U.S. patent application number 15/846083 was filed with the patent office on 2018-12-06 for compositions and methods for the diagnosis and treatment of tumor.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to Mark DENNIS, William MALLET, Paul POLAKIS.
Application Number | 20180344864 15/846083 |
Document ID | / |
Family ID | 37595671 |
Filed Date | 2018-12-06 |
United States Patent
Application |
20180344864 |
Kind Code |
A1 |
DENNIS; Mark ; et
al. |
December 6, 2018 |
COMPOSITIONS AND METHODS FOR THE DIAGNOSIS AND TREATMENT OF
TUMOR
Abstract
The invention is directed to antibody drug conjugate
compositions of matter useful for the diagnosis and treatment of
tumors in mammals and to methods of using those compositions of
matter for the same.
Inventors: |
DENNIS; Mark; (San Carlos,
CA) ; MALLET; William; (Redwood City, CA) ;
POLAKIS; Paul; (Mill Valley, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
37595671 |
Appl. No.: |
15/846083 |
Filed: |
December 18, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15590873 |
May 9, 2017 |
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15846083 |
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14945980 |
Nov 19, 2015 |
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15590873 |
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14108820 |
Dec 17, 2013 |
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14945980 |
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13945052 |
Jul 18, 2013 |
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14108820 |
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13357913 |
Jan 25, 2012 |
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13945052 |
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13045722 |
Mar 11, 2011 |
8449883 |
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13357913 |
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11452990 |
Jun 14, 2006 |
7989595 |
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13045722 |
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60793951 |
Apr 21, 2006 |
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60692092 |
Jun 20, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 5/00 20180101; A61K
31/4196 20130101; A61K 31/7068 20130101; A61K 47/6817 20170801;
A61K 47/6851 20170801; A61K 2039/505 20130101; A61P 1/18 20180101;
A61K 38/08 20130101; C07K 16/3069 20130101; C07K 2317/55 20130101;
A61P 11/00 20180101; A61K 31/337 20130101; A61K 31/555 20130101;
A61K 31/4025 20130101; A61P 15/00 20180101; C07K 16/3092 20130101;
A61K 31/519 20130101; A61K 31/7072 20130101; A61K 31/513 20130101;
C07K 16/303 20130101; A61P 43/00 20180101; C07K 2317/565 20130101;
A61K 31/537 20130101; Y10T 428/13 20150115; A61P 35/00 20180101;
A61K 38/07 20130101; A61K 31/517 20130101; A61K 47/6835 20170801;
A61K 45/06 20130101; A61K 47/6803 20170801; C07K 16/3015 20130101;
C07K 2317/24 20130101 |
International
Class: |
A61K 47/68 20170101
A61K047/68; A61K 31/7068 20060101 A61K031/7068; A61K 31/337
20060101 A61K031/337; C07K 16/30 20060101 C07K016/30; A61K 45/06
20060101 A61K045/06; A61K 38/08 20060101 A61K038/08; A61K 38/07
20060101 A61K038/07; A61K 31/7072 20060101 A61K031/7072; A61K
31/555 20060101 A61K031/555; A61K 31/537 20060101 A61K031/537; A61K
31/519 20060101 A61K031/519; A61K 31/517 20060101 A61K031/517; A61K
31/513 20060101 A61K031/513; A61K 31/4196 20060101 A61K031/4196;
A61K 31/4025 20060101 A61K031/4025 |
Claims
1-78. (canceled)
79. A pharmaceutical formulation comprising an antibody drug
conjugate, and a pharmaceutically acceptable diluent, carrier or
excipient, wherein the antibody drug conjugate comprises an
antibody covalently attached by a linker to one or more toxin drug
moieties, the conjugate having the formula: Ab-(L-D).sub.p or a
pharmaceutically acceptable salt or solvate thereof, wherein: Ab is
an antibody that comprises three light chain hypervariable regions
(HVR-L1, HVR-L2 and HVR-L3) and three heavy chain hypervariable
regions (HVR-H1, HVR-H2 and HVR-H3) wherein (a) HVR-L1 comprises
the amino acid sequence of SEQ ID NO:119; (b) HVR-L2 comprises the
amino acid sequence of SEQ ID NO:121; (c) HVR-L3 comprises the
amino acid sequence of SEQ ID NO:122; (d) HVR-H1 comprises the
amino acid sequence of SEQ ID NO:123; (e) HVR-H2 comprises the
amino acid sequence of SEQ ID NO:125; and (f) HVR-H3 comprises the
amino acid sequence of SEQ ID NO:183; L is a linker; D is a toxin
drug moiety; and p is 1 to about 20.
80. The pharmaceutical formulation of claim 79 further comprising a
therapeutically effective amount of a chemotherapeutic agent
selected from letrozole, oxaliplatin, doxetaxel, 5-FU, leucovorin,
lapatinib, and gemcitabine.
81. An article of manufacture comprising an antibody-drug conjugate
comprising an antibody covalently attached by a linker to one or
more toxin drug moieties, the conjugate having the formula:
Ab-(L-D).sub.p or a pharmaceutically acceptable salt or solvate
thereof, wherein: Ab is an antibody that comprises three light
chain hypervariable regions (HVR-L1, HVR-L2 and HVR-L3) and three
heavy chain hypervariable regions (HVR-H1, HVR-H2 and HVR-H3)
wherein (a) HVR-L1 comprises the amino acid sequence of SEQ ID
NO:119; (b) HVR-L2 comprises the amino acid sequence of SEQ ID
NO:121; (c) HVR-L3 comprises the amino acid sequence of SEQ ID
NO:122; (d) HVR-H1 comprises the amino acid sequence of SEQ ID
NO:123; (e) HVR-H2 comprises the amino acid sequence of SEQ ID
NO:125; and (f) HVR-H3 comprises the amino acid sequence of SEQ ID
NO:183; L is a linker; D is a toxin drug moiety; and p is 1 to
about 20; a container; and a package insert or label indicating
that the compound can be used to treat cancer characterized by the
overexpression of a TAT10772 polypeptide.
82. The article of manufacture of claim 81 wherein the cancer is
prostate cancer, cancer of the urinary tract, pancreatic cancer,
lung cancer, breast cancer, colon cancer or ovarian cancer.
83. The article of manufacture of claim 81, wherein the antibody
further comprises a V.sub.H acceptor human consensus framework
sequence of any one of SEQ ID NOS:184-193.
84. The article of manufacture of claim 81, wherein the antibody
further comprises a V.sub.L acceptor human consensus framework
sequence of any one of SEQ ID NOS:194-197.
85. The article of manufacture of claim 81, wherein the antibody
comprises a V.sub.H acceptor human consensus framework sequence of
any one of SEQ ID NOS:184-193 and a V.sub.L acceptor human
consensus framework sequence of any one of SEQ ID NOS:194-197.
86. The article of manufacture of claim 81, wherein the antibody is
an antibody fragment.
87. The article of manufacture of claim 81, wherein the antibody is
a chimeric or a humanized antibody.
88. The article of manufacture of claim 81, wherein the antibody
comprises the V.sub.H sequence shown as SEQ ID NO:208.
89. The article of manufacture of claim 81, wherein the antibody
comprises the V.sub.L sequence shown as SEQ ID NO:211.
90. The article of manufacture of claim 81, wherein the antibody
comprises the V.sub.H sequence shown as SEQ ID NO:208 and the
V.sub.L sequence shown as SEQ ID NO:211.
91. The article of manufacture of claim 81, wherein D is an
auristatin.
92. The article of manufacture of claim 91, wherein the auristatin
is MMAE or MMAF.
93. The article of manufacture of claim 81, wherein L is
MC-val-cit-PAB or MC.
94. The article of manufacture of claim 81, wherein L is SMCC, SPP,
or BMPEO.
95. The article of manufacture of claim 81, wherein the antibody
drug conjugate is selected from the formula:
Ab-MC-val-cit-PAB-MMAE, Ab-MC-val-cit-PAB-MMAF, Ab-MC-MMAE,
Ab-MC-MMAF, Ab-SPP-DM1, and Ab-SMCC-DM1.
96. The article of manufacture of claim 81, wherein the antibody
drug conjugate has the formula Ab-MC-val-cit-PAB-MMAE.
97. The article of manufacture of claim 81, wherein the antibody is
attached to the linker through a non-native cysteine amino acid
residue.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/945,052, filed on Jul. 18, 2013, which is a
divisional of U.S. patent application Ser. No. 13/357,913, filed on
Jan. 25, 2012, now abandoned, which is a continuation of U.S.
patent application Ser. No. 13/045,722, filed on Mar. 11, 2011,
issued on May 28, 2013, as U.S. Pat. No. 8,449,883, which is a
divisional of U.S. patent application Ser. No. 11/452,990, filed on
Jun. 14, 2006, issued on Aug. 2, 2011 as U.S. Pat. No. 7,989,595,
which claims the benefit of priority under 35 U.S.C. .sctn. 119(e)
to provisional applications Ser. No. 60/692,092 filed Jun. 20,
2005, and Ser. No. 60/793,951 filed Apr. 21, 2006, the contents of
which are incorporated herein in their entirety by reference.
FIELD OF THE INVENTION
[0002] 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
[0003] 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.
[0004] 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.
[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 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 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 cellular polypeptides (and their
encoding nucleic acids or fragments thereof) which are expressed to
a greater degree on the surface of one or more types of cancer
cell(s) as compared to on the surface of one or more types of
normal non-cancer cells. These polypeptides are herein referred to
as Tumor-associated Antigenic Target polypeptides ("TAT"
polypeptides) and are expected to serve as effective targets for
cancer therapy and diagnosis in mammals.
[0007] Accordingly, in one embodiment of the present invention, the
invention provides an isolated nucleic acid molecule having a
nucleotide sequence that encodes a tumor-associated antigenic
target polypeptide or fragment thereof (a "TAT" polypeptide).
[0008] In certain aspects, the isolated nucleic acid molecule
comprises a nucleotide sequence having at least about 80% nucleic
acid sequence identity, alternatively at least about 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA
molecule encoding a full-length TAT polypeptide having an amino
acid sequence as disclosed herein, a TAT polypeptide amino acid
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a transmembrane TAT polypeptide, with or
without the signal peptide, as disclosed herein or any other
specifically defined fragment of a full-length TAT polypeptide
amino acid sequence as disclosed herein, or (b) the complement of
the DNA molecule of (a).
[0009] In other aspects, the isolated nucleic acid molecule
comprises a nucleotide sequence having at least about 80% nucleic
acid sequence identity, alternatively at least about 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99% or 100% nucleic acid sequence identity, to (a) a DNA
molecule comprising the coding sequence of a full-length TAT
polypeptide cDNA as disclosed herein, the coding sequence of a TAT
polypeptide lacking the signal peptide as disclosed herein, the
coding sequence of an extracellular domain of a transmembrane TAT
polypeptide, with or without the signal peptide, as disclosed
herein or the coding sequence of any other specifically defined
fragment of the full-length TAT polypeptide amino acid sequence as
disclosed herein, or (b) the complement of the DNA molecule of
(a).
[0010] In further aspects, the invention concerns an isolated
nucleic acid molecule comprising a nucleotide sequence having at
least about 80% nucleic acid sequence identity, alternatively at
least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid
sequence identity, to (a) a DNA molecule that encodes the same
mature polypeptide encoded by the full-length coding region of any
of the human protein cDNAs deposited with the ATCC as disclosed
herein, or (b) the complement of the DNA molecule of (a).
[0011] Another aspect of the invention provides an isolated nucleic
acid molecule comprising a nucleotide sequence encoding a TAT
polypeptide which is either transmembrane domain-deleted or
transmembrane domain-inactivated, or is complementary to such
encoding nucleotide sequence, wherein the transmembrane domain(s)
of such polypeptide(s) are disclosed herein. Therefore, soluble
extracellular domains of the herein described TAT polypeptides are
contemplated.
[0012] In other aspects, the present invention is directed to
isolated nucleic acid molecules which hybridize to (a) a nucleotide
sequence encoding a TAT polypeptide having a full-length amino acid
sequence as disclosed herein, a TAT polypeptide amino acid sequence
lacking the signal peptide as disclosed herein, an extracellular
domain of a transmembrane TAT polypeptide, with or without the
signal peptide, as disclosed herein or any other specifically
defined fragment of a full-length TAT polypeptide amino acid
sequence as disclosed herein, or (b) the complement of the
nucleotide sequence of (a). In this regard, an embodiment of the
present invention is directed to fragments of a full-length TAT
polypeptide coding sequence, or the complement thereof, as
disclosed herein, that may find use as, for example, hybridization
probes useful as, for example, diagnostic probes, PCR primers,
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.
Moreover, such nucleic acid fragments are usually comprised of
consecutive nucleotides derived from the full-length coding
sequence of a TAT polypeptide or the complement thereof. It is
noted that novel fragments of a TAT polypeptide-encoding nucleotide
sequence, or the complement thereof, 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), or the
complement thereof, are novel. All of such novel fragments of TAT
polypeptide-encoding nucleotide sequences, or the complement
thereof, are contemplated herein. Also contemplated are the TAT
polypeptide fragments encoded by these nucleotide molecule
fragments, preferably those TAT polypeptide fragments that comprise
a binding site for an anti-TAT antibody, a TAT binding oligopeptide
or other small organic molecule that binds to a TAT
polypeptide.
[0013] In another embodiment, the invention provides isolated TAT
polypeptides encoded by any of the isolated nucleic acid sequences
hereinabove identified.
[0014] In a certain aspect, the invention concerns an isolated TAT
polypeptide, comprising an amino acid sequence having at least
about 80% amino acid sequence identity, alternatively at least
about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence
identity, to a TAT polypeptide having a full-length amino acid
sequence as disclosed herein, a TAT polypeptide amino acid sequence
lacking the signal peptide as disclosed herein, an extracellular
domain of a transmembrane TAT polypeptide protein, with or without
the signal peptide, as disclosed herein, an amino acid sequence
encoded by any of the nucleic acid sequences disclosed herein or
any other specifically defined fragment of a full-length TAT
polypeptide amino acid sequence as disclosed herein.
[0015] In a further aspect, the invention concerns an isolated TAT
polypeptide comprising an amino acid sequence having at least about
80% amino acid sequence identity, alternatively at least about 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to an
amino acid sequence encoded by any of the human protein cDNAs
deposited with the ATCC as disclosed herein.
[0016] In a yet further aspect, the invention concerns an isolated
TAT polypeptide comprising an amino acid sequence that is encoded
by a nucleotide sequence that hybridizes to the complement of a DNA
molecule encoding (a) a TAT polypeptide having a full-length amino
acid sequence as disclosed herein, (b) a TAT polypeptide amino acid
sequence lacking the signal peptide as disclosed herein, (c) an
extracellular domain of a transmembrane TAT polypeptide protein,
with or without the signal peptide, as disclosed herein, (d) an
amino acid sequence encoded by any of the nucleic acid sequences
disclosed herein or (e) any other specifically defined fragment of
a full-length TAT polypeptide amino acid sequence as disclosed
herein.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] 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.
[0021] 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 inhibit the growth or proliferation of or induce the
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.
[0022] 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.
[0023] 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 inhibit the growth or proliferation of or
induce the 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.
[0024] 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.
[0025] 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 inhibit the growth or
proliferation of or induce the 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.
[0026] 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.
[0027] 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.
[0028] 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.
[0029] Other embodiments of the present invention are directed to
any isolated antibody comprising one or more of the HVR-L1, HVR-L2,
HVR-L3, HVR-H1, HVR-H2, or HVR-H3 sequences disclosed herein, or
any antibody that binds to the same epitope as any such
antibody.
B. Additional Embodiments
[0030] 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.
[0031] 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.
[0032] 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.
[0033] 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.
[0034] 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.
[0035] 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.
[0036] 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. In preferred embodiments, the
antibody is labeled with a molecule or compound that is useful for
qualitatively and/or quantitatively determining the location and/or
amount of binding of the antibody, oligopeptide or small organic
molecule to the cell.
[0037] 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.
[0038] 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.
[0039] 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
[0040] In yet further embodiments, the invention is directed to the
following:
[0041] An isolated nucleic acid having a nucleotide sequence that
has at least 80% nucleic acid sequence identity to:
[0042] (a) a DNA molecule encoding the amino acid sequence shown as
SEQ ID NO:2;
[0043] (b) a DNA molecule encoding the amino acid sequence shown as
SEQ ID NO:2, lacking its associated signal peptide;
[0044] (c) a DNA molecule encoding an extracellular domain of the
polypeptide shown as SEQ ID NO:2, with its associated signal
peptide;
[0045] (d) a DNA molecule encoding an extracellular domain of the
polypeptide shown as SEQ ID NO:2, lacking its associated signal
peptide;
[0046] (e) the nucleotide sequence shown as SEQ ID NO:1;
[0047] (f) the full-length coding sequence of the nucleotide
sequence shown as SEQ ID NO:1; or
[0048] (g) the complement of (a), (b), (c), (d), (e) or (f).
