttttatt 1750 cacaaaattt
ttgaaatcgt ggtaatatgg tttgaaacct gtatcttaat 1800 tatttttttt
ttaaattgag acagggtctc actctgtcac tcaatctgga 1850 atgcagtggc
acaatcttgc ctcactgcaa cgcctgcctc tcaggctcaa
1900 gcaaacctct cacctcagcc tgctgagtag ctgggactac aggcacatgc 1950
caccaaactt ggccattttt tgtcttacgt agagacaaga tttcaccgtt 2000
ttgcccaggc tggtctcaaa ctcctgggct caagcaatgt attgaatttt 2050
aaaataacca ggcactcact cttatgaatt aataaacatt tggaggtata 2100
taaagtaaaa agttaaagtc tttcctgtaa gttaacacaa atgttaacta 2150
ttgttaaaaa ctttacaggt agctctctag atatttttct atttttgtat 2200
gtatacttat gcatacatgt aagtatataa acatttagaa gtgtacctat 2250
ctaacaaact attatgaaat actttcaaat ctgtaaatag atctattata 2300
ctattttaaa agtctctata gtagtgtgtt atatagataa atcataactt 2350
ttttcttttt ttattgtagt aaatatgcac aacataaaat tgatcatttt 2400
aaccattttt aagtgtacaa ttcagtggca ttaagtacta tcataatata 2450
ttttaatcct tctcatcact ggtggacatt aaggagactc tcaaaaaatt 2500
catattataa aaacaaagtt caaacaaatg tctttgtact agcatattat 2550
ggcactcctg ctggattatc tgaaggataa atttgtaaat ctagtattgc 2600
tagattatgc atattaaata ttcttgttaa atagtcaaaa aaaaaaaaaa 2650 aa 2652
8 3459 DNA Homo sapiens 8 ctcaatcagc tttatgcaga gaagaagctt
actgagctca ctgctggtgc 50 tggtgtaggc aagtgctgct ttggcaatct
gggctgacct ggcttgtctc 100 ctcagaactc cttctccaac cctggagcag
gcttccatgc tgctgtgggc 150 gtccttgctg gcctttgctc cagtctgtgg
acaatctgca gctgcacaca 200 aacctgtgat ttccgtccat cctccatgga
ccacattctt caaaggagag 250 agagtgactc tgacttgcaa tggatttcag
ttctatgcaa cagagaaaac 300 aacatggtat catcggcact actggggaga
aaagttgacc ctgaccccag 350 gaaacaccct cgaggttcgg gaatctggac
tgtacagatg ccaggcccgg 400 ggctccccac gaagtaaccc tgtgcgcttg
ctcttttctt cagactcctt 450 aatcctgcag gcaccatatt ctgtgtttga
aggtgacaca ttggttctga 500 gatgccacag aagaaggaaa gagaaattga
ctgctgtgaa atatacttgg 550 aatggaaaca ttctttccat ttctaataaa
agctgggatc ttcttatccc 600 acaagcaagt tcaaataaca atggcaatta
tcgatgcatt ggatatggag 650 atgagaatga tgtatttaga tcaaatttca
aaataattaa aattcaagaa 700 ctatttccac atccagagct gaaagctaca
gactctcagc ctacagaggg 750 gaattctgta aacctgagct gtgaaacaca
gcttcctcca gagcggtcag 800 acaccccact tcacttcaac ttcttcagag
atggcgaggt catcctgtca 850 gactggagca cgtacccgga actccagctc
ccaaccgtct ggagagaaaa 900 ctcaggatcc tattggtgtg gtgctgaaac
agtgaggggt aacatccaca 950 agcacagtcc ctcgctacag atccatgtgc
agcggatccc tgtgtctggg 1000 gtgctcctgg agacccagcc ctcagggggc
caggctgttg aaggggagat 1050 gctggtcctt gtctgctccg tggctgaagg
cacaggggat accacattct 1100 cctggcaccg agaggacatg caggagagtc
tggggaggaa aactcagcgt 1150 tccctgagag cagagctgga gctccctgcc
atcagacaga gccatgcagg 1200 gggatactac tgtacagcag acaacagcta
cggccctgtc cagagcatgg 1250 tgctgaatgt cactgtgaga gagaccccag
gcaacagaga tggccttgtc 1300 gccgcgggag ccactggagg gctgctcagt
gctcttctcc tggctgtggc 1350 cctgctgttt cactgctggc gtcggaggaa
gtcaggagtt ggtttcttgg 1400 gagacgaaac caggctccct cccgctccag
gcccaggaga gtcctcccat 1450 tccatctgcc ctgcccaggt ggagcttcag
tcgttgtatg ttgatgtaca 1500 ccccaaaaag ggagatttgg tatactctga
gatccagact actcagctgg 1550 gagaagaaga ggaagctaat acctccagga
cacttctaga ggataaggat 1600 gtctcagttg tctactctga ggtaaagaca
caacacccag ataactcagc 1650 tggaaagatc agctctaagg atgaagaaag
ttaagagaat gaaaagttac 1700 gggaacgtcc tactcatgtg atttctccct
tgtccaaagt cccaggccca 1750 gtgcagtcct tgcggcacct ggaatgatca
actcattcca gctttctaat 1800 tcttctcatg catatgcatt cactcccagg
aatactcatt cgtctactct 1850 gatgttggga tggaatggcc tctgaaagac
ttcactaaaa tgaccaggat 1900 ccacagttaa gagaagaccc tgtagtattt
gctgtgggcc tgacctaatg 1950 cattccctag ggtctgcttt agagaagggg
gataaagaga gagaaggact 2000 gttatgaaaa acagaagcac aaattttggt
gaattgggat ttgcagagat 2050 gaaaaagact gggtgacctg gatctctgct
taatacatct acaaccattg 2100 tctcactgga gactcacttg catcagtttg
tttaactgtg agtggctgca 2150 caggcactgt gcaaacaatg aaaagcccct
tcacttctgc ctgcacagct 2200 tacactgtca ggattcagtt gcagattaaa
gaacccatct ggaatggttt 2250 acagagagag gaatttaaaa gaggacatca
gaagagctgg agatgcaagc 2300 tctaggctgc gcttccaaaa gcaaatgata
attatgttaa tgtcattagt 2350 gacaaagatt tgcaacatta gagaaaagag
acacaaatat aaaattaaaa 2400 acttaagtac caactctcca aaactaaatt
tgaacttaaa atattagtat 2450 aaactcataa taaactctgc ctttaaaaaa
agataaatat ttcctacgtc 2500 tgttcactga aataattacc aaccccttag
caataagcac tccttgcaga 2550 gaggttttat tctctaaata ccattccctt
ctcaaaggaa ataaggttgc 2600 ttttcttgta ggaactgtgt ctttgagtta
ctaattagtt tatatgagaa 2650 taattcttgc aataaatgaa gaaggaataa
aagaaatagg aagccacaaa 2700 tttgtatgga tatttcatga tacacctact
ggttaaataa ttgacaaaaa 2750 ccagcagcca aatattagag gtctcctgat
ggaagtgtac aataccacct 2800 acaaattatc catgccccaa gtgttaaaac
tgaatccatt caagtctttc 2850 taactgaata cttgttttat agaaaatgca
tggagaaaag gaatttgttt 2900 aaataacatt atgggattgc aaccagcaaa
acataaactg agaaaaagtt 2950 ctatagggca aatcacctgg cttctataac
aaataaatgg gaaaaaaatg 3000 aaataaaaag aagagaggga ggaagaaagg
gagagagaag aaaagaaaaa 3050 tgaagaaaag taattagaat attttcaaca
taaagaaaag acgaatattt 3100 aaggtgacag atatcccaac tacgctgatt
tgatctttac aaattatatg 3150 agtgtatgaa tttgtcacat gtatcacccc
caaaaaaaga gaaaaagaaa 3200 aatagaagac atataaatta aatgagacga
gacatgtcga ccaaaaggaa 3250 tgtgtgggtc ttgtttggat cctgactcaa
attaagaaaa aataaaacta 3300 cctacgaaat actaagaaaa atttgtatac
taatattaag aaattgttgt 3350 gtgttttgga tataagtgat agtttattgt
agtgatgttt ttataaaagc 3400 aaaaggatat tcactttcag cgcttatact
gaagtattag attaaagctt 3450 attaacgta 3459 9 1869 DNA Homo sapiens 9
gccgagctga gcggatcctc acatgactgt gatccgattc tttccagcgg 50
cttctgcaac caagcgggtc ttacccccgg tcctccgcgt ctccagtcct 100
cgcacctgga accccaacgt ccccgagagt ccccgaatcc ccgctcccag 150
gctacctaag aggatgagcg gtgctccgac ggccggggca gccctgatgc 200
tctgcgccgc caccgccgtg ctactgagcg ctcagggcgg acccgtgcag 250
tccaagtcgc cgcgctttgc gtcctgggac gagatgaatg tcctggcgca 300
cggactcctg cagctcggcc aggggctgcg cgaacacgcg gagcgcaccc 350
gcagtcagct gagcgcgctg gagcggcgcc tgagcgcgtg cgggtccgcc 400
tgtcagggaa ccgaggggtc caccgacctc ccgttagccc ctgagagccg 450
ggtggaccct gaggtccttc acagcctgca gacacaactc aaggctcaga 500
acagcaggat ccagcaactc ttccacaagg tggcccagca gcagcggcac 550
ctggagaagc agcacctgcg aattcagcat ctgcaaagcc agtttggcct 600
cctggaccac aagcacctag accatgaggt ggccaagcct gcccgaagaa 650
agaggctgcc cgagatggcc cagccagttg acccggctca caatgtcagc 700
cgcctgcacc ggctgcccag ggattgccag gagctgttcc aggttgggga 750
gaggcagagt ggactatttg aaatccagcc tcaggggtct ccgccatttt 800
tggtgaactg caagatgacc tcagatggag gctggacagt aattcagagg 850
cgccacgatg gctcagtgga cttcaaccgg ccctgggaag cctacaaggc 900
ggggtttggg gatccccacg gcgagttctg gctgggtctg gagaaggtgc 950
atagcatcac gggggaccgc aacagccgcc tggccgtgca gctgcgggac 1000
tgggatggca acgccgagtt gctgcagttc tccgtgcacc tgggtggcga 1050
ggacacggcc tatagcctgc agctcactgc acccgtggcc ggccagctgg 1100
gcgccaccac cgtcccaccc agcggcctct ccgtaccctt ctccacttgg 1150
gaccaggatc acgacctccg cagggacaag aactgcgcca agagcctctc 1200
tggaggctgg tggtttggca cctgcagcca ttccaacctc aacggccagt 1250
acttccgctc catcccacag cagcggcaga agcttaagaa gggaatcttc 1300
tggaagacct ggcggggccg ctactacccg ctgcaggcca ccaccatgtt 1350
gatccagccc atggcagcag aggcagcctc ctagcgtcct ggctgggcct 1400
ggtcccaggc ccacgaaaga cggtgactct tggctctgcc cgaggatgtg 1450
gccgttccct gcctgggcag gggctccaag gaggggccat ctggaaactt 1500
gtggacagag aagaagacca cgactggaga agcccccttt ctgagtgcag 1550
gggggctgca tgcgttgcct cctgagatcg aggctgcagg atatgctcag 1600
actctagagg cgtggaccaa ggggcatgga gcttcactcc ttgctggcca 1650
gggagttggg gactcagagg gaccacttgg ggccagccag actggcctca 1700
atggcggact cagtcacatt gactgacggg gaccagggct tgtgtgggtc 1750
gagagcgccc tcatggtgct ggtgctgttg tgtgtaggtc ccctggggac 1800
acaagcaggc gccaatggta tctgggcgga gctcacagag ttcttggaat 1850
aaaagcaacc tcagaacac 1869 10 685 DNA Homo sapiens 10 gatgtgctcc
ttggagctgg tgtgcagtgt cctgactgta agatcaagtc 50 caaacctgtt
ttggaattga ggaaacttct cttttgatct cagcccttgg 100 tggtccaggt
cttcatgctg ctgtgggtga tattactggt cctggctcct 150 gtcagtggac
agtttgcaag gacacccagg cccattattt tcctccagcc 200 tccatggacc
acagtcttcc aaggagagag agtgaccctc acttgcaagg 250 gatttcgctt
ctactcacca cagaaaacaa aatggtacca tcggtacctt 300 gggaaagaaa
tactaagaga aaccccagac aatatccttg aggttcagga 350 atctggagag
tacagatgcc aggcccaggg ctcccctctc agtagccctg 400 tgcacttgga
tttttcttca gagatgggat ttcctcatgc tgcccaggct 450 aatgttgaac
tcctgggctc aagtgatctg ctcacctagg cctctcaaag 500 cgctgggatt
acagcttcgc tgatcctgca agctccactt tctgtgtttg 550 aaggagactc
tgtggttctg aggtgccggg caaaggcgga agtaacactg 600 aataatacta
tttacaagaa tgataatgtc ctggcattcc ttaataaaag 650 aactgacttc
caaaaaaaaa aaaaaaaaaa aaaaa 685 11 5645 DNA Homo sapiens 11
cgcccgcgcg ctgcagcccc atctcctagc ggcagcccag gcgcggaggg 50
agcgagtccg ccccgaggta ggtccaggac gggcgcacag cagcagccga 100
ggctggccgg gagagggagg aagaggatgg cagggccacg ccccagccca 150
tgggccaggc tgctcctggc agccttgatc agcgtcagcc tctctgggac 200
cttggcaaac cgctgcaaga aggccccagt gaagagctgc acggagtgtg 250
tccgtgtgga taaggactgc gcctactgca cagacgagat gttcagggac 300
cggcgctgca acacccaggc ggagctgctg gccgcgggct gccagcggga 350
gagcatcgtg gtcatggaga gcagcttcca aatcacagag gagacccaga 400
ttgacaccac cctgcggcgc agccagatgt ccccccaagg cctgcgggtc 450
cgtctgcggc ccggtgagga gcggcatttt gagctggagg tgtttgagcc 500
actggagagc cccgtggacc tgtacatcct catggacttc tccaactcca 550
tgtccgatga tctggacaac ctcaagaaga tggggcagaa cctggctcgg 600
gtcctgagcc agctcaccag cgactacact attggatttg gcaagtttgt 650
ggacaaagtc agcgtcccgc agacggacat gaggcctgag aagctgaagg 700
agccctggcc caacagtgac ccccccttct ccttcaagaa cgtcatcagc 750
ctgacagaag atgtggatga gttccggaat aaactgcagg gagagcggat 800
ctcaggcaac ctggatgctc ctgagggcgg cttcgatgcc atcctgcaga 850
cagctgtgtg cacgagggac attggctggc gcccggacag cacccacctg 900
ctggtcttct ccaccgagtc agccttccac tatgaggctg atggcgccaa 950
cgtgctggct ggcatcatga gccgcaacga tgaacggtgc cacctggaca 1000
ccacgggcac ctacacccag tacaggacac aggactaccc gtcggtgccc 1050
accctggtgc gcctgctcgc caagcacaac atcatcccca tctttgctgt 1100
caccaactac tcctatagct actacgagaa gcttcacacc tatttccctg 1150
tctcctcact gggggtgctg caggaggact cgtccaacat cgtggagctg 1200
ctggaggagg ccttcaatcg gatccgctcc aacctggaca tccgggccct 1250
agacagcccc cgaggccttc ggacagaggt cacctccaag atgttccaga 1300
agacgaggac tgggtccttt cacatccggc ggggggaagt gggtatatac 1350
caggtgcagc tgcgggccct tgagcacgtg gatgggacgc acgtgtgcca 1400
gctgccggag gaccagaagg gcaacatcca tctgaaacct tccttctccg 1450
acggcctcaa gatggacgcg ggcatcatct gtgatgtgtg cacctgcgag 1500
ctgcaaaaag aggtgcggtc agctcgctgc agcttcaacg gagacttcgt 1550
gtgcggacag tgtgtgtgca gcgagggctg gagtggccag acctgcaact 1600
gctccaccgg ctctctgagt gacattcagc cctgcctgcg ggagggcgag 1650
gacaagccgt gctccggccg tggggagtgc cagtgcgggc actgtgtgtg 1700
ctacggcgaa ggccgctacg agggtcagtt ctgcgagtat gacaacttcc 1750
agtgtccccg cacttccggg ttcctctgca atgaccgagg acgctgctcc 1800
atgggccagt gtgtgtgtga gcctggttgg acaggcccaa gctgtgactg 1850
tcccctcagc aatgccacct gcatcgacag caatgggggc atctgtaatg 1900
gacgtggcca ctgtgagtgt ggccgctgcc actgccacca gcagtcgctc 1950
tacacggaca ccatctgcga gatcaactac tcggcgatcc acccgggcct 2000
ctgcgaggac ctacgctcct gcgtgcagtg ccaggcgtgg ggcaccggcg 2050
agaagaaggg gcgcacgtgt gaggaatgca acttcaaggt caagatggtg 2100
gacgagctta agagagccga ggaggtggtg gtgcgctgct ccttccggga 2150
cgaggatgac gactgcacct acagctacac catggaaggt gacggcgccc 2200
ctgggcccaa cagcactgtc ctggtgcaca agaagaagga ctgccctccg 2250
ggctccttct ggtggctcat ccccctgctc ctcctcctcc tgccgctcct 2300
ggccctgcta ctgctgctat gctggaagta ctgtgcctgc tgcaaggcct 2350
gcctggcact tctcccgtgc tgcaaccgag gtcacatggt gggctttaag 2400
gaagaccact acatgctgcg ggagaacctg atggcctctg accacttgga 2450
cacgcccatg ctgcgcagcg ggaacctcaa gggccgtgac gtggtccgct 2500
ggaaggtcac caacaacatg cagcggcctg gctttgccac tcatgccgcc 2550
agcatcaacc ccacagagct ggtgccctac gggctgtcct tgcgcctggc 2600
ccgcctttgc accgagaacc tgctgaagcc tgacactcgg gagtgcgccc 2650
agctgcgcca ggaggtggag gagaacctga acgaggtcta caggcagatc 2700
tccggtgtac acaagctcca gcagaccaag ttccggcagc agcccaatgc 2750
cgggaaaaag caagaccaca ccattgtgga cacagtgctg atggcgcccc 2800
gctcggccaa gccggccctg ctgaagctta cagagaagca ggtggaacag 2850
agggccttcc acgacctcaa ggtggccccc ggctactaca ccctcactgc 2900
agaccaggac gcccggggca tggtggagtt ccaggagggc gtggagctgg 2950
tggacgtacg ggtgcccctc tttatccggc ctgaggatga cgacgagaag 3000
cagctgctgg tggaggccat cgacgtgccc gcaggcactg ccaccctcgg 3050
ccgccgcctg gtaaacatca ccatcatcaa ggagcaagcc agagacgtgg 3100
tgtcctttga gcagcctgag ttctcggtca gccgcgggga ccaggtggcc 3150
cgcatccctg tcatccggcg tgtcctggac ggcgggaagt cccaggtctc 3200
ctaccgcaca caggatggca ccgcgcaggg caaccgggac tacatccccg 3250
tggagggtga gctgctgttc cagcctgggg aggcctggaa agagctgcag 3300
gtgaagctcc tggagctgca agaagttgac tccctcctgc ggggccgcca 3350
ggtccgccgt ttccacgtcc agctcagcaa ccctaagttt ggggcccacc 3400
tgggccagcc ccactccacc accatcatca tcagggaccc agatgaactg 3450
gaccggagct tcacgagtca gatgttgtca tcacagccac cccctcacgg 3500
cgacctgggc gccccgcaga accccaatgc taaggccgct gggtccagga 3550
agatccattt caactggctg cccccttctg gcaagccaat ggggtacagg 3600
gtaaagtact ggattcaggg tgactccgaa tccgaagccc acctgctcga 3650
cagcaaggtg ccctcagtgg agctcaccaa cctgtacccg tattgcgact 3700
atgagatgaa ggtgtgcgcc tacggggctc agggcgaggg accctacagc 3750
tccctggtgt cctgccgcac ccaccaggaa gtgcccagcg agccagggcg 3800
tctggccttc aatgtcgtct cctccacggt gacccagctg agctgggctg 3850
agccggctga gaccaacggt gagatcacag cctacgaggt ctgctatggc 3900
ctggtcaacg atgacaaccg acctattggg cccatgaaga aagtgctggt 3950
tgacaaccct aagaaccgga tgctgcttat tgagaacctt cgggagtccc 4000
agccctaccg ctacacggtg aaggcgcgca acggggccgg ctgggggcct 4050
gagcgggagg ccatcatcaa cctggccacc cagcccaaga ggcccatgtc 4100
catccccatc atccctgaca tccctatcgt ggacgcccag agcggggagg 4150
actacgacag cttccttatg tacagcgatg acgttctacg ctctccatcg 4200
ggcagccaga ggcccagcgt ctccgatgac actgagcacc tggtgaatgg 4250
ccggatggac tttgccttcc cgggcagcac caactccctg cacaggatga 4300
ccacgaccag tgctgctgcc tatggcaccc acctgagccc acacgtgccc 4350
caccgcgtgc taagcacatc ctccaccctc acacgggact acaactcact 4400
gacccgctca gaacactcac actcgaccac actgccgagg gactactcca 4450
ccctcacctc cgtctcctcc cacgactctc gcctgactgc tggtgtgccc 4500
gacacgccca cccgcctggt gttctctgcc ctggggccca catctctcag 4550
agtgagctgg caggagccgc ggtgcgagcg gccgctgcag ggctacagtg 4600
tggagtacca gctgctgaac ggcggtgagc tgcatcggct caacatcccc 4650