[0049] An isolated nucleic acid having:
[0050] (a) a nucleotide sequence that encodes the amino acid
sequence shown as SEQ ID NO:2;
[0051] (b) a nucleotide sequence that encodes the amino acid
sequence shown as SEQ ID NO:2, lacking its associated signal
peptide;
[0052] (c) a nucleotide sequence that encodes an extracellular
domain of the polypeptide shown as SEQ ID NO:2, with its associated
signal peptide;
[0053] (d) a nucleotide sequence that encodes an extracellular
domain of the polypeptide shown as SEQ ID NO:2, lacking its
associated signal peptide;
[0054] (e) the nucleotide sequence shown as SEQ ID NO:1;
[0055] (f) the full-length coding region of the nucleotide sequence
shown as SEQ ID NO:1; or
[0056] (g) the complement of (a), (b), (c), (d), (e) or (f).
[0057] An isolated nucleic acid that hybridizes to:
[0058] (a) a nucleic acid that encodes the amino acid sequence
shown as SEQ ID NO:2;
[0059] (b) a nucleic acid that encodes the amino acid sequence
shown as SEQ ID NO:2, lacking its associated signal peptide;
[0060] (c) a nucleic acid that encodes an extracellular domain of
the polypeptide shown as SEQ ID NO:2, with its associated signal
peptide;
[0061] (d) a nucleic acid that encodes an extracellular domain of
the polypeptide shown as SEQ ID NO:2, lacking its associated signal
peptide;
[0062] (e) the nucleotide sequence shown as SEQ ID NO:1;
[0063] (f) the full-length coding region of the nucleotide sequence
shown as SEQ ID NO:1; or
[0064] (g) the complement of (a), (b), (c), (d), (e) or (f).
[0065] In some embodiments, the hybridization occurs under
stringent conditions.
[0066] In some embodiments, the nucleic acid is at least about 5
nucleotides in length.
[0067] The invention also provides an expression vector comprising
the foregoing nucleic acid molecules.
[0068] In some embodiments, of the expression vectors, the nucleic
acid is operably linked to control sequences recognized by a host
cell transformed with the vector.
[0069] The invention also provides host cell comprising such
expression vectors. The host cell may be, for example, a CHO cell,
an E. coli cell or a yeast cell.
[0070] The host cells may be used in a process for producing a
polypeptide comprising culturing the host cell under conditions
suitable for expression of said polypeptide and recovering said
polypeptide from the cell culture.
[0071] The invention also provides an isolated polypeptide having
at least 80% amino acid sequence identity to:
[0072] (a) the polypeptide shown as SEQ ID NO:2;
[0073] (b) the polypeptide shown as SEQ ID NO:2, lacking its
associated signal peptide;
[0074] (c) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, with its associated signal peptide;
[0075] (d) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, lacking its associated signal peptide;
[0076] (e) a polypeptide encoded by the nucleotide sequence shown
as SEQ ID NO:1; or
[0077] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown as SEQ ID NO:1.
[0078] The invention further provides an isolated polypeptide
having:
[0079] (a) the amino acid sequence shown as SEQ ID NO:2;
[0080] (b) the amino acid sequence shown as SEQ ID NO:2, lacking
its associated signal peptide sequence;
[0081] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, with its associated signal
peptide sequence;
[0082] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, lacking its associated signal
peptide sequence;
[0083] (e) an amino acid sequence encoded by the nucleotide
sequence shown as SEQ ID NO:1; or
[0084] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown as SEQ ID NO:1.
[0085] The invention also provides a chimeric polypeptide
comprising a polypeptide fused to a heterologous polypeptide. Such
heterologous polypeptide is an epitope tag sequence or an Fc region
of an immunoglobulin.
[0086] The invention further provides an isolated antibody that
binds to a polypeptide having at least 80% amino acid sequence
identity to:
[0087] (a) the polypeptide shown as SEQ ID NO:2;
[0088] (b) the polypeptide shown as SEQ ID NO:2, lacking its
associated signal peptide;
[0089] (c) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, with its associated signal peptide;
[0090] (d) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, lacking its associated signal peptide;
[0091] (e) a polypeptide encoded by the nucleotide sequence shown
as SEQ ID NO:1; or
[0092] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown as SEQ ID NO:1.
[0093] The invention also provides an isolated antibody that binds
to a polypeptide having:
[0094] (a) the amino acid sequence shown as SEQ ID NO:2;
[0095] (b) the amino acid sequence shown as SEQ ID NO:2, lacking
its associated signal peptide sequence;
[0096] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, with its associated signal
peptide sequence;
[0097] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, lacking its associated signal
peptide sequence;
[0098] (e) an amino acid sequence encoded by the nucleotide
sequence shown as SEQ ID NO:1; or
[0099] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown as SEQ ID NO:1.
[0100] An antibody as set forth in the preceding two paragraphs may
be a monoclonal antibody, an antibody fragment, a chimeric or a
humanized antibody, conjugated to a growth inhibitory agent, or
conjugated to a cytotoxic agent. The cytotoxic agent is selected
from the group consisting of toxins, antibiotics, radioactive
isotopes and nucleolytic enzymes. In some embodiments for example,
the cytotoxic agent is a toxin. In some embodiments, the toxin is
selected from the group consisting of maytansinoid and
calicheamicin. In some embodiments, the toxin is a maytansinoid. In
some embodiments, the antibody is produced in bacteria. In some
embodiments, the antibody is produced in CHO cells. Such an
antibody can induce death of a cell to which it binds. In addition,
the antibodies may be detectably labeled. The invention thus also
provides isolated nucleic acid molecules having nucleotide
sequences that encode such antibodies, expression vectors
comprising the nucleic acid molecules encoding the antibodies
operably linked to control sequences recognized by a host cell
transformed with the vector, and host cells comprising the
expression vectors.
[0101] The host cell may be for example a CHO cell, an E. coli cell
or a yeast cell.
[0102] The invention also provides a process for producing an
antibody comprising culturing the host cell under conditions
suitable for expression of said antibody and recovering said
antibody from the cell culture.
[0103] The invention further provides an isolated oligopeptide that
binds to a polypeptide having at least 80% amino acid sequence
identity to:
[0104] (a) the polypeptide shown as SEQ ID NO:2;
[0105] (b) the polypeptide shown as SEQ ID NO:2, lacking its
associated signal peptide;
[0106] (c) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, with its associated signal peptide;
[0107] (d) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, lacking its associated signal peptide;
[0108] (e) a polypeptide encoded by the nucleotide sequence shown
as SEQ ID NO:1; or
[0109] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown as SEQ ID NO:1.
[0110] The invention also provides an isolated oligopeptide that
binds to a polypeptide having:
[0111] (a) the amino acid sequence shown as SEQ ID NO:2;
[0112] (b) the amino acid sequence shown as SEQ ID NO:2, lacking
its associated signal peptide sequence;
[0113] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, with its associated signal
peptide sequence;
[0114] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, lacking its associated signal
peptide sequence;
[0115] (e) an amino acid sequence encoded by the nucleotide
sequence shown as SEQ ID NO:1; or
[0116] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown as SEQ ID NO:1.
[0117] These oligopeptides may be conjugated to a growth inhibitory
agent or a cytotoxic agent. The cytotoxic agent may be selected
from the group consisting of toxins, antibiotics, radioactive
isotopes and nucleolytic enzymes. In some embodiments, the
cytotoxic agent is a toxin selected from the group consisting of
maytansinoid and calicheamicin. In some embodiments, the toxin is a
maytansinoid. In some embodiments, the oligopeptide induces death
of a cell to which it binds. In some embodiments, the oligopeptide
is detectably labeled.
[0118] The invention also provides a TAT binding organic molecule
that binds to a polypeptide having at least 80% amino acid sequence
identity to:
[0119] (a) the polypeptide shown as SEQ ID NO:2;
[0120] (b) the polypeptide shown as SEQ ID NO:2, lacking its
associated signal peptide;
[0121] (c) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, with its associated signal peptide;
[0122] (d) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, lacking its associated signal peptide;
[0123] (e) a polypeptide encoded by the nucleotide sequence shown
as SEQ ID NO:1; or
[0124] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown as SEQ ID NO:1.
[0125] In some embodiments, the organic molecule binds to a
polypeptide having:
[0126] (a) the amino acid sequence shown as SEQ ID NO:2;
[0127] (b) the amino acid sequence shown as SEQ ID NO:2, lacking
its associated signal peptide sequence;
[0128] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, with its associated signal
peptide sequence;
[0129] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, lacking its associated signal
peptide sequence;
[0130] (e) an amino acid sequence encoded by the nucleotide
sequence shown as SEQ ID NO:1; or
[0131] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown as SEQ ID NO:1.
[0132] In some embodiments, the organic molecule is conjugated to a
growth inhibitory agent, or a cytotoxic agent. The cytotoxic agent
may be selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes. In some embodiments,
the cytotoxic agent is a toxin. In some embodiments, the toxin is
selected from the group consisting of maytansinoid and
calicheamicin. In some embodiments, the toxin is a maytansinoid. In
some embodiments, the organic molecule induces death of a cell to
which it binds. In some embodiments, the organic molecule is
detectably labeled.
[0133] The invention also provides a composition of matter
comprising any foregoing polypeptide, chimeric polypeptide,
antibody, oligopeptide, or TAT binding organic molecule in
combination with a carrier such as a pharmaceutically acceptable
carrier.
[0134] The invention also provides an article of manufacture
comprising:
[0135] (a) a container; and
[0136] (b) the composition of matter of the invention contained
within said container. The article of manufacture may further
comprise 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.
[0137] The invention also provides a method of inhibiting the
growth of a cell that expresses a protein having at least 80% amino
acid sequence identity to:
[0138] (a) the polypeptide shown as SEQ ID NO:2;
[0139] (b) the polypeptide shown as SEQ ID NO:2, lacking its
associated signal peptide;
[0140] (c) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, with its associated signal peptide;
[0141] (d) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, lacking its associated signal peptide;
[0142] (e) a polypeptide encoded by the nucleotide sequence shown
as SEQ ID NO:1; or
[0143] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown as SEQ ID NO:1, 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.
[0144] In some embodiments of this method, the antibody is a
monoclonal antibody, an antibody fragment, a chimeric antibody or a
humanized antibody.
[0145] In some embodiments of the method, the antibody,
oligopeptide or organic molecule is conjugated to a growth
inhibitory agent, or a cytotoxic agent.
[0146] In some embodiments, the cytotoxic agent is selected from
the group consisting of toxins, antibiotics, radioactive isotopes
and nucleolytic enzymes. In some embodiments, the cytotoxic agent
is a toxin. In some embodiments, the toxin is selected from the
group consisting of maytansinoid and calicheamicin. In some
embodiments, the toxin is a maytansinoid.
[0147] In some embodiments, antibody is produced in bacteria. In
other embodiments, the antibody is produced in CHO cells.
[0148] In this method of the invention, the cell is a cancer cell,
such as a cancer cell 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. In
some embodiments, the cancer cell is further exposed to radiation
treatment or a chemotherapeutic agent.
[0149] In this method, the protein is more abundantly expressed by
said cancer cell as compared to a normal cell of the same tissue
origin. In some embodiments, this method causes the death of said
cell.
[0150] In some embodiments of this method, the protein has:
[0151] (a) the amino acid sequence shown as SEQ ID NO:2;
[0152] (b) the amino acid sequence shown as SEQ ID NO:2, lacking
its associated signal peptide sequence;
[0153] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, with its associated signal
peptide sequence;
[0154] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, lacking its associated signal
peptide sequence;
[0155] (e) an amino acid sequence encoded by the nucleotide
sequence shown as SEQ ID NO:1; or
[0156] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown as SEQ ID NO:1.
[0157] The invention further provides 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:
[0158] (a) the polypeptide shown as SEQ ID NO:2;
[0159] (b) the polypeptide shown as SEQ ID NO:2, lacking its
associated signal peptide;
[0160] (c) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, with its associated signal peptide;
[0161] (d) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, lacking its associated signal peptide;
[0162] (e) a polypeptide encoded by the nucleotide sequence shown
as SEQ ID NO:1; or
[0163] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown as SEQ ID NO:1, 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.
[0164] In some embodiments of this method, the protein is a
monoclonal antibody, an antibody fragment, a chimeric antibody or a
humanized antibody. In some embodiments, the antibody, oligopeptide
or organic molecule is conjugated to a growth inhibitory agent or a
cytotoxic agent. In some embodiments, the cytotoxic agent is
selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes. In some embodiments,
the cytotoxic agent is a toxin. In some embodiments, the toxin is
selected from the group consisting of maytansinoid and
calicheamicin. In some embodiments, the toxin is a maytansinoid. In
some embodiments, the antibody is produced in bacteria. In some
embodiments, the antibody is produced in CHO cells. In some
embodiments of this method, the tumor is further exposed to
radiation treatment or a chemotherapeutic agent.
[0165] The tumor may be 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. In
some embodiments, the protein is more abundantly expressed by the
cancerous cells of said tumor as compared to a normal cell of the
same tissue origin.
[0166] In this method, the protein has:
[0167] (a) the amino acid sequence shown as SEQ ID NO:2;
[0168] (b) the amino acid sequence shown as SEQ ID NO:2, lacking
its associated signal peptide sequence;
[0169] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, with its associated signal
peptide sequence;
[0170] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, lacking its associated signal
peptide sequence;
[0171] (e) an amino acid sequence encoded by the nucleotide
sequence shown as SEQ ID NO:1; or
[0172] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown as SEQ ID NO:1.
[0173] The invention further provides 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:
[0174] (a) the polypeptide shown as SEQ ID NO:2;
[0175] (b) the polypeptide shown as SEQ ID NO:2, lacking its
associated signal peptide;
[0176] (c) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, with its associated signal peptide;
[0177] (d) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, lacking its associated signal peptide;
[0178] (e) a polypeptide encoded by the nucleotide sequence shown
as SEQ ID NO:1; or
[0179] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown as SEQ ID NO:1, 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.
[0180] In some embodiments of this method of the invention the
sample comprises a cell suspected of expressing said protein. In
some embodiments, the cell is a cancer cell. In some embodiments,
the antibody, oligopeptide or organic molecule is detectably
labeled.
[0181] In some embodiments, the protein has:
[0182] (a) the amino acid sequence shown as SEQ ID NO:2;
[0183] (b) the amino acid sequence shown as SEQ ID NO:2, lacking
its associated signal peptide sequence;
[0184] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, with its associated signal
peptide sequence;
[0185] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, lacking its associated signal
peptide sequence;
[0186] (e) an amino acid sequence encoded by the nucleotide
sequence shown as SEQ ID NO:1; or
[0187] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown as SEQ ID NO:1.
[0188] The invention also provides 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:
[0189] (a) the polypeptide shown as SEQ ID NO:2;
[0190] (b) the polypeptide shown as SEQ ID NO:2, lacking its
associated signal peptide;
[0191] (c) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, with its associated signal peptide;
[0192] (d) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, lacking its associated signal peptide;
[0193] (e) a polypeptide encoded by the nucleotide sequence shown
as SEQ ID NO:1; or
[0194] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown as SEQ ID NO:1, 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.
[0195] In this method, the step of determining the level of
expression of a gene encoding said protein may comprise employing
an oligonucleotide in an in situ hybridization or RT-PCR analysis
or an antibody in an immunohistochemistry or Western blot
analysis.
[0196] In this method, the protein may have:
[0197] (a) the amino acid sequence shown as SEQ ID NO:2;
[0198] (b) the amino acid sequence shown as SEQ ID NO:2, lacking
its associated signal peptide sequence;
[0199] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, with its associated signal
peptide sequence;
[0200] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, lacking its associated signal
peptide sequence;
[0201] (e) an amino acid sequence encoded by the nucleotide
sequence shown as SEQ ID NO:1; or
[0202] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown as SEQ ID NO:1.
[0203] The invention further provides 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:
[0204] (a) the polypeptide shown as SEQ ID NO:2;
[0205] (b) the polypeptide shown as SEQ ID NO:2, lacking its
associated signal peptide;
[0206] (c) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, with its associated signal peptide;
[0207] (d) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, lacking its associated signal peptide;
[0208] (e) a polypeptide encoded by the nucleotide sequence shown
as SEQ ID NO:1; or
[0209] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown as SEQ ID NO:1, 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.
[0210] In this method, the antibody, oligopeptide or organic
molecule may be detectably labeled. In some embodiments, the test
sample of tissue cells is obtained from an individual suspected of
having a cancerous tumor.
[0211] In some embodiments of this method, the protein has:
[0212] (a) the amino acid sequence shown as SEQ ID NO:2;
[0213] (b) the amino acid sequence shown as SEQ ID NO:2, lacking
its associated signal peptide sequence;
[0214] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, with its associated signal
peptide sequence;
[0215] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, lacking its associated signal
peptide sequence;
[0216] (e) an amino acid sequence encoded by the nucleotide
sequence shown as SEQ ID NO:1; or
[0217] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown as SEQ ID NO:1.
[0218] The invention also provides 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:
[0219] (a) the polypeptide shown as SEQ ID NO:2;
[0220] (b) the polypeptide shown as SEQ ID NO:2, lacking its
associated signal peptide;
[0221] (c) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, with its associated signal peptide;
[0222] (d) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, lacking its associated signal peptide;
[0223] (e) a polypeptide encoded by the nucleotide sequence shown
as SEQ ID NO:1; or
[0224] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown as SEQ ID NO:1, 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.
[0225] In some embodiments, the cell proliferative disorder is
cancer.
[0226] In some embodiments, the antagonist is an anti-TAT
polypeptide antibody, TAT binding oligopeptide, TAT binding organic
molecule or antisense oligonucleotide.
[0227] The invention also provides 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:
[0228] (a) the polypeptide shown as SEQ ID NO:2;
[0229] (b) the polypeptide shown as SEQ ID NO:2, lacking its
associated signal peptide;
[0230] (c) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, with its associated signal peptide;
[0231] (d) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, lacking its associated signal peptide;
[0232] (e) a polypeptide encoded by the nucleotide sequence shown
as SEQ ID NO:1; or
[0233] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown as SEQ ID NO:1, 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.
[0234] In some embodiments, the antibody is a monoclonal antibody.
In some embodiments, the antibody is an antibody fragment. In some
embodiments, the antibody is a chimeric or a humanized antibody. In
some embodiments, the antibody, oligopeptide or organic molecule is
conjugated to a growth inhibitory agent. In some embodiments, the
antibody, oligopeptide or organic molecule is conjugated to a
cytotoxic agent. In some embodiments, the cytotoxic agent is
selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes. In some embodiments,
the cytotoxic agent is a toxin. In some embodiments, the toxin is
selected from the group consisting of maytansinoid and
calicheamicin. In some embodiments, the toxin is a maytansinoid. In
some embodiments, the antibody is produced in bacteria. In some
embodiments, the antibody is produced in CHO cells. In some
embodiments, the cell is a cancer cell. In some embodiments, the
cancer cell is further exposed to radiation treatment or a
chemotherapeutic agent.
[0235] In this method, the cancer cell may be 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.
[0236] In some embodiments, the protein is more abundantly
expressed by said cancer cell as compared to a normal cell of the
same tissue origin. In some embodiments, the method causes the
death of said cell.
[0237] The invention also provides for the use of the foregoing
nucleic acids of the invention in the preparation of a medicament
for the therapeutic treatment or diagnostic detection of a
cancer.
[0238] The invention also provides for the use of the foregoing
nucleic acids of the invention in the preparation of a medicament
for treating a tumor.
[0239] The invention also provides for the use of the foregoing
nucleic acids of the invention in the preparation of a medicament
for treatment or prevention of a cell proliferative disorder.
[0240] The invention also provides for the use of the foregoing
expression vectors of the invention in the preparation of a
medicament for the therapeutic treatment or diagnostic detection of
a cancer.
[0241] The invention also provides for the use of the foregoing
expression vectors of the invention in the preparation of
medicament for treating a tumor.
[0242] The invention also provides for the use of the foregoing
expression vectors of the invention in the preparation of a
medicament for treatment or prevention of a cell proliferative
disorder.
[0243] The invention also provides for the use of the foregoing
host cells of the invention in the preparation of a medicament for
the therapeutic treatment or diagnostic detection of a cancer.
[0244] The invention also provides for the use of the foregoing
host cells of the invention in the preparation of a medicament for
treating a tumor.
[0245] The invention also provides for the use of the foregoing
host cells of the invention in the preparation of a medicament for
treatment or prevention of a cell proliferative disorder.
[0246] The invention also provides for the use of the foregoing
polypeptides of the invention in the preparation of a medicament
for the therapeutic treatment or diagnostic detection of a
cancer.
[0247] The invention also provides for the use of the foregoing
polypeptides of the invention in the preparation of a medicament
for treating a tumor.
[0248] The invention also provides for the use of the foregoing
polypeptides of the invention in the preparation of a medicament
for treatment or prevention of a cell proliferative disorder.
[0249] The invention also provides for the use of the foregoing
antibodies of the invention in the preparation of a medicament for
the therapeutic treatment or diagnostic detection of a cancer. The
invention also provides for the use of the foregoing antibodies of
the invention in the preparation of a medicament for treating a
tumor.
[0250] The invention also provides for the use of the foregoing
antibodies of the invention in the preparation of a medicament for
treatment or prevention of a cell proliferative disorder.
[0251] The invention also provides for the use of the foregoing
oligopeptides of the invention in the preparation of a medicament
for the therapeutic treatment or diagnostic detection of a
cancer.
[0252] The invention also provides for the use of the foregoing
oligopeptides of the invention in the preparation of a medicament
for treating a tumor.
[0253] The invention also provides for the use of the foregoing
oligopeptides of the invention in the preparation of a medicament
for treatment or prevention of a cell proliferative disorder.
[0254] Attorney Docket No. P05040-US-12
[0255] The invention also provides for the use of the foregoing TAT
binding organic molecules of the invention in the preparation of a
medicament for the therapeutic treatment or diagnostic detection of
a cancer.
[0256] The invention also provides for the use of the foregoing TAT
binding organic molecules of the invention in the preparation of a
medicament for treating a tumor.
[0257] The invention also provides for the use of the foregoing TAT
binding organic molecules of the invention in the preparation of a
medicament for treatment or prevention of a cell proliferative
disorder.
[0258] The invention also provides for the use of the foregoing
compositions of the invention of matter in the preparation of a
medicament for the therapeutic treatment or diagnostic detection of
a cancer.
[0259] The invention also provides for the use of the foregoing
compositions of the invention of matter in the preparation of a
medicament for treating a tumor.
[0260] The invention also provides for the use of the foregoing
compositions of the invention of matter in the preparation of a
medicament for treatment or prevention of a cell proliferative
disorder.
[0261] The invention also provides for the use of the foregoing
articles of manufacture of the invention of matter in the
preparation of a medicament for the therapeutic treatment or
diagnostic detection of a cancer.
[0262] The invention also provides for the use of the foregoing
articles of manufacture of the invention of matter in the
preparation of a medicament for treating a tumor.
[0263] The invention also provides for the use of the foregoing
articles of manufacture of the invention of matter in the
preparation of a medicament for treatment or prevention of a cell
proliferative disorder.
[0264] The invention further provides 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:
[0265] (a) the polypeptide shown as SEQ ID NO:2;
[0266] (b) the polypeptide shown as SEQ ID NO:2, lacking its
associated signal peptide;
[0267] (c) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, with its associated signal peptide;
[0268] (d) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, lacking its associated signal peptide;
[0269] (e) a polypeptide encoded by the nucleotide sequence shown
as SEQ ID NO:1; or
[0270] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown as SEQ ID NO:1, 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.
[0271] In some embodiments, the cell is a cancer cell. In some
embodiments, the protein is expressed by said cell. In some
embodiments, the binding of said antibody, oligopeptide or organic
molecule to said protein antagonizes a cell growth-potentiating
activity of said protein. In some embodiments, the binding of said
antibody, oligopeptide or organic molecule to said protein induces
the death of said cell. In some embodiments, the antibody is a
monoclonal antibody, an antibody fragment, a chimeric antibody or a
humanized antibody.
[0272] In some embodiments, the antibody, oligopeptide or organic
molecule is conjugated to a growth inhibitory agent. In some
embodiments, the antibody, oligopeptide or organic molecule is
conjugated to a cytotoxic agent. In some embodiments, the cytotoxic
agent is selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes. In some embodiments,
the cytotoxic agent is a toxin. In some embodiments, the toxin is
selected from the group consisting of maytansinoid and
calicheamicin. In some embodiments, the toxin is a
maytansinoid.
[0273] In some embodiments, the antibody is produced in bacteria.
In some embodiments, the antibody is produced in CHO cells.
[0274] In some embodiments, the protein has:
[0275] (a) the amino acid sequence shown as SEQ ID NO:2;
[0276] (b) the amino acid sequence shown as SEQ ID NO:2, lacking
its associated signal peptide sequence;
[0277] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, with its associated signal
peptide sequence;
[0278] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, lacking its associated signal
peptide sequence;
[0279] (e) an amino acid sequence encoded by the nucleotide
sequence shown as SEQ ID NO:1; or
[0280] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown as SEQ ID NO:1.
[0281] The invention also provides 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:
[0282] (a) the polypeptide shown in as SEQ ID NO:2;
[0283] (b) the polypeptide shown as SEQ ID NO:2, lacking its
associated signal peptide;
[0284] (c) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, with its associated signal peptide;
[0285] (d) an extracellular domain of the polypeptide shown as SEQ
ID NO:2, lacking its associated signal peptide;
[0286] (e) a polypeptide encoded by the nucleotide sequence shown
as SEQ ID NO:1; or
[0287] (f) a polypeptide encoded by the full-length coding region
of the nucleotide sequence shown as SEQ ID NO:1, said method
comprising contacting said protein with an antibody, oligopeptide
or organic molecule that binds to said protein, thereby effectively
treating said tumor.
[0288] In some embodiments of this method, the protein is expressed
by cells of said tumor. In some embodiments, the binding of said
antibody, oligopeptide or organic molecule to said protein
antagonizes a cell growth-potentiating activity of said protein. In
some embodiments, the antibody is a monoclonal antibody. In some
embodiments, the antibody is an antibody fragment. In some
embodiments, the antibody is a chimeric or a humanized antibody. In
some embodiments, the antibody, oligopeptide or organic molecule is
conjugated to a growth inhibitory agent. In some embodiments, the
antibody, oligopeptide or organic molecule is conjugated to a
cytotoxic agent. In some embodiments, the cytotoxic agent is
selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes. In some embodiments,
the cytotoxic agent is a toxin. In some embodiments, the toxin is
selected from the group consisting of maytansinoid and
calicheamicin. In some embodiments, the toxin is a maytansinoid. In
some embodiments, the antibody is produced in bacteria. In some
embodiments, the antibody is produced in CHO cells.
[0289] In some embodiments, the protein has:
[0290] (a) the amino acid sequence shown as SEQ ID NO:2;
[0291] (b) the amino acid sequence shown as SEQ ID NO:2, lacking
its associated signal peptide sequence;
[0292] (c) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, with its associated signal
peptide sequence;
[0293] (d) an amino acid sequence of an extracellular domain of the
polypeptide shown as SEQ ID NO:2, lacking its associated signal
peptide sequence;
[0294] (e) an amino acid sequence encoded by the nucleotide
sequence shown as SEQ ID NO:1; or
[0295] (f) an amino acid sequence encoded by the full-length coding
region of the nucleotide sequence shown as SEQ ID NO:1.
[0296] The invention further provides an isolated antibody that
binds to the same epitope bound by an antibody produced by any of
the hybridoma cell lines shown in Table 11.
[0297] In some embodiments, the antibody is a monoclonal antibody,
an antibody fragment, a chimeric or a humanized antibody. In some
embodiments, the antibody may be conjugated to a growth inhibitory
agent. In some embodiments, the antibody is conjugated to a
cytotoxic agent. In some embodiments, the cytotoxic agent is
selected from the group consisting of toxins, antibiotics,
radioactive isotopes and nucleolytic enzymes. In some embodiments,
the cytotoxic agent is a toxin. In some embodiments, the toxin is
selected from the group consisting of maytansinoid and
calicheamicin. In some embodiments, the toxin is a
maytansinoid.
[0298] In some embodiments, the antibody is produced in bacteria.
In some embodiments, the antibody is produced in CHO cells. In some
embodiments, the antibody induces death of a cell to which it
binds. In some embodiments, the antibody is detectably labeled. In
some embodiments, the antibody comprises at least one of the
complementarity determining regions of any antibody produced by any
of the hybridoma cell lines shown in Table 11.
[0299] The invention also provides a monoclonal antibody produced
by any of the hybridoma cells shown in Table 11.
[0300] The invention also provides a hybridoma cell which produces
a monoclonal antibody that binds to a TAT polypeptide.
[0301] The invention also provides a method of identifying an
antibody that binds to an epitope bound by an antibody produced by
any of the hybridoma cell lines shown in Table 11, said method
comprising determining the ability of a first antibody to block
binding of a second antibody produced by any of the hybridoma cell
lines shown in Table 11 to a TAT polypeptide, wherein the ability
of said first antibody to block the binding of said second antibody
to said TAT polypeptide by at least 40% and at equal antibody
concentrations is indicative of said first antibody being capable
of binding to an epitope bound by said second antibody.
[0302] 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
[0303] FIGS. 1A-E show a nucleotide sequence (SEQ ID NO:1) of a
TAT10772 cDNA, wherein SEQ ID NO:1 is a clone designated herein as
"DNA772".
[0304] FIGS. 2A-B show the amino acid sequence (SEQ ID NO:2)
derived from the coding sequence of SEQ ID NO:1 shown in FIG.
1.
[0305] FIG. 3 shows alignment of amino acid sequences of the
variable light chains for the following: light chain human subgroup
I consensus sequence (huKI; SEQ ID NO:3), murine 11D10
anti-TAT10772 antibody (mu11D10-L; SEQ ID NO:4), and 11D10
anti-TAT10772 grafted "humanized" antibody (11D10-graft; SEQ ID
NO:5).
[0306] FIG. 4 shows alignment of amino acid sequences of the
variable heavy chains for the following: heavy chain human subgroup
III consensus sequence (hum III; SEQ ID NO:6), murine 11D10
anti-TAT10772 antibody (mu11D10-H; SEQ ID NO:7), and 11D10
anti-TAT10772 grafted "humanized" antibody (11D10-graft; SEQ ID
NO:8).
[0307] FIG. 5 shows alignment of amino acid sequences of the
variable light chains for the following: light chain human subgroup
I consensus sequence (huKI; SEQ ID NO:3), murine 3A5 anti-TAT10772
antibody (mu3A5-L; SEQ ID NO:9), and 3A5 anti-TAT10772 grafted
"humanized" antibody (3A5-graft; SEQ ID NO:10).
[0308] FIG. 6 shows alignment of amino acid sequences of the
variable heavy chains for the following: heavy chain human subgroup
III consensus sequence (hum III; SEQ ID NO:6), murine 3A5
anti-TAT10772 antibody (mu3A5-H; SEQ ID NO:11), 3A5 anti-TAT10772
grafted "humanized" antibody "L variant" (3A5.L-graft; SEQ ID
NO:12), and 3A5 anti-TAT10772 grafted "humanized" antibody "F
variant" (3A5.F-graft; SEQ ID NO:13).
[0309] FIG. 7 shows various HVR-L1 sequences (SEQ ID NOS:14-34) of
selected affinity-matured 11D10-derived antibodies.
[0310] FIG. 8 shows various HVR-L2 sequences (SEQ ID NOS:35-58) of
selected affinity-matured 11D10-derived antibodies.
[0311] FIG. 9 shows various HVR-L3 sequences (SEQ ID NOS:59-73) of
selected affinity-matured 11D10-derived antibodies.
[0312] FIG. 10 shows various HVR-H1 sequences (SEQ ID NOS:74-93) of
selected affinity-matured 11D10-derived antibodies.
[0313] FIG. 11 shows various HVR-H2 sequences (SEQ ID NOS:94-112)
of selected affinity-matured 11D10-derived antibodies.
[0314] FIG. 12 shows various HVR-H3 sequences (SEQ ID NOS:113-118)
of selected affinity-matured 11D10-derived antibodies.
[0315] FIG. 13 shows an HVR-L1 sequence (SEQ ID NO:119) of a
selected affinity-matured 3A5-derived antibody.
[0316] FIG. 14 shows various HVR-L2 sequences (SEQ ID NOS:120-121)
of selected affinity-matured 3A5-derived antibodies.
[0317] FIG. 15 shows an HVR-L3 sequence (SEQ ID NO:122) of a
selected affinity-matured 3A5-derived antibody.
[0318] FIG. 16 shows an HVR-H1 sequence (SEQ ID NO:123) of a
selected affinity-matured 3A5-derived antibody.
[0319] FIG. 17 shows various HVR-H2 sequences (SEQ ID NOS:124-127)
of selected affinity-matured 3A5-derived antibodies.
[0320] FIGS. 18A-B show various HVR-H3 sequences (SEQ ID
NOS:128-183) of selected affinity-matured 3A5-derived
antibodies.