aaccctgccc agacctcggt ggtggtggaa gacctcctgc ccaaccactc 4700
ctacgtgttc cgcgtgcggg cccagagcca ggaaggctgg ggccgagagc 4750
gtgagggtgt catcaccatt gaatcccagg tgcacccgca gagcccactg 4800
tgtcccctgc caggctccgc cttcactttg agcactccca gtgccccagg 4850
cccgctggtg ttcactgccc tgagcccaga ctcgctgcag ctgagctggg 4900
agcggccacg gaggcccaat ggggatatcg tcggctacct ggtgacctgt 4950
gagatggccc aaggaggagg gccagccacc gcattccggg tggatggaga 5000
cagccccgag agccggctga ccgtgccggg cctcagcgag aacgtgccct 5050
acaagttcaa ggtgcaggcc aggaccactg agggcttcgg gccagagcgc 5100
gagggcatca tcaccataga gtcccaggat ggaggaccct tcccgcagct 5150
gggcagccgt gccgggctct tccagcaccc gctgcaaagc gagtacagca 5200
gcatcaccac cacccacacc agcgccaccg agcccttcct agtggatggg 5250
ccgaccctgg gggcccagca cctggaggca ggcggctccc tcacccggca 5300
tgtgacccag gagtttgtga gccggacact gaccaccagc ggaaccctta 5350
gcacccacat ggaccaacag ttcttccaaa cttgaccgca ccctgcccca 5400
cccccgccat gtcccactag gcgtcctccc gactcctctc ccggagcctc 5450
ctcagctact ccatccttgc acccctgggg gcccagccca cccgcatgca 5500
cagagcaggg gctaggtgtc tcctgggagg catgaagggg gcaaggtccg 5550
tcctctgtgg
gcccaaacct atttgtaacc aaagagctgg gagcagcaca 5600 aggacccagc
ctttgttctg cacttaataa atggttttgc tactg 5645 12 4381 DNA Homo
sapiens 12 gccttcaact accatcccac cacctgctga ggagaaaaat tcttcaagac
50 tcagagcaca cagccagcac cagaggcccc atgaccctgg acagaccagg 100
ggagggggcc accatgctga agacattcac tgttttgctc ttttgcattc 150
ggatgagtct gggtatgaca tcgatagtga tggaccctca accggagttg 200
tggatagagt ccaactaccc ccaggcccct tgggagaaca tcacgctttg 250
gtgccgaagc ccctctcgga tatcaagcaa gttcctgctg ctgaaggata 300
agacacaaat gacctggatc cgcccttccc acaagacctt ccaagtttca 350
ttccttatag gtgcccttac tgagtccaat gcaggtcttt accggtgctg 400
ctactggaag gagacaggct ggtcaaagcc cagtaaagtt ctagagttgg 450
aggcaccagg ccaactgccc aagcccatct tctggattca ggctgagacc 500
cccgctcttc ctgggtgtaa tgttaacatc ctctgccatg gctggctgca 550
ggatttggta ttcatgctgt ttaaagaggg atatgcagag cctgtggatt 600
accaagtccc aactgggaca atggccatat tctccattga caacctgaca 650
cctgaggatg aaggggttta catctgccgc actcatatcc agatgctccc 700
caccctgtgg tcagagccca gcaaccccct gaagctggtt gtagcaggac 750
tctaccccaa accaactttg acagcccatc ctgggcccat catggcacct 800
ggagaaagcc tgaatctcag gtgccaaggg ccaatctatg gaatgacctt 850
tgctctaatg agggttgaag acttggagaa gtccttttac cacaagaaga 900
caataaaaaa tgaggcaaat ttcttcttcc agtctttgaa gatccaagat 950
actggacatt acctctgttt ttactatgac gcatcatata gaggttcact 1000
ccttagtgat gtcctgaaaa tctgggtaac tgacactttc cccaagacct 1050
ggctacttgc tcggcccagt gctgtggtcc aaatgggtca gaatgtgagc 1100
ctacggtgtc gaggaccagt ggatggagtg ggtcttgcac tctataagaa 1150
aggagaagac aaaccacttc aatttttgga tgccaccagc atcgatgaca 1200
acacatcatt cttcctcaac aatgtaacct acagtgatac tggcatctat 1250
agctgccact atcttctcac ctggaagacc tccattagga tgccatcaca 1300
caacactgtg gagcttatgg ttgtagataa gccccccaaa ccctccctgt 1350
cagcttggcc aagcactgtg ttcaagctag gaaaggccat cacccttcag 1400
tgccgagtat ctcatccagt actggaattt tctctggaat gggaagaaag 1450
agaaacattc caaagattct cagtaaacgg agacttcatc atcagtaatg 1500
ttgacgggaa aggcacaggg acctacagtt gcagctatcg cgtagagaca 1550
catcctaaca tgtggtcaca tcgcagtgag cccctgaagc tgatggggcc 1600
agcaggctat ctcacctgga attacgttct gaatgaagct atcaggttgt 1650
ctctaatcat gcagcttgtt gccttgctgt tggtagtgct gtggataagg 1700
tggaagtgtc ggagactcag aatcagagaa gcctggttgc tgggaacagc 1750
tcaaggggtc accatgctct tcatagtcac ggcccttctc tgctgtggac 1800
tgtgcaatgg ggtattgata gaagagactg aaatagtcat gccaacccct 1850
aagcctgagc tgtgggcaga gaccaacttt cctctggccc cgtggaagaa 1900
cttaaccctc tggtgcagaa gcccttctgg ctcaactaag gagtttgtgt 1950
tgctgaagga tgggaccggg tggatcgcca ctcgcccggc ctcagagcag 2000
gtccgggctg ccttccccct tggcgccctg acccagagcc acaccgggag 2050
ctaccactgc cattcatggg aggagatggc tgtatcggag cccagtgagg 2100
cacttgagct ggtggggaca gacatcctcc ccaaacctgt catttctgct 2150
tcccccacaa tccggggcca ggaactacaa ctccggtgca aaggatggct 2200
ggcaggcatg gggtttgctc tgtataagga gggagagcaa gaacctgtcc 2250
agcaacttgg tgctgttgga agagaagcct tctttacaat ccagagaatg 2300
gaggataaag acgaaggcaa ttacagctgc cgcactcaca ctgaaaaact 2350
ccccttcaag tggtctgagc ccagtgagcc gctggagctt gtcataaaag 2400
aaatgtaccc taagcccttc ttcaagacat gggccagccc tgtggtcacc 2450
cctggtgccc gagtgacttt caattgctcc accccccacc agcatatgag 2500
ctttattctt tacaaagatg gaagtgaaat agcatccagt gacaggtcct 2550
gggcaagtcc gggggccagt gcagctcact ttctaatcat ttcggtgggc 2600
attggtgatg gagggaatta cagctgccga tattatgact tttctatctg 2650
gtctgagccc agcgaccctg tggagctcgt ggtgacagaa ttctacccca 2700
aacccactct cctggcacag ccaggtcctg tggtgtttcc tgggaagagt 2750
gtgatcctgc gctgccaagg gactttccag ggcatgaggt tcgccctctt 2800
gcaggaggga gcccatgttc ccttacagtt tcggagtgtc tcagggaact 2850
cagctgactt ccttctccac actgttggag cagaggactc tgggaactat 2900
agctgtatct actatgagac aaccatgtca aacagggggt catatctcag 2950
tatgcccctt atgatctggg tgactgacac attccctaag ccatggttgt 3000
ttgctgagcc cagttctgtg gttcccatgg ggcagaatgt tactctctgg 3050
tgccgagggc cggtccatgg agtaggatac attctgcaca aagaaggaga 3100
agccacttca atgcagctct ggggatccac cagtaatgac ggggcattcc 3150
ccatcaccaa tatatctggt actagcatgg ggcgttacag ctgctgctac 3200
caccctgact ggaccagttc tatcaagata caacctagca acaccctgga 3250
actcctagtc acaggcttac tccccaaacc cagcctatta gcccagcctg 3300
gtcccatggt ggcccctggc gaaaatatga ctcttcagtg tcaaggggaa 3350
ctgccagact caacatttgt gctgttgaag gagggggctc aggagccttt 3400
agagcaacag aggccaagtg ggtacagggc tgacttctgg atgccagcag 3450
tgagaggtga agactctggg atctatagct gtgtttatta tttggactct 3500
actccctttg cagcttcaaa tcacagtgac tccctggaga tctgggtgac 3550
tgataagccc cctaaaccct ctctgtcagc ctggcccagc accatgttca 3600
agttagggaa ggacatcacc cttcagtgcc gaggacccct gccaggtgtt 3650
gaatttgttc tagaacatga tggagaagaa gcacctcagc agttttcaga 3700
ggatggagac tttgtcatca acaacgtaga aggaaaaggc attggaaact 3750
acagctgcag ctaccgcctc caggcctacc ctgatatctg gtcagagcct 3800
agtgatcccc tggagctggt gggggcagca gggcctgttg ctcaggagtg 3850
cactgtaggg aacattgtcc gaagtagcct aatcgtggtg gttgttgtag 3900
ccttgggggt agtgctagcc atagagtgga agaagtggcc tcgactgcga 3950
accagaggct cagagacaga cggaagagac cagaccattg cccttgaaga 4000
gtgtaaccaa gaaggagaac caggcacccc tgccaattct ccttcatcaa 4050
cctctcagag aatctctgtg gaactgcccg ttccaatata ataatctcct 4100
cctttacaag agctttcctc tcctctctct tgctctcaga gacctataaa 4150
tccaaccagt taccctgcaa gtcagcccca tctgctgttc cttggtctct 4200
aatcacctga gctgggtaaa ggggattctg ggagttgaga gctctgccag 4250
ggtgagatgt ttcctgaaga gaggttcccc acccctgtaa ctcctcactg 4300
tactgattta ctggcgcatg aaattctatt aaaaatgcat tcttctgaat 4350
aaaaagagta ttcactattt aacttcaatt t 4381 13 1068 DNA Homo sapiens 13
cgccggagga gttctgcgtc tcggggtggt gactgggtcc agaatggctt 50
cggattcggg gaaccagggg accctctgca cgttggagtt cgcggtgcag 100
atgacctgtc agagctgtgt ggacgcggtg cgcaaatccc tgcaaggggt 150
ggcaggtgtc caggatgtgg aggtgcactt ggaggaccag atggtcttgg 200
tacacaccac tctacccagc caggaggtgc aggctctcct ggaaggcacg 250
gggcggcagg cggtactcaa gggcatgggc agcggccagt tgcagaatct 300
gggggcagca gtggccatcc tgggggggcc tggcaccgtg cagggggtgg 350
tgcgcttcct acagctgacc cctgagcgct gcctcatcga gggaactatt 400
gacggcctgg agcctgggct gcatggactc cacgtccatc agtacgggga 450
ccttacaaac aactgcaaca gctgtgggaa tcactttaac cctgatggag 500
catctcatgg gggcccccag gactctgacc ggcaccgcgg agacctgggc 550
aatgtccgtg ctgatgctga cggccgcgcc atcttcagaa tggaggatga 600
gcagctgaag gtgtgggatg tgattggccg cagcctgatt attgatgagg 650
gagaagatga cctgggccgg ggaggccatc ccttatccaa gatcacaggg 700
aactccgggg agaggttggc ctgtggcatc attgcacgct ccgctggcct 750
tttccagaac cccaagcaga tctgctcttg cgatggcctc accatctggg 800
aggagcgagg ccggcccatc gctggcaagg gccgaaagga gtcagcgcag 850
ccccctgccc acctttgagc aggacctcac cttggctctg ttgctgtcct 900
ccagggcgag cactttccac ttccagaggg ggccagaggg actttgcctg 950
cccagtcttt ggagagctca gtacagggca ggagctgctg tggtgttccc 1000
ttggcaaatg aaagttttat tttcgtttgg gaaaaaaaaa aaaaaaaaaa 1050
aaaaaaaaaa aaaaaaaa 1068 14 2402 DNA Homo sapiens Unsure 2138
Unknown base 14 ggagaggtgc gggccgaatc cgagccgagc gagaggaatc
cggcagtaga 50 gagcggactc cagccggcgg accctgcagc cctcgcctgg
gacagcggcg 100 cgctgggcag gcgcccaaga gagcatcgag cagcggaacc
cgcgaagccg 150 gcccgcagcc gcgacccgcg cagcctgccg ctctcccgcc
gccggtccgg 200 gcagcatgag gcgcgcggcg ctctggctct ggctgtgcgc
gctggcgctg 250 agcctgcagc tggccctgcc gcaaattgtg gctactaatt
tgccccctga 300 agatcaagat ggctctgggg atgactctga caacttctcc
ggctcaggtg 350 caggtgcttt gcaagatatc accttgtcac agcagacccc
ctccacttgg 400 aaggacacgc agctcctgac ggctattccc acgtctccag
aacccaccgg 450 cctggaggct acagctgcct ccacctccac cctgccggct
ggagaggggc 500 ccaaggaggg agaggctgta gtcctgccag aagtggagcc
tggcctcacc 550 gcccgggagc aggaggccac cccccgaccc agggagacca
cacagctccc 600 gaccactcat caggcctcaa cgaccacagc caccacggcc
caggagcccg 650 ccacctccca cccccacagg gacatgcagc ctggccacca
tgagacctca 700 acccctgcag gacccagcca agctgacctt cacactcccc
acacagagga 750 tggaggtcct tctgccaccg agagggctgc tgaggatgga
gcctccagtc 800 agctcccagc agcagagggc tctggggagc aggacttcac
ctttgaaacc 850 tcgggggaga atacggctgt agtggccgtg gagcctgacc
gccggaacca 900 gtccccagtg gatcaggggg ccacgggggc ctcacagggc
ctcctggaca 950 ggaaagaggt gctgggaggg gtcattgccg gaggcctcgt
ggggctcatc 1000 tttgctgtgt gcctggtggg tttcatgctg taccgcatga
agaagaagga 1050 cgaaggcagc tactccttgg aggagccgaa acaagccaac
ggcggggcct 1100 accagaagcc caccaaacag gaggaattct atgcctgacg
cgggagccat 1150 gcgccccctc cgccctgcca ctcactaggc ccccacttgc
ctcttccttg 1200 aagaactgca ggccctggcc tcccctgcca ccaggccacc
tccccagcat 1250 tccagcccct ctggtcgctc ctgcccacgg agtcgtgggt
gtgctgggag 1300 ctccactctg cttctctgac ttctgcctgg agacttaggg
caccaggggt 1350 ttctcgcata ggacctttcc accacagcca gcacctggca
tcgcaccatt 1400 ctgactcggt ttctccaaac tgaagcagcc tctccccagg
tccagctctg 1450 gaggggaggg ggatccgact gctttggacc taaatggcct
catgtggctg 1500 gaagatctgc gggtggggct tggggctcac acacctgtag
cacttactgg 1550 taggaccaag catcttgggg gggtggccgc tgagtggcag
ggacaggagt 1600 cactttgttt cgtggggagg tctaatctag atatcgactt
gtttttgcac 1650 atgtttcctc tagttctttg ttcatagccc agtagacctt
gttacttctg 1700 aggtaagtta agtaagttga ttcggtatcc ccccatcttg
cttccctaat 1750 ctatggtcgg gagacagcat cagggttaag aagacttttt
tttttttttt 1800 ttaaactagg agaaccaaat ctggaagcca aaatgtaggc
ttagtttgtg 1850 tgttgtctct tgagtttgtc gctcatgtgt gcaacagggt
atggactatc 1900 tgtctggtgg ccccgtttct ggtggtctgt tggcaggctg
gccagtccag 1950 gctgccgtgg ggccgccgcc tctttcaagc agtcgtgcct
gtgtccatgc 2000 gctcagggcc atgctgaggc ctgggccgct gccacgttgg
agaagcccgt 2050 gtgagaagtg aatgctggga ctcagccttc agacagagag
gactgtaggg 2100 agggcggcag gggcctggag atcctcctgc agaccacncc
cgtcctgcct 2150 gtgcgccgtc tccaggggct gcttcctcct ggaaattgac
gaggggtgtc 2200 ttgggcagag ctggctctga gcgcctccat ccaaggccag
gttctccgtt 2250 agctcctgtg gccccaccct gggccctggg ctggaatcag
gaatattttc 2300 caaagagtga tagtcttttg cttttggcaa aactctactt
aatccaatgg 2350 gtttttccct gtacagtaga ttttccaaat gtaataaact
ttaatataaa 2400 gt 2402 15 1145 DNA Homo sapiens 15 gctccggcca
gccgcggtcc agagcgcgcg aggttcgggg agctccgcca 50 ggctgctggt
acctgcgtcc gcccggcgag caggacaggc tgctttggtt 100 tgtgacctcc
aggcaggacg gccatcctct ccagaatgaa gatcttcttg 150 ccagtgctgc
tggctgccct tctgggtgtg gagcgagcca gctcgctgat 200 gtgcttctcc
tgcttgaacc agaagagcaa tctgtactgc ctgaagccga 250 ccatctgctc
cgaccaggac aactactgcg tgactgtgtc tgctagtgcc 300 ggcattggga
atctcgtgac atttggccac agcctgagca agacctgttc 350 cccggcctgc
cccatcccag aaggcgtcaa tgttggtgtg gcttccatgg 400 gcatcagctg
ctgccagagc tttctgtgca atttcagtgc ggccgatggc 450 gggctgcggg
caagcgtcac cctgctgggt gccgggctgc tgctgagcct 500 gctgccggcc
ctgctgcggt ttggcccctg accgcccaga ccctgtcccc 550 cgatccccca
gctcaggaag gaaagcccag ccctttctgg atcccacagt 600 gtatgggagc
ccctgactcc tcacgtgcct gatctgtgcc cttggtccca 650 ggtcaggccc
accccctgca cctccacctg ccccagcccc tgcctctgcc 700 caagtgggcc
agctgccctc acttctgggg tggatgatgt gaccttcctt 750 gggggactgc
ggaagggacg agggttccct ggagtcttac ggtccaacat 800 cagaccaagt
cccatggaca tgctgacagg gtccccaggg agaccgtgtc 850 agtagggatg
tgtgcctggc tgtgtacgtg ggtgtgcagt gcacgtgaga 900 gcacgtggcg
gcttctgggg gccatgtttg gggagggagg tgtgccagca 950 gcctggagag
cctcagtccc tgtagccccc tgccctggca cagctgcatg 1000 cacttcaagg
gcagcctttg ggggttgggg tttctgccac ttccgggtct 1050 aggccctgcc
caaatccagc cagtcctgcc ccagcccacc cccacattgg 1100 agccctcctg
ctgctttggt gcctcaaata aatacagatg tcccc 1145 16 1743 DNA Homo
sapiens 16 cagcaggtca cagcccctcg aggcgacagc ggccccgccg caccagagca
50 gtggtacagg catggatggg aagaaatgca gcgtatggat gttcctacct 100
cttgtattta ctttgtttac ttcagctgga ttgtggatag tatacttcat 150
agctgtggaa gatgacaaaa ttttaccatt aaattcagct gaaaggaaac 200
ctggtgtgaa gcatgcacca tatataagca ttgcaggtga tgatcctcct 250
gcaagctgtg tgtttagtca agttatgaac atggcagcct tcctagccct 300
tgtggtagct gttctgcgct tcatacaact gaaaccgaag gttttaaacc 350
cgtggctgaa tattagtgga ttggtggctc tgtgtctggc ttccttcgga 400
atgaccttac ttggtaattt tcagctcaca aatgatgaag aaatccataa 450
cgtcggaact tccttgacct ttggatttgg cacattgacc tgctggatcc 500
aggctgcgct gacactcaag gtcaacatca agaatgaagg acggagagtt 550
ggaattccac gggttattct gtcggcatct atcactctct gtgtggtcct 600
ctacttcatc ctcatggccc aaagcatcca catgtatgca gccagggtcc 650
agtggggcct ggtcatgtgc ttcctgtctt attttggcac ctttgccgtg 700
gagttccggc attaccgcta tgagattgtt tgctctgagt accaggagaa 750
tttcctaagc ttctcagaaa gcctgtcaga agcttctgaa tatcagactg 800
accaggtgta aaccatcagt ttttccttgc tggtgaggtg ggtgtgacag 850
tgggggaggg gccagtagga cacactcaca ggacttgaca tagaacctca 900
tttcacacac acacacacac acacattcat ggccacattt gccaaatgag 950
cttttcaggg cgagttattt ctttaatgaa aaagcacaag cccttatgtg 1000
tcgaaataca cgctgttaca ctgaaaatat atgcacgaca gagcaagaag 1050
cttgtgcatg atcacttctt atccgtcccc ttcccagcac tccctcctct 1100
tcccattctc tccacatgtc tcaagcaccc taccgagtag ggcaggccaa 1150
atgttccttg ggagtaatgc caactcccga cgttgccttc aggtccaaag 1200
ggcttggaac cagctcgtga ggaagttctg aatctggcac taatattctt 1250
gagtggataa tagtgtatca tagaatagga cggaaattgt attgagatgt 1300
gaccctgtgt cgcctgtgga aaggcatagt gagaagaact ttcccacgaa 1350
agcccccttc atcgttgttc agtggtcggc tgtgtggatc ccaggagaga 1400
catatgccac agactgtgag agcaaagccc gccgctgtga tctggacttg 1450
atgcactgtg actgagaatg atttccaaat gtgaatatgt gtagggacgt 1500
ggtctatcag gcctggaaca agatgggggc agtgaaggta tggtttagtg 1550
tttgctttca tagtatgcca tgtacaatgt tttatatttc atagtttctt 1600
ttaagtaact accatgagtc tctctaagcc tcatggacaa agatgtagac 1650
caaatgcaag agctgagctt gctttgggtt caaccatgat caaagaaaaa 1700
ctgaggtcac ctgcaggctt acgtgggaag ctaagacaat atc 1743 17 1939 DNA
Homo sapiens 17 ctgcctccac tgctctgtgc tgggatcatg gaacttgcac
tgctgtgtgg 50 gctggtggtg atggctggtg tgattccaat ccagggcggg
atcctgaacc 100 tgaacaagat ggtcaagcaa gtgactggga aaatgcccat
cctctcctac 150 tggccctacg gctgtcactg cggactaggt ggcagaggcc
aacccaaaga 200 tgccacggac tggtgctgcc agacccatga ctgctgctat
gaccacctga 250 agacccaggg gtgcggcatc tacaaggact attacagata
caacttttcc 300 caggggaaca tccactgctc tgacaaggga agctggtgtg
agcagcagct 350 gtgtgcctgt gacaaggagg tggccttctg cctgaagcgc
aacctggaca 400 cctaccagaa gcgactgcgt ttctactggc ggccccactg
ccgggggcag 450 acccctgggt gctagaagcc cacaccctct accctgttcc
tcagcatgga 500 gctctggcat ccccacctca gtatctaacc tgaaccagcc
tggcttttca 550 aacactccgg ggggaggtag tcccagcctc ccccggaacc
ctctaccaat 600 gccttctgac cttctgaagc tttccgaatc ctcccagttg
aggcagtagc 650 tgtgtcctct gagggtggat gggaatcttg ggagaagccc
aagcaaggga 700 gccctcagag gtggtgtttg gaccaaagca tcggggtggg
ggaggggtct 750 gccgctgtcc cccacctgct ggcccccttg tccttcctca
ccccctccaa 800 tatagtctcg gagctacaac tgcagcagcc actataaagg
gcaatattga 850 tctttctgtc catgtggctc tatcttttaa aacctcaagg
ccctccactg 900 tcctaagata aagcctctca taggcactgg ggaccctgca
cagtctggcc 950 atgtgaccct ctccccaggc aagctctgaa gtccctgcag
gtggaggcca 1000 tgcctgtctt aaactcagtt gcatccctgg tgcccaaagc
aacaccagaa 1050 ccaagaagga gctccataaa tccttcttgg gtgaagccta
gacaaagccg 1100 ccaggtcttg tggctccagg caccagagcc ttgagtactt
tctcctgcct 1150 ccaggcattg gctcagggtg aattacaagg ggctactgaa
tggctattac 1200 tttcatcacg actgatcccc acctcctcag ggtcaaaggg
ctactttctg 1250 gaagtctccc caggctgact ccttctccct gactgcaagg
gctcactccc 1300 tcctccaagc tcccacaatg cttcatggct ctgccgctta
cctagcttgg 1350 cctagagtgg caaatggaac ttctctgatc tcccccaact
agactggagc 1400 ccccgaagga tggagaccat gtctgtgcca tctctgtttc
ccctgttttc 1450 ccacatacta ggtgctcaat tcatgcctgt
gaatggcgtg agcccataat 1500 ggatacacag aggttgcagc agatggtgtg
ggtacctcac ccagatatct 1550 tccaggccca aggcccctct ccctgagtga
ggccaggtgt tggcagccaa 1600 ctgctccaat ctgcctcctt cccctaaata
ctgccctggt ctagtgggag 1650 ctgccttccc cctgccccac ctctcccacc
aagaggccac ctgtcactca 1700 tggccaggag agtgacacca tggagggtac
aattgccagc tcccccgtgt 1750 ctgtgcagca ttgtctgggt tgaatgacac
tctcaaattg ttcctgggat 1800 cgggctgagg ccaggcctct cctggaacca
cctctctgct tggtctgacc 1850 ccttggccta tccagttttc ctggttccct
cacaggtttc tccagaaagt 1900 actccctcag taaagcattt gcacaagaaa
aaaaaaaaa 1939 18 5420 DNA Homo sapiens Unsure 5094 Unknown base 18
ggctgaaaga gcctgagctg tgcctctcca ttccactgct gtggcagggt 50
cagaaatctt ggatagagaa aaccttttgc aaacgggaat gtatctttgt 100
aattcctagc acgaaagact ctaacaggtg ttgctgtggc cagttcacca 150
accagcatat cccccctctg ccaagtgcaa cacccagcaa aaatgaagag 200
gaaagcaaac aggtggagac tcagcctgag aaatggtctg ttgccaagca 250
cacccagagc tacccaacag attcctatgg agttcttgaa ttccagggtg 300
gcggatattc caataaagcc atgtatatcc gtgtatccta tgacaccaag 350
ccagactcac tgctccatct catggtgaaa gattggcagc tggaactccc 400
caagctctta atatctgtgc atggaggcct ccagaacttt gagatgcagc 450
ccaagctgaa acaagtcttt gggaaaggcc tgatcaaggc tgctatgacc 500
accggggcct ggatcttcac cgggggtgtc agcacaggtg ttatcagcca 550
cgtaggggat gccttgaaag accactcctc caagtccaga ggccgggttt 600
gtgctatagg aattgctcca tggggcatcg tggagaataa ggaagacctg 650
gttggaaagg atgtaacaag agtgtaccag accatgtcca accctctaag 700
taagctctct gtgctcaaca actcccacac ccacttcatc ctggctgaca 750
atggcaccct gggcaagtat ggcgccgagg tgaagctgcg aaggctgctg 800
gaaaagcaca tctccctcca gaagatcaac acaagactgg ggcagggcgt 850
gcccctcgtg ggtctcgtgg tggagggggg ccctaacgtg gtgtccatcg 900
tcttggaata cctgcaagaa gagcctccca tccctgtggt gatttgtgat 950
ggcagcggac gtgcctcgga catcctgtcc tttgcgcaca agtactgtga 1000
agaaggcgga ataataaatg agtccctcag ggagcagctt ctagttacca 1050
ttcagaaaac atttaattat aataaggcac aatcacatca gctgtttgca 1100
attataatgg agtgcatgaa gaagaaagaa ctcgtcactg tgttcagaat 1150
gggttctgag ggccagcagg acatcgagat ggcaatttta actgccctgc 1200
tgaaaggaac aaacgtatct gctccagatc agctgagctt ggcactggct 1250
tggaaccgcg tggacatagc acgaagccag atctttgtct ttgggcccca 1300
ctggacgccc ctgggaagcc tggcaccccc gacggacagc aaagccacgg 1350
agaaggagaa gaagccaccc atggccacca ccaagggagg aagaggaaaa 1400
gggaaaggca agaagaaagg gaaagtgaaa gaggaagtgg aggaagaaac 1450
tgacccccgg aagatagagc tgctgaactg ggtgaatgct ttggagcaag 1500
cgatgctaga tgctttagtc ttagatcgtg tcgactttgt gaagctcctg 1550
attgaaaacg gagtgaacat gcaacacttt ctgaccattc cgaggctgga 1600
ggagctctat aacacaagac tgggtccacc aaacacactt catctgctgg 1650
tgagggatgt gaaaaagagc aaccttccgc ctgattacca catcagcctc 1700
atagacatcg ggctcgtgct ggagtacctc atgggaggag cctaccgctg 1750
caactacact cggaaaaact ttcggaccct ttacaacaac ttgtttggac 1800
caaagaggcc taaagctctt aaacttctgg gaatggaaga tgatgagcct 1850
ccagctaaag ggaagaaaaa aaaaaaaaag aaaaaggagg aagagatcga 1900
cattgatgtg gacgaccctg ccgtgagtcg gttccagtat cccttccacg 1950
agctgatggt gtgggcagtg ctgatgaaac gccagaaaat ggcagtgttc 2000
ctctggcagc gaggggaaga gagcatggcc aaggccctgg tggcctgcaa 2050
gctctacaag gccatggccc acgagtcctc cgagagtgat ctggtggatg 2100
acatctccca ggacttggat aacaattcca aagacttcgg ccagcttgct 2150
ttggagttat tagaccagtc ctataagcat gacgagcaga tcgctatgaa 2200
actcctgacc tacgagctga aaaactggag caactcgacc tgcctcaaac 2250
tggccgtggc agccaaacac cgggacttca ttgctcacac ctgcagccag 2300
atgctgctga ccgatatgtg gatgggaaga ctgcggatgc ggaagaaccc 2350
cggcctgaag gttatcatgg ggattcttct accccccacc atcttgtttt 2400
tggaatttcg cacatatgat gatttctcgt atcaaacatc caaggaaaac 2450
gaggatggca aagaaaaaga agaggaaaat acggatgcaa atgcagatgc 2500
tggctcaaga aagggggatg aggagaacga gcataaaaaa cagagaagta 2550
ttcccatcgg aacaaagatc tgtgaattct ataacgcgcc cattgtcaag 2600
ttctggtttt acacaatatc atacttgggc tacctgctgc tgtttaacta 2650
cgtcatcctg gtgcggatgg atggctggcc gtccctccag gagtggatcg 2700
tcatctccta catcgtgagc ctggcgttag agaagatacg agagatcctc 2750
atgtcagaac caggcaaact cagccagaaa atcaaagttt ggcttcagga 2800
gtactggaac atcacagatc tcgtggccat ttccacattc atgattggag 2850
caattcttcg cctacagaac cagccctaca tgggctatgg ccgggtgatc 2900
tactgtgtgg atatcatctt ctggtacatc cgtgtcctgg acatctttgg 2950
tgtcaacaag tatctggggc catacgtgat gatgattgga aagatgatga 3000
tcgacatgct gtactttgtg gtcatcatgc tggtcgtgct catgagtttc 3050
ggagtagccc gtcaagccat tctgcatcca gaggagaagc cctcttggaa 3100
actggcccga aacatcttct acatgcccta ctggatgatc tatggagagg 3150
tgtttgcaga ccagatagac ctctacgcca tggaaattaa tcctccttgt 3200
ggtgagaacc tatatgatga ggagggcaag cggcttcctc cctgtatccc 3250
cggcgcctgg ctcactccag cactcatggc gtgctatcta ctggtcgcca 3300
acatcctgct ggtgaacctg ctgattgctg tgttcaacaa tactttcttt 3350
gaagtaaaat caatatccaa ccaggtgtgg aagttccagc gatatcagct 3400
gattatgaca tttcatgaca ggccagtcct gcccccaccg atgatcattt 3450
taagccacat ctacatcatc attatgcgtc tcagcggccg ctgcaggaaa 3500
aagagagaag gggaccaaga ggaacgggat cgtggattga agctcttcct 3550
tagcgacgag gagctaaaga ggctgcatga gttcgaggag cagtgcgtgc 3600
aggagcactt ccgggagaag gaggatgagc agcagtcgtc cagcgacgag 3650
cgcatccggg tcacttctga aagagttgaa aatatgtcaa tgaggttgga 3700
agaaatcaat gaaagagaaa cttttatgaa aacttccctg cagactgttg 3750
accttcgact tgctcagcta gaagaattat ctaacagaat ggtgaatgct 3800
cttgaaaatc ttgcgggaat cgacaggtct gacctgatcc aggcacggtc 3850
ccgggcttct tctgaatgtg aggcaacgta tcttctccgg caaagcagca 3900
tcaatagcgc tgatggctac agcttgtatc gatatcattt taacggagaa 3950
gagttattat ttgaggatac atctctctcc acgtcaccag ggacaggagt 4000
caggaaaaaa acctgttcct tccgtataaa ggaagagaag gacgtgaaaa 4050
cgcacctagt cccagaatgt cagaacagtc ttcacctttc actgggcaca 4100
agcacatcag caaccccaga tggcagtcac cttgcagtag atgacttaaa 4150
gaacgctgaa gagtcaaaat taggtccaga tattgggatt tcaaaggaag 4200
atgatgaaag acagacagac tctaaaaaag aagaaactat ttccccaagt 4250
ttaaataaaa cagatgtgat acatggacag gacaaatcag atgttcaaaa 4300
cactcagcta acagtggaaa cgacaaatat agaaggcact atttcctatc 4350
ccctggaaga aaccaaaatt acacgctatt tccccgatga aacgatcaat 4400
gcttgtaaaa caatgaagtc cagaagcttc gtctattccc ggggaagaaa 4450
gctggtcggt ggggttaacc aggatgtaga gtacagttca atcacggacc 4500
agcaattgac gacggaatgg caatgccaag ttcaaaagat cacgcgctct 4550
catagcacag atattcctta cattgtgtcg gaagctgcag tgcaagctga 4600
gcaaaaagag cagtttgcag atatgcaaga tgaacaccat gtcgctgaag 4650
caattcctcg aatccctcgc ttgtccctaa ccattactga cagaaatggg 4700
atggaaaact tactgtctgt gaagccagat caaactttgg gattcccatc 4750
tctcaggtca aaaagtttac atggacatcc taggaatgtg aaatccattc 4800
agggaaagtt agacagatct ggacatgcca gtagtgtaag cagcttagta 4850
attgtgtctg gaatgacagc agaagaaaaa aaggttaaga aagagaaagc 4900
ttccacagaa actgaatgct agtctgtttt gtttctttaa tttttttttt 4950
taacagtcag aaacccacta atgggtgtca tcttggccca tcctaaacac 5000
atmtccaatt tcctaaaaac attttccctt aaaaaatttt ggaaattcag 5050
acttgattta caatttaatg cactaaaagt agtattttgt tagnatatgt 5100
tagtaggctt agttttttca gttgcagtag tatcaaatga aagtgatgat 5150
actgtaacga agataaattg gctaatcagt atacaagatt atacaatctc 5200
tttattactg agggccacca aatagcctag gaagtgccct cgagcactga 5250
agtcaccatt aggtcactca agaagtaagc aactagctgg gcacagtggc 5300
tcatgcctgt aatcctagca ctttgggagg ccaaggcaga aagatagctt 5350
gagtccagga gtttgagacc agcctgggca acatagtgat accccatctc 5400
ttaaaaaaaa aaaaaaaaaa 5420 19 1664 DNA Homo sapiens 19 ctgaatcttc
gtttctctcc cagggaccct ccattttcca tatccaggaa 50 aatgtgatgc
gccacaggta tcagcgtctg gatcgccact tcacgtttta 100 gccacaagtg
actcagtgga agatccagag tcaacagagg ctcgtcagga 150 agatgtctac
agaaaaggta gaccaaaagg aggaagctgg ggaaaaagag 200 gtgtgcggag
accagatcaa aggaccggac aaagaggagg aaccaccagc 250 tgctgcatcc
catggccagg ggtggcgtcc aggtggcaga gcagctagga 300 acgcaaggcc
tgaacctggg gccagacacc ctgctctccc ggccatggtc 350 aacgaccctc
cagtacctgc cttactgtgg gcccaggagg tgggccaagt 400 cttggcaggc
cgtgcccgca ggctgctgct gcagtttggg gtgctcttct 450 gcaccatcct
ccttttgctc tgggtgtctg tcttcctcta tggctccttc 500 tactattcct
atatgccgac agtcagccac ctcagccctg tgcatttcta 550 ctacaggacc
gactgtgatt cctccaccac ctcactctgc tccttccctg 600 ttgccaatgt
ctcgctgact aagggtggac gtgatcgggt gctgatgtat 650 ggacagccgt
atcgtgttac cttagagctt gagctgccag agtcccctgt 700 gaatcaagat
ttgggcatgt tcttggtcac catttcctgc tacaccagag 750 gtggccgaat
catctccact tcttcgcgtt cggtgatgct gcattaccgc 800 tcagacctgc
tccagatgct ggacacactg gtcttctcta gcctcctgct 850 atttggcttt
gcagagcaga agcagctgct ggaggtggaa ctctacgcag 900 actatagaga
gaactcgtac gtgccgacca ctggagcgat cattgagatc 950 cacagcaagc
gcatccagct gtatggagcc tacctccgca tccacgcgca 1000 cttcactggg
ctcagatacc tgctatacaa cttcccgatg acctgcgcct 1050 tcataggtgt
tgccagcaac ttcaccttcc tcagcgtcat cgtgctcttc 1100 agctacatgc
agtgggtgtg ggggggcatc tggccccgac accgcttctc 1150 tttgcaggtt
aacatccgaa aaagagacaa ttcccggaag gaagtccaac 1200 gaaggatctc
tgctcatcag ccagggcctg aaggccagga ggagtcaact 1250 ccgcaatcag
atgttacaga ggatggtgag agccctgaag atccctcagg 1300 gacagagggt
cagctgtccg aggaggagaa accagatcag cagcccctga 1350 gcggagaaga
ggagctagag cctgaggcca gtgatggttc aggctcctgg 1400 gaagatgcag
ctttgctgac ggaggccaac ctgcctgctc ctgctcctgc 1450 ttctgcttct
gcccctgtcc tagagactct gggcagctct gaacctgctg 1500 ggggtgctct
ccgacagcgc cccacctgct ctagttcctg aagaaaaggg 1550 gcagactcct
cacattccag cactttccca cctgactcct ctcccctcgt 1600 ttttccttca
ataaactatt ttgtgtcagc ttcaaaaaaa aaaaaaaaaa 1650 aaaaaaaaaa aaaa
1664 20 4719 DNA Homo sapiens 20 aaccggctgc ggggatgggg ccaccgctcc
cgctgctgct gctgctactg 50 ctgctgctgc cgccacgcgt cctgcctgcc
gccccttcgt ccgtcccccg 100 cggccggcag ctcccggggc gtctgggctg
cctgctcgag gagggcctct 150 gcggagcgtc cgaggcctgt gtgaacgatg
gagtgtttgg aaggtgccag 200 aaggttccgg caatggactt ttaccgctac
gaggtgtcgc ccgtggccct 250 gcagcgcctg cgcgtggcgt tgcagaagct
ttccggcaca ggtttcacgt 300 ggcaggatga ctatactcag tatgtgatgg
accaggaact tgcagacctc 350 ccgaaaacct acctgaggcg tcctgaagca
tccagcccag ccaggccctc 400 aaaacacagc gttggcagcg agaggaggta
cagtcgggag ggcggtgctg 450 ccctggccaa cgccctccga cgccacctgc
ccttcctgga ggccctgtcc 500 caggccccag cctcagacgt gctcgccagg
acccatacgg cgcaggacag 550 accccccgct gagggtgatg accgcttctc
cgagagcatc ctgacctatg 600 tggcccacac gtctgcgctg acgtaccctc
ccgggccccg gacccagctc 650 cgggaggacc tcctgccgcg gaccctcggc
cagctccagc cagatgagct 700 cagccctaag gtggacagtg gtgtggacag
acaccatctg atggcggccc 750 tcagtgccta tgctgcccag aggcccccag
ctccccccgg ggagggcagc 800 ctggagccac agtaccttct gcgtgcaccc
tcaagaatgc ccaggccttt 850 gctggcacca gccgcccccc agaagtggcc
ttcacctctg ggagattccg 900 aagacccctc cagcacaggc gatggagcac
ggattcatac cctcctgaag 950 gacctgcaga ggcagccggc tgaggtgagg
ggcctgagtg gcctggagct 1000 ggacggcatg gctgagctga tggctggcct
gatgcaaggc gtggaccatg 1050 gagtagctcg aggcagccct gggagagcgg
ccctgggaga gtctggagaa 1100 caggcggatg gccccaaggc caccctccgt
ggagacagct ttccagatga 1150 cggagtgcag gacgacgatg atagacttta
ccaagaggtc caccgtctga 1200 gtgccacact cgggggcctc ctgcaggacc
acgggtctcg actcttacct 1250 ggagccctcc cctttgcaag gcccctcgac
atggagagga agaagtccga 1300 gcaccctgag tcttccctgt cttcagaaga
ggagactgcc ggagtggaga 1350 acgtcaagag ccagacgtat tccaaagatc
tgctggggca gcagccgcat 1400 tcggagcccg gggccgctgc gtttggggag
ctccaaaacc agatgcctgg 1450 gccctcgaag gaggagcaga gccttccagc
gggtgctcag gaggccctca 1500 gcgacggcct gcaattggag gtccagcctt
ccgaggaaga ggcgcggggc 1550 tacatcgtga cagacagaga ccccctgcgc
cccgaggaag gaaggcggct 1600 ggtggaggac gtcgcccgcc tcctgcaggt
gcccagcagt gcgttcgctg 1650 acgtggaggt tctcggacca gcagtgacct
tcaaagtgag cgccaatgtc 1700 caaaacgtga ccactgagga tgtggagaag
gccacagttg acaacaaaga 1750 caaactggag gaaacctctg gactgaaaat
tcttcaaacc ggagtcgggt 1800 cgaaaagcaa actcaagttc ctgcctcctc
aggcggagca agaagactcc 1850 accaagttca tcgcgctcac cctggtctcc
ctcgcctgca tcctgggcgt 1900 cctcctggcc tctggcctca tctactgcct
ccgccatagc tctcagcaca 1950 ggctgaagga gaagctctcg ggactagggg
gcgacccagg tgcagatgcc 2000 actgccgcct accaggagct gtgccgccag
cgtatggcca cgcggccacc 2050 agaccgacct gagggcccgc acacgtcacg
catcagcagc gtctcatccc 2100 agttcagcga cgggccgatc cccagcccct
ccgcacgcag cagcgcctca 2150 tcctggtccg aggagcctgt gcagtccaac
atggacatct ccaccggcca 2200 catgatcctg tcctacatgg aggaccacct
gaagaacaag aaccggctgg 2250 agaaggagtg ggaagcgctg tgcgcctacc