[0321] FIG. 19 shows exemplary acceptor human consensus framework
sequences for use in practicing the instant invention with the
sequence identifiers as follows: human VH subgroup I consensus
framework minus Kabat CDRs (SEQ ID NO:184), human VH subgroup I
consensus framework minus extended hypervariable regions (SEQ ID
NOS:185-187), human VH subgroup II consensus framework minus Kabat
CDRs (SEQ ID NO:188), human VH subgroup II consensus framework
minus extended hypervariable regions (SEQ ID NOS:189-191), human VH
subgroup III consensus framework minus Kabat CDRs "L-variant" (SEQ
ID NO:192), and human VH subgroup III consensus framework minus
Kabat CDRs "F-variant" (SEQ ID NO:193).
[0322] FIG. 20 shows exemplary acceptor human consensus framework
sequences for use in practicing the instant invention with the
sequence identifiers as follows: human VL kappa subgroup I
consensus framework minus Kabat CDRs (SEQ ID NO:194), human VL
kappa subgroup II consensus framework minus Kabat CDRs (SEQ ID
NO:195), human VL kappa subgroup III consensus framework minus
Kabat CDRs (SEQ ID NO:196), and human VL kappa subgroup IV
consensus framework minus Kabat CDRs (SEQ ID NO:197).
[0323] FIGS. 21A-B shows the complete variable heavy chain
sequences for the following antibodies: 3A5v1 (SEQ ID NO:198),
3A5v2 (SEQ ID NO:199), 3A5v3 (SEQ ID NO:200), 3A5v4 (SEQ ID
NO:201), 3A5v5 (SEQ ID NO:202), 3A5v6 (SEQ ID NO:203), 3A5v7 (SEQ
ID NO:204), 3A5v8 (SEQ ID NO:205), 3A5v1b.52 (SEQ ID NO:206),
3A5v1b.54 (SEQ ID NO:207), 3A5v4b.52 (SEQ ID NO:208), and 3A5v4b.54
(SEQ ID NO:209). All of these antibodies contain the huKI variable
light chain amino acid sequence of SEQ ID NO:3.
[0324] FIG. 22 shows the complete variable light chain sequences
(SEQ ID NOS:210-211) employed for certain anti-TAT10772 antibodies
described herein.
[0325] FIG. 23 shows the ability of various humanized 3A5
antibodies to inhibit the binding of ruthenium-labeled chimeric 3A5
to a biotinylated 5'-domain TAT10772 polypeptide target. "h2H7 ctr"
is a negative control antibody that does not specifically bind to
TAT10772.
[0326] FIG. 24 shows the ability of various humanized 3A5
antibodies to inhibit the binding of ruthenium-labeled chimeric 3A5
to a biotinylated CA125 polypeptide. "h2H7 ctr" is a negative
control antibody that does not specifically bind to TAT10772.
[0327] FIG. 25 shows the results from an ELISA analysis using
various humanized 3A5 antibodies to measure binding to OVCAR-3
cells. "h2H7 ctr" is a negative control antibody that does not
specifically bind to TAT10772.
[0328] FIG. 26 shows in vitro proliferation of OVCAR-3 cells (which
endogenously express TAT10772 polypeptide) following treatment with
chimeric 11D10-vc-MMAF or chimeric 3A5-vc-MMAF antibodies.
[0329] FIG. 27 shows in vitro proliferation of OVCAR-3 cells (which
endogenously express TAT10772 polypeptide) following treatment with
chimeric 11D10-vc-MMAE or chimeric 3A5-vc-MMAE antibodies.
[0330] FIG. 28 shows in vitro proliferation of OVCAR-3 cells (which
endogenously express TAT10772 polypeptide) following treatment with
chimeric 11D10-MC-MMAF or chimeric 3A5-MC-MMAF antibodies.
[0331] FIG. 29 shows in vitro proliferation of PC3 cells
transfected with a vector allowing those cells to express TAT10772
polypeptide (PC3/A5.3B2) or PC3 cells which do not express TAT10772
polypeptide (PC3/neo) following treatment with chimeric
11D10-vc-MMAF or chimeric 3A5-vc-MMAF antibodies.
[0332] FIG. 30 shows in vitro proliferation of PC3 cells
transfected with a vector allowing those cells to express TAT10772
polypeptide (PC3/A5.3B2) or PC3 cells which do not express TAT10772
polypeptide (PC3/neo) following treatment with chimeric
11D10-vc-PAB-MMAE or chimeric 3A5-vc-PAB-MMAE antibodies.
[0333] FIG. 31 shows in vitro proliferation of PC3 cells
transfected with a vector allowing those cells to express TAT10772
polypeptide (PC3/A5.3B2) or PC3 cells which do not express TAT10772
polypeptide (PC3/neo) following treatment with chimeric
11D10-MC-MMAF or chimeric 3A5-MC-MMAf antibodies.
[0334] FIG. 32 shows in vivo mean tumor volume measurements
(subcutaneous injection model) in PC3/A5.3B2-derived tumors
following treatment with various toxin-conjugated chimeric 3A5
antibodies, control antibodies or vehicle alone.
[0335] FIG. 33 shows in vivo mean tumor volume measurements
(mammary fat pad transplant SCID beige mouse model) in
OVCAR-3-derived tumors following treatment with various
toxin-conjugated chimeric 3A5 antibodies, control antibodies or
vehicle alone.
[0336] FIG. 34 shows in vivo mean tumor volume measurements
(mammary fat pad transplant SCID beige mouse model) in
OVCAR-3-derived tumors following treatment with various
toxin-conjugated chimeric 3A5 antibodies, control antibodies or
vehicle alone.
[0337] FIG. 35 shows in vivo mean tumor volume measurements
(mammary fat pad transplant SCID beige mouse model) in
OVCAR-3-derived tumors following treatment with various
toxin-conjugated chimeric 3A5 antibodies, control antibodies or
vehicle alone.
[0338] FIG. 36 shows in vivo mean tumor volume measurements
(xenograft tumors in nude mice, 10 million cells per mouse) in
PC3/A5.3B2-derived tumors following treatment with various
toxin-conjugated chimeric 3A5 antibodies, control antibodies or
vehicle alone.
[0339] FIG. 37 shows in vivo mean tumor volume measurements
(mammary fat pad transplant SCID beige mouse model) in
OVCAR-3-derived tumors following treatment with various
toxin-conjugated chimeric 3A5 antibodies, control antibodies or
vehicle alone.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0340] 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.
[0341] 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 1 in the
figures may be employed as the starting amino acid residue for the
TAT polypeptides.
[0342] 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.
[0343] 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.
[0344] "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.
[0345] "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.
[0346] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A. As examples of
% amino acid sequence identity calculations using this method,
Tables 2 and 3 demonstrate how to calculate the % amino acid
sequence identity of the amino acid sequence designated "Comparison
Protein" to the amino acid sequence designated "TAT", wherein "TAT"
represents the amino acid sequence of a hypothetical TAT
polypeptide of interest, "Comparison Protein" represents the amino
acid sequence of a polypeptide against which the "TAT" polypeptide
of interest is being compared, and "X, "Y" and "Z" each represent
different hypothetical amino acid residues. Unless specifically
stated otherwise, all % amino acid sequence identity values used
herein are obtained as described in the immediately preceding
paragraph using the ALIGN-2 computer program.
[0347] "TAT variant polynucleotide" or "TAT variant nucleic acid
sequence" means a nucleic acid molecule which encodes a TAT
polypeptide, preferably an active TAT polypeptide, as defined
herein and which has at least about 80% nucleic acid sequence
identity with a nucleotide acid sequence encoding a full-length
native sequence TAT polypeptide sequence as disclosed herein, a
full-length native sequence TAT polypeptide sequence lacking the
signal peptide as disclosed herein, an extracellular domain of a
TAT polypeptide, with or without the signal peptide, as disclosed
herein or any other fragment of a full-length TAT polypeptide
sequence as disclosed herein (such as those encoded by a nucleic
acid that represents only a portion of the complete coding sequence
for a full-length TAT polypeptide). Ordinarily, a TAT variant
polynucleotide will have at least about 80% nucleic acid sequence
identity, alternatively at least about 81%, 82%, 83%, 84%, 85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or
99% nucleic acid sequence identity with a nucleic acid sequence
encoding a full-length native sequence TAT polypeptide sequence as
disclosed herein, a full-length native sequence TAT polypeptide
sequence lacking the signal peptide as disclosed herein, an
extracellular domain of a TAT polypeptide, with or without the
signal sequence, as disclosed herein or any other fragment of a
full-length TAT polypeptide sequence as disclosed herein. Variants
do not encompass the native nucleotide sequence.
[0348] 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.
[0349] "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.
[0350] In situations where ALIGN-2 is employed for nucleic acid
sequence comparisons, the % nucleic acid sequence identity of a
given nucleic acid sequence C to, with, or against a given nucleic
acid sequence D (which can alternatively be phrased as a given
nucleic acid sequence C that has or comprises a certain % nucleic
acid sequence identity to, with, or against a given nucleic acid
sequence D) is calculated as follows:
100 times the fraction W/Z
where W is the number of nucleotides scored as identical matches by
the sequence alignment program ALIGN-2 in that program's alignment
of C and D, and where Z is the total number of nucleotides in D. It
will be appreciated that where the length of nucleic acid sequence
C is not equal to the length of nucleic acid sequence D, the %
nucleic acid sequence identity of C to D will not equal the %
nucleic acid sequence identity of D to C. As examples of % nucleic
acid sequence identity calculations, Tables 4 and 5, demonstrate
how to calculate the % nucleic acid sequence identity of the
nucleic acid sequence designated "Comparison DNA" to the nucleic
acid sequence designated "TAT-DNA", wherein "TAT-DNA" represents a
hypothetical TAT-encoding nucleic acid sequence of interest,
"Comparison DNA" represents the nucleotide sequence of a nucleic
acid molecule against which the "TAT-DNA" nucleic acid molecule of
interest is being compared, and "N", "L" and "V" each represent
different hypothetical nucleotides. Unless specifically stated
otherwise, all % nucleic acid sequence identity values used herein
are obtained as described in the immediately preceding paragraph
using the ALIGN-2 computer program.
[0351] 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.
[0352] 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).
[0353] "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.
[0354] 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.
[0355] 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.
[0356] 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.
[0357] "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).
[0358] "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 EC; (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 EC; 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
EC, with a 10 minute wash at 42 EC in 0.2.times.SSC (sodium
chloride/sodium citrate) followed by a 10 minute high-stringency
wash consisting of 0.1.times.SSC containing EDTA at 55 EC.
[0359] "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 EC 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 EC.
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.
[0360] 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).
[0361] "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.
[0362] 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.
[0363] "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.
[0364] 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).
[0365] "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.
[0366] "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.
[0367] Administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order.
[0368] "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..
[0369] 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.
[0370] 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.
[0371] A "small" molecule or "small" organic molecule is defined
herein to have a molecular weight below about 500 Daltons.
[0372] 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.
[0373] 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.
[0374] 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.
[0375] 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.
[0376] 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.
[0377] 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.
[0378] 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, CT,
1994, page 71 and Chapter 6.
[0379] 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.
[0380] The term "variable" refers to the fact that certain segments
of the variable domains differ extensively in sequence among
antibodies. The V domain mediates antigen binding and define
specificity of a particular antibody for its particular antigen.
However, the variability is not evenly distributed across the
110-amino acid span of the variable domains. Instead, the V regions
consist of relatively invariant stretches called framework regions
(FRs) of 15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-12 amino
acids long. The variable domains of native heavy and light chains
each comprise four FRs, largely adopting a .beta.-sheet
configuration, connected by three hypervariable regions, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The hypervariable regions in each chain are
held together in close proximity by the FRs and, with the
hypervariable regions from the other chain, contribute to the
formation of the antigen-binding site of antibodies (see Kabat et
al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991)). The constant domains are not involved directly in binding
an antibody to an antigen, but exhibit various effector functions,
such as participation of the antibody in antibody dependent
cellular cytotoxicity (ADCC).
[0381] 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.
[0382] 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.
[0383] 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.
[0384] "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.
[0385] 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.
[0386] 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.
[0387] "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.
[0388] "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, N.Y., pp. 269-315
(1994); Borrebaeck 1995, infra.
[0389] 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).
[0390] "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).
[0391] 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.-7M, 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.
[0392] The term "variable domain residue numbering as in Kabat" or
"amino acid position numbering as in Kabat", and variations
thereof, refers to the numbering system used for heavy chain
variable domains or light chain variable domains of the compilation
of antibodies in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991). Using this numbering
system, the actual linear amino acid sequence may contain fewer or
additional amino acids corresponding to a shortening of, or
insertion into, a FR or CDR of the variable domain. For example, a
heavy chain variable domain may include a single amino acid insert
(residue 52a according to Kabat) after residue 52 of H2 and
inserted residues (e.g. residues 82a, 82b, and 82c, etc according
to Kabat) after heavy chain FR residue 82. The Kabat numbering of
residues may be determined for a given antibody by alignment at
regions of homology of the sequence of the antibody with a
"standard" Kabat numbered sequence.
[0393] The phrase "substantially similar," or "substantially the
same", as used herein, denotes a sufficiently high degree of
similarity between two numeric values (generally one associated
with an antibody of the invention and the other associated with a
reference/comparator antibody) such that one of skill in the art
would consider the difference between the two values to be of
little or no biological and/or statistical significance within the
context of the biological characteristic measured by said values
(e.g., Kd values). The difference between said two values is
preferably less than about 50%, preferably less than about 40%,
preferably less than about 30%, preferably less than about 20%,
preferably less than about 10% as a function of the value for the
reference/comparator antibody.
[0394] "Binding affinity" generally refers to the strength of the
sum total of noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Low-affinity antibodies generally
bind antigen slowly and tend to dissociate readily, whereas
high-affinity antibodies generally bind antigen faster and tend to
remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used for
purposes of the present invention. Specific illustrative
embodiments are described in the following.
[0395] In one embodiment, the "Kd" or "Kd value" according to this
invention is measured by a radiolabeled antigen binding assay (RIA)
performed with the Fab version of an antibody of interest and its
antigen as described by the following assay that measures solution
binding affinity of Fabs for antigen by equilibrating Fab with a
minimal concentration of (.sup.125I)-labeled antigen in the
presence of a titration series of unlabeled antigen, then capturing
bound antigen with an anti-Fab antibody-coated plate (Chen, et al.,
(1999) J. Mol Biol 293:865-881). To establish conditions for the
assay, microtiter plates (Dynex) are coated overnight with 5 ug/ml
of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium
carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine
serum albumin in PBS for two to five hours at room temperature
(approximately 23.degree. C.). In a non-adsorbant plate (Nunc
#269620), 100 pM or 26 pM [.sup.125I]-antigen are mixed with serial
dilutions of a Fab of interest (e.g., consistent with assessment of
an anti-VEGF antibody, Fab-12, in Presta et al., (1997) Cancer Res.
57:4593-4599). The Fab of interest is then incubated overnight;
however, the incubation may continue for a longer period (e.g., 65
hours) to insure that equilibrium is reached. Thereafter, the
mixtures are transferred to the capture plate for incubation at
room temperature (e.g., for one hour). The solution is then removed
and the plate washed eight times with 0.1% Tween-20 in PBS. When
the plates have dried, 150 ul/well of scintillant (MicroScint-20;
Packard) is added, and the plates are counted on a Topcount gamma
counter (Packard) for ten minutes. Concentrations of each Fab that
give less than or equal to 20% of maximal binding are chosen for
use in competitive binding assays. According to another embodiment
the Kd or Kd value is measured by using surface plasmon resonance
assays using a BIAcore.TM.-2000 or a BIAcore.TM.-3000 (BIAcore,
Inc., Piscataway, N.J.) at 25.degree. C. with immobilized antigen
CM5 chips at .about.10 response units (RU). Briefly,
carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.) are
activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the
supplier's instructions. Antigen is diluted with 10mM sodium
acetate, pH 4.8, into 5 ug/ml (.about.0.2uM) before injection at a
flow rate of 5 ul/minute to achieve approximately 10 response units
(RU) of coupled protein. Following the injection of antigen, 1M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% Tween 20 (PBST) at 25.degree. C. at
a flow rate of approximately 25 ul/min. Association rates
(k.sub.on) and dissociation rates (k.sub.off) are calculated using
a simple one-to-one Langmuir binding model (BIAcore Evaluation
Software version 3.2) by simultaneous fitting the association and
dissociation sensorgram. The equilibrium dissociation constant (Kd)
is calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen, Y.,
et al., (1999) J. Mol Biol 293:865-881. If the on-rate exceeds
10.sup.6M.sup.-1 S.sup.-1 by the surface plasmon resonance assay
above, then the on-rate can be determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340
nm, 16 nm band-pass) at 25.degree. C. of a 20 nM anti-antigen
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as measured in a spectrometer, such as a
stop-flow equipped spectrophometer (Aviv Instruments) or a
8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a
stir red cuvette.