aggcggagcc caacagctcg 2300 ttcgtggccc agagggagga gaacgtgccc
aagaaccgct ccctggctgt 2350 gctgacctat gaccactccc gggtcctgct
gaaggcggag aacagccaca 2400 gccactcaga ctacatcaac gctagcccca
tcatggatca cgacccgagg 2450 aaccccgcgt acatcgccac ccagggaccg
ctgcccgcca ccgtggctga 2500 cttttggcag atggtgtggg agagcggctg
cgtggtgatc gtcatgctga 2550 cacccctcgc ggagaacggc gtccggcagt
gctaccacta ctggccggat 2600 gaaggctcca atctctacca catctatgag
gtgaacctgg tctccgagca 2650 catctggtgt gaggacttcc tggtgaggag
cttctatctg aagaacctgc 2700 agaccaacga gacgcgcacc gtgacgcagt
tccacttcct gagttggtat 2750 gaccgaggag tcccttcctc ctcaaggtcc
ctcctggact tccgcagaaa 2800 agtaaacaaa tgctacaggg gccgttcttg
tccgataatt gttcattgca 2850 gtgacggtgc aggccggagc ggcacctacg
tcctgatcga catggttctc 2900 aacaagatgg ccaaaggtgc taaagagatt
gatatcgcag cgaccctgga 2950 gcacttgagg gaccagagac ccggcatggt
ccagacgaag gagcagtttg 3000 agttcgcgct gacagccgtg gctgaggagg
tgaacgccat cctcaaggcc 3050 cttccccagt gagcggcagc gtcaggggcc
tcaggggagc ccccacccca 3100 cggatgttgt caggaatcat gatctgactt
taattgtgtg tcttctatta 3150 taactgcata gtaatagggc ccttagctct
cccgtagtca gcgcagttta 3200 gcagttaaaa gtgtattttt gtttaatcaa
acaataataa agagagattt 3250 gtggaaaaat ccagttacgg gtggagggga
atcggttcat caattttcac 3300 ttgcttaaaa aaaatacttt ttcttaaagc
acccgttcac cttcttggtt 3350 gaagttgtgt taacaatgca gtagccagca
cgttcgaggc ggtttccagg 3400 aagagtgtgc ttgtcatctg ccactttcgg
gagggtggat ccactgtgca 3450 ggagtggccg gggaagctgg cagcactcag
tgaggccgcc cggcacacaa 3500 ggcacgtttg gcatttctct ttgagagagt
ttatcattgg gagaagccgc 3550 ggggacagaa ctgaacgtcc tgcagcttcg
gggcaagtga gacaatcaca 3600 gctcctcgct gcgtctccat caacactgcg
ccgggtacca tggacggccc 3650 cgtcagccac accggtcagc ccaagcagag
tgattcaggg gctccccggg 3700 ggcagacacc tgtgcacccc atgagtagtg
cccacttgag gctggcactc 3750 ccctgacctc acctttgcaa agttacagat
gcaccccaac attgagatgt 3800 gtttttaatg ttaaaatatt gatttctacg
ttatgaaaac agatgccccc 3850 gtgaatgctt acctgtgaga taaccacaac
caggaagaac aaatctgggc 3900 attgagcaag ctatgagggt ccccgggagc
acacgaaccc tgccaggccc 3950 ccgctggctc ctccaggcac gtcccggacc
tgtggggccc cagagagggg 4000 acatttccct cctgggagag aaggagatca
gggcaactcg gagagggctg 4050 cgagcatttc cctcccggga gaggaaatca
gggcgacctg cacgcactgc 4100 gtagagcctg gaagggaagt gagaaaccag
ccgaccggcc ctgcccctct 4150 tcccgggatc acttaatgaa ccacgtgttt
tgacatcatg ttaacctaag 4200 cacgtacaga tgattccgga tttgacaaaa
taacatttga gtatccgatt 4250 cgccatcacc cctacccccg aaataggaca
actcacttca ttgaccagga 4300 tgatcacatg gaaggcggcg cagaggcagc
tgtgtgggct gcagatttcc 4350 tgtgtggggt tcagcgtata aaacgcacct
ccatcccgcc cttcccacag 4400 cattcctcca tcttagatag atggtactct
ccaaaggccc taccagaggg 4450 aacacggcct actgagcgga cagaatgatg
ccaaaatatt gcttatgtct 4500 ctacatggta ttgtaatgaa tatctgcttt
aatatagcta tcatttcttt 4550 tccaaaatta cttctcttta tctggaattt
aattaatcga aatgaattta 4600 tctgaatata ggaagcatat gcctacttgt
aatttctaac tacttatgtt 4650 tgaagagaaa cctccggtgt gagatataca
aatatattta attgtgtcat 4700 attaaacttc ccggaattc 4719 21 1453 DNA
Homo sapiens 21 gcatctggtt tgtcagatcc gagaggctct gaaactgcgg
agcggccacc 50 ggacgccttc tggagcaggt agcagcatgc
agccgcctcc aagtctgtgc 100 ggacgcgccc tggttgcgct ggttcttgcc
tgcggcctgt cgcggatctg 150 gggagaggag agaggcttcc cgcctgacag
ggccactccg cttttgcaaa 200 ccgcagagat aatgacgcca cccactaaga
ccttatggcc caagggttcc 250 aacgccagtc tggcgcggtc gttggcacct
gcggaggtgc ctaaaggaga 300 caggacggca ggatctccgc cacgcaccat
ctcccctccc ccgtgccaag 350 gacccatcga gatcaaggag actttcaaat
acatcaacac ggttgtgtcc 400 tgccttgtgt tcgtgctggg gatcatcggg
aactccacac ttctgagaat 450 tatctacaag aacaagtgca tgcgaaacgg
tcccaatatc ttgatcgcca 500 gcttggctct gggagacctg ctgcacatcg
tcattgacat ccctatcaat 550 gtctacaagc tgctggcaga ggactggcca
tttggagctg agatgtgtaa 600 gctggtgcct ttcatacaga aagcctccgt
gggaatcact gtgctgagtc 650 tatgtgctct gagtattgac agatatcgag
ctgttgcttc ttggagtaga 700 attaaaggaa ttggggttcc aaaatggaca
gcagtagaaa ttgttttgat 750 ttgggtggtc tctgtggttc tggctgtccc
tgaagccata ggttttgata 800 taattacgat ggactacaaa ggaagttatc
tgcgaatctg cttgcttcat 850 cccgttcaga agacagcttt catgcagttt
tacaagacag caaaagattg 900 gtggctgttc agtttctatt tctgcttgcc
attggccatc actgcatttt 950 tttatacact aatgacctgt gaaatgttga
gaaagaaaag tggcatgcag 1000 attgctttaa atgatcacct aaagcagaga
cgggaagtgg ccaaaaccgt 1050 cttttgcctg gtccttgtct ttgccctctg
ctggcttccc cttcacctca 1100 gcaggattct gaagctcact ctttataatc
agaatgatcc caatagatgt 1150 gaacttttga gctttctgtt ggtattggac
tatattggta tcaacatggc 1200 ttcactgaat tcctgcatta acccaattgc
tctgtatttg gtgagcaaaa 1250 gattcaaaaa ctgctttaag tcatgcttat
gctgctggtg ccagtcattt 1300 gaagaaaaac agtccttgga ggaaaagcag
tcgtgcttaa agttcaaagc 1350 taatgatcac ggatatgaca acttccgttc
cagtaataaa tacagctcat 1400 cttgaaagaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 1450 aaa 1453 22 796 PRT Homo sapiens 22 Met
Lys Glu Asn Tyr Cys Leu Gln Ala Ala Leu Val Cys Leu Gly 1 5 10 15
Met Leu Cys His Ser His Ala Phe Ala Pro Glu Arg Arg Gly His 20 25
30 Leu Arg Pro Ser Phe His Gly His His Glu Lys Gly Lys Glu Gly 35
40 45 Gln Val Leu Gln Arg Ser Lys Arg Gly Trp Val Trp Asn Gln Phe
50 55 60 Phe Val Ile Glu Glu Tyr Thr Gly Pro Asp Pro Val Leu Val
Gly 65 70 75 Arg Leu His Ser Asp Ile Asp Ser Gly Asp Gly Asn Ile
Lys Tyr 80 85 90 Ile Leu Ser Gly Glu Gly Ala Gly Thr Ile Phe Val
Ile Asp Asp 95 100 105 Lys Ser Gly Asn Ile His Ala Thr Lys Thr Leu
Asp Arg Glu Glu 110 115 120 Arg Ala Gln Tyr Thr Leu Met Ala Gln Ala
Val Asp Arg Asp Thr 125 130 135 Asn Arg Pro Leu Glu Pro Pro Ser Glu
Phe Ile Val Lys Val Gln 140 145 150 Asp Ile Asn Asp Asn Pro Pro Glu
Phe Leu His Glu Thr Tyr His 155 160 165 Ala Asn Val Pro Glu Arg Ser
Asn Val Gly Thr Ser Val Ile Gln 170 175 180 Val Thr Ala Ser Asp Ala
Asp Asp Pro Thr Tyr Gly Asn Ser Ala 185 190 195 Lys Leu Val Tyr Ser
Ile Leu Glu Gly Gln Pro Tyr Phe Ser Val 200 205 210 Glu Ala Gln Thr
Gly Ile Ile Arg Thr Ala Leu Pro Asn Met Asp 215 220 225 Arg Glu Ala
Lys Glu Glu Tyr His Val Val Ile Gln Ala Lys Asp 230 235 240 Met Gly
Gly His Met Gly Gly Leu Ser Gly Thr Thr Lys Val Thr 245 250 255 Ile
Thr Leu Thr Asp Val Asn Asp Asn Pro Pro Lys Phe Pro Gln 260 265 270
Arg Leu Tyr Gln Met Ser Val Ser Glu Ala Ala Val Pro Gly Glu 275 280
285 Glu Val Gly Arg Val Lys Ala Lys Asp Pro Asp Ile Gly Glu Asn 290
295 300 Gly Leu Val Thr Tyr Asn Ile Val Asp Gly Asp Gly Met Glu Ser
305 310 315 Phe Glu Ile Thr Thr Asp Tyr Glu Thr Gln Glu Gly Val Ile
Lys 320 325 330 Leu Lys Lys Pro Val Asp Phe Glu Thr Glu Arg Ala Tyr
Ser Leu 335 340 345 Lys Val Glu Ala Ala Asn Val His Ile Asp Pro Lys
Phe Ile Ser 350 355 360 Asn Gly Pro Phe Lys Asp Thr Val Thr Val Lys
Ile Ser Val Glu 365 370 375 Asp Ala Asp Glu Pro Pro Met Phe Leu Ala
Pro Ser Tyr Ile His 380 385 390 Glu Val Gln Glu Asn Ala Ala Ala Gly
Thr Val Val Gly Arg Val 395 400 405 His Ala Lys Asp Pro Asp Ala Ala
Asn Ser Pro Ile Arg Tyr Ser 410 415 420 Ile Asp Arg His Thr Asp Leu
Asp Arg Phe Phe Thr Ile Asn Pro 425 430 435 Glu Asp Gly Phe Ile Lys
Thr Thr Lys Pro Leu Asp Arg Glu Glu 440 445 450 Thr Ala Trp Leu Asn
Ile Thr Val Phe Ala Ala Glu Ile His Asn 455 460 465 Arg His Gln Glu
Ala Gln Val Pro Val Ala Ile Arg Val Leu Asp 470 475 480 Val Asn Asp
Asn Ala Pro Lys Phe Ala Ala Pro Tyr Glu Gly Phe 485 490 495 Ile Cys
Glu Ser Asp Gln Thr Lys Pro Leu Ser Asn Gln Pro Ile 500 505 510 Val
Thr Ile Ser Ala Asp Asp Lys Asp Asp Thr Ala Asn Gly Pro 515 520 525
Arg Phe Ile Phe Ser Leu Pro Pro Glu Ile Ile His Asn Pro Asn 530 535
540 Phe Thr Val Arg Asp Asn Arg Asp Asn Thr Ala Gly Val Tyr Ala 545
550 555 Arg Arg Gly Gly Phe Ser Arg Gln Lys Gln Asp Leu Tyr Leu Leu
560 565 570 Pro Ile Val Ile Ser Asp Gly Gly Ile Pro Pro Met Ser Ser
Thr 575 580 585 Asn Thr Leu Thr Ile Lys Val Cys Gly Cys Asp Val Asn
Gly Ala 590 595 600 Leu Leu Ser Cys Asn Ala Glu Ala Tyr Ile Leu Asn
Ala Gly Leu 605 610 615 Ser Thr Gly Ala Leu Ile Ala Ile Leu Ala Cys
Ile Val Ile Leu 620 625 630 Leu Val Ile Val Val Leu Phe Val Thr Leu
Arg Arg Gln Lys Lys 635 640 645 Glu Pro Leu Ile Val Phe Glu Glu Glu
Asp Val Arg Glu Asn Ile 650 655 660 Ile Thr Tyr Asp Asp Glu Gly Gly
Gly Glu Glu Asp Thr Glu Ala 665 670 675 Phe Asp Ile Ala Thr Leu Gln
Asn Pro Asp Gly Ile Asn Gly Phe 680 685 690 Ile Pro Arg Lys Asp Ile
Lys Pro Glu Tyr Gln Tyr Met Pro Arg 695 700 705 Pro Gly Leu Arg Pro
Ala Pro Asn Ser Val Asp Val Asp Asp Phe 710 715 720 Ile Asn Thr Arg
Ile Gln Glu Ala Asp Asn Asp Pro Thr Ala Pro 725 730 735 Pro Tyr Asp
Ser Ile Gln Ile Tyr Gly Tyr Glu Gly Arg Gly Ser 740 745 750 Val Ala
Gly Ser Leu Ser Ser Leu Glu Ser Ala Thr Thr Asp Ser 755 760 765 Asp
Leu Asp Tyr Asp Tyr Leu Gln Asn Trp Gly Pro Arg Phe Lys 770 775 780
Lys Leu Ala Asp Leu Tyr Gly Ser Lys Asp Thr Phe Asp Asp Asp 785 790
795 Ser 23 215 PRT Homo sapiens 23 Met Tyr Gly Lys Ser Ser Thr Arg
Ala Val Leu Leu Leu Leu Gly 1 5 10 15 Ile Gln Leu Thr Ala Leu Trp
Pro Ile Ala Ala Val Glu Ile Tyr 20 25 30 Thr Ser Arg Val Leu Glu
Ala Val Asn Gly Thr Asp Ala Arg Leu 35 40 45 Lys Cys Thr Phe Ser
Ser Phe Ala Pro Val Gly Asp Ala Leu Thr 50 55 60 Val Thr Trp Asn
Phe Arg Pro Leu Asp Gly Gly Pro Glu Gln Phe 65 70 75 Val Phe Tyr
Tyr His Ile Asp Pro Phe Gln Pro Met Ser Gly Arg 80 85 90 Phe Lys
Asp Arg Val Ser Trp Asp Gly Asn Pro Glu Arg Tyr Asp 95 100 105 Ala
Ser Ile Leu Leu Trp Lys Leu Gln Phe Asp Asp Asn Gly Thr 110 115 120
Tyr Thr Cys Gln Val Lys Asn Pro Pro Asp Val Asp Gly Val Ile 125 130
135 Gly Glu Ile Arg Leu Ser Val Val His Thr Val Arg Phe Ser Glu 140
145 150 Ile His Phe Leu Ala Leu Ala Ile Gly Ser Ala Cys Ala Leu Met
155 160 165 Ile Ile Ile Val Ile Val Val Val Leu Phe Gln His Tyr Arg
Lys 170 175 180 Lys Arg Trp Ala Glu Arg Ala His Lys Val Val Glu Ile
Lys Ser 185 190 195 Lys Glu Glu Glu Arg Leu Asn Gln Glu Lys Lys Val
Ser Val Tyr 200 205 210 Leu Glu Asp Thr Asp 215 24 796 PRT Homo
sapiens 24 Met Lys Glu Asn Tyr Cys Leu Gln Ala Ala Leu Val Cys Leu
Gly 1 5 10 15 Met Leu Cys His Ser His Ala Phe Ala Pro Glu Arg Arg
Gly His 20 25 30 Leu Arg Pro Ser Phe His Gly His His Glu Lys Gly
Lys Glu Gly 35 40 45 Gln Val Leu Gln Arg Ser Lys Arg Gly Trp Val
Trp Asn Gln Phe 50 55 60 Phe Val Ile Glu Glu Tyr Thr Gly Pro Asp
Pro Val Leu Val Gly 65 70 75 Arg Leu His Ser Asp Ile Asp Ser Gly
Asp Gly Asn Ile Lys Tyr 80 85 90 Ile Leu Ser Gly Glu Gly Ala Gly
Thr Ile Phe Val Ile Asp Asp 95 100 105 Lys Ser Gly Asn Ile His Ala
Thr Lys Thr Leu Asp Arg Glu Glu 110 115 120 Arg Ala Gln Tyr Thr Leu
Met Ala Gln Ala Val Asp Arg Asp Thr 125 130 135 Asn Arg Pro Leu Glu
Pro Pro Ser Glu Phe Ile Val Lys Val Gln 140 145 150 Asp Ile Asn Asp
Asn Pro Pro Glu Phe Leu His Glu Thr Tyr His 155 160 165 Ala Asn Val
Pro Glu Arg Ser Asn Val Gly Thr Ser Val Ile Gln 170 175 180 Val Thr
Ala Ser Asp Ala Asp Asp Pro Thr Tyr Gly Asn Ser Ala 185 190 195 Lys
Leu Val Tyr Ser Ile Leu Glu Gly Gln Pro Tyr Phe Ser Val 200 205 210
Glu Ala Gln Thr Gly Ile Ile Arg Thr Ala Leu Pro Asn Met Asp 215 220
225 Arg Glu Ala Lys Glu Glu Tyr His Val Val Ile Gln Ala Lys Asp 230
235 240 Met Gly Gly His Met Gly Gly Leu Ser Gly Thr Thr Lys Val Thr
245 250 255 Ile Thr Leu Thr Asp Val Asn Asp Asn Pro Pro Lys Phe Pro
Gln 260 265 270 Arg Leu Tyr Gln Met Ser Val Ser Glu Ala Ala Val Pro
Gly Glu 275 280 285 Glu Val Gly Arg Val Lys Ala Lys Asp Pro Asp Ile
Gly Glu Asn 290 295 300 Gly Leu Val Thr Tyr Asn Ile Val Asp Gly Asp
Gly Met Glu Ser 305 310 315 Phe Glu Ile Thr Thr Asp Tyr Glu Thr Gln
Glu Gly Val Ile Lys 320 325 330 Leu Lys Lys Pro Val Asp Phe Glu Thr
Glu Arg Ala Tyr Ser Leu 335 340 345 Lys Val Glu Ala Ala Asn Val His
Ile Asp Pro Lys Phe Ile Ser 350 355 360 Asn Gly Pro Phe Lys Asp Thr
Val Thr Val Lys Ile Ser Val Glu 365 370 375 Asp Ala Asp Glu Pro Pro
Met Phe Leu Ala Pro Ser Tyr Ile His 380 385 390 Glu Val Gln Glu Asn
Ala Ala Ala Gly Thr Val Val Gly Arg Val 395 400 405 His Ala Lys Asp
Pro Asp Ala Ala Asn Ser Pro Ile Arg Tyr Ser 410 415 420 Ile Asp Arg
His Thr Asp Leu Asp Arg Phe Phe Thr Ile Asn Pro 425 430 435 Glu Asp
Gly Phe Ile Lys Thr Thr Lys Pro Leu Asp Arg Glu Glu 440 445 450 Thr
Ala Trp Leu Asn Ile Thr Val Phe Ala Ala Glu Ile His Asn 455 460 465
Arg His Gln Glu Ala Gln Val Pro Val Ala Ile Arg Val Leu Asp 470 475
480 Val Asn Asp Asn Ala Pro Lys Phe Ala Ala Pro Tyr Glu Gly Phe 485
490 495 Ile Cys Glu Ser Asp Gln Thr Lys Pro Leu Ser Asn Gln Pro Ile
500 505 510 Val Thr Ile Ser Ala Asp Asp Lys Asp Asp Thr Ala Asn Gly
Pro 515 520 525 Arg Phe Ile Phe Ser Leu Pro Pro Glu Ile Ile His Asn
Pro Asn 530 535 540 Phe Thr Val Arg Asp Asn Arg Asp Asn Thr Ala Gly
Val Tyr Ala 545 550 555 Arg Arg Gly Gly Phe Ser Arg Gln Lys Gln Asp
Leu Tyr Leu Leu 560 565 570 Pro Ile Val Ile Ser Asp Gly Gly Ile Pro
Pro Met Ser Ser Thr 575 580 585 Asn Thr Leu Thr Ile Lys Val Cys Gly
Cys Asp Val Asn Gly Ala 590 595 600 Leu Leu Ser Cys Asn Ala Glu Ala
Tyr Ile Leu Asn Ala Gly Leu 605 610 615 Ser Thr Gly Ala Leu Ile Ala
Ile Leu Ala Cys Ile Val Ile Leu 620 625 630 Leu Val Ile Val Val Leu
Phe Val Thr Leu Arg Arg Gln Lys Lys 635 640 645 Glu Pro Leu Ile Val
Phe Glu Glu Glu Asp Val Arg Glu Asn Ile 650 655 660 Ile Thr Tyr Asp
Asp Glu Gly Gly Gly Glu Glu Asp Thr Glu Ala 665 670 675 Phe Asp Ile
Ala Thr Leu Gln Asn Pro Asp Gly Ile Asn Gly Phe 680 685 690 Ile Pro
Arg Lys Asp Ile Lys Pro Glu Tyr Gln Tyr Met Pro Arg 695 700 705 Pro
Gly Leu Arg Pro Ala Pro Asn Ser Val Asp Val Asp Asp Phe 710 715 720
Ile Asn Thr Arg Ile Gln Glu Ala Asp Asn Asp Pro Thr Ala Pro 725 730
735 Pro Tyr Asp Ser Ile Gln Ile Tyr Gly Tyr Glu Gly Arg Gly Ser 740
745 750 Val Ala Gly Ser Leu Ser Ser Leu Glu Ser Ala Thr Thr Asp Ser
755 760 765 Asp Leu Asp Tyr Asp Tyr Leu Gln Asn Trp Gly Pro Arg Phe
Lys 770 775 780 Lys Leu Ala Asp Leu Tyr Gly Ser Lys Asp Thr Phe Asp
Asp Asp 785 790 795 Ser 25 123 PRT Homo sapiens 25 Met Ala Thr Ala
Ala Gly Ala Thr Tyr Phe Gln Arg Gly Ser Leu 1 5 10 15 Phe Trp Phe
Thr Val Ile Thr Leu Ser Phe Gly Tyr Tyr Thr Trp 20 25 30 Val Val
Phe Trp Pro Gln Ser Ile Pro Tyr Gln Asn Leu Gly Pro 35 40 45 Leu
Gly Pro Phe Thr Gln Tyr Leu Val Asp His His His Thr Leu 50 55 60
Leu Cys Asn Gly Tyr Trp Leu Ala Trp Leu Ile His Val Gly Glu 65 70
75 Ser Leu Tyr Ala Ile Ala Leu Cys Lys His Lys Gly Ile Thr Ser 80
85 90 Gly Arg Ala Gln Leu Leu Trp Phe Leu Gln Thr Phe Phe Phe Gly
95 100 105 Ile Ala Ser Leu Thr Ile Leu Ile Ala Tyr Lys Arg Lys Arg
Gln 110 115 120 Lys Gln Thr 26 257 PRT Homo sapiens 26 Met Ala Ser
Lys Ile Gly Ser Arg Arg Trp Met Leu Gln Leu Ile 1 5 10 15 Met Gln
Leu Gly Ser Val Leu Leu Thr Arg Cys Pro Phe Trp Gly 20 25 30 Cys
Phe Ser Gln Leu Met Leu Tyr Ala Glu Arg Ala Glu Ala Arg 35 40 45
Arg Lys Pro Asp Ile Pro Val Pro Tyr Leu Tyr Phe Asp Met Gly 50 55
60 Ala Ala Val Leu Cys Ala Ser Phe Met Ser Phe Gly Val Lys Arg 65
70 75 Arg Trp Phe Ala Leu Gly Ala Ala Leu Gln Leu Ala Ile Ser Thr
80 85 90 Tyr Ala Ala Tyr Ile Gly Gly Tyr Val
His Tyr Gly Asp Trp Leu 95 100 105 Lys Val Arg Met Tyr Ser Arg Thr
Val Ala Ile Ile Gly Gly Phe 110 115 120 Leu Val Leu Ala Ser Gly Ala
Gly Glu Leu Tyr Arg Arg Lys Pro 125 130 135 Arg Ser Arg Ser Leu Gln
Ser Thr Gly Gln Val Phe Leu Gly Ile 140 145 150 Tyr Leu Ile Cys Val
Ala Tyr Ser Leu Gln His Ser Lys Glu Asp 155 160 165 Arg Leu Ala Tyr
Leu Asn His Leu Pro Gly Gly Glu Leu Met Ile 170 175 180 Gln Leu Phe
Phe Val Leu Tyr Gly Ile Leu Ala Leu Ala Phe Leu 185 190 195 Ser Gly
Tyr Tyr Val Thr Leu Ala Ala Gln Ile Leu Ala Val Leu 200 205 210 Leu
Pro Pro Val Met Leu Leu Ile Asp Gly Asn Val Ala Tyr Trp 215 220 225
His Asn Thr Arg Arg Val Glu Phe Trp Asn Gln Met Lys Leu Leu 230 235
240 Gly Glu Ser Val Gly Ile Phe Gly Thr Ala Val Ile Leu Ala Thr 245
250 255 Asp Gly 27 460 PRT Homo sapiens Unsure 381 Unknown amino
acid 27 Gly Arg Gly Ser Pro Leu Ala Leu Leu Ile Arg Met Lys Thr Leu
1 5 10 15 Leu Phe Gly Val Trp Ala Leu Leu Ala Leu Ile Leu Cys Pro
Gly 20 25 30 Val Pro Glu Glu Leu Phe Glu Val Ser Ile Trp Pro Ser
Gln Ala 35 40 45 Leu Val Glu Phe Gly Gln Ser Leu Val Cys Asn Cys
Ser Thr Thr 50 55 60 Cys Pro Asp Pro Gly Pro Ser Gly Ile Glu Thr
Phe Leu Lys Lys 65 70 75 Thr Gln Val Asp Lys Gly Pro Gln Trp Lys
Glu Phe Leu Leu Glu 80 85 90 Asp Val Thr Glu Asn Ser Ile Leu Gln
Cys Phe Phe Ser Cys Ala 95 100 105 Gly Ile Gln Lys Asp Thr Ser Leu
Gly Ile Thr Val Tyr Gln Pro 110 115 120 Pro Glu Gln Val Ile Leu Glu
Leu Gln Pro Ala Trp Val Ala Val 125 130 135 Asp Glu Ala Phe Thr Val
Lys Cys His Val Pro Ser Val Ala Pro 140 145 150 Leu Glu Ser Leu Thr
Leu Ala Leu Leu Gln Gly Asn Gln Glu Leu 155 160 165 His Arg Lys Asn
Phe Thr Ser Leu Ala Val Ala Ser Gln Arg Ala 170 175 180 Glu Val Ile
Ile Ser Val Arg Ala Gln Lys Glu Asn Asp Arg Cys 185 190 195 Asn Ser
Ser Cys His Ala Glu Leu Asp Leu Ser Leu Gln Gly Gly 200 205 210 Arg
Leu Phe Gln Gly Ser Ser Pro Ile Arg Ile Val Arg Ile Phe 215 220 225
Glu Phe Ser Gln Ser Pro His Ile Trp Val Ser Ser Leu Leu Glu 230 235
240 Ala Gly Met Ala Glu Thr Val Ser Cys Glu Val Ala Arg Val Phe 245
250 255 Pro Ala Lys Glu Val Met Phe His Met Phe Leu Glu Asp Gln Glu
260 265 270 Leu Ser Ser Ser Leu Ser Trp Glu Gly Asp Thr Ala Trp Ala
Asn 275 280 285 Ala Thr Ile Arg Thr Met Glu Ala Gly Asp Gln Glu Leu
Ser Cys 290 295 300 Phe Ala Ser Leu Gly Ala Met Glu Gln Lys Thr Arg
Lys Leu Val 305 310 315 His Ser Tyr Ser Phe Pro Pro Pro Ile Leu Glu
Leu Lys Glu Ser 320 325 330 Tyr Pro Leu Ala Gly Thr Asp Ile Asn Val
Thr Cys Ser Gly His 335 340 345 Val Leu Thr Ser Pro Ser Pro Thr Leu
Arg Leu Gln Gly Ala Pro 350 355 360 Asp Leu Pro Ala Gly Glu Pro Ala
Trp Leu Leu Leu Thr Ala Arg 365 370 375 Glu Glu Asp Asp Gly Xaa Asn
Phe Ser Cys Glu Ala Ser Leu Val 380 385 390 Val Gln Gly Gln Arg Leu
Met Lys Thr Thr Val Ile Gln Leu His 395 400 405 Ile Leu Lys Pro Gln
Leu Glu Glu Ser Ser Cys Pro Gly Lys Gln 410 415 420 Thr Trp Leu Glu
Gly Met Glu His Thr Leu Ala Cys Val Pro Lys 425 430 435 Gly Asn Pro
Ala Pro Ala Leu Val Cys Thr Trp Asn Gly Val Val 440 445 450 Phe Asp
Leu Glu Val Pro Gln Lys Ala Thr 455 460 28 332 PRT Homo sapiens 28
Met Leu Pro Arg Leu Leu Leu Leu Ile Cys Ala Pro Leu Cys Glu 1 5 10
15 Pro Ala Glu Leu Phe Leu Ile Ala Ser Pro Ser His Pro Thr Glu 20
25 30 Gly Ser Pro Val Thr Leu Thr Cys Lys Met Pro Phe Leu Gln Ser
35 40 45 Ser Asp Ala Gln Phe Gln Phe Cys Phe Phe Arg Asp Thr Arg
Ala 50 55 60 Leu Gly Pro Gly Trp Ser Ser Ser Pro Lys Leu Gln Ile
Ala Ala 65 70 75 Met Trp Lys Glu Asp Thr Gly Ser Tyr Trp Cys Glu
Ala Gln Thr 80 85 90 Met Ala Ser Lys Val Leu Arg Ser Arg Arg Ser
Gln Ile Asn Val 95 100 105 His Ile Pro Val Ser Arg Pro Ile Leu Met
Leu Arg Ala Pro Arg 110 115 120 Ala Gln Ala Ala Val Glu Asp Val Leu
Glu Leu His Cys Glu Ala 125 130 135 Leu Arg Gly Ser Pro Pro Ile Leu
Tyr Trp Phe Tyr His Glu Asp 140 145 150 Ile Thr Leu Gly Ser Arg Ser
Ala Pro Ser Gly Gly Gly Ala Ser 155 160 165 Phe Asn Leu Ser Leu Thr
Glu Glu His Ser Gly Asn Tyr Ser Cys 170 175 180 Glu Ala Asn Asn Gly
Leu Gly Ala Gln Arg Ser Glu Ala Val Thr 185 190 195 Leu Asn Phe Thr
Val Pro Thr Gly Ala Arg Ser Asn His Leu Thr 200 205 210 Ser Gly Val
Ile Glu Gly Leu Leu Ser Thr Leu Gly Pro Ala Thr 215 220 225 Val Ala
Leu Leu Phe Cys Tyr Gly Leu Lys Arg Lys Ile Gly Arg 230 235 240 Arg
Ser Ala Arg Asp Pro Leu Arg Ser Leu Pro Ala Leu Pro Gln 245 250 255
Glu Phe Thr Tyr Leu Asn Ser Pro Thr Pro Gly Gln Leu Gln Pro 260 265
270 Ile Tyr Glu Asn Val Asn Val Val Ser Gly Asp Glu Val Tyr Ser 275
280 285 Leu Ala Tyr Tyr Asn Gln Pro Glu Gln Glu Ser Val Ala Ala Glu
290 295 300 Thr Leu Gly Thr His Met Glu Asp Lys Val Ser Leu Asp Ile
Tyr 305 310 315 Ser Arg Leu Arg Lys Ala Asn Ile Thr Asp Val Asp Tyr
Glu Asp 320 325 330 Ala Met 29 515 PRT Homo sapiens 29 Met Leu Leu
Trp Ala Ser Leu Leu Ala Phe Ala Pro Val Cys Gly 1 5 10 15 Gln Ser
Ala Ala Ala His Lys Pro Val Ile Ser Val His Pro Pro 20 25 30 Trp
Thr Thr Phe Phe Lys Gly Glu Arg Val Thr Leu Thr Cys Asn 35 40 45
Gly Phe Gln Phe Tyr Ala Thr Glu Lys Thr Thr Trp Tyr His Arg 50 55
60 His Tyr Trp Gly Glu Lys Leu Thr Leu Thr Pro Gly Asn Thr Leu 65
70 75 Glu Val Arg Glu Ser Gly Leu Tyr Arg Cys Gln Ala Arg Gly Ser
80 85 90 Pro Arg Ser Asn Pro Val Arg Leu Leu Phe Ser Ser Asp Ser
Leu 95 100 105 Ile Leu Gln Ala Pro Tyr Ser Val Phe Glu Gly Asp Thr
Leu Val 110 115 120 Leu Arg Cys His Arg Arg Arg Lys Glu Lys Leu Thr
Ala Val Lys 125 130 135 Tyr Thr Trp Asn Gly Asn Ile Leu Ser Ile Ser
Asn Lys Ser Trp 140 145 150 Asp Leu Leu Ile Pro Gln Ala Ser Ser Asn
Asn Asn Gly Asn Tyr 155 160 165 Arg Cys Ile Gly Tyr Gly Asp Glu Asn
Asp Val Phe Arg Ser Asn 170 175 180 Phe Lys Ile Ile Lys Ile Gln Glu
Leu Phe Pro His Pro Glu Leu 185 190 195 Lys Ala Thr Asp Ser Gln Pro
Thr Glu Gly Asn Ser Val Asn Leu 200 205 210 Ser Cys Glu Thr Gln Leu
Pro Pro Glu Arg Ser Asp Thr Pro Leu 215 220 225 His Phe Asn Phe Phe
Arg Asp Gly Glu Val Ile Leu Ser Asp Trp 230 235 240 Ser Thr Tyr Pro
Glu Leu Gln Leu Pro Thr Val Trp Arg Glu Asn 245 250 255 Ser Gly Ser
Tyr Trp Cys Gly Ala Glu Thr Val Arg Gly Asn Ile 260 265 270 His Lys
His Ser Pro Ser Leu Gln Ile His Val Gln Arg Ile Pro 275 280 285 Val
Ser Gly Val Leu Leu Glu Thr Gln Pro Ser Gly Gly Gln Ala 290 295 300
Val Glu Gly Glu Met Leu Val Leu Val Cys Ser Val Ala Glu Gly 305 310
315 Thr Gly Asp Thr Thr Phe Ser Trp His Arg Glu Asp Met Gln Glu 320
325 330 Ser Leu Gly Arg Lys Thr Gln Arg Ser Leu Arg Ala Glu Leu Glu
335 340 345 Leu Pro Ala Ile Arg Gln Ser His Ala Gly Gly Tyr Tyr Cys
Thr 350 355 360 Ala Asp Asn Ser Tyr Gly Pro Val Gln Ser Met Val Leu
Asn Val 365 370 375 Thr Val Arg Glu Thr Pro Gly Asn Arg Asp Gly Leu
Val Ala Ala 380 385 390 Gly Ala Thr Gly Gly Leu Leu Ser Ala Leu Leu
Leu Ala Val Ala 395 400 405 Leu Leu Phe His Cys Trp Arg Arg Arg Lys
Ser Gly Val Gly Phe 410 415 420 Leu Gly Asp Glu Thr Arg Leu Pro Pro
Ala Pro Gly Pro Gly Glu 425 430 435 Ser Ser His Ser Ile Cys Pro Ala
Gln Val Glu Leu Gln Ser Leu 440 445 450 Tyr Val Asp Val His Pro Lys
Lys Gly Asp Leu Val Tyr Ser Glu 455 460 465 Ile Gln Thr Thr Gln Leu
Gly Glu Glu Glu Glu Ala Asn Thr Ser 470 475 480 Arg Thr Leu Leu Glu
Asp Lys Asp Val Ser Val Val Tyr Ser Glu 485 490 495 Val Lys Thr Gln
His Pro Asp Asn Ser Ala Gly Lys Ile Ser Ser 500 505 510 Lys Asp Glu
Glu Ser 515 30 453 PRT Homo sapiens 30 Met Thr Val Ile Arg Phe Phe
Pro Ala Ala Ser Ala Thr Lys Arg 1 5 10 15 Val Leu Pro Pro Val Leu
Arg Val Ser Ser Pro Arg Thr Trp Asn 20 25 30 Pro Asn Val Pro Glu
Ser Pro Arg Ile Pro Ala Pro Arg Leu Pro 35 40 45 Lys Arg Met Ser
Gly Ala Pro Thr Ala Gly Ala Ala Leu Met Leu 50 55 60 Cys Ala Ala
Thr Ala Val Leu Leu Ser Ala Gln Gly Gly Pro Val 65 70 75 Gln Ser
Lys Ser Pro Arg Phe Ala Ser Trp Asp Glu Met Asn Val 80 85 90 Leu
Ala His Gly Leu Leu Gln Leu Gly Gln Gly Leu Arg Glu His 95 100 105
Ala Glu Arg Thr Arg Ser Gln Leu Ser Ala Leu Glu Arg Arg Leu 110 115
120 Ser Ala Cys Gly Ser Ala Cys Gln Gly Thr Glu Gly Ser Thr Asp 125
130 135 Leu Pro Leu Ala Pro Glu Ser Arg Val Asp Pro Glu Val Leu His
140 145 150 Ser Leu Gln Thr Gln Leu Lys Ala Gln Asn Ser Arg Ile Gln
Gln 155 160 165 Leu Phe His Lys Val Ala Gln Gln Gln Arg His Leu Glu
Lys Gln 170 175 180 His Leu Arg Ile Gln His Leu Gln Ser Gln Phe Gly
Leu Leu Asp 185 190 195 His Lys His Leu Asp His Glu Val Ala Lys Pro
Ala Arg Arg Lys 200 205 210 Arg Leu Pro Glu Met Ala Gln Pro Val Asp
Pro Ala His Asn Val 215 220 225 Ser Arg Leu His Arg Leu Pro Arg Asp
Cys Gln Glu Leu Phe Gln 230 235 240 Val Gly Glu Arg Gln Ser Gly Leu
Phe Glu Ile Gln Pro Gln Gly 245 250 255 Ser Pro Pro Phe Leu Val Asn
Cys Lys Met Thr Ser Asp Gly Gly 260 265 270 Trp Thr Val Ile Gln Arg
Arg His Asp Gly Ser Val Asp Phe Asn 275 280 285 Arg Pro Trp Glu Ala
Tyr Lys Ala Gly Phe Gly Asp Pro His Gly 290 295 300 Glu Phe Trp Leu
Gly Leu Glu Lys Val His Ser Ile Thr Gly Asp 305 310 315 Arg Asn Ser
Arg Leu Ala Val Gln Leu Arg Asp Trp Asp Gly Asn 320 325 330 Ala Glu
Leu Leu Gln Phe Ser Val His Leu Gly Gly Glu Asp Thr 335 340 345 Ala
Tyr Ser Leu Gln Leu Thr Ala Pro Val Ala Gly Gln Leu Gly 350 355 360
Ala Thr Thr Val Pro Pro Ser Gly Leu Ser Val Pro Phe Ser Thr 365 370
375 Trp Asp Gln Asp His Asp Leu Arg Arg Asp Lys Asn Cys Ala Lys 380
385 390 Ser Leu Ser Gly Gly Trp Trp Phe Gly Thr Cys Ser His Ser Asn
395 400 405 Leu Asn Gly Gln Tyr Phe Arg Ser Ile Pro Gln Gln Arg Gln
Lys 410 415 420 Leu Lys Lys Gly Ile Phe Trp Lys Thr Trp Arg Gly Arg
Tyr Tyr 425 430 435 Pro Leu Gln Ala Thr Thr Met Leu Ile Gln Pro Met
Ala Ala Glu 440 445 450 Ala Ala Ser 31 124 PRT Homo sapiens 31 Met
Leu Leu Trp Val Ile Leu Leu Val Leu Ala Pro Val Ser Gly 1 5 10 15
Gln Phe Ala Arg Thr Pro Arg Pro Ile Ile Phe Leu Gln Pro Pro 20 25
30 Trp Thr Thr Val Phe Gln Gly Glu Arg Val Thr Leu Thr Cys Lys 35
40 45 Gly Phe Arg Phe Tyr Ser Pro Gln Lys Thr Lys Trp Tyr His Arg
50 55 60 Tyr Leu Gly Lys Glu Ile Leu Arg Glu Thr Pro Asp Asn Ile
Leu 65 70 75 Glu Val Gln Glu Ser Gly Glu Tyr Arg Cys Gln Ala Gln
Gly Ser 80 85 90 Pro Leu Ser Ser Pro Val His Leu Asp Phe Ser Ser
Glu Met Gly 95 100 105 Phe Pro His Ala Ala Gln Ala Asn Val Glu Leu
Leu Gly Ser Ser 110 115 120 Asp Leu Leu Thr 32 1752 PRT Homo
sapiens 32 Met Ala Gly Pro Arg Pro Ser Pro Trp Ala Arg Leu Leu Leu
Ala 1 5 10 15 Ala Leu Ile Ser Val Ser Leu Ser Gly Thr Leu Ala Asn
Arg Cys 20 25 30 Lys Lys Ala Pro Val Lys Ser Cys Thr Glu Cys Val
Arg Val Asp 35 40 45 Lys Asp Cys Ala Tyr Cys Thr Asp Glu Met Phe
Arg Asp Arg Arg 50 55 60 Cys Asn Thr Gln Ala Glu Leu Leu Ala Ala
Gly Cys Gln Arg Glu 65 70 75 Ser Ile Val Val Met Glu Ser Ser Phe
Gln Ile Thr Glu Glu Thr 80 85 90 Gln Ile Asp Thr Thr Leu Arg Arg
Ser Gln Met Ser Pro Gln Gly 95 100 105 Leu Arg Val Arg Leu Arg Pro
Gly Glu Glu Arg His Phe Glu Leu 110 115 120 Glu Val Phe Glu Pro Leu
Glu Ser Pro Val Asp Leu Tyr Ile Leu 125 130 135 Met Asp Phe Ser Asn
Ser Met Ser Asp Asp Leu Asp Asn Leu Lys 140 145 150 Lys Met Gly Gln
Asn Leu Ala Arg Val Leu Ser Gln Leu Thr Ser 155 160 165 Asp Tyr Thr
Ile Gly Phe Gly Lys Phe Val Asp Lys Val Ser Val 170 175 180 Pro Gln
Thr Asp Met Arg Pro Glu Lys Leu Lys Glu Pro Trp Pro 185 190 195 Asn
Ser Asp Pro Pro Phe Ser Phe Lys Asn Val Ile Ser Leu Thr 200 205 210
Glu Asp Val Asp Glu Phe Arg Asn Lys Leu Gln Gly Glu Arg Ile 215 220
225 Ser Gly Asn Leu Asp Ala Pro Glu Gly Gly Phe Asp Ala Ile Leu
230 235 240 Gln Thr Ala Val Cys Thr Arg Asp Ile Gly Trp Arg Pro Asp
Ser 245 250 255 Thr His Leu Leu Val Phe Ser Thr Glu Ser Ala Phe His
Tyr Glu 260 265 270 Ala Asp Gly Ala Asn Val Leu Ala Gly Ile Met Ser
Arg Asn Asp 275 280 285 Glu Arg Cys His Leu Asp Thr Thr Gly Thr Tyr
Thr Gln Tyr Arg 290 295 300 Thr Gln Asp Tyr Pro Ser Val Pro Thr Leu
Val Arg Leu Leu Ala 305 310 315 Lys His Asn Ile Ile Pro Ile Phe Ala
Val Thr Asn Tyr Ser Tyr 320 325 330 Ser Tyr Tyr Glu Lys Leu His Thr
Tyr Phe Pro Val Ser Ser Leu 335 340 345 Gly Val Leu Gln Glu Asp Ser
Ser Asn Ile Val Glu Leu Leu Glu 350 355 360 Glu Ala Phe Asn Arg Ile
Arg Ser Asn Leu Asp Ile Arg Ala Leu 365 370 375 Asp Ser Pro Arg Gly
Leu Arg Thr Glu Val Thr Ser Lys Met Phe 380 385 390 Gln Lys Thr Arg
Thr Gly Ser Phe His Ile Arg Arg Gly Glu Val 395 400 405 Gly Ile Tyr
Gln Val Gln Leu Arg Ala Leu Glu His Val Asp Gly 410 415 420 Thr His
Val Cys Gln Leu Pro Glu Asp Gln Lys Gly Asn Ile His 425 430 435 Leu
Lys Pro Ser Phe Ser Asp Gly Leu Lys Met Asp Ala Gly Ile 440 445 450
Ile Cys Asp Val Cys Thr Cys Glu Leu Gln Lys Glu Val Arg Ser 455 460
465 Ala Arg Cys Ser Phe Asn Gly Asp Phe Val Cys Gly Gln Cys Val 470
475 480 Cys Ser Glu Gly Trp Ser Gly Gln Thr Cys Asn Cys Ser Thr Gly
485 490 495 Ser Leu Ser Asp Ile Gln Pro Cys Leu Arg Glu Gly Glu Asp
Lys 500 505 510 Pro Cys Ser Gly Arg Gly Glu Cys Gln Cys Gly His Cys
Val Cys 515 520 525 Tyr Gly Glu Gly Arg Tyr Glu Gly Gln Phe Cys Glu
Tyr Asp Asn 530 535 540 Phe Gln Cys Pro Arg Thr Ser Gly Phe Leu Cys
Asn Asp Arg Gly 545 550 555 Arg Cys Ser Met Gly Gln Cys Val Cys Glu
Pro Gly Trp Thr Gly 560 565 570 Pro Ser Cys Asp Cys Pro Leu Ser Asn
Ala Thr Cys Ile Asp Ser 575 580 585 Asn Gly Gly Ile Cys Asn Gly Arg
Gly His Cys Glu Cys Gly Arg 590 595 600 Cys His Cys His Gln Gln Ser
Leu Tyr Thr Asp Thr Ile Cys Glu 605 610 615 Ile Asn Tyr Ser Ala Ile
His Pro Gly Leu Cys Glu Asp Leu Arg 620 625 630 Ser Cys Val Gln Cys
Gln Ala Trp Gly Thr Gly Glu Lys Lys Gly 635 640 645 Arg Thr Cys Glu
Glu Cys Asn Phe Lys Val Lys Met Val Asp Glu 650 655 660 Leu Lys Arg
Ala Glu Glu Val Val Val Arg Cys Ser Phe Arg Asp 665 670 675 Glu Asp
Asp Asp Cys Thr Tyr Ser Tyr Thr Met Glu Gly Asp Gly 680 685 690 Ala
Pro Gly Pro Asn Ser Thr Val Leu Val His Lys Lys Lys Asp 695 700 705
Cys Pro Pro Gly Ser Phe Trp Trp Leu Ile Pro Leu Leu Leu Leu 710 715