[0396] An "on-rate" or "rate of association" or "association rate"
or "k.sub.on" according to this invention can also be determined
with the same surface plasmon resonance technique described above
using a BIAcore.TM.-2000 or a BIAcore.TM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) at 25.degree. C. with immobilized antigen CMS
chips at .about.10 response units (RU). Briefly, carboxymethylated
dextran biosensor chips (CMS, BIAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NETS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
into 5 ug/ml (.about.0.2 uM) before injection at a flow rate of 5
ul/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of 1M ethanolamine to
block unreacted groups. For kinetics measurements, two-fold serial
dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%
Tween 20 (PBST) at 25.degree. C. at a flow rate of approximately 25
ul/min. Association rates (k.sub.on) and dissociation rates
(k.sub.off) are calculated using a simple one-to-one Langmuir
binding model (BIAcore Evaluation Software version 3.2) by
simultaneous fitting the association and dissociation sensorgram.
The equilibrium dissociation constant (Kd) was calculated as the
ratio koff/kon. See, e.g., Chen, Y., et al., (1999) J. Mol Biol
293:865-881. However, if the on-rate exceeds 10.sup.6 M.sup.-1
S.sup.-1 by the surface plasmon resonance assay above, then the
on-rate is preferably determined by using a fluorescent quenching
technique that measures the increase or decrease in fluorescence
emission intensity (excitation=295 nm; emission=340 nm, 16 nm
band-pass) at 25.degree. C. of a 20 nM anti-antigen antibody (Fab
form) in PBS, pH 7.2, in the presence of increasing concentrations
of antigen as measured in a spectrometer, such as a stop-flow
equipped spectrophometer (Aviv Instruments) or a 8000-series
SLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred
cuvette. The "Kd" or "Kd value" according to this invention is in
one embodiment measured by a radiolabeled antigen binding assay
(RIA) performed with the Fab version of the antibody and antigen
molecule as described by the following assay that measures solution
binding affinity of Fabs for antigen by equilibrating Fab with a
minimal concentration of (.sup.125I)-labeled antigen in the
presence of a titration series of unlabeled antigen, then capturing
bound antigen with an anti-Fab antibody-coated plate (Chen, et al.,
(1999) J. Mol Biol 293:865-881). To establish conditions for the
assay, microtiter plates (Dynex) are coated overnight with 5 ug/ml
of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium
carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine
serum albumin in PBS for two to five hours at room temperature
(approximately 23t). In a non-adsorbant plate (Nunc #269620), 100
pM or 26 pM [.sup.125I]-antigen are mixed with serial dilutions of
a Fab of interest (consistent with assessment of an anti-VEGF
antibody, Fab-12, in Presta et al., (1997) Cancer Res.
57:4593-4599). The Fab of interest is then incubated overnight;
however, the incubation may continue for a longer period (e.g., 65
hours) to insure that equilibrium is reached. Thereafter, the
mixtures are transferred to the capture plate for incubation at
room temperature for one hour. The solution is then removed and the
plate washed eight times with 0.1% Tween-20 in PBS. When the plates
have dried, 150 ul/well of scintillant (MicroScint-20; Packard) is
added, and the plates are counted on a Topcount gamma counter
(Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of maximal binding are chosen for use in
competitive binding assays. According to another embodiment, the Kd
or Kd value is measured by using surface plasmon resonance assays
using a BIAcore.TM.-2000 or a BIAcore.TM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) at 25.degree. C. with immobilized antigen CM5
chips at .about.10 response units (RU). Briefly, carboxymethylated
dextran biosensor chips (CMS, BIAcore Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10mM sodium acetate, pH 4.8,
into 5 ug/ml (.about.0.2uM) before injection at a flow rate of 5
ul/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% Tween 20 (PBST) at 25.degree. C. at
a flow rate of approximately 25 ul/min. Association rates
(k.sub.on) and dissociation rates (k.sub.off) are calculated using
a simple one-to-one Langmuir binding model (BIAcore Evaluation
Software version 3.2) by simultaneous fitting the association and
dissociation sensorgram. The equilibrium dissociation constant (Kd)
is calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen, Y.,
et al., (1999) J. Mol Biol 293:865-881. If the on-rate exceeds
10.sup.6 M.sup.-1 S.sup.-1 by the surface plasmon resonance assay
above, then the on-rate can be determined by using a fluorescent
quenching technique that measures the increase or decrease in
fluorescence emission intensity (excitation=295 nm; emission=340
nm, 16 nm band-pass) at 25.degree. C. of a 20 nM anti-antigen
antibody (Fab form) in PBS, pH 7.2, in the presence of increasing
concentrations of antigen as measured in a spectrometer, such as a
stop-flow equipped spectrophometer (Aviv Instruments) or a
8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with a
stir red cuvette.
[0397] In one embodiment, an "on-rate" or "rate of association" or
"association rate" or "kon" according to this invention is
determined with the same surface plasmon resonance technique
described above using a BIAcore.TM.-2000 or a BIAcore.TM.-3000
(BIAcore, Inc., Piscataway, N.J.) at 25.degree. C. with immobilized
antigen CMS chips at .about.10 response units (RU). Briefly,
carboxymethylated dextran biosensor chips (CMS, BIAcore Inc.) are
activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NETS) according to
the supplier's instructions. Antigen is diluted with 10mM sodium
acetate, pH 4.8, into 5 ug/ml (.about.0.2 uM) before injection at a
flow rate of 5 ul/minute to achieve approximately 10 response units
(RU) of coupled protein. Following the injection of 1M ethanolamine
to block unreacted groups. For kinetics measurements, two-fold
serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS
with 0.05% Tween 20 (PBST) at 25.degree. C. at a flow rate of
approximately 25 ul/min. Association rates (k.sub.on) and
dissociation rates (k.sub.off) are calculated using a simple
one-to-one Langmuir binding model (BIAcore Evaluation Software
version 3.2) by simultaneous fitting the association and
dissociation sensorgram. The equilibrium dissociation constant (Kd)
was calculated as the ratio k.sub.off/k.sub.on. See, e.g., Chen,
Y., et al., (1999) J. Mol Biol 293:865-881. However, if the on-rate
exceeds 10.sup.6 M.sup.-1 S.sup.-1 by the surface plasmon resonance
assay above, then the on-rate is preferably determined by using a
fluorescent quenching technique that measures the increase or
decrease in fluorescence emission intensity (excitation=295 nm;
emission=340 nm, 16 nm band-pass) at 25.degree. C. of a 20 nM
anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of
increasing concentrations of antigen as measured in a spectrometer,
such as a stop-flow equipped spectrophometer (Aviv Instruments) or
a 8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with
a stirred cuvette.
[0398] The phrase "substantially reduced," or "substantially
different", as used herein, denotes a sufficiently high degree of
difference between two numeric values (generally one associated
with an antibody of the invention and the other associated with a
reference/comparator antibody) such that one of skill in the art
would consider the difference between the two values to be of
statistical significance within the context of the biological
characteristic measured by said values (e.g., Kd values, HAMA
response). The difference between said two values is preferably
greater than about 10%, preferably greater than about 20%,
preferably greater than about 30%, preferably greater than about
40%, preferably greater than about 50% as a function of the value
for the reference/comparator antibody.
[0399] An "antigen" is a predetermined antigen to which an antibody
can selectively bind. The target antigen may be polypeptide,
carbohydrate, nucleic acid, lipid, hapten or other naturally
occurring or synthetic compound. Preferably, the target antigen is
a polypeptide. An "acceptor human framework" for the purposes
herein is a framework comprising the amino acid sequence of a VL or
VH framework derived from a human immunoglobulin framework, or from
a human consensus framework. An acceptor human framework "derived
from" a human immunoglobulin framework or human consensus framework
may comprise the same amino acid sequence thereof, or may contain
pre-existing amino acid sequence changes. Where pre-existing amino
acid changes are present, preferably no more than 5 and preferably
4 or less, or 3 or less, pre-existing amino acid changes are
present. Where pre-existing amino acid changes are present in a VH,
preferably those changes are only at three, two or one of positions
71H, 73H and 78H; for instance, the amino acid residues at those
positions may be 71A, 73T and/or 78A. In one embodiment, the VL
acceptor human framework is identical in sequence to the VL human
immunoglobulin framework sequence or human consensus framework
sequence.
[0400] Antibodies of the present invention may be able to compete
for binding to the same epitope as is bound by a second antibody.
Monoclonal antibodies are considered to share the "same epitope" if
each blocks binding of the other by 40% or greater at the same
antibody concentration in a standard in vitro antibody competition
binding analysis.
[0401] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residue in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al.
[0402] A "VH subgroup III consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable heavy subgroup III of Kabat et al.
[0403] A "VL subgroup I consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable light kappa subgroup I of Kabat et al.
[0404] An "unmodified human framework" is a human framework which
has the same amino acid sequence as the acceptor human framework,
e.g. lacking human to non-human amino acid substitution(s) in the
acceptor human framework.
[0405] An "altered hypervariable region" for the purposes herein is
a hypervariable region comprising one or more (e.g. one to about
16) amino acid substitution(s) therein.
[0406] An "un-modified hypervariable region" for the purposes
herein is a hypervariable region having the same amino acid
sequence as a non-human antibody from which it was derived, i.e.
one which lacks one or more amino acid substitutions therein.
[0407] The term "hypervariable region", "HVR", or "HV", when used
herein refers to the regions of an antibody variable domain which
are hypervariable in sequence and/or form structurally defined
loops. Generally, antibodies comprise six hypervariable regions;
three in the VH (H1, H2, H3), and three in the VL (L1, L2, L3). A
number of hypervariable region delineations are in use and are
encompassed herein. The Kabat Complementarity Determining Regions
(CDRs) are based on sequence variability and are the most commonly
used (Kabat et al., Sequences of Proteins of Immunological
Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md. (1991)). Chothia refers instead to the
location of the structural loops (Chothia and Lesk J. Mol. Biol.
196:901-917 (1987)). The "contact" hypervariable regions are based
on an analysis of the available complex crystal structures. The
residues from each of these hypervariable regions are noted below.
Unless otherwise denoted, Kabat numbering will be employed.
Hypervariable region locations are generally as follows: amino
acids 24-34 (HVR-L1), amino acids 49-56 (HVR-L2), amino acids 89-97
(HVR-L3), amino acids 26-35A (HVR-H1), amino acids 49-65 (HVR-H2),
and amino acids 93-102 (HVR-H3).
[0408] Hypervariable regions may also comprise "extended
hypervariable regions" as follows: amino acids 24-36 (L1), and
amino acids 46-56 (L2) in the VL. The variable domain residues are
numbered according to Kabat et al., supra for each of these
definitions.
[0409] "Framework" or "FR" residues are those variable domain
residues other than the hypervariable region residues as herein
defined.
[0410] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues.
[0411] An "affinity matured" antibody is one with one or more
alterations in one or more CDRs thereof which result in an
improvement in the affinity of the antibody for antigen, compared
to a parent antibody which does not possess those alteration(s).
Preferred affinity matured antibodies will have nanomolar or even
picomolar affinities for the target antigen.
[0412] Affinity matured antibodies are produced by procedures known
in the art. Marks et al. Bio/Technology 10:779-783 (1992) describes
affinity maturation by VH and VL domain shuffling. Random
mutagenesis of CDR and/or framework residues is described by:
Barbas et al. Proc Nat. Acad. Sci, USA 91:3809-3813 (1994); Schier
et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol.
155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9
(1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).
[0413] A "blocking" antibody or an "antagonist" antibody is one
which inhibits or reduces biological activity of the antigen it
bind. Preferred blocking antibodies or antagonist antibodies
substantially or completely inhibit the biological activity of the
antigen.
[0414] 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 W084/03564; 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).
[0415] 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).
[0416] 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.-10 M,
alternatively at least about 10.sup.-11 M, alternatively at least
about 10.sup.-12 M, or greater. In one embodiment, the term
"specific binding" refers to binding where a molecule binds to a
particular polypeptide or epitope on a particular polypeptide
without substantially binding to any other polypeptide or
polypeptide epitope.
[0417] 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.
[0418] An antibody, oligopeptide or other organic molecule which
"induces apoptosis" is one which induces programmed cell death as
determined by binding of annexin V, fragmentation of DNA, cell
shrinkage, dilation of endoplasmic reticulum, cell fragmentation,
and/or formation of membrane vesicles (called apoptotic bodies).
The cell is usually one which overexpresses a TAT polypeptide.
Preferably the cell is a tumor cell, e.g., a prostate, breast,
ovarian, stomach, endometrial, lung, kidney, colon, bladder cell.
Various methods are available for evaluating the cellular events
associated with apoptosis. For example, phosphatidyl serine (PS)
translocation can be measured by annexin binding; DNA fragmentation
can be evaluated through DNA laddering; and nuclear/chromatin
condensation along with DNA fragmentation can be evaluated by any
increase in hypodiploid cells. Preferably, the antibody,
oligopeptide or other organic molecule which induces apoptosis is
one which results in about 2 to 50 fold, preferably about 5 to 50
fold, and most preferably about 10 to 50 fold, induction of annexin
binding relative to untreated cell in an annexin binding assay.
[0419] 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.
[0420] "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).
[0421] "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)).
[0422] "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.
[0423] "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.
[0424] 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.
[0425] 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.
[0426] "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.
[0427] 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.
[0428] A "TAT-expressing cell" is a cell which expresses an
endogenous or transfected TAT polypeptide either on the cell
surface or in a secreted form. A "TAT-expressing cancer" is a
cancer comprising cells that have a TAT polypeptide present on the
cell surface or that produce and secrete a TAT polypeptide. A
"TAT-expressing cancer" optionally produces sufficient levels of
TAT polypeptide on the surface of cells thereof, such that an
anti-TAT antibody, oligopeptide or other organic molecule can bind
thereto and have a therapeutic effect with respect to the cancer.
In another embodiment, a "TAT-expressing cancer" optionally
produces and secretes sufficient levels of TAT polypeptide, such
that an anti-TAT antibody, oligopeptide or other organic molecule
antagonist can bind thereto and have a therapeutic effect with
respect to the cancer. With regard to the latter, the antagonist
may be an antisense oligonucleotide which reduces, inhibits or
prevents production and secretion of the secreted TAT polypeptide
by tumor cells. A cancer which "overexpresses" a TAT polypeptide is
one which has significantly higher levels of TAT polypeptide at the
cell surface thereof, or produces and secretes, compared to a
noncancerous cell of the same tissue type. Such overexpression may
be caused by gene amplification or by increased transcription or
translation. TAT polypeptide overexpression may be determined in a
diagnostic or prognostic assay by evaluating increased levels of
the TAT protein present on the surface of a cell, or secreted by
the cell (e.g., via an immunohistochemistry assay using anti-TAT
antibodies prepared against an isolated TAT polypeptide which may
be prepared using recombinant DNA technology from an isolated
nucleic acid encoding the TAT polypeptide; FACS analysis, etc.).
Alternatively, or additionally, one may measure levels of TAT
polypeptide-encoding nucleic acid or mRNA in the cell, e.g., via
fluorescent in situ hybridization using a nucleic acid based probe
corresponding to a TAT-encoding nucleic acid or the complement
thereof; (FISH; see WO98/45479 published October, 1998), Southern
blotting, Northern blotting, or polymerase chain reaction (PCR)
techniques, such as real time quantitative PCR (RT-PCR). One may
also study TAT polypeptide overexpression by measuring shed antigen
in a biological fluid such as serum, e.g, using antibody-based
assays (see also, e.g., U.S. Pat. No. 4,933,294 issued Jun. 12,
1990; WO91/05264 published Apr. 18, 1991; U.S. Pat. No. 5,401,638
issued Mar. 28, 1995; and Sias et al., J. Immunol. Methods
132:73-80 (1990)). Aside from the above assays, various in vivo
assays are available to the skilled practitioner. For example, one
may expose cells within the body of the patient to an antibody
which is optionally labeled with a detectable label, e.g., a
radioactive isotope, and binding of the antibody to cells in the
patient can be evaluated, e.g., by external scanning for
radioactivity or by analyzing a biopsy taken from a patient
previously exposed to the antibody.
[0429] 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.
[0430] 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.
[0431] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g., At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents, 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.