720 Leu Leu Pro Leu Leu Ala Leu Leu Leu Leu Leu Cys Trp Lys Tyr 725
730 735 Cys Ala Cys Cys Lys Ala Cys Leu Ala Leu Leu Pro Cys Cys Asn
740 745 750 Arg Gly His Met Val Gly Phe Lys Glu Asp His Tyr Met Leu
Arg 755 760 765 Glu Asn Leu Met Ala Ser Asp His Leu Asp Thr Pro Met
Leu Arg 770 775 780 Ser Gly Asn Leu Lys Gly Arg Asp Val Val Arg Trp
Lys Val Thr 785 790 795 Asn Asn Met Gln Arg Pro Gly Phe Ala Thr His
Ala Ala Ser Ile 800 805 810 Asn Pro Thr Glu Leu Val Pro Tyr Gly Leu
Ser Leu Arg Leu Ala 815 820 825 Arg Leu Cys Thr Glu Asn Leu Leu Lys
Pro Asp Thr Arg Glu Cys 830 835 840 Ala Gln Leu Arg Gln Glu Val Glu
Glu Asn Leu Asn Glu Val Tyr 845 850 855 Arg Gln Ile Ser Gly Val His
Lys Leu Gln Gln Thr Lys Phe Arg 860 865 870 Gln Gln Pro Asn Ala Gly
Lys Lys Gln Asp His Thr Ile Val Asp 875 880 885 Thr Val Leu Met Ala
Pro Arg Ser Ala Lys Pro Ala Leu Leu Lys 890 895 900 Leu Thr Glu Lys
Gln Val Glu Gln Arg Ala Phe His Asp Leu Lys 905 910 915 Val Ala Pro
Gly Tyr Tyr Thr Leu Thr Ala Asp Gln Asp Ala Arg 920 925 930 Gly Met
Val Glu Phe Gln Glu Gly Val Glu Leu Val Asp Val Arg 935 940 945 Val
Pro Leu Phe Ile Arg Pro Glu Asp Asp Asp Glu Lys Gln Leu 950 955 960
Leu Val Glu Ala Ile Asp Val Pro Ala Gly Thr Ala Thr Leu Gly 965 970
975 Arg Arg Leu Val Asn Ile Thr Ile Ile Lys Glu Gln Ala Arg Asp 980
985 990 Val Val Ser Phe Glu Gln Pro Glu Phe Ser Val Ser Arg Gly Asp
995 1000 1005 Gln Val Ala Arg Ile Pro Val Ile Arg Arg Val Leu Asp
Gly Gly 1010 1015 1020 Lys Ser Gln Val Ser Tyr Arg Thr Gln Asp Gly
Thr Ala Gln Gly 1025 1030 1035 Asn Arg Asp Tyr Ile Pro Val Glu Gly
Glu Leu Leu Phe Gln Pro 1040 1045 1050 Gly Glu Ala Trp Lys Glu Leu
Gln Val Lys Leu Leu Glu Leu Gln 1055 1060 1065 Glu Val Asp Ser Leu
Leu Arg Gly Arg Gln Val Arg Arg Phe His 1070 1075 1080 Val Gln Leu
Ser Asn Pro Lys Phe Gly Ala His Leu Gly Gln Pro 1085 1090 1095 His
Ser Thr Thr Ile Ile Ile Arg Asp Pro Asp Glu Leu Asp Arg 1100 1105
1110 Ser Phe Thr Ser Gln Met Leu Ser Ser Gln Pro Pro Pro His Gly
1115 1120 1125 Asp Leu Gly Ala Pro Gln Asn Pro Asn Ala Lys Ala Ala
Gly Ser 1130 1135 1140 Arg Lys Ile His Phe Asn Trp Leu Pro Pro Ser
Gly Lys Pro Met 1145 1150 1155 Gly Tyr Arg Val Lys Tyr Trp Ile Gln
Gly Asp Ser Glu Ser Glu 1160 1165 1170 Ala His Leu Leu Asp Ser Lys
Val Pro Ser Val Glu Leu Thr Asn 1175 1180 1185 Leu Tyr Pro Tyr Cys
Asp Tyr Glu Met Lys Val Cys Ala Tyr Gly 1190 1195 1200 Ala Gln Gly
Glu Gly Pro Tyr Ser Ser Leu Val Ser Cys Arg Thr 1205 1210 1215 His
Gln Glu Val Pro Ser Glu Pro Gly Arg Leu Ala Phe Asn Val 1220 1225
1230 Val Ser Ser Thr Val Thr Gln Leu Ser Trp Ala Glu Pro Ala Glu
1235 1240 1245 Thr Asn Gly Glu Ile Thr Ala Tyr Glu Val Cys Tyr Gly
Leu Val 1250 1255 1260 Asn Asp Asp Asn Arg Pro Ile Gly Pro Met Lys
Lys Val Leu Val 1265 1270 1275 Asp Asn Pro Lys Asn Arg Met Leu Leu
Ile Glu Asn Leu Arg Glu 1280 1285 1290 Ser Gln Pro Tyr Arg Tyr Thr
Val Lys Ala Arg Asn Gly Ala Gly 1295 1300 1305 Trp Gly Pro Glu Arg
Glu Ala Ile Ile Asn Leu Ala Thr Gln Pro 1310 1315 1320 Lys Arg Pro
Met Ser Ile Pro Ile Ile Pro Asp Ile Pro Ile Val 1325 1330 1335 Asp
Ala Gln Ser Gly Glu Asp Tyr Asp Ser Phe Leu Met Tyr Ser 1340 1345
1350 Asp Asp Val Leu Arg Ser Pro Ser Gly Ser Gln Arg Pro Ser Val
1355 1360 1365 Ser Asp Asp Thr Glu His Leu Val Asn Gly Arg Met Asp
Phe Ala 1370 1375 1380 Phe Pro Gly Ser Thr Asn Ser Leu His Arg Met
Thr Thr Thr Ser 1385 1390 1395 Ala Ala Ala Tyr Gly Thr His Leu Ser
Pro His Val Pro His Arg 1400 1405 1410 Val Leu Ser Thr Ser Ser Thr
Leu Thr Arg Asp Tyr Asn Ser Leu 1415 1420 1425 Thr Arg Ser Glu His
Ser His Ser Thr Thr Leu Pro Arg Asp Tyr 1430 1435 1440 Ser Thr Leu
Thr Ser Val Ser Ser His Asp Ser Arg Leu Thr Ala 1445 1450 1455 Gly
Val Pro Asp Thr Pro Thr Arg Leu Val Phe Ser Ala Leu Gly 1460 1465
1470 Pro Thr Ser Leu Arg Val Ser Trp Gln Glu Pro Arg Cys Glu Arg
1475 1480 1485 Pro Leu Gln Gly Tyr Ser Val Glu Tyr Gln Leu Leu Asn
Gly Gly 1490 1495 1500 Glu Leu His Arg Leu Asn Ile Pro Asn Pro Ala
Gln Thr Ser Val 1505 1510 1515 Val Val Glu Asp Leu Leu Pro Asn His
Ser Tyr Val Phe Arg Val 1520 1525 1530 Arg Ala Gln Ser Gln Glu Gly
Trp Gly Arg Glu Arg Glu Gly Val 1535 1540 1545 Ile Thr Ile Glu Ser
Gln Val His Pro Gln Ser Pro Leu Cys Pro 1550 1555 1560 Leu Pro Gly
Ser Ala Phe Thr Leu Ser Thr Pro Ser Ala Pro Gly 1565 1570 1575 Pro
Leu Val Phe Thr Ala Leu Ser Pro Asp Ser Leu Gln Leu Ser 1580 1585
1590 Trp Glu Arg Pro Arg Arg Pro Asn Gly Asp Ile Val Gly Tyr Leu
1595 1600 1605 Val Thr Cys Glu Met Ala Gln Gly Gly Gly Pro Ala Thr
Ala Phe 1610 1615 1620 Arg Val Asp Gly Asp Ser Pro Glu Ser Arg Leu
Thr Val Pro Gly 1625 1630 1635 Leu Ser Glu Asn Val Pro Tyr Lys Phe
Lys Val Gln Ala Arg Thr 1640 1645 1650 Thr Glu Gly Phe Gly Pro Glu
Arg Glu Gly Ile Ile Thr Ile Glu 1655 1660 1665 Ser Gln Asp Gly Gly
Pro Phe Pro Gln Leu Gly Ser Arg Ala Gly 1670 1675 1680 Leu Phe Gln
His Pro Leu Gln Ser Glu Tyr Ser Ser Ile Thr Thr 1685 1690 1695 Thr
His Thr Ser Ala Thr Glu Pro Phe Leu Val Asp Gly Pro Thr 1700 1705
1710 Leu Gly Ala Gln His Leu Glu Ala Gly Gly Ser Leu Thr Arg His
1715 1720 1725 Val Thr Gln Glu Phe Val Ser Arg Thr Leu Thr Thr Ser
Gly Thr 1730 1735 1740 Leu Ser Thr His Met Asp Gln Gln Phe Phe Gln
Thr 1745 1750 33 1336 PRT Homo sapiens 33 Met Thr Leu Asp Arg Pro
Gly Glu Gly Ala Thr Met Leu Lys Thr 1 5 10 15 Phe Thr Val Leu Leu
Phe Cys Ile Arg Met Ser Leu Gly Met Thr 20 25 30 Ser Ile Val Met
Asp Pro Gln Pro Glu Leu Trp Ile Glu Ser Asn 35 40 45 Tyr Pro Gln
Ala Pro Trp Glu Asn Ile Thr Leu Trp Cys Arg Ser 50 55 60 Pro Ser
Arg Ile Ser Ser Lys Phe Leu Leu Leu Lys Asp Lys Thr 65 70 75 Gln
Met Thr Trp Ile Arg Pro Ser His Lys Thr Phe Gln Val Ser 80 85 90
Phe Leu Ile Gly Ala Leu Thr Glu Ser Asn Ala Gly Leu Tyr Arg 95 100
105 Cys Cys Tyr Trp Lys Glu Thr Gly Trp Ser Lys Pro Ser Lys Val 110
115 120 Leu Glu Leu Glu Ala Pro Gly Gln Leu Pro Lys Pro Ile Phe Trp
125 130 135 Ile Gln Ala Glu Thr Pro Ala Leu Pro Gly Cys Asn Val Asn
Ile 140 145 150 Leu Cys His Gly Trp Leu Gln Asp Leu Val Phe Met Leu
Phe Lys 155 160 165 Glu Gly Tyr Ala Glu Pro Val Asp Tyr Gln Val Pro
Thr Gly Thr 170 175 180 Met Ala Ile Phe Ser Ile Asp Asn Leu Thr Pro
Glu Asp Glu Gly 185 190 195 Val Tyr Ile Cys Arg Thr His Ile Gln Met
Leu Pro Thr Leu Trp 200 205 210 Ser Glu Pro Ser Asn Pro Leu Lys Leu
Val Val Ala Gly Leu Tyr 215 220 225 Pro Lys Pro Thr Leu Thr Ala His
Pro Gly Pro Ile Met Ala Pro 230 235 240 Gly Glu Ser Leu Asn Leu Arg
Cys Gln Gly Pro Ile Tyr Gly Met 245 250 255 Thr Phe Ala Leu Met Arg
Val Glu Asp Leu Glu Lys Ser Phe Tyr 260 265 270 His Lys Lys Thr Ile
Lys Asn Glu Ala Asn Phe Phe Phe Gln Ser 275 280 285 Leu Lys Ile Gln
Asp Thr Gly His Tyr Leu Cys Phe Tyr Tyr Asp 290 295 300 Ala Ser Tyr
Arg Gly Ser Leu Leu Ser Asp Val Leu Lys Ile Trp 305 310 315 Val Thr
Asp Thr Phe Pro Lys Thr Trp Leu Leu Ala Arg Pro Ser 320 325 330 Ala
Val Val Gln Met Gly Gln Asn Val Ser Leu Arg Cys Arg Gly 335 340 345
Pro Val Asp Gly Val Gly Leu Ala Leu Tyr Lys Lys Gly Glu Asp 350 355
360 Lys Pro Leu Gln Phe Leu Asp Ala Thr Ser Ile Asp Asp Asn Thr 365
370 375 Ser Phe Phe Leu Asn Asn Val Thr Tyr Ser Asp Thr Gly Ile Tyr
380 385 390 Ser Cys His Tyr Leu Leu Thr Trp Lys Thr Ser Ile Arg Met
Pro 395 400 405 Ser His Asn Thr Val Glu Leu Met Val Val Asp Lys Pro
Pro Lys 410 415 420 Pro Ser Leu Ser Ala Trp Pro Ser Thr Val Phe Lys
Leu Gly Lys 425 430 435 Ala Ile Thr Leu Gln Cys Arg Val Ser His Pro
Val Leu Glu Phe 440 445 450 Ser Leu Glu Trp Glu Glu Arg Glu Thr Phe
Gln Arg Phe Ser Val 455 460 465 Asn Gly Asp Phe Ile Ile Ser Asn Val
Asp Gly Lys Gly Thr Gly 470 475 480 Thr Tyr Ser Cys Ser Tyr Arg Val
Glu Thr His Pro Asn Met Trp 485 490 495 Ser His Arg Ser Glu Pro Leu
Lys Leu Met Gly Pro Ala Gly Tyr 500 505 510 Leu Thr Trp Asn Tyr Val
Leu Asn Glu Ala Ile Arg Leu Ser Leu 515 520 525 Ile Met Gln Leu Val
Ala Leu Leu Leu Val Val Leu Trp Ile Arg 530 535 540 Trp Lys Cys Arg
Arg Leu Arg Ile Arg Glu Ala Trp Leu Leu Gly 545 550 555 Thr Ala Gln
Gly Val Thr Met Leu Phe Ile Val Thr Ala Leu Leu 560 565 570 Cys Cys
Gly Leu Cys Asn Gly Val Leu Ile Glu Glu Thr Glu Ile 575 580 585 Val
Met Pro Thr Pro Lys Pro Glu Leu Trp Ala Glu Thr Asn Phe 590 595 600
Pro Leu Ala Pro Trp Lys Asn Leu Thr Leu Trp Cys Arg Ser Pro 605 610
615 Ser Gly Ser Thr Lys Glu Phe Val Leu Leu Lys Asp Gly Thr Gly 620
625 630 Trp Ile Ala Thr Arg Pro Ala Ser Glu Gln Val Arg Ala Ala Phe
635 640 645 Pro Leu Gly Ala Leu Thr Gln Ser His Thr Gly Ser Tyr His
Cys 650 655 660 His Ser Trp Glu Glu Met Ala Val Ser Glu Pro Ser Glu
Ala Leu 665 670 675 Glu Leu Val Gly Thr Asp Ile Leu Pro Lys Pro Val
Ile Ser Ala 680 685 690 Ser Pro Thr Ile Arg Gly Gln Glu Leu Gln Leu
Arg Cys Lys Gly 695 700 705 Trp Leu Ala Gly Met Gly Phe Ala Leu Tyr
Lys Glu Gly Glu Gln 710 715 720 Glu Pro Val Gln Gln Leu Gly Ala Val
Gly Arg Glu Ala Phe Phe 725 730 735 Thr Ile Gln Arg Met Glu Asp Lys
Asp Glu Gly Asn Tyr Ser Cys 740 745 750 Arg Thr His Thr Glu Lys Leu
Pro Phe Lys Trp Ser Glu Pro Ser 755 760 765 Glu Pro Leu Glu
Leu Val Ile Lys Glu Met Tyr Pro Lys Pro Phe 770 775 780 Phe Lys Thr
Trp Ala Ser Pro Val Val Thr Pro Gly Ala Arg Val 785 790 795 Thr Phe
Asn Cys Ser Thr Pro His Gln His Met Ser Phe Ile Leu 800 805 810 Tyr
Lys Asp Gly Ser Glu Ile Ala Ser Ser Asp Arg Ser Trp Ala 815 820 825
Ser Pro Gly Ala Ser Ala Ala His Phe Leu Ile Ile Ser Val Gly 830 835
840 Ile Gly Asp Gly Gly Asn Tyr Ser Cys Arg Tyr Tyr Asp Phe Ser 845
850 855 Ile Trp Ser Glu Pro Ser Asp Pro Val Glu Leu Val Val Thr Glu
860 865 870 Phe Tyr Pro Lys Pro Thr Leu Leu Ala Gln Pro Gly Pro Val
Val 875 880 885 Phe Pro Gly Lys Ser Val Ile Leu Arg Cys Gln Gly Thr
Phe Gln 890 895 900 Gly Met Arg Phe Ala Leu Leu Gln Glu Gly Ala His
Val Pro Leu 905 910 915 Gln Phe Arg Ser Val Ser Gly Asn Ser Ala Asp
Phe Leu Leu His 920 925 930 Thr Val Gly Ala Glu Asp Ser Gly Asn Tyr
Ser Cys Ile Tyr Tyr 935 940 945 Glu Thr Thr Met Ser Asn Arg Gly Ser
Tyr Leu Ser Met Pro Leu 950 955 960 Met Ile Trp Val Thr Asp Thr Phe
Pro Lys Pro Trp Leu Phe Ala 965 970 975 Glu Pro Ser Ser Val Val Pro
Met Gly Gln Asn Val Thr Leu Trp 980 985 990 Cys Arg Gly Pro Val His
Gly Val Gly Tyr Ile Leu His Lys Glu 995 1000 1005 Gly Glu Ala Thr
Ser Met Gln Leu Trp Gly Ser Thr Ser Asn Asp 1010 1015 1020 Gly Ala
Phe Pro Ile Thr Asn Ile Ser Gly Thr Ser Met Gly Arg 1025 1030 1035
Tyr Ser Cys Cys Tyr His Pro Asp Trp Thr Ser Ser Ile Lys Ile 1040
1045 1050 Gln Pro Ser Asn Thr Leu Glu Leu Leu Val Thr Gly Leu Leu
Pro 1055 1060 1065 Lys Pro Ser Leu Leu Ala Gln Pro Gly Pro Met Val
Ala Pro Gly 1070 1075 1080 Glu Asn Met Thr Leu Gln Cys Gln Gly Glu
Leu Pro Asp Ser Thr 1085 1090 1095 Phe Val Leu Leu Lys Glu Gly Ala
Gln Glu Pro Leu Glu Gln Gln 1100 1105 1110 Arg Pro Ser Gly Tyr Arg
Ala Asp Phe Trp Met Pro Ala Val Arg 1115 1120 1125 Gly Glu Asp Ser
Gly Ile Tyr Ser Cys Val Tyr Tyr Leu Asp Ser 1130 1135 1140 Thr Pro
Phe Ala Ala Ser Asn His Ser Asp Ser Leu Glu Ile Trp 1145 1150 1155
Val Thr Asp Lys Pro Pro Lys Pro Ser Leu Ser Ala Trp Pro Ser 1160
1165 1170 Thr Met Phe Lys Leu Gly Lys Asp Ile Thr Leu Gln Cys Arg
Gly 1175 1180 1185 Pro Leu Pro Gly Val Glu Phe Val Leu Glu His Asp
Gly Glu Glu 1190 1195 1200 Ala Pro Gln Gln Phe Ser Glu Asp Gly Asp
Phe Val Ile Asn Asn 1205 1210 1215 Val Glu Gly Lys Gly Ile Gly Asn
Tyr Ser Cys Ser Tyr Arg Leu 1220 1225 1230 Gln Ala Tyr Pro Asp Ile
Trp Ser Glu Pro Ser Asp Pro Leu Glu 1235 1240 1245 Leu Val Gly Ala
Ala Gly Pro Val Ala Gln Glu Cys Thr Val Gly 1250 1255 1260 Asn Ile
Val Arg Ser Ser Leu Ile Val Val Val Val Val Ala Leu 1265 1270 1275
Gly Val Val Leu Ala Ile Glu Trp Lys Lys Trp Pro Arg Leu Arg 1280
1285 1290 Thr Arg Gly Ser Glu Thr Asp Gly Arg Asp Gln Thr Ile Ala
Leu 1295 1300 1305 Glu Glu Cys Asn Gln Glu Gly Glu Pro Gly Thr Pro
Ala Asn Ser 1310 1315 1320 Pro Ser Ser Thr Ser Gln Arg Ile Ser Val
Glu Leu Pro Val Pro 1325 1330 1335 Ile 34 1907 PRT Homo sapiens 34
Met Ala Pro Glu Pro Ala Pro Gly Arg Thr Met Val Pro Leu Val 1 5 10
15 Pro Ala Leu Val Met Leu Gly Leu Val Ala Gly Ala His Gly Asp 20
25 30 Ser Lys Pro Val Phe Ile Lys Val Pro Glu Asp Gln Thr Gly Leu
35 40 45 Ser Gly Gly Val Ala Ser Phe Val Cys Gln Ala Thr Gly Glu
Pro 50 55 60 Lys Pro Arg Ile Thr Trp Met Lys Lys Gly Lys Lys Val
Ser Ser 65 70 75 Gln Arg Phe Glu Val Ile Glu Phe Asp Asp Gly Ala
Gly Ser Val 80 85 90 Leu Arg Ile Gln Pro Leu Arg Val Gln Arg Asp
Glu Ala Ile Tyr 95 100 105 Glu Cys Thr Ala Thr Asn Ser Leu Gly Glu
Ile Asn Thr Ser Ala 110 115 120 Lys Leu Ser Val Leu Glu Glu Glu Gln
Leu Pro Pro Gly Phe Pro 125 130 135 Ser Ile Asp Met Gly Pro Gln Leu
Lys Val Val Glu Lys Ala Arg 140 145 150 Thr Ala Thr Met Leu Cys Ala
Ala Gly Gly Asn Pro Asp Pro Glu 155 160 165 Ile Ser Trp Phe Lys Asp
Phe Leu Pro Val Asp Pro Ala Thr Ser 170 175 180 Asn Gly Arg Ile Lys
Gln Leu Arg Ser Gly Ala Leu Gln Ile Glu 185 190 195 Ser Ser Glu Glu
Ser Asp Gln Gly Lys Tyr Glu Cys Val Ala Thr 200 205 210 Asn Ser Ala
Gly Thr Arg Tyr Ser Ala Pro Ala Asn Leu Tyr Val 215 220 225 Arg Val
Arg Arg Val Ala Pro Arg Phe Ser Ile Pro Pro Ser Ser 230 235 240 Gln
Glu Val Met Pro Gly Gly Ser Val Asn Leu Thr Cys Val Ala 245 250 255
Val Gly Ala Pro Met Pro Tyr Val Lys Trp Met Met Gly Ala Glu 260 265
270 Glu Leu Thr Lys Glu Asp Glu Met Pro Val Gly Arg Asn Val Leu 275
280 285 Glu Leu Ser Asn Val Val Arg Ser Ala Asn Tyr Thr Cys Val Ala
290 295 300 Ile Ser Ser Leu Gly Met Ile Glu Ala Thr Ala Gln Val Thr
Val 305 310 315 Lys Ala Leu Pro Lys Pro Pro Ile Asp Leu Val Val Thr
Glu Thr 320 325 330 Thr Ala Thr Ser Val Thr Leu Thr Trp Asp Ser Gly
Asn Ser Glu 335 340 345 Pro Val Thr Tyr Tyr Gly Ile Gln Tyr Arg Ala
Ala Gly Thr Glu 350 355 360 Gly Pro Phe Gln Glu Val Asp Gly Val Ala
Thr Thr Arg Tyr Ser 365 370 375 Ile Gly Gly Leu Ser Pro Phe Ser Glu
Tyr Ala Phe Arg Val Leu 380 385 390 Ala Val Asn Ser Ile Gly Arg Gly
Pro Pro Ser Glu Ala Val Arg 395 400 405 Ala Arg Thr Gly Glu Gln Ala
Pro Ser Ser Pro Pro Arg Arg Val 410 415 420 Gln Ala Arg Met Leu Ser
Ala Ser Thr Met Leu Val Gln Trp Glu 425 430 435 Pro Pro Glu Glu Pro
Asn Gly Leu Val Arg Gly Tyr Arg Val Tyr 440 445 450 Tyr Thr Pro Asp
Ser Arg Arg Pro Pro Asn Ala Trp His Lys His 455 460 465 Asn Thr Asp
Ala Gly Leu Leu Thr Thr Val Gly Ser Leu Leu Pro 470 475 480 Gly Ile
Thr Tyr Ser Leu Arg Val Leu Ala Phe Thr Ala Val Gly 485 490 495 Asp
Gly Pro Pro Ser Pro Thr Ile Gln Val Lys Thr Gln Gln Gly 500 505 510
Val Pro Ala Gln Pro Ala Asp Phe Gln Ala Glu Val Glu Ser Asp 515 520
525 Thr Arg Ile Gln Leu Ser Trp Leu Leu Pro Pro Gln Glu Arg Ile 530
535 540 Ile Met Tyr Glu Leu Val Tyr Trp Ala Ala Glu Asp Glu Asp Gln
545 550 555 Gln His Lys Val Thr Phe Asp Pro Thr Ser Ser Tyr Thr Leu
Glu 560 565 570 Asp Leu Lys Pro Asp Thr Leu Tyr Arg Phe Gln Leu Ala
Ala Arg 575 580 585 Ser Asp Met Gly Val Gly Val Phe Thr Pro Thr Ile
Glu Ala Arg 590 595 600 Thr Ala Gln Ser Thr Pro Ser Ala Pro Pro Gln
Lys Val Met Cys 605 610 615 Val Ser Met Gly Ser Thr Thr Val Arg Val
Ser Trp Val Pro Pro 620 625 630 Pro Ala Asp Ser Arg Asn Gly Val Ile
Thr Gln Tyr Ser Val Ala 635 640 645 His Glu Ala Val Asp Gly Glu Asp
Arg Gly Arg His Val Val Asp 650 655 660 Gly Ile Ser Arg Glu His Ser
Ser Trp Asp Leu Val Gly Leu Glu 665 670 675 Lys Trp Thr Glu Tyr Arg
Val Trp Val Arg Ala His Thr Asp Val 680 685 690 Gly Pro Gly Pro Glu
Ser Ser Pro Val Leu Val Arg Thr Asp Glu 695 700 705 Asp Val Pro Ser
Gly Pro Pro Arg Lys Val Glu Val Glu Pro Leu 710 715 720 Asn Ser Thr
Ala Val His Val Tyr Trp Lys Leu Pro Val Pro Ser 725 730 735 Lys Gln
His Gly Gln Ile Arg Gly Tyr Gln Val Thr Tyr Val Arg 740 745 750 Leu
Glu Asn Gly Glu Pro Arg Gly Leu Pro Ile Ile Gln Asp Val 755 760 765
Met Leu Ala Glu Ala Gln Trp Arg Pro Glu Glu Ser Glu Asp Tyr 770 775
780 Glu Thr Thr Ile Ser Gly Leu Thr Pro Glu Thr Thr Tyr Ser Val 785
790 795 Thr Val Ala Ala Tyr Thr Thr Lys Gly Asp Gly Ala Arg Ser Lys
800 805 810 Pro Lys Ile Val Thr Thr Thr Gly Ala Val Pro Gly Arg Pro
Thr 815 820 825 Met Met Ile Ser Thr Thr Ala Met Asn Thr Ala Leu Leu
Gln Trp 830 835 840 His Pro Pro Lys Glu Leu Pro Gly Glu Leu Leu Gly
Tyr Arg Leu 845 850 855 Gln Tyr Cys Arg Ala Asp Glu Ala Arg Pro Asn
Thr Ile Asp Phe 860 865 870 Gly Lys Asp Asp Gln His Phe Thr Val Thr
Gly Leu His Lys Gly 875 880 885 Thr Thr Tyr Ile Phe Arg Leu Ala Ala
Lys Asn Arg Ala Gly Leu 890 895 900 Gly Glu Glu Phe Glu Lys Glu Ile
Arg Thr Pro Glu Asp Leu Pro 905 910 915 Ser Gly Phe Pro Gln Asn Leu
His Val Thr Gly Leu Thr Thr Ser 920 925 930 Thr Thr Glu Leu Ala Trp
Asp Pro Pro Val Leu Ala Glu Arg Asn 935 940 945 Gly Arg Ile Ile Ser
Tyr Thr Val Val Phe Arg Asp Ile Asn Ser 950 955 960 Gln Gln Glu Leu
Gln Asn Ile Thr Thr Asp Thr Arg Phe Thr Leu 965 970 975 Thr Gly Leu
Lys Pro Asp Thr Thr Tyr Asp Ile Lys Val Arg Ala 980 985 990 Trp Thr
Ser Lys Gly Ser Gly Pro Leu Ser Pro Ser Ile Gln Ser 995 1000 1005
Arg Thr Met Pro Val Glu Gln Val Phe Ala Lys Asn Phe Arg Val 1010
1015 1020 Ala Ala Ala Met Lys Thr Ser Val Leu Leu Ser Trp Glu Val
Pro 1025 1030 1035 Asp Ser Tyr Lys Ser Ala Val Pro Phe Lys Ile Leu
Tyr Asn Gly 1040 1045 1050 Gln Ser Val Glu Val Asp Gly His Ser Met
Arg Lys Leu Ile Ala 1055 1060 1065 Asp Leu Gln Pro Asn Thr Glu Tyr
Ser Phe Val Leu Met Asn Arg 1070 1075 1080 Gly Ser Ser Ala Gly Gly
Leu Gln His Leu Val Ser Ile Arg Thr 1085 1090 1095 Ala Pro Asp Leu
Leu Pro His Lys Pro Leu Pro Ala Ser Ala Tyr 1100 1105 1110 Ile Glu
Asp Gly Arg Phe Asp Leu Ser Met Pro His Val Gln Asp 1115 1120 1125
Pro Ser Leu Val Arg Trp Phe Tyr Ile Val Val Val Pro Ile Asp 1130
1135 1140 Arg Val Gly Gly Ser Met Leu Thr Pro Arg Trp Ser Thr Pro
Glu 1145 1150 1155 Glu Leu Glu Leu Asp Glu Leu Leu Glu Ala Ile Glu
Gln Gly Gly 1160 1165 1170 Glu Glu Gln Arg Arg Arg Arg Arg Gln Ala
Glu Arg Leu Lys Pro 1175 1180 1185 Tyr Val Ala Ala Gln Leu Asp Val
Leu Pro Glu Thr Phe Thr Leu 1190 1195 1200 Gly Asp Lys Lys Asn Tyr
Arg Gly Phe Tyr Asn Arg Pro Leu Ser 1205 1210 1215 Pro Asp Leu Ser
Tyr Gln Cys Phe Val Leu Ala Ser Leu Lys Glu 1220 1225 1230 Pro Met
Asp Gln Lys Arg Tyr Ala Ser Ser Pro Tyr Ser Asp Glu 1235 1240 1245
Ile Val Val Gln Val Thr Pro Ala Gln Gln Gln Glu Glu Pro Glu 1250
1255 1260 Met Leu Trp Val Thr Gly Pro Val Leu Ala Val Ile Leu Ile
Ile 1265 1270 1275 Leu Ile Val Ile Ala Ile Leu Leu Phe Lys Arg Lys
Arg Thr His 1280 1285 1290 Ser Pro Ser Ser Lys Asp Glu Gln Ser Ile
Gly Leu Lys Asp Ser 1295 1300 1305 Leu Leu Ala His Ser Ser Asp Pro
Val Glu Met Arg Arg Leu Asn 1310 1315 1320 Tyr Gln Thr Pro Gly Met
Arg Asp His Pro Pro Ile Pro Ile Thr 1325 1330 1335 Asp Leu Ala Asp
Asn Ile Glu Arg Leu Lys Ala Asn Asp Gly Leu 1340 1345 1350 Lys Phe
Ser Gln Glu Tyr Glu Ser Ile Asp Pro Gly Gln Gln Phe 1355 1360 1365
Thr Trp Glu Asn Ser Asn Leu Glu Val Asn Lys Pro Lys Asn Arg 1370
1375 1380 Tyr Ala Asn Val Ile Ala Tyr Asp His Ser Arg Val Ile Leu
Thr 1385 1390 1395 Ser Ile Asp Gly Val Pro Gly Ser Asp Tyr Ile Asn
Ala Asn Tyr 1400 1405 1410 Ile Asp Gly Tyr Arg Lys Gln Asn Ala Tyr
Ile Ala Thr Gln Gly 1415 1420 1425 Pro Leu Pro Glu Thr Met Gly Asp
Phe Trp Arg Met Val Trp Glu 1430 1435 1440 Gln Arg Thr Ala Thr Val
Val Met Met Thr Arg Leu Glu Glu Lys 1445 1450 1455 Ser Arg Val Lys
Cys Asp Gln Tyr Trp Pro Ala Arg Gly Thr Glu 1460 1465 1470 Thr Cys
Gly Leu Ile Gln Val Thr Leu Leu Asp Thr Val Glu Leu 1475 1480 1485
Ala Thr Tyr Thr Val Arg Thr Phe Ala Leu His Lys Ser Gly Ser 1490
1495 1500 Ser Glu Lys Arg Glu Leu Arg Gln Phe Gln Phe Met Ala Trp
Pro 1505 1510 1515 Asp His Gly Val Pro Glu Tyr Pro Thr Pro Ile Leu
Ala Phe Leu 1520 1525 1530 Arg Arg Val Lys Ala Cys Asn Pro Leu Asp
Ala Gly Pro Met Val 1535 1540 1545 Val His Cys Ser Ala Gly Val Gly
Arg Thr Gly Cys Phe Ile Val 1550 1555 1560 Ile Asp Ala Met Leu Glu
Arg Met Lys His Glu Lys Thr Val Asp 1565 1570 1575 Ile Tyr Gly His
Val Thr Cys Met Arg Ser Gln Arg Asn Tyr Met 1580 1585 1590 Val Gln
Thr Glu Asp Gln Tyr Val Phe Ile His Glu Ala Leu Leu 1595 1600 1605
Glu Ala Ala Thr Cys Gly His Thr Glu Val Pro Ala Arg Asn Leu 1610
1615 1620 Tyr Ala His Ile Gln Lys Leu Gly Gln Val Pro Pro Gly Glu
Ser 1625 1630 1635 Val Thr Ala Met Glu Leu Glu Phe Lys Leu Leu Ala
Ser Ser Lys 1640 1645 1650 Ala His Thr Ser Arg Phe Ile Ser Ala Asn
Leu Pro Cys Asn Lys 1655 1660 1665 Phe Lys Asn Arg Leu Val Asn Ile
Met Pro Tyr Glu Leu Thr Arg 1670 1675 1680 Val Cys Leu Gln Pro Ile
Arg Gly Val Glu Gly Ser Asp Tyr Ile 1685 1690 1695 Asn Ala Ser Phe
Leu Asp Gly Tyr Arg Gln Gln Lys Ala Tyr Ile 1700 1705
1710 Ala Thr Gln Gly Pro Leu Ala Glu Ser Thr Glu Asp Phe Trp Arg
1715 1720 1725 Met Leu Trp Glu His Asn Ser Thr Ile Ile Val Met Leu
Thr Lys 1730 1735 1740 Leu Arg Glu Met Gly Arg Glu Lys Cys His Gln
Tyr Trp Pro Ala 1745 1750 1755 Glu Arg Ser Ala Arg Tyr Gln Tyr Phe
Val Val Asp Pro Met Ala 1760 1765 1770 Glu Tyr Asn Met Pro Gln Tyr
Ile Leu Arg Glu Phe Lys Val Thr 1775 1780 1785 Asp Ala Arg Asp Gly
Gln Ser Arg Thr Ile Arg Gln Phe Gln Phe 1790 1795 1800 Thr Asp Trp
Pro Glu Gln Gly Val Pro Lys Thr Gly Glu Gly Phe 1805 1810 1815 Ile
Asp Phe Ile Gly Gln Val His Lys Thr Lys Glu Gln Phe Gly 1820 1825
1830 Gln Asp Gly Pro Ile Thr Val His Cys Ser Ala Gly Val Gly Arg
1835 1840 1845 Thr Gly Val Phe Ile Thr Leu Ser Ile Val Leu Glu Arg
Met Arg 1850 1855 1860 Tyr Glu Gly Val Val Asp Met Phe Gln Thr Val
Lys Thr Leu Arg 1865 1870 1875 Thr Gln Arg Pro Ala Met Val Gln Thr
Glu Asp Gln Tyr Gln Leu 1880 1885 1890 Cys Tyr Arg Ala Ala Leu Glu
Tyr Leu Gly Ser Phe Asp His Tyr 1895 1900 1905 Ala Thr 35 310 PRT
Homo sapiens 35 Met Arg Arg Ala Ala Leu Trp Leu Trp Leu Cys Ala Leu
Ala Leu 1 5 10 15 Ser Leu Gln Leu Ala Leu Pro Gln Ile Val Ala Thr
Asn Leu Pro 20 25 30 Pro Glu Asp Gln Asp Gly Ser Gly Asp Asp Ser
Asp Asn Phe Ser 35 40 45 Gly Ser Gly Ala Gly Ala Leu Gln Asp Ile
Thr Leu Ser Gln Gln 50 55 60 Thr Pro Ser Thr Trp Lys Asp Thr Gln
Leu Leu Thr Ala Ile Pro 65 70 75 Thr Ser Pro Glu Pro Thr Gly Leu
Glu Ala Thr Ala Ala Ser Thr 80 85 90 Ser Thr Leu Pro Ala Gly Glu
Gly Pro Lys Glu Gly Glu Ala Val 95 100 105 Val Leu Pro Glu Val Glu
Pro Gly Leu Thr Ala Arg Glu Gln Glu 110 115 120 Ala Thr Pro Arg Pro
Arg Glu Thr Thr Gln Leu Pro Thr Thr His 125 130 135 Gln Ala Ser Thr
Thr Thr Ala Thr Thr Ala Gln Glu Pro Ala Thr 140 145 150 Ser His Pro
His Arg Asp Met Gln Pro Gly His His Glu Thr Ser 155 160 165 Thr Pro
Ala Gly Pro Ser Gln Ala Asp Leu His Thr Pro His Thr 170 175 180 Glu
Asp Gly Gly Pro Ser Ala Thr Glu Arg Ala Ala Glu Asp Gly 185 190 195
Ala Ser Ser Gln Leu Pro Ala Ala Glu Gly Ser Gly Glu Gln Asp 200 205
210 Phe Thr Phe Glu Thr Ser Gly Glu Asn Thr Ala Val Val Ala Val 215
220 225 Glu Pro Asp Arg Arg Asn Gln Ser Pro Val Asp Gln Gly Ala Thr
230 235 240 Gly Ala Ser Gln Gly Leu Leu Asp Arg Lys Glu Val Leu Gly
Gly 245 250 255 Val Ile Ala Gly Gly Leu Val Gly Leu Ile Phe Ala Val
Cys Leu 260 265 270 Val Gly Phe Met Leu Tyr Arg Met Lys Lys Lys Asp
Glu Gly Ser 275 280 285 Tyr Ser Leu Glu Glu Pro Lys Gln Ala Asn Gly
Gly Ala Tyr Gln 290 295 300 Lys Pro Thr Lys Gln Glu Glu Phe Tyr Ala
305 310 36 131 PRT Homo sapiens 36 Met Lys Ile Phe Leu Pro Val Leu
Leu Ala Ala Leu Leu Gly Val 1 5 10 15 Glu Arg Ala Ser Ser Leu Met
Cys Phe Ser Cys Leu Asn Gln Lys 20 25 30 Ser Asn Leu Tyr Cys Leu
Lys Pro Thr Ile Cys Ser Asp Gln Asp 35 40 45 Asn Tyr Cys Val Thr
Val Ser Ala Ser Ala Gly Ile Gly Asn Leu 50 55 60 Val Thr Phe Gly
His Ser Leu Ser Lys Thr Cys Ser Pro Ala Cys 65 70 75 Pro Ile Pro
Glu Gly Val Asn Val Gly Val Ala Ser Met Gly Ile 80 85 90 Ser Cys
Cys Gln Ser Phe Leu Cys Asn Phe Ser Ala Ala Asp Gly 95 100 105 Gly
Leu Arg Ala Ser Val Thr Leu Leu Gly Ala Gly Leu Leu Leu 110 115 120
Ser Leu Leu Pro Ala Leu Leu Arg Phe Gly Pro 125 130 37 249 PRT Homo
sapiens 37 Met Asp Gly Lys Lys Cys Ser Val Trp Met Phe Leu Pro Leu
Val 1 5 10 15 Phe Thr Leu Phe Thr Ser Ala Gly Leu Trp Ile Val Tyr
Phe Ile 20 25 30 Ala Val Glu Asp Asp Lys Ile Leu Pro Leu Asn Ser
Ala Glu Arg 35 40 45 Lys Pro Gly Val Lys His Ala Pro Tyr Ile Ser
Ile Ala Gly Asp 50 55 60 Asp Pro Pro Ala Ser Cys Val Phe Ser Gln
Val Met Asn Met Ala 65 70 75 Ala Phe Leu Ala Leu Val Val Ala Val
Leu Arg Phe Ile Gln Leu 80 85 90 Lys Pro Lys Val Leu Asn Pro Trp
Leu Asn Ile Ser Gly Leu Val 95 100 105 Ala Leu Cys Leu Ala Ser Phe
Gly Met Thr Leu Leu Gly Asn Phe 110 115 120 Gln Leu Thr Asn Asp Glu
Glu Ile His Asn Val Gly Thr Ser Leu 125 130 135 Thr Phe Gly Phe Gly
Thr Leu Thr Cys Trp Ile Gln Ala Ala Leu 140 145 150 Thr Leu Lys Val
Asn Ile Lys Asn Glu Gly Arg Arg Val Gly Ile 155 160 165 Pro Arg Val
Ile Leu Ser Ala Ser Ile Thr Leu Cys Val Val Leu 170 175 180 Tyr Phe
Ile Leu Met Ala Gln Ser Ile His Met Tyr Ala Ala Arg 185 190 195 Val
Gln Trp Gly Leu Val Met Cys Phe Leu Ser Tyr Phe Gly Thr 200 205 210
Phe Ala Val Glu Phe Arg His Tyr Arg Tyr Glu Ile Val Cys Ser 215 220
225 Glu Tyr Gln Glu Asn Phe Leu Ser Phe Ser Glu Ser Leu Ser Glu 230
235 240 Ala Ser Glu Tyr Gln Thr Asp Gln Val 245 38 145 PRT Homo
sapiens 38 Met Glu Leu Ala Leu Leu Cys Gly Leu Val Val Met Ala Gly
Val 1 5 10 15 Ile Pro Ile Gln Gly Gly Ile Leu Asn Leu Asn Lys Met
Val Lys 20 25 30 Gln Val Thr Gly Lys Met Pro Ile Leu Ser Tyr Trp
Pro Tyr Gly 35 40 45 Cys His Cys Gly Leu Gly Gly Arg Gly Gln Pro
Lys Asp Ala Thr 50 55 60 Asp Trp Cys Cys Gln Thr His Asp Cys Cys
Tyr Asp His Leu Lys 65 70 75 Thr Gln Gly Cys Gly Ile Tyr Lys Asp
Tyr Tyr Arg Tyr Asn Phe 80 85 90 Ser Gln Gly Asn Ile His Cys Ser
Asp Lys Gly Ser Trp Cys Glu 95 100 105 Gln Gln Leu Cys Ala Cys Asp
Lys Glu Val Ala Phe Cys Leu Lys 110 115 120 Arg Asn Leu Asp Thr Tyr
Gln Lys Arg Leu Arg Phe Tyr Trp Arg 125 130 135 Pro His Cys Arg Gly
Gln Thr Pro Gly Cys 140 145 39 1533 PRT Homo sapiens 39 Met Tyr Ile
Arg Val Ser Tyr Asp Thr Lys Pro Asp Ser Leu Leu 1 5 10 15 His Leu
Met Val Lys Asp Trp Gln Leu Glu Leu Pro Lys Leu Leu 20 25 30 Ile
Ser Val His Gly Gly Leu Gln Asn Phe Glu Met Gln Pro Lys 35 40 45
Leu Lys Gln Val Phe Gly Lys Gly Leu Ile Lys Ala Ala Met Thr 50 55
60 Thr Gly Ala Trp Ile Phe Thr Gly Gly Val Ser Thr Gly Val Ile 65
70 75 Ser His Val Gly Asp Ala Leu Lys Asp His Ser Ser Lys Ser Arg
80 85 90 Gly Arg Val Cys Ala Ile Gly Ile Ala Pro Trp Gly Ile Val
Glu 95 100 105 Asn Lys Glu Asp Leu Val Gly Lys Asp Val Thr Arg Val
Tyr Gln 110 115 120 Thr Met Ser Asn Pro Leu Ser Lys Leu Ser Val Leu
Asn Asn Ser 125 130 135 His Thr His Phe Ile Leu Ala Asp Asn Gly Thr
Leu Gly Lys Tyr 140 145 150 Gly Ala Glu Val Lys Leu Arg Arg Leu Leu
Glu Lys His Ile Ser 155 160 165 Leu Gln Lys Ile Asn Thr Arg Leu Gly
Gln Gly Val Pro Leu Val 170 175 180 Gly Leu Val Val Glu Gly Gly Pro
Asn Val Val Ser Ile Val Leu 185 190 195 Glu Tyr Leu Gln Glu Glu Pro
Pro Ile Pro Val Val Ile Cys Asp 200 205 210 Gly Ser Gly Arg Ala Ser
Asp Ile Leu Ser Phe Ala His Lys Tyr 215 220 225 Cys Glu Glu Gly Gly
Ile Ile Asn Glu Ser Leu Arg Glu Gln Leu 230 235 240 Leu Val Thr Ile
Gln Lys Thr Phe Asn Tyr Asn Lys Ala Gln Ser 245 250 255 His Gln Leu
Phe Ala Ile Ile Met Glu Cys Met Lys Lys Lys Glu 260 265 270 Leu Val
Thr Val Phe Arg Met Gly Ser Glu Gly Gln Gln Asp Ile 275 280 285 Glu
Met Ala Ile Leu Thr Ala Leu Leu Lys Gly Thr Asn Val Ser 290 295 300
Ala Pro Asp Gln Leu Ser Leu Ala Leu Ala Trp Asn Arg Val Asp 305 310
315 Ile Ala Arg Ser Gln Ile Phe Val Phe Gly Pro His Trp Thr Pro 320
325 330 Leu Gly Ser Leu Ala Pro Pro Thr Asp Ser Lys Ala Thr Glu Lys
335 340 345 Glu Lys Lys Pro Pro Met Ala Thr Thr Lys Gly Gly Arg Gly
Lys 350 355 360 Gly Lys Gly Lys Lys Lys Gly Lys Val Lys Glu Glu Val
Glu Glu 365 370 375 Glu Thr Asp Pro Arg Lys Ile Glu Leu Leu Asn Trp
Val Asn Ala 380 385 390 Leu Glu Gln Ala Met Leu Asp Ala Leu Val Leu
Asp Arg Val Asp 395 400 405 Phe Val Lys Leu Leu Ile Glu Asn Gly Val
Asn Met Gln His Phe 410 415 420 Leu Thr Ile Pro Arg Leu Glu Glu Leu
Tyr Asn Thr Arg Leu Gly 425 430 435 Pro Pro Asn Thr Leu His Leu Leu
Val Arg Asp Val Lys Lys Ser 440 445 450 Asn Leu Pro Pro Asp Tyr His
Ile Ser Leu Ile Asp Ile Gly Leu 455 460 465 Val Leu Glu Tyr Leu Met
Gly Gly Ala Tyr Arg Cys Asn Tyr Thr 470 475 480 Arg Lys Asn Phe Arg
Thr Leu Tyr Asn Asn Leu Phe Gly Pro Lys 485 490 495 Arg Pro Lys Ala
Leu Lys Leu Leu Gly Met Glu Asp Asp Glu Pro 500 505 510 Pro Ala Lys
Gly Lys Lys Lys Lys Lys Lys Lys Lys Glu Glu Glu 515 520 525 Ile Asp
Ile Asp Val Asp Asp Pro Ala Val Ser Arg Phe Gln Tyr 530 535 540 Pro
Phe His Glu Leu Met Val Trp Ala Val Leu Met Lys Arg Gln 545 550 555
Lys Met Ala Val Phe Leu Trp Gln Arg Gly Glu Glu Ser Met Ala 560 565
570 Lys Ala Leu Val Ala Cys Lys Leu Tyr Lys Ala Met Ala His Glu 575
580 585 Ser Ser Glu Ser Asp Leu Val Asp Asp Ile Ser Gln Asp Leu Asp