[0432] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include alkylating agents such as thiotepa and CYTOXAN.RTM.
cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan
and piposulfan; aziridines such as benzodopa, carboquone,
meturedopa, and uredopa; ethylenimines and methylamelamines
including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); delta-9-tetrahydrocannabinol (dronabinol,
MARINOL.RTM.); beta-lapachone; lapachol; colchicines; betulinic
acid; a camptothecin (including the synthetic analogue topotecan
(HYCAMTIN.RTM.), CPT-11 (irinotecan, CAMPTOSAR.RTM.),
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
bryostatin; callystatin; CC-1065 (including its adozelesin,
carzelesin and bizelesin synthetic analogues); podophyllotoxin;
podophyllinic acid; teniposide; cryptophycins (particularly
cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin
(including the synthetic analogues, KW-2189 and CB1-TM1);
eleutherobin; pancratistatin; a sarcodictyin; spongistatin;
nitrogen mustards such as chlorambucil, chlornaphazine,
cholophosphamide, estramustine, ifosfamide, mechlorethamine,
mechlorethamine oxide hydrochloride, melphalan, novembichin,
phenesterine, prednimustine, trofosfamide, uracil mustard;
nitrosoureas such as carmustine, chlorozotocin, fotemustine,
lomustine, nimustine, and ranimnustine; antibiotics such as the
enediyne antibiotics (e. g., calicheamicin, especially
calicheamicin gammaI1 and calicheamicin omegaI1 (see, e.g., Agnew,
Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; an esperamicin; as well as neocarzinostatin
chromophore and related chromoprotein enediyne antibiotic
chromophores), aclacinomysins, actinomycin, authramycin, azaserine,
bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin,
chromomycinis, dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, ADRIAMYCIN.RTM. doxorubicin (including
morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalamycin, olivomycins, peplomycin,
porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin,
streptozocin, tubericidin, ubenimex, zinostatin, zorubicin;
anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);
folic acid analogues such as denopterin, methotrexate, pteropterin,
trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine,
thiamiprine, thioguanine; pyrimidine analogs such as ancitabine,
azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine,
doxifluridine, enocitabine, floxuridine; androgens such as
calusterone, dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elfornithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide;
procarbazine; PSK.RTM. polysaccharide complex (JHS Natural
Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran;
spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine
(ELDISINE.RTM., FILDESIN.RTM.); dacarbazine; mannomustine;
mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside
("Ara-C"); thiotepa; taxoids, e.g., TAXOL.RTM. paclitaxel
(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE.RTM.
Cremophor-free, albumin-engineered nanoparticle formulation of
paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),
and TAXOTERE.RTM. docetaxel (Rhone-Poulenc Rorer, Antony, France);
chlorambucil; gemcitabine (GEMZAR.RTM.); 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine (VELBAN.RTM.); platinum; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine (ONCOVIN.RTM.);
oxaliplatin; leucovovin; vinorelbine (NAVELBINE.RTM.); novantrone;
edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase
inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such
as retinoic acid; capecitabine (XELODA.RTM.); pharmaceutically
acceptable salts, acids or derivatives of any of the above; as well
as combinations of two or more of the above such as CHOP, an
abbreviation for a combined therapy of cyclophosphamide,
doxorubicin, vincristine, and prednisolone, and FOLFOX, an
abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovovin.
[0433] Also included in this definition are anti-hormonal agents
that act to regulate, reduce, block, or inhibit the effects of
hormones that can promote the growth of cancer, and are often in
the form of systemic, or whole-body treatment. They may be hormones
themselves. Examples include anti-estrogens and selective estrogen
receptor modulators (SERMs), including, for example, tamoxifen
(including NOLVADEX.RTM. tamoxifen), EVISTA.RTM. raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and FARESTON.RTM. toremifene; anti-progesterones;
estrogen receptor down-regulators (ERDs); agents that function to
suppress or shut down the ovaries, for example, leutinizing
hormone-releasing hormone (LHRH) agonists such as LUPRON.RTM. and
ELIGARD.RTM. leuprolide acetate, goserelin acetate, buserelin
acetate and tripterelin; other anti-androgens such as flutamide,
nilutamide and bicalutamide; and aromatase inhibitors that inhibit
the enzyme aromatase, which regulates estrogen production in the
adrenal glands, such as, for example, 4(5)-imidazoles,
aminoglutethimide, MEGASE.RTM. megestrol acetate, AROMASIN.RTM.
exemestane, formestanie, fadrozole, RIVISOR.RTM. vorozole,
FEMARA.RTM. letrozole, and ARIMIDEX.RTM. anastrozole. In addition,
such definition of chemotherapeutic agents includes bisphosphonates
such as clodronate (for example, BONEFOS.RTM. or OSTAC.RTM.),
DIDROCAL.RTM. etidronate, NE-58095, ZOMETA.RTM. zoledronic
acid/zoledronate, FOSAMAX.RTM. alendronate, AREDIA.RTM.
pamidronate, SKELID.RTM. tiludronate, or ACTONEL.RTM. risedronate;
as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine
analog); antisense oligonucleotides, particularly those that
inhibit expression of genes in signaling pathways implicated in
abherant cell proliferation, such as, for example, PKC-alpha, Raf,
H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such
as THERATOPE.RTM. vaccine and gene therapy vaccines, for example,
ALLOVECTIN.RTM. vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM.
vaccine; LURTOTECAN.RTM. topoisomerase 1 inhibitor; ABARELIX.RTM.
rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosine kinase
small-molecule inhibitor also known as GW572016); and
pharmaceutically acceptable salts, acids or derivatives of any of
the above.
[0434] 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.
[0435] "Doxorubicin" is an anthracycline antibiotic. The full
chemical name of doxorubicin is
(8S-cis)-10-[(3-amino-2,3,6-trideoxy-.alpha.-L-lyxo-hexapyranosyl)oxy]-7,-
8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-napht-
hacenedione.
[0436] 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.
[0437] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications and/or warnings
concerning the use of such therapeutic products.
TABLE-US-00001 TABLE 2 TAT XXXXXXXXXXXXXXX (Length = 15 amino
acids) Comparison XXXXXYYYYYYY (Length = 12 amino acids) Protein %
amino acid sequence identity = (the number of identically matching
amino acid residues between the two polypeptide sequences as
determined by ALIGN-2) divided by (the total number of amino acid
residues of the TAT polypeptide) = 5 divided by 15 = 33.3%
TABLE-US-00002 TABLE 3 TAT XXXXXXXXXX (Length = 10 amino acids)
Comparison XXXXXYYYYYYZZYZ (Length = 15 amino acids) Protein %
amino acid sequence identity = (the number of identically matching
amino acid residues between the two polypeptide sequences as
determined by ALIGN-2) divided by (the total number of amino acid
residues of the TAT polypeptide) = 5 divided by 10 = 50%
TABLE-US-00003 TABLE 4 TAT-DNA NNNNNNNNNNNNNN (Length = 14
nucleotides) Comparison NNNNNNLLLLLLLLLL (Length = 16 nucleotides)
DNA % nucleic acid sequence identity = (the number of identically
matching nucleotides between the two nucleic acid sequences as
determined by ALIGN-2) divided by (the total number of nucleotides
of the TAT-DNA nucleic acid sequence) = 6 divided by 14 = 42.9%
TABLE-US-00004 TABLE 5 TAT-DNA NNNNNNNNNNNN (Length = 12
nucleotides) Comparison 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%
II. Compositions and Methods of the Invention
[0438] A. Anti-TAT Antibodies
[0439] 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.
[0440] 1. Polyclonal Antibodies
[0441] 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.
[0442] Animals are immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later, the
animals are boosted with 1/5 to 1/10 the original amount of peptide
or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later, the animals
are bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Conjugates also can be made in
recombinant cell culture as protein fusions. Also, aggregating
agents such as alum are suitably used to enhance the immune
response.
[0443] 2. Monoclonal Antibodies
[0444] 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).
[0445] In the hybridoma method, a mouse or other appropriate host
animal, such as a hamster, is immunized as described above to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the protein used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
After immunization, lymphocytes are isolated and then fused with a
myeloma cell line using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal
Antibodies: Principles and Practice, pp.59-103 (Academic Press,
1986)).
[0446] 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.
[0447] 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)).
[0448] 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).
[0449] 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).
[0450] 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.
[0451] 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.
[0452] 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).
[0453] 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.
[0454] 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 CO 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.
[0455] 3. Human and Humanized Antibodies
[0456] 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)].
[0457] 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.
[0458] 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)).
[0459] 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.
[0460] 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.
[0461] As an alternative to humanization, human antibodies can be
generated. For example, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array into such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggemann et al., Year in Immuno. 7:33
(1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669 (all of
GenPharm); 5,545,807; and WO 97/17852.
[0462] 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.
[0463] 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).
[0464] 4. Antibody Fragments
[0465] 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.
[0466] Various techniques have been developed for the production of
antibody fragments. Traditionally, these fragments were derived via
proteolytic digestion of intact antibodies (see, e.g., Morimoto et
al., Journal of Biochemical and Biophysical Methods 24:107-117
(1992); and Brennan et al., Science, 229:81 (1985)). However, these
fragments can now be produced directly by recombinant host cells.
Fab, Fv and ScFv antibody fragments can all be expressed in and
secreted from E. coli, thus allowing the facile production of large
amounts of these fragments. Antibody fragments can be isolated from
the antibody phage libraries discussed above. Alternatively,
Fab'-SH fragments can be directly recovered from E. coli and
chemically coupled to form F(ab').sub.2 fragments (Carter et al.,
Bio/Technology 10:163-167 (1992)). According to another approach,
F(ab').sub.2 fragments can be isolated directly from recombinant
host cell culture. Fab and F(ab').sub.2 fragment with increased in
vivo half-life comprising a salvage receptor binding epitope
residues are described in U.S. Pat. No. 5,869,046. Other techniques
for the production of antibody fragments will be apparent to the
skilled practitioner. In other embodiments, the antibody of choice
is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Pat.
No. 5,571,894; and U.S. Pat. No. 5,587,458. Fv and sFv are the only
species with intact combining sites that are devoid of constant
regions; thus, they are suitable for reduced nonspecific binding
during in vivo use. sFv fusion proteins may be constructed to yield
fusion of an effector protein at either the amino or the carboxy
terminus of an sFv. See Antibody Engineering, ed. Borrebaeck,
supra. The antibody fragment may also be a "linear antibody", e.g.,
as described in U.S. Pat. No. 5,641,870 for example. Such linear
antibody fragments may be monospecific or bispecific.
[0467] 5. Bispecific Antibodies
[0468] 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).
[0469] WO 96/16673 describes a bispecific
anti-ErbB2/anti-Fc.gamma.RIII antibody and U.S. Pat. No. 5,837,234
discloses a bispecific anti-ErbB2/anti-Fc.gamma.RI antibody. A
bispecific anti-ErbB2/Fc.alpha. antibody is shown in WO98/02463.
U.S. Pat. No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3
antibody.
[0470] 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).
[0471] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences.
Preferably, the fusion is with an Ig heavy chain constant domain,
comprising at least part of the hinge, C.sub.H2, and C.sub.H3
regions. It is preferred to have the first heavy-chain constant
region (C.sub.H1) containing the site necessary for light chain
bonding, present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host cell. This
provides for greater flexibility in adjusting the mutual
proportions of the three polypeptide fragments in embodiments when
unequal ratios of the three polypeptide chains used in the
construction provide the optimum yield of the desired bispecific
antibody. It is, however, possible to insert the coding sequences
for two or all three polypeptide chains into a single expression
vector when the expression of at least two polypeptide chains in
equal ratios results in high yields or when the ratios have no
significant affect on the yield of the desired chain
combination.
[0472] 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).
[0473] 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.
[0474] 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.
[0475] 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.
[0476] 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.
[0477] 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).
[0478] Antibodies with more than two valencies are contemplated.
For example, trispecific antibodies can be prepared. Tutt et al.,
J. Immunol. 147:60 (1991).
[0479] 6. Heteroconjugate Antibodies
[0480] 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.
[0481] 7. Multivalent Antibodies
[0482] 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.
[0483] 8. Effector Function Engineering
[0484] 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.
[0485] 9. Immunoconjugates
[0486] 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).
[0487] 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.
[0488] Conjugates of an antibody and one or more small molecule
toxins, such as a calicheamicin, maytansinoids, a trichothene, and
CC1065, and the derivatives of these toxins that have toxin
activity, are also contemplated herein.
Maytansine and Maytansinoids
[0489] In one preferred embodiment, an anti-TAT antibody (full
length or fragments) of the invention is conjugated to one or more
maytansinoid molecules.
[0490] Maytansinoids are mitototic inhibitors which act by
inhibiting tubulin polymerization. Maytansine was first isolated
from the east African shrub Maytenus serrata (U.S. Pat. No.
3,896,111). Subsequently, it was discovered that certain microbes
also produce maytansinoids, such as maytansinol and C-3 maytansinol
esters (U.S. Pat. No. 4,151,042). Synthetic maytansinol and
derivatives and analogues thereof are disclosed, for example, in
U.S. Pat. Nos. 4,137,230; 4,248,870; 4,256,746; 4,260,608;
4,265,814; 4,294,757; 4,307,016; 4,308,268; 4,308,269; 4,309,428;
4,313,946; 4,315,929; 4,317,821; 4,322,348; 4,331,598; 4,361,650;
4,364,866; 4,424,219; 4,450,254; 4,362,663; and 4,371,533, the
disclosures of which are hereby expressly incorporated by
reference.
Maytansinoid-Antibody Conjugates
[0491] In an attempt to improve their therapeutic index, maytansine
and maytansinoids have been conjugated to antibodies specifically
binding to tumor cell antigens. Immunoconjugates containing
maytansinoids and their therapeutic use are disclosed, for example,
in U.S. Pat. Nos. 5,208,020, 5,416,064 and European Patent EP 0 425
235 B1, the disclosures of which are hereby expressly incorporated
by reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623
(1996) described immunoconjugates comprising a maytansinoid
designated DM1 linked to the monoclonal antibody C242 directed
against human colorectal cancer. The conjugate was found to be
highly cytotoxic towards cultured colon cancer cells, and showed
antitumor activity in an in vivo tumor growth assay. Chari et al.,
Cancer Research 52:127-131 (1992) describe immunoconjugates in
which a maytansinoid was conjugated via a disulfide linker to the
murine antibody A7 binding to an antigen on human colon cancer cell
lines, or to another murine monoclonal antibody TA.1 that binds the
HER-2/neu oncogene. The cytotoxicity of the TA.1-maytansonoid
conjugate was tested in vitro on the human breast cancer cell line
SK-BR-3, which expresses 3.times.10.sup.5 HER-2 surface antigens
per cell. The drug conjugate achieved a degree of cytotoxicity
similar to the free maytansinoid drug, which could be increased by
increasing the number of maytansinoid molecules per antibody
molecule. The A7-maytansinoid conjugate showed low systemic
cytotoxicity in mice.
Anti-TAT Polypeptide Antibody-Maytansinoid Conjugates
(Immunoconjugates)
[0492] 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.
[0493] 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 0425235B1, Chari
et al., Cancer Research 52:127-131 (1992), and U.S. patent
application Ser. No. 10/960,602, filed Oct. 8, 2004, the
disclosures of which are hereby expressly incorporated by
reference. Antibody-maytansinoid conjugates comprising the linker
component SMCC may be prepared as disclosed in U.S. patent
application Ser. No. 10/960,602, filed Oct. 8, 2004. The linking
groups include disulfide groups, thioether groups, acid labile
groups, photolabile groups, peptidase labile groups, or esterase
labile groups, as disclosed in the above-identified patents,
disulfide and thioether groups being preferred. Additional linking
groups are described and exemplified herein.
[0494] Conjugates of the antibody and maytansinoid may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).
Particularly preferred coupling agents include
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlsson et
al., Biochem. J. 173:723-737 [1978]) and
N-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for a
disulfide linkage.
[0495] The linker may be attached to the maytansinoid molecule at
various positions, depending on the type of the link. For example,
an ester linkage may be formed by reaction with a hydroxyl group
using conventional coupling techniques. The reaction may occur at
the C-3 position having a hydroxyl group, the C-14 position
modified with hydroxymethyl, the C-15 position modified with a
hydroxyl group, and the C-20 position having a hydroxyl group. In a
preferred embodiment, the linkage is formed at the C-3 position of
maytansinol or a maytansinol analogue.
Auristatins and Dolastatins
[0496] In some embodiments, the immunoconjugate comprises an
antibody of the invention conjugated to dolastatins or dolastatin
peptidic analogs and derivatives, the auristatins (U.S. Pat. Nos.
5,635,483; 5,780,588). Dolastatins and auristatins have been shown
to interfere with microtubule dynamics, GTP hydrolysis, and nuclear
and cellular division (Woyke et al (2001) Antimicrob. Agents and
Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No.
5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob.
Agents Chemother. 42:2961-2965). The dolastatin or auristatin drug
moiety may be attached to the antibody through the N (amino)
terminus or the C (carboxyl) terminus of the peptidic drug moiety
(WO 02/088172).