590 595 600 Asn Asn Ser Lys Asp Phe Gly Gln Leu Ala Leu Glu Leu Leu
Asp 605 610 615 Gln Ser Tyr Lys His Asp Glu Gln Ile Ala Met Lys Leu
Leu Thr 620 625 630 Tyr Glu Leu Lys Asn Trp Ser Asn Ser Thr Cys Leu
Lys Leu Ala 635 640 645 Val Ala Ala Lys His Arg Asp Phe Ile Ala His
Thr Cys Ser Gln 650 655 660 Met Leu Leu Thr Asp Met Trp Met Gly Arg
Leu Arg Met Arg Lys 665 670 675 Asn Pro Gly Leu Lys Val Ile Met Gly
Ile Leu Leu Pro Pro Thr 680 685 690 Ile Leu Phe Leu Glu Phe Arg Thr
Tyr Asp Asp Phe Ser Tyr Gln 695 700 705 Thr Ser Lys Glu Asn Glu Asp
Gly Lys Glu Lys Glu Glu Glu Asn 710 715 720 Thr Asp Ala Asn Ala Asp
Ala Gly Ser Arg Lys Gly Asp Glu Glu 725 730 735 Asn Glu His Lys Lys
Gln Arg Ser Ile Pro Ile Gly Thr Lys Ile 740 745 750 Cys Glu Phe Tyr
Asn Ala Pro Ile Val Lys Phe Trp Phe Tyr Thr 755 760 765 Ile Ser Tyr
Leu Gly Tyr Leu Leu Leu Phe Asn Tyr Val Ile Leu 770 775 780 Val Arg
Met Asp Gly Trp Pro Ser Leu Gln Glu Trp Ile Val Ile 785 790 795 Ser
Tyr Ile Val Ser Leu Ala Leu Glu Lys Ile Arg Glu Ile Leu 800 805 810
Met Ser Glu Pro Gly Lys Leu Ser Gln Lys Ile Lys Val Trp Leu 815 820
825 Gln Glu Tyr Trp Asn Ile Thr Asp Leu Val Ala Ile Ser Thr Phe 830
835 840 Met Ile Gly Ala Ile Leu Arg Leu Gln Asn Gln Pro Tyr Met Gly
845 850 855 Tyr Gly Arg Val Ile Tyr Cys Val Asp Ile Ile Phe Trp Tyr
Ile 860 865 870 Arg Val Leu Asp Ile Phe Gly Val Asn Lys Tyr Leu Gly
Pro Tyr 875 880 885 Val Met Met Ile Gly Lys Met Met Ile Asp Met Leu
Tyr Phe Val 890 895 900 Val Ile Met Leu Val Val Leu Met Ser Phe Gly
Val Ala Arg Gln 905 910 915 Ala Ile Leu His Pro Glu Glu Lys Pro Ser
Trp Lys Leu Ala Arg 920 925 930 Asn Ile Phe Tyr Met Pro Tyr Trp Met
Ile Tyr Gly Glu Val Phe 935 940 945 Ala Asp Gln Ile Asp Leu Tyr Ala
Met Glu Ile Asn Pro Pro Cys 950 955 960 Gly Glu Asn Leu Tyr Asp Glu
Glu Gly Lys Arg Leu Pro Pro Cys 965 970 975 Ile Pro Gly Ala Trp Leu
Thr Pro Ala Leu Met Ala Cys Tyr Leu 980 985 990 Leu Val Ala Asn Ile
Leu Leu Val Asn Leu Leu Ile Ala Val Phe 995 1000 1005 Asn Asn Thr
Phe Phe Glu Val Lys Ser Ile Ser Asn Gln Val Trp 1010 1015 1020 Lys
Phe Gln Arg Tyr Gln Leu Ile Met Thr Phe His Asp Arg Pro 1025 1030
1035 Val Leu Pro Pro Pro Met Ile Ile Leu Ser His Ile Tyr Ile Ile
1040 1045 1050 Ile Met Arg Leu Ser Gly Arg Cys Arg Lys Lys Arg Glu
Gly Asp 1055 1060 1065 Gln Glu Glu Arg Asp Arg Gly Leu Lys Leu Phe
Leu Ser Asp Glu 1070 1075 1080 Glu Leu Lys Arg Leu His Glu Phe Glu
Glu Gln Cys Val Gln Glu 1085 1090 1095 His Phe Arg Glu Lys Glu Asp
Glu Gln Gln Ser Ser Ser Asp Glu 1100 1105 1110 Arg Ile Arg Val Thr
Ser Glu Arg Val Glu Asn Met Ser Met Arg 1115 1120 1125 Leu Glu Glu
Ile Asn Glu Arg Glu Thr Phe Met Lys Thr Ser Leu 1130 1135 1140 Gln
Thr Val Asp Leu Arg Leu Ala Gln Leu Glu Glu Leu Ser Asn 1145 1150
1155 Arg Met Val Asn Ala Leu Glu Asn Leu Ala Gly Ile Asp Arg Ser
1160 1165 1170 Asp Leu Ile Gln Ala Arg Ser Arg Ala Ser Ser Glu Cys
Glu Ala 1175 1180 1185 Thr Tyr Leu Leu Arg Gln Ser Ser Ile Asn Ser
Ala Asp Gly Tyr 1190 1195 1200 Ser Leu Tyr Arg Tyr His Phe Asn Gly
Glu Glu Leu Leu Phe Glu 1205 1210 1215 Asp Thr Ser Leu Ser Thr Ser
Pro Gly Thr Gly Val Arg Lys Lys 1220 1225 1230 Thr Cys Ser Phe Arg
Ile Lys Glu Glu Lys Asp Val Lys Thr His 1235 1240 1245 Leu
Val Pro Glu Cys Gln Asn Ser Leu His Leu Ser Leu Gly Thr 1250 1255
1260 Ser Thr Ser Ala Thr Pro Asp Gly Ser His Leu Ala Val Asp Asp
1265 1270 1275 Leu Lys Asn Ala Glu Glu Ser Lys Leu Gly Pro Asp Ile
Gly Ile 1280 1285 1290 Ser Lys Glu Asp Asp Glu Arg Gln Thr Asp Ser
Lys Lys Glu Glu 1295 1300 1305 Thr Ile Ser Pro Ser Leu Asn Lys Thr
Asp Val Ile His Gly Gln 1310 1315 1320 Asp Lys Ser Asp Val Gln Asn
Thr Gln Leu Thr Val Glu Thr Thr 1325 1330 1335 Asn Ile Glu Gly Thr
Ile Ser Tyr Pro Leu Glu Glu Thr Lys Ile 1340 1345 1350 Thr Arg Tyr
Phe Pro Asp Glu Thr Ile Asn Ala Cys Lys Thr Met 1355 1360 1365 Lys
Ser Arg Ser Phe Val Tyr Ser Arg Gly Arg Lys Leu Val Gly 1370 1375
1380 Gly Val Asn Gln Asp Val Glu Tyr Ser Ser Ile Thr Asp Gln Gln
1385 1390 1395 Leu Thr Thr Glu Trp Gln Cys Gln Val Gln Lys Ile Thr
Arg Ser 1400 1405 1410 His Ser Thr Asp Ile Pro Tyr Ile Val Ser Glu
Ala Ala Val Gln 1415 1420 1425 Ala Glu Gln Lys Glu Gln Phe Ala Asp
Met Gln Asp Glu His His 1430 1435 1440 Val Ala Glu Ala Ile Pro Arg
Ile Pro Arg Leu Ser Leu Thr Ile 1445 1450 1455 Thr Asp Arg Asn Gly
Met Glu Asn Leu Leu Ser Val Lys Pro Asp 1460 1465 1470 Gln Thr Leu
Gly Phe Pro Ser Leu Arg Ser Lys Ser Leu His Gly 1475 1480 1485 His
Pro Arg Asn Val Lys Ser Ile Gln Gly Lys Leu Asp Arg Ser 1490 1495
1500 Gly His Ala Ser Ser Val Ser Ser Leu Val Ile Val Ser Gly Met
1505 1510 1515 Thr Ala Glu Glu Lys Lys Val Lys Lys Glu Lys Ala Ser
Thr Glu 1520 1525 1530 Thr Glu Cys 40 462 PRT Homo sapiens 40 Met
Ser Thr Glu Lys Val Asp Gln Lys Glu Glu Ala Gly Glu Lys 1 5 10 15
Glu Val Cys Gly Asp Gln Ile Lys Gly Pro Asp Lys Glu Glu Glu 20 25
30 Pro Pro Ala Ala Ala Ser His Gly Gln Gly Trp Arg Pro Gly Gly 35
40 45 Arg Ala Ala Arg Asn Ala Arg Pro Glu Pro Gly Ala Arg His Pro
50 55 60 Ala Leu Pro Ala Met Val Asn Asp Pro Pro Val Pro Ala Leu
Leu 65 70 75 Trp Ala Gln Glu Val Gly Gln Val Leu Ala Gly Arg Ala
Arg Arg 80 85 90 Leu Leu Leu Gln Phe Gly Val Leu Phe Cys Thr Ile
Leu Leu Leu 95 100 105 Leu Trp Val Ser Val Phe Leu Tyr Gly Ser Phe
Tyr Tyr Ser Tyr 110 115 120 Met Pro Thr Val Ser His Leu Ser Pro Val
His Phe Tyr Tyr Arg 125 130 135 Thr Asp Cys Asp Ser Ser Thr Thr Ser
Leu Cys Ser Phe Pro Val 140 145 150 Ala Asn Val Ser Leu Thr Lys Gly
Gly Arg Asp Arg Val Leu Met 155 160 165 Tyr Gly Gln Pro Tyr Arg Val
Thr Leu Glu Leu Glu Leu Pro Glu 170 175 180 Ser Pro Val Asn Gln Asp
Leu Gly Met Phe Leu Val Thr Ile Ser 185 190 195 Cys Tyr Thr Arg Gly
Gly Arg Ile Ile Ser Thr Ser Ser Arg Ser 200 205 210 Val Met Leu His
Tyr Arg Ser Asp Leu Leu Gln Met Leu Asp Thr 215 220 225 Leu Val Phe
Ser Ser Leu Leu Leu Phe Gly Phe Ala Glu Gln Lys 230 235 240 Gln Leu
Leu Glu Val Glu Leu Tyr Ala Asp Tyr Arg Glu Asn Ser 245 250 255 Tyr
Val Pro Thr Thr Gly Ala Ile Ile Glu Ile His Ser Lys Arg 260 265 270
Ile Gln Leu Tyr Gly Ala Tyr Leu Arg Ile His Ala His Phe Thr 275 280
285 Gly Leu Arg Tyr Leu Leu Tyr Asn Phe Pro Met Thr Cys Ala Phe 290
295 300 Ile Gly Val Ala Ser Asn Phe Thr Phe Leu Ser Val Ile Val Leu
305 310 315 Phe Ser Tyr Met Gln Trp Val Trp Gly Gly Ile Trp Pro Arg
His 320 325 330 Arg Phe Ser Leu Gln Val Asn Ile Arg Lys Arg Asp Asn
Ser Arg 335 340 345 Lys Glu Val Gln Arg Arg Ile Ser Ala His Gln Pro
Gly Pro Glu 350 355 360 Gly Gln Glu Glu Ser Thr Pro Gln Ser Asp Val
Thr Glu Asp Gly 365 370 375 Glu Ser Pro Glu Asp Pro Ser Gly Thr Glu
Gly Gln Leu Ser Glu 380 385 390 Glu Glu Lys Pro Asp Gln Gln Pro Leu
Ser Gly Glu Glu Glu Leu 395 400 405 Glu Pro Glu Ala Ser Asp Gly Ser
Gly Ser Trp Glu Asp Ala Ala 410 415 420 Leu Leu Thr Glu Ala Asn Leu
Pro Ala Pro Ala Pro Ala Ser Ala 425 430 435 Ser Ala Pro Val Leu Glu
Thr Leu Gly Ser Ser Glu Pro Ala Gly 440 445 450 Gly Ala Leu Arg Gln
Arg Pro Thr Cys Ser Ser Ser 455 460 41 1015 PRT Homo sapiens 41 Met
Gly Pro Pro Leu Pro Leu Leu Leu Leu Leu Leu Leu Leu Leu 1 5 10 15
Pro Pro Arg Val Leu Pro Ala Ala Pro Ser Ser Val Pro Arg Gly 20 25
30 Arg Gln Leu Pro Gly Arg Leu Gly Cys Leu Leu Glu Glu Gly Leu 35
40 45 Cys Gly Ala Ser Glu Ala Cys Val Asn Asp Gly Val Phe Gly Arg
50 55 60 Cys Gln Lys Val Pro Ala Met Asp Phe Tyr Arg Tyr Glu Val
Ser 65 70 75 Pro Val Ala Leu Gln Arg Leu Arg Val Ala Leu Gln Lys
Leu Ser 80 85 90 Gly Thr Gly Phe Thr Trp Gln Asp Asp Tyr Thr Gln
Tyr Val Met 95 100 105 Asp Gln Glu Leu Ala Asp Leu Pro Lys Thr Tyr
Leu Arg Arg Pro 110 115 120 Glu Ala Ser Ser Pro Ala Arg Pro Ser Lys
His Ser Val Gly Ser 125 130 135 Glu Arg Arg Tyr Ser Arg Glu Gly Gly
Ala Ala Leu Ala Asn Ala 140 145 150 Leu Arg Arg His Leu Pro Phe Leu
Glu Ala Leu Ser Gln Ala Pro 155 160 165 Ala Ser Asp Val Leu Ala Arg
Thr His Thr Ala Gln Asp Arg Pro 170 175 180 Pro Ala Glu Gly Asp Asp
Arg Phe Ser Glu Ser Ile Leu Thr Tyr 185 190 195 Val Ala His Thr Ser
Ala Leu Thr Tyr Pro Pro Gly Pro Arg Thr 200 205 210 Gln Leu Arg Glu
Asp Leu Leu Pro Arg Thr Leu Gly Gln Leu Gln 215 220 225 Pro Asp Glu
Leu Ser Pro Lys Val Asp Ser Gly Val Asp Arg His 230 235 240 His Leu
Met Ala Ala Leu Ser Ala Tyr Ala Ala Gln Arg Pro Pro 245 250 255 Ala
Pro Pro Gly Glu Gly Ser Leu Glu Pro Gln Tyr Leu Leu Arg 260 265 270
Ala Pro Ser Arg Met Pro Arg Pro Leu Leu Ala Pro Ala Ala Pro 275 280
285 Gln Lys Trp Pro Ser Pro Leu Gly Asp Ser Glu Asp Pro Ser Ser 290
295 300 Thr Gly Asp Gly Ala Arg Ile His Thr Leu Leu Lys Asp Leu Gln
305 310 315 Arg Gln Pro Ala Glu Val Arg Gly Leu Ser Gly Leu Glu Leu
Asp 320 325 330 Gly Met Ala Glu Leu Met Ala Gly Leu Met Gln Gly Val
Asp His 335 340 345 Gly Val Ala Arg Gly Ser Pro Gly Arg Ala Ala Leu
Gly Glu Ser 350 355 360 Gly Glu Gln Ala Asp Gly Pro Lys Ala Thr Leu
Arg Gly Asp Ser 365 370 375 Phe Pro Asp Asp Gly Val Gln Asp Asp Asp
Asp Arg Leu Tyr Gln 380 385 390 Glu Val His Arg Leu Ser Ala Thr Leu
Gly Gly Leu Leu Gln Asp 395 400 405 His Gly Ser Arg Leu Leu Pro Gly
Ala Leu Pro Phe Ala Arg Pro 410 415 420 Leu Asp Met Glu Arg Lys Lys
Ser Glu His Pro Glu Ser Ser Leu 425 430 435 Ser Ser Glu Glu Glu Thr
Ala Gly Val Glu Asn Val Lys Ser Gln 440 445 450 Thr Tyr Ser Lys Asp
Leu Leu Gly Gln Gln Pro His Ser Glu Pro 455 460 465 Gly Ala Ala Ala
Phe Gly Glu Leu Gln Asn Gln Met Pro Gly Pro 470 475 480 Ser Lys Glu
Glu Gln Ser Leu Pro Ala Gly Ala Gln Glu Ala Leu 485 490 495 Ser Asp
Gly Leu Gln Leu Glu Val Gln Pro Ser Glu Glu Glu Ala 500 505 510 Arg
Gly Tyr Ile Val Thr Asp Arg Asp Pro Leu Arg Pro Glu Glu 515 520 525
Gly Arg Arg Leu Val Glu Asp Val Ala Arg Leu Leu Gln Val Pro 530 535
540 Ser Ser Ala Phe Ala Asp Val Glu Val Leu Gly Pro Ala Val Thr 545
550 555 Phe Lys Val Ser Ala Asn Val Gln Asn Val Thr Thr Glu Asp Val
560 565 570 Glu Lys Ala Thr Val Asp Asn Lys Asp Lys Leu Glu Glu Thr
Ser 575 580 585 Gly Leu Lys Ile Leu Gln Thr Gly Val Gly Ser Lys Ser
Lys Leu 590 595 600 Lys Phe Leu Pro Pro Gln Ala Glu Gln Glu Asp Ser
Thr Lys Phe 605 610 615 Ile Ala Leu Thr Leu Val Ser Leu Ala Cys Ile
Leu Gly Val Leu 620 625 630 Leu Ala Ser Gly Leu Ile Tyr Cys Leu Arg
His Ser Ser Gln His 635 640 645 Arg Leu Lys Glu Lys Leu Ser Gly Leu
Gly Gly Asp Pro Gly Ala 650 655 660 Asp Ala Thr Ala Ala Tyr Gln Glu
Leu Cys Arg Gln Arg Met Ala 665 670 675 Thr Arg Pro Pro Asp Arg Pro
Glu Gly Pro His Thr Ser Arg Ile 680 685 690 Ser Ser Val Ser Ser Gln
Phe Ser Asp Gly Pro Ile Pro Ser Pro 695 700 705 Ser Ala Arg Ser Ser
Ala Ser Ser Trp Ser Glu Glu Pro Val Gln 710 715 720 Ser Asn Met Asp
Ile Ser Thr Gly His Met Ile Leu Ser Tyr Met 725 730 735 Glu Asp His
Leu Lys Asn Lys Asn Arg Leu Glu Lys Glu Trp Glu 740 745 750 Ala Leu
Cys Ala Tyr Gln Ala Glu Pro Asn Ser Ser Phe Val Ala 755 760 765 Gln
Arg Glu Glu Asn Val Pro Lys Asn Arg Ser Leu Ala Val Leu 770 775 780
Thr Tyr Asp His Ser Arg Val Leu Leu Lys Ala Glu Asn Ser His 785 790
795 Ser His Ser Asp Tyr Ile Asn Ala Ser Pro Ile Met Asp His Asp 800
805 810 Pro Arg Asn Pro Ala Tyr Ile Ala Thr Gln Gly Pro Leu Pro Ala
815 820 825 Thr Val Ala Asp Phe Trp Gln Met Val Trp Glu Ser Gly Cys
Val 830 835 840 Val Ile Val Met Leu Thr Pro Leu Ala Glu Asn Gly Val
Arg Gln 845 850 855 Cys Tyr His Tyr Trp Pro Asp Glu Gly Ser Asn Leu
Tyr His Ile 860 865 870 Tyr Glu Val Asn Leu Val Ser Glu His Ile Trp
Cys Glu Asp Phe 875 880 885 Leu Val Arg Ser Phe Tyr Leu Lys Asn Leu
Gln Thr Asn Glu Thr 890 895 900 Arg Thr Val Thr Gln Phe His Phe Leu
Ser Trp Tyr Asp Arg Gly 905 910 915 Val Pro Ser Ser Ser Arg Ser Leu
Leu Asp Phe Arg Arg Lys Val 920 925 930 Asn Lys Cys Tyr Arg Gly Arg
Ser Cys Pro Ile Ile Val His Cys 935 940 945 Ser Asp Gly Ala Gly Arg
Ser Gly Thr Tyr Val Leu Ile Asp Met 950 955 960 Val Leu Asn Lys Met
Ala Lys Gly Ala Lys Glu Ile Asp Ile Ala 965 970 975 Ala Thr Leu Glu
His Leu Arg Asp Gln Arg Pro Gly Met Val Gln 980 985 990 Thr Lys Glu
Gln Phe Glu Phe Ala Leu Thr Ala Val Ala Glu Glu 995 1000 1005 Val
Asn Ala Ile Leu Lys Ala Leu Pro Gln 1010 1015 42 442 PRT Homo
sapiens 42 Met Gln Pro Pro Pro Ser Leu Cys Gly Arg Ala Leu Val Ala
Leu 1 5 10 15 Val Leu Ala Cys Gly Leu Ser Arg Ile Trp Gly Glu Glu
Arg Gly 20 25 30 Phe Pro Pro Asp Arg Ala Thr Pro Leu Leu Gln Thr
Ala Glu Ile 35 40 45 Met Thr Pro Pro Thr Lys Thr Leu Trp Pro Lys
Gly Ser Asn Ala 50 55 60 Ser Leu Ala Arg Ser Leu Ala Pro Ala Glu
Val Pro Lys Gly Asp 65 70 75 Arg Thr Ala Gly Ser Pro Pro Arg Thr
Ile Ser Pro Pro Pro Cys 80 85 90 Gln Gly Pro Ile Glu Ile Lys Glu
Thr Phe Lys Tyr Ile Asn Thr 95 100 105 Val Val Ser Cys Leu Val Phe
Val Leu Gly Ile Ile Gly Asn Ser 110 115 120 Thr Leu Leu Arg Ile Ile
Tyr Lys Asn Lys Cys Met Arg Asn Gly 125 130 135 Pro Asn Ile Leu Ile
Ala Ser Leu Ala Leu Gly Asp Leu Leu His 140 145 150 Ile Val Ile Asp
Ile Pro Ile Asn Val Tyr Lys Leu Leu Ala Glu 155 160 165 Asp Trp Pro
Phe Gly Ala Glu Met Cys Lys Leu Val Pro Phe Ile 170 175 180 Gln Lys
Ala Ser Val Gly Ile Thr Val Leu Ser Leu Cys Ala Leu 185 190 195 Ser
Ile Asp Arg Tyr Arg Ala Val Ala Ser Trp Ser Arg Ile Lys 200 205 210
Gly Ile Gly Val Pro Lys Trp Thr Ala Val Glu Ile Val Leu Ile 215 220
225 Trp Val Val Ser Val Val Leu Ala Val Pro Glu Ala Ile Gly Phe 230
235 240 Asp Ile Ile Thr Met Asp Tyr Lys Gly Ser Tyr Leu Arg Ile Cys
245 250 255 Leu Leu His Pro Val Gln Lys Thr Ala Phe Met Gln Phe Tyr
Lys 260 265 270 Thr Ala Lys Asp Trp Trp Leu Phe Ser Phe Tyr Phe Cys
Leu Pro 275 280 285 Leu Ala Ile Thr Ala Phe Phe Tyr Thr Leu Met Thr
Cys Glu Met 290 295 300 Leu Arg Lys Lys Ser Gly Met Gln Ile Ala Leu
Asn Asp His Leu 305 310 315 Lys Gln Arg Arg Glu Val Ala Lys Thr Val
Phe Cys Leu Val Leu 320 325 330 Val Phe Ala Leu Cys Trp Leu Pro Leu
His Leu Ser Arg Ile Leu 335 340 345 Lys Leu Thr Leu Tyr Asn Gln Asn
Asp Pro Asn Arg Cys Glu Leu 350 355 360 Leu Ser Phe Leu Leu Val Leu
Asp Tyr Ile Gly Ile Asn Met Ala 365 370 375 Ser Leu Asn Ser Cys Ile
Asn Pro Ile Ala Leu Tyr Leu Val Ser 380 385 390 Lys Arg Phe Lys Asn
Cys Phe Lys Ser Cys Leu Cys Cys Trp Cys 395 400 405 Gln Ser Phe Glu
Glu Lys Gln Ser Leu Glu Glu Lys Gln Ser Cys 410 415 420 Leu Lys Phe
Lys Ala Asn Asp His Gly Tyr Asp Asn Phe Arg Ser 425 430 435 Ser Asn
Lys Tyr Ser Ser Ser 440
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