[0497] Exemplary auristatin embodiments include the N-terminus
linked monomethylauristatin drug moieties DE and DF (i.e., MMAE and
MMAF), disclosed in "Senter et al, Proceedings of the American
Association for Cancer Research, Volume 45, Abstract Number 623,
presented Mar. 28, 2004, the disclosure of which is expressly
incorporated by reference in its entirety.
[0498] Typically, peptide-based drug moieties can be prepared by
forming a peptide bond between two or more amino acids and/or
peptide fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see E. Schroder and
K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic
Press) that is well known in the field of peptide chemistry. The
auristatin/dolastatin drug moieties may be prepared according to
the methods of: US 5,635,483; US 5,780,588; Pettit et al (1989) J.
Am. Chem. Soc. 111:5463-5465; Pettit et al (1998) Anti-Cancer Drug
Design 13:243-277; Pettit, G. R., et al. Synthesis, 1996, 719-725;
Pettit et al (1996) J. Chem. Soc. Perkin Trans. 1 5:859-863; and
Doronina (2003) Nat Biotechnol 21(7):778-784.
Calicheamicin
[0499] 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.1, .alpha..sub.2.sup.1,
.alpha..sub.3.sup.1, N-acetyl-.gamma..sub.1.sup.1, PSAG and
.theta..sup.1.sub.1 (Hinman et al., Cancer Research 53:3336-3342
(1993), Lode et al., Cancer Research 58:2925-2928 (1998) and the
aforementioned U.S. patents to American Cyanamid). Another
anti-tumor drug that the antibody can be conjugated is QFA which is
an antifolate. Both calicheamicin and QFA have intracellular sites
of action and do not readily cross the plasma membrane. Therefore,
cellular uptake of these agents through antibody mediated
internalization greatly enhances their cytotoxic effects.
Other Cytotoxic Agents
[0500] 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).
[0501] 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.
[0502] 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).
[0503] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
anti-TAT antibodies. Examples include At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of Lu.
When the conjugate is used for diagnosis, it may comprise a
radioactive atom for scintigraphic studies, for example tc.sup.99m
or I.sup.123, or a spin label for nuclear magnetic resonance (NMR)
imaging (also known as magnetic resonance imaging, mri), such as
iodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,
nitrogen-15, oxygen-17, gadolinium, manganese or iron.
[0504] 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.
[0505] Conjugates of the antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate,
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis (p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolylene 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.
[0506] The compounds of the invention expressly contemplate, but
are not limited to, ADC prepared with cross-linker reagents: BMPS,
EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB,
SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,
sulfo-SMCC, and sulfo-SMPB, and SVSB
(succinimidyl-(4-vinylsulfone)benzoate) which are commercially
available (e.g., from Pierce Biotechnology, Inc., Rockford, Ill.,
U.S.A). See pages 467-498, 2003-2004 Applications Handbook and
Catalog.
[0507] 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.
[0508] 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).
[0509] 10. Immunoliposomes
[0510] The anti-TAT antibodies disclosed herein may also be
formulated as immunoliposomes. A "liposome" is a small vesicle
composed of various types of lipids, phospholipids and/or
surfactant which is useful for delivery of a drug to a mammal. The
components of the liposome are commonly arranged in a bilayer
formation, similar to the lipid arrangement of biological
membranes. Liposomes containing the antibody are prepared by
methods known in the art, such as described in Epstein et al.,
Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl
Acad. Sci. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and
4,544,545; and WO97/38731 published Oct. 23, 1997. Liposomes with
enhanced circulation time are disclosed in U.S. Pat. No.
5,013,556.
[0511] Particularly useful liposomes can be generated by the
reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the present invention
can be conjugated to the liposomes as described in Martin et al.,
J. Biol. Chem. 257:286-288 (1982) via a disulfide interchange
reaction. A chemotherapeutic agent is optionally contained within
the liposome. See Gabizon et al., J. National Cancer Inst.
81(19):1484 (1989).
[0512] B. TAT Binding Oligopeptides
[0513] 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).
[0514] 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.
[0515] 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.
[0516] Many other improvements and variations of the basic phage
display concept have now been developed. These improvements enhance
the ability of display systems to screen peptide libraries for
binding to selected target molecules and to display functional
proteins with the potential of screening these proteins for desired
properties. Combinatorial reaction devices for phage display
reactions have been developed (WO 98/14277) and phage display
libraries have been used to analyze and control bimolecular
interactions (WO 98/20169; WO 98/20159) and properties of
constrained helical peptides (WO 98/20036). WO 97/35196 describes a
method of isolating an affinity ligand in which a phage display
library is contacted with one solution in which the ligand will
bind to a target molecule and a second solution in which the
affinity ligand will not bind to the target molecule, to
selectively isolate binding ligands. WO 97/46251 describes a method
of biopanning a random phage display library with an affinity
purified antibody and then isolating binding phage, followed by a
micropanning process using microplate wells to isolate high
affinity binding phage. The use of Staphlylococcus aureus protein A
as an affinity tag has also been reported (Li et al. (1998) Mol
Biotech., 9:187). WO 97/47314 describes the use of substrate
subtraction libraries to distinguish enzyme specificities using a
combinatorial library which may be a phage display library. A
method for selecting enzymes suitable for use in detergents using
phage display is described in WO 97/09446. Additional methods of
selecting specific binding proteins are described in U.S. Pat. Nos.
5,498,538, 5,432,018, and WO 98/15833.
[0517] 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.
[0518] C. TAT Binding Organic Molecules
[0519] 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.
[0520] D. Screening for Anti-TAT Antibodies, TAT Binding
Oligopeptides and TAT Binding Organic Molecules With the Desired
Properties
[0521] 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.
[0522] 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 cells in vivo can be determined in various ways known in the
art. Preferably, the tumor cell is one that overexpresses a TAT
polypeptide. Preferably, the anti-TAT antibody, TAT binding
oligopeptide or TAT binding organic molecule will inhibit cell
proliferation of a TAT-expressing tumor cell in vitro or in vivo by
about 25-100% compared to the untreated tumor cell, more
preferably, by about 30-100%, and even more preferably by about
50-100% or 70-100%, in one embodiment, at an antibody concentration
of about 0.5 to 30 .mu.g/ml. Growth inhibition can be measured at
an antibody concentration of about 0.5 to 30 .mu.g/ml or about 0.5
nM to 200 nM in cell culture, where the growth inhibition is
determined 1-10 days after exposure of the tumor cells to the
antibody. The antibody is growth inhibitory in vivo if
administration of the anti-TAT antibody at about 1 .mu.g/kg to
about 100 mg/kg body weight results in reduction in tumor size or
reduction of tumor cell proliferation within about 5 days to 3
months from the first administration of the antibody, preferably
within about 5 to 30 days.
[0523] 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.m/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.
[0524] To screen for antibodies, oligopeptides or other organic
molecules which bind to an epitope on a TAT polypeptide bound by an
antibody of interest, a routine cross-blocking assay such as that
described in Antibodies, A Laboratory Manual, Cold Spring Harbor
Laboratory, Ed Harlow and David Lane (1988), can be performed. This
assay can be used to determine if a test antibody, oligopeptide or
other organic molecule binds the same site or epitope as a known
anti-TAT antibody. Alternatively, or additionally, epitope mapping
can be performed by methods known in the art . For example, the
antibody sequence can be mutagenized such as by alanine scanning,
to identify contact residues. The mutant antibody is initially
tested for binding with polyclonal antibody to ensure proper
folding. In a different method, peptides corresponding to different
regions of a TAT polypeptide can be used in competition assays with
the test antibodies or with a test antibody and an antibody with a
characterized or known epitope.
[0525] E. Antibody Dependent Enzyme Mediated Prodrug Therapy
(ADEPT)
[0526] 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.
[0527] 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.
[0528] 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.
[0529] 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).
[0530] F. Full-Length TAT Polypeptides
[0531] 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.
[0532] 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.
[0533] G. Anti-TAT Antibody and TAT Polypeptide Variants
[0534] 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.
[0535] 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.
[0536] 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.
[0537] 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.
[0538] In particular embodiments, conservative substitutions of
interest are shown in Table 6 under the heading of preferred
substitutions. If such substitutions result in a change in
biological activity, then more substantial changes, denominated
exemplary substitutions in Table 6, or as further described below
in reference to amino acid classes, are introduced and the products
screened.
TABLE-US-00005 TABLE 6 Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp; Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser, Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp, Gln Asp
Gly (G) 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) Trp; Leu; Val; Ile; Ala; Tyr Leu Pro (P) Ala Ala Ser
(S) Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp;
Phe; Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala;
Norleucine
[0539] 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: [0540] (1) hydrophobic: Norleucine,
Met, Ala, Val, Leu, Ile; [0541] (2) neutral hydrophilic: Cys, Ser,
Thr; Asn; Gln [0542] (3) acidic: Asp, Glu; [0543] (4) basic: His,
Lys, Arg; [0544] (5) residues that influence chain orientation:
Gly, Pro; and [0545] (6) aromatic: Trp, Tyr, Phe.
[0546] 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.
[0547] 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.
[0548] 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.
[0549] 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).
[0550] 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-di splayed 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.
[0551] 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.
[0552] H. Modifications of Anti-TAT Antibodies and TAT
Polypeptides
[0553] Covalent modifications of anti-TAT antibodies and TAT
polypeptides are included within the scope of this invention. One
type of covalent modification includes reacting targeted amino acid
residues of an anti-TAT antibody or TAT polypeptide with an organic
derivatizing agent that is capable of reacting with selected side
chains or the N- or C-terminal residues of the anti-TAT antibody or
TAT polypeptide. Derivatization with bifunctional agents is useful,
for instance, for crosslinking anti-TAT antibody or TAT polypeptide
to a water-insoluble support matrix or surface for use in the
method for purifying anti-TAT antibodies, and vice-versa. Commonly
used crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides
such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate.
[0554] 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 a-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.
[0555] 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.
[0556] 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-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid,
most commonly serine or threonine, although 5-hydroxyproline or
5-hydroxylysine may also be used.
[0557] 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.
[0558] 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
Sept. 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp.
259-306 (1981).
[0559] 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).
[0560] 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).
[0561] 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.
[0562] In one embodiment, such a chimeric molecule comprises a
fusion of the anti-TAT antibody or TAT polypeptide with a tag
polypeptide which provides an epitope to which an anti-tag antibody
can selectively bind. The epitope tag is generally placed at the
amino- or carboxyl-terminus of the anti-TAT antibody or TAT
polypeptide. The presence of such epitope-tagged forms of the
anti-TAT antibody or TAT polypeptide can be detected using an
antibody against the tag polypeptide. Also, provision of the
epitope tag enables the anti-TAT antibody or TAT polypeptide to be
readily purified by affinity purification using an anti-tag
antibody or another type of affinity matrix that binds to the
epitope tag. Various tag polypeptides and their respective
antibodies are well known in the art. Examples include
poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly)
tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et
al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the
8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al.,
Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes
Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et
al., Protein Engineering, 3(6):547-553 (1990)]. Other tag
polypeptides include the Flag-peptide [Hopp et al., BioTechnology,
6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al.,
Science, 255:192-194 (1992)]; an .alpha.-tubulin epitope peptide
[Skinner et al., J. Biol. Chem., 266:15163-15166 (1991)]; and the
T7 gene 10 protein peptide tag [Lutz-Freyermuth et al., Proc. Natl.
Acad. Sci. USA, 87:6393-6397 (1990)].
[0563] In an alternative embodiment, the chimeric molecule may
comprise a fusion of the anti-TAT antibody or TAT polypeptide with
an immunoglobulin or a particular region of an immunoglobulin. For
a bivalent form of the chimeric molecule (also referred to as an
"immunoadhesin"), such a fusion could be to the Fc region of an IgG
molecule. The Ig fusions preferably include the substitution of a
soluble (transmembrane domain deleted or inactivated) form of an
anti-TAT antibody or TAT polypeptide in place of at least one
variable region within an Ig molecule. In a particularly preferred
embodiment, the immunoglobulin fusion includes the hinge, CH.sub.2
and CH.sub.3, or the hinge, CH.sub.1, CH.sub.2 and CH.sub.3 regions
of an IgG1 molecule. For the production of immunoglobulin fusions
see also U.S. Pat. No. 5,428,130 issued Jun. 27, 1995.
[0564] I. Preparation of Anti-TAT Antibodies and TAT
Polypeptides
[0565] 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.
[0566] 1. Isolation of DNA Encoding Anti-TAT Antibody or TAT
Polypeptide
[0567] 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).
[0568] 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)].
[0569] Techniques for screening a cDNA library are well known in
the art. The oligonucleotide sequences selected as probes should be
of sufficient length and sufficiently unambiguous that false
positives are minimized. The oligonucleotide is preferably labeled
such that it can be detected upon hybridization to DNA in the
library being screened. Methods of labeling are well known in the
art, and include the use of radiolabels like .sup.32P-labeled ATP,
biotinylation or enzyme labeling. Hybridization conditions,
including moderate stringency and high stringency, are provided in
Sambrook et al., supra.
[0570] 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.
[0571] 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.
[0572] 2. Selection and Transformation of Host Cells
[0573] 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.
[0574] Methods of eukaryotic cell transfection and prokaryotic cell
transformation are known to the ordinarily skilled artisan, for
example, CaCl.sub.2, CaPO.sub.4, liposome-mediated and
electroporation. Depending on the host cell used, transformation is
performed using standard techniques appropriate to such cells. The
calcium treatment employing calcium chloride, as described in
Sambrook et al., supra, or electroporation is generally used for
prokaryotes. Infection with Agrobacterium tumefaciens is used for
transformation of certain plant cells, as described by Shaw et al.,
Gene, 23:315 (1983) and WO 89/05859 published 29 Jun. 1989. For
mammalian cells without such cell walls, the calcium phosphate
precipitation method of Graham and van der Eb, Virology, 52:456-457
(1978) can be employed. General aspects of mammalian cell host
system transfections have been described in U.S. Pat. No.
4,399,216. Transformations into yeast are typically carried out
according to the method of Van Solingen et al., J. Bact., 130:946
(1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829
(1979). However, other methods for introducing DNA into cells, such
as by nuclear microinjection, electroporation, bacterial protoplast
fusion with intact cells, or polycations, e.g., polybrene,
polyornithine, may also be used. For various techniques for
transforming mammalian cells, see Keown et al., Methods in
Enzymology, 185:527-537 (1990) and Mansour et al., Nature,
336:348-352 (1988).
[0575] 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, Enwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published 12 Apr. 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. These examples are illustrative rather than limiting.
Strain W3110 is one particularly preferred host or parent host
because it is a common host strain for recombinant DNA product
fermentations. Preferably, the host cell secretes minimal amounts
of proteolytic enzymes. For example, strain W3110 may be modified
to effect a genetic mutation in the genes encoding proteins
endogenous to the host, with examples of such hosts including E.
coli W3110 strain 1A2, which has the complete genotype tonA; E.
coli W3110 strain 9E4, which has the complete genotype tonA ptr3;
E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete
genotype tonA ptr3 phoA E15 (argF-lac)169 degP ompT kan.sup.r; E.
coli W3110 strain 37D6, which has the complete genotype tonA ptr3
phoA E15 (argF-lac)169 degP ompT rbs7 ilvG kan.sup.r; E. coli W3110
strain 40B4, which is strain 37D6 with a non-kanamycin resistant
degP deletion mutation; and an E. coli strain having mutant
periplasmic protease disclosed in U.S. Pat. No. 4,946,783 issued 7
Aug. 1990. Alternatively, in vitro methods of cloning, e.g., PCR or
other nucleic acid polymerase reactions, are suitable.
[0576] 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.
[0577] 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; Louvencourt et al., J. Bacteriol.,
154(2):737-742 [1983]), K. fragilis (ATCC 12,424), K. bulgaricus
(ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC
56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al.,
Bio/Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus;
yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et
al., J. Basic Microbiol., 28:265-278 [1988]); Candida; Trichoderma
reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl.
Acad. Sci. USA, 76:5259-5263 [1979]); Schwanniomyces such as
Schwanniomyces occidentalis (EP 394,538 published 31 Oct. 1990);
and filamentous fungi such as, e.g., Neurospora, Penicillium,
Tolypocladium (WO 91/00357 published 10 Jan. 1991), and Aspergillus
hosts such as A. nidulans (Ballance et al., Biochem. Biophys. Res.
Commun., 112:284-289 [1983]; Tilburn et al., Gene, 26:205-221
[1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474
[1984]) and A. niger (Kelly and Hynes, EMBO 1, 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).
[0578] 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 frupperda (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
frupperda cells.
[0579] However, interest has been greatest in vertebrate cells, and
propagation of vertebrate cells in culture (tissue culture) has
become a routine procedure. Examples of useful mammalian host cell
lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC
CRL 1651); human embryonic kidney line (293 or 293 cells subcloned
for growth in suspension culture, Graham et al., J. Gen Virol.
36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);
Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl.
Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather,
Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL
70); African green monkey kidney cells (VERO-76, ATCC CRL-1587);
human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney
cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC
CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells
(Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51);
TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982));
MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
[0580] 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.
[0581] 3. Selection and Use of a Replicable Vector
[0582] 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.
[0583] 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,
1pp, 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.
[0584] 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.
[0585] 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.
[0586] An example of suitable selectable markers for mammalian
cells are those that enable the identification of cells competent
to take up the anti-TAT antibody- or TAT polypeptide-encoding
nucleic acid, such as DHFR or thymidine kinase. An appropriate host
cell when wild-type DHFR is employed is the CHO cell line deficient
in DHFR activity, prepared and propagated as described by Urlaub et
al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitable
selection gene for use in yeast is the trp1 gene present in the
yeast plasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979);
Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157
(1980)]. The trp1 gene provides a selection marker for a mutant
strain of yeast lacking the ability to grow in tryptophan, for
example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85:12
(1977)].
[0587] 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.
[0588] 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.
[0589] 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.
[0590] 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.
[0591] 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,
a-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.
[0592] 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.
[0593] 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.
[0594] 4. Culturing the Host Cells
[0595] 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; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or
U.S. Pat. No. Re. 30,985 may be used as culture media for the host
cells. Any of these media may be supplemented as necessary with
hormones and/or other growth factors (such as insulin, transferrin,
or epidermal growth factor), salts (such as sodium chloride,
calcium, magnesium, and phosphate), buffers (such as HEPES),
nucleotides (such as adenosine and thymidine), antibiotics (such as
GENTAMYCIN.TM. drug), trace elements (defined as inorganic
compounds usually present at final concentrations in the micromolar
range), and glucose or an equivalent energy source. Any other
necessary supplements may also be included at appropriate
concentrations that would be known to those skilled in the art. The
culture conditions, such as temperature, pH, and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan.
[0596] 5. Detecting Gene Amplification/Expression
[0597] 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.
[0598] 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.
[0599] 6. Purification of Anti-TAT Antibody and TAT Polypeptide
[0600] 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.
[0601] 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, N.Y. (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.
[0602] 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.
[0603] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma.1, .gamma.2 or .gamma.4 heavy chains
(Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is
recommended for all mouse isotypes and for human .gamma.3 (Guss et
al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity
ligand is attached is most often agarose, but other matrices are
available. Mechanically stable matrices such as controlled pore
glass or poly(styrenedivinyl)benzene allow for faster flow rates
and shorter processing times than can be achieved with agarose.
Where the antibody comprises a C.sub.H3 domain, the Bakerbond
ABX.TM.resin (J. T. Baker, Phillipsburg, N.J.) is useful for
purification. Other techniques for protein purification such as
fractionation on an ion-exchange column, ethanol precipitation,
Reverse Phase HPLC, chromatography on silica, chromatography on
heparin SEPHAROSE.TM. chromatography on an anion or cation exchange
resin (such as a polyaspartic acid column), chromatofocusing,
SDS-PAGE, and ammonium sulfate precipitation are also available
depending on the antibody to be recovered.
[0604] 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).
[0605] J. Pharmaceutical Formulations
[0606] 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.
[0607] 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.
[0608] 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).
[0609] 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 y 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.
[0610] The formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes.
[0611] K. Diagnosis and Treatment with Anti-TAT Antibodies, TAT
Binding Oligopeptides and TAT Binding Organic Molecules
[0612] To determine TAT expression in the cancer, various
diagnostic assays are available. In one embodiment, TAT polypeptide
overexpression may be analyzed by immunohistochemistry
[0613] 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:
[0614] Score 0--no staining is observed or membrane staining is
observed in less than 10% of tumor cells.
[0615] 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.
[0616] Score 2+--a weak to moderate complete membrane staining is
observed in more than 10% of the tumor cells.
[0617] Score 3+--a moderate to strong complete membrane staining is
observed in more than 10% of the tumor cells.
[0618] 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.
[0619] 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.
[0620] 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.
[0621] 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.
[0622] 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. (paclitaxel), estramustine and mitoxantrone are used in
treating cancer, in particular, in good risk patients. In the
present method of the invention for treating or alleviating cancer,
the cancer patient can be administered anti-TAT antibody,
oligopeptide or organic molecule in conjunction with treatment with
the one or more of the preceding chemotherapeutic agents. In
particular, combination therapy with palictaxel and modified
derivatives (see, e.g., EP0600517) is contemplated. The anti-TAT
antibody, oligopeptide or organic molecule will be administered
with a therapeutically effective dose of the chemotherapeutic
agent. In another embodiment, the anti-TAT antibody, oligopeptide
or organic molecule is administered in conjunction with
chemotherapy to enhance the activity and efficacy of the
chemotherapeutic agent, e.g., paclitaxel. The Physicians' Desk
Reference (PDR) discloses dosages of these agents that have been
used in treatment of various cancers. The dosing regimen and
dosages of these aforementioned chemotherapeutic drugs that are
therapeutically effective will depend on the particular cancer
being treated, the extent of the disease and other factors familiar
to the physician of skill in the art and can be determined by the
physician.
[0623] 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.
[0624] 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.
[0625] 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.
[0626] 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.
[0627] 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 docetaxel) 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).
[0628] 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.
[0629] 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.
[0630] For the prevention or treatment of disease, the dosage and
mode of administration will be chosen by the physician according to
known criteria. The appropriate dosage of antibody, oligopeptide or
organic molecule will depend on the type of disease to be treated,
as defined above, the severity and course of the disease, whether
the antibody, oligopeptide or organic molecule is administered for
preventive or therapeutic purposes, previous therapy, the patient's
clinical history and response to the antibody, oligopeptide or
organic molecule, and the discretion of the attending physician.
The antibody, oligopeptide or organic molecule is suitably
administered to the patient at one time or over a series of
treatments. Preferably, the antibody, oligopeptide or organic
molecule is administered by intravenous infusion or by subcutaneous
injections. Depending on the type and severity of the disease,
about 1 .mu.g/kg to about 50 mg/kg body weight (e.g., about 0.1-15
mg/kg/dose) of antibody can be an initial candidate dosage for
administration to the patient, whether, for example, by one or more
separate administrations, or by continuous infusion. A dosing
regimen can comprise administering an initial loading dose of about
4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of
the anti-TAT antibody. However, other dosage regimens may be
useful. A typical daily dosage might range from about 1 .mu.g/kg to
100 mg/kg or more, depending on the factors mentioned above. For
repeated administrations over several days or longer, depending on
the condition, the treatment is sustained until a desired
suppression of disease symptoms occurs. The progress of this
therapy can be readily monitored by conventional methods and assays
and based on criteria known to the physician or other persons of
skill in the art.
[0631] 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.
[0632] There are two major approaches to getting the nucleic acid
(optionally contained in a vector) into the patient's cells; in
vivo and ex vivo. For in vivo delivery the nucleic acid is injected
directly into the patient, usually at the site where the antibody
is required. For ex vivo treatment, the patient's cells are
removed, the nucleic acid is introduced into these isolated cells
and the modified cells are administered to the patient either
directly or, for example, encapsulated within porous membranes
which are implanted into the patient (see, e.g., U.S. Pat. Nos.
4,892,538 and 5,283,187). There are a variety of techniques
available for introducing nucleic acids into viable cells. The
techniques vary depending upon whether the nucleic acid is
transferred into cultured cells in vitro, or in vivo in the cells
of the intended host. Techniques suitable for the transfer of
nucleic acid into mammalian cells in vitro include the use of
liposomes, electroporation, microinjection, cell fusion,
DEAE-dextran, the calcium phosphate precipitation method, etc. A
commonly used vector for ex vivo delivery of the gene is a
retroviral vector.
[0633] 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.
[0634] 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.
[0635] 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.
[0636] Methods of producing the above antibodies are described in
detail herein.
[0637] 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.
[0638] 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.
[0639] 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.
[0640] 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.
[0641] 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.
[0642] L. Articles of Manufacture and Kits
[0643] 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.
[0644] 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.
[0645] M. Uses for TAT Polypeptides and TAT-Polypeptide Encoding
Nucleic Acids
[0646] 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.
[0647] 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.
[0648] Other useful fragments of the TAT-encoding nucleic acids
include antisense or sense oligonucleotides comprising a
singe-stranded nucleic acid sequence (either RNA or DNA) capable of
binding to target TAT mRNA (sense) or TAT DNA (antisense)
sequences. Antisense or sense oligonucleotides, according to the
present invention, comprise a fragment of the coding region of TAT
DNA. Such a fragment generally comprises at least about 14
nucleotides, preferably from about 14 to 30 nucleotides. The
ability to derive an antisense or a sense oligonucleotide, based
upon a cDNA sequence encoding a given protein is described in, for
example, Stein and Cohen (Cancer Res. 48:2659, 1988) and van der
Krol et al. (BioTechniques 6:958, 1988).
[0649] Binding of anti sense 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.
[0650] 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.
[0651] 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, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates, 5'-alkylene phosphonates and chiral phosphonates,
phosphinates, phosphoramidates including 3'-amino phosphoramidate
and aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters,
selenophosphates and borano-phosphates having normal 3'-5'
linkages, 2'-5' linked analogs of these, and those having inverted
polarity wherein one or more internucleotide linkages is a 3' to
3', 5' to 5' or 2' to 2' linkage. Preferred oligonucleotides having
inverted polarity comprise a single 3' to 3' linkage at the 3'-most
internucleotide linkage i.e. a single inverted nucleoside residue
which may be abasic (the nucleobase is missing or has a hydroxyl
group in place thereof). Various salts, mixed salts and free acid
forms are also included. Representative United States patents that
teach the preparation of phosphorus-containing linkages include,
but are not limited to, U.S. Pat. Nos.: 3,687,808; 4,469,863;
4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;
5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;
5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;
5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555;
5,527,899; 5,721,218; 5,672,697 and 5,625,050, each of which is
herein incorporated by reference.
[0652] 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.
[0653] 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.
[0654] Preferred antisense oligonucleotides incorporate
phosphorothioate backbones and/or heteroatom backbones, and in
particular --CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--]
described in the above referenced U.S. Pat. No. 5,489,677, and the
amide backbones of the above referenced U.S. Pat. No. 5,602,240.
Also preferred are antisense oligonucleotides having morpholino
backbone structures of the above-referenced U.S. Pat. No.
5,034,506.
[0655] Modified oligonucleotides may also contain one or more
substituted sugar moieties. Preferred oligonucleotides comprise one
of the following at the 2' position: OH; F; O-alkyl, S-alkyl, or
N-alkyl; O-alkenyl, S-alkenyl, or N-alkenyl; O-alkynyl, S-alkynyl
or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and
alkynyl may be substituted or unsubstituted C.sub.1 to C.sub.10
alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Particularly
preferred are O[(CH.sub.2).sub.nO].sub.mCH.sub.3,
O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m
are from 1 to about 10. Other preferred antisense oligonucleotides
comprise one of the following at the 2' position: C.sub.1 to
C.sub.10 lower alkyl, substituted lower alkyl, alkenyl, alkynyl,
alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl,
Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3, SO.sub.2 CH.sub.3,
ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2, heterocycloalkyl,
heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted
silyl, an RNA cleaving group, a reporter group, an intercalator, a
group for improving the pharmacokinetic properties of an
oligonucleotide, or a group for improving the pharmacodynamic
properties of an oligonucleotide, and other substituents having
similar properties. A preferred modification includes
2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Hely. 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).
[0656] A further preferred modification includes Locked Nucleic
Acids (LNAs) in which the 2'-hydroxyl group is linked to the 3' or
4' carbon atom of the sugar ring thereby forming a bicyclic sugar
moiety. The linkage is preferably a methylene (--CH.sub.2--).sub.n
group bridging the 2' oxygen atom and the 4' carbon atom wherein n
is 1 or 2. LNAs and preparation thereof are described in WO
98/39352 and WO 99/14226.
[0657] Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-aminopropoxy (2'-OCH.sub.2CH.sub.2CH.sub.2
NH.sub.2), 2'-allyl (2'-CH.sub.2--CH.dbd.CH.sub.2), 2'-O-allyl
(2'-O--CH.sub.2--CH.dbd.CH.sub.2) and 2'-fluoro (2'-F). The
2'-modification may be in the arabino (up) position or ribo (down)
position. A preferred 2'-arabino modification is 2'-F. Similar
modifications may also be made at other positions on the
oligonucleotide, particularly the 3' position of the sugar on the
3' terminal nucleotide or in 2'-5' linked oligonucleotides and the
5' position of 5' terminal nucleotide. Oligonucleotides may also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative United States patents that
teach the preparation of such modified sugar structures include,
but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;
5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;
5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747;
and 5,700,920, each of which is herein incorporated by reference in
its entirety.
[0658] Oligonucleotides may also include nucleobase (often referred
to in the art simply as "base") modifications or substitutions. As
used herein, "unmodified" or "natural" nucleobases include the
purine bases adenine (A) and guanine (G), and the pyrimidine bases
thymine (T), cytosine (C) and uracil (U). Modified nucleobases
include other synthetic and natural nucleobases such as
5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,
hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives
of adenine and guanine, 2-propyl and other alkyl derivatives of
adenine and guanine, 2-thiouracil, 2-thiothymine and
2-thiocytosine, 5-halouracil and cytosine, 5-propynyl
(--C.ident.C--CH.sub.3 or --CH.sub.2--C.ident.CH) uracil and
cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo
uracil, cytosine and thymine, 5-uracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and
other 8-substituted adenines and guanines, 5-halo particularly
5-bromo, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine,
2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and
7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further
modified nucleobases include tricyclic pyrimidines such as
phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
phenothiazine cytidine
(1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as a
substituted phenoxazine cytidine (e.g.
9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole
cytidine (H-pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one).
Modified nucleobases may also include those in which the purine or
pyrimidine base is replaced with other heterocycles, for example
7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone.
Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, those disclosed in The Concise Encyclopedia Of Polymer
Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John
Wiley & Sons, 1990, and those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613. Certain of
these nucleobases are particularly useful for increasing the
binding affinity of the oligomeric compounds of the invention.
These include 5-substituted pyrimidines, 6-azapyrimidines and N-2,
N-6 and O-6 substituted purines, including 2-aminopropyl adenine,
5-propynyluracil and 5-propynylcytosine. 5-methyl cytosine
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.
[0659] 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, dansyl
sarcosine, 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.
[0660] 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, oligonucleotides and/or oligonucleotide
mimetics as described above. Preferred chimeric antisense
oligonucleotides incorporate at least one 2' modified sugar
(preferably 2'-O--(CH.sub.2).sub.2--O--CH.sub.3) at the 3' terminal
to confer nuclease resistance and a region with at least 4
contiguous 2'-H sugars to confer RNase H activity. Such compounds
have also been referred to in the art as hybrids or gapmers.
Preferred gapmers have a region of 2' modified sugars (preferably
2'-O--(CH.sub.2).sub.2--O--CH.sub.3) at the 3'-terminal and at the
5' terminal separated by at least one region having at least 4
contiguous 2'-H sugars and preferably incorporate phosphorothioate
backbone linkages. Representative United States patents that teach
the preparation of such hybrid structures include, but are not
limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007;
5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;
5,652,355; 5,652,356; and 5,700,922, each of which is herein
incorporated by reference in its entirety.
[0661] 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.
[0662] 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.
[0663] 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, DCTSB and DCTSC (see WO 90/13641).
[0664] 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.
[0665] 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.
[0666] 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.
[0667] The probes may also be employed in PCR techniques to
generate a pool of sequences for identification of closely related
TAT coding sequences.
[0668] 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.
[0669] 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.
[0670] 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.
[0671] 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.
[0672] Nucleic acid encoding the TAT polypeptides may also be used
in gene therapy. In gene therapy applications, genes are introduced
into cells in order to achieve in vivo synthesis of a
therapeutically effective genetic product, for example for
replacement of a defective gene. "Gene therapy" includes both
conventional gene therapy where a lasting effect is achieved by a
single treatment, and the administration of gene therapeutic
agents, which involves the one time or repeated administration of a
therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can
be used as therapeutic agents for blocking the expression of
certain genes in vivo. It has already been shown that short
antisense oligonucleotides can be imported into cells where they
act as inhibitors, despite their low intracellular concentrations
caused by their restricted uptake by the cell membrane. (Zamecnik
et al., Proc. Natl. Acad. Sci. USA 83:4143-4146 [1986]). The
oligonucleotides can be modified to enhance their uptake, e.g. by
substituting their negatively charged phosphodiester groups by
uncharged groups.
[0673] 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).
[0674] 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.
[0675] 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.
[0676] 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.
[0677] 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.
[0678] 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.
[0679] 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.
[0680] 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 GAL1-lacZ
reporter gene under control of a GAL4-activated promoter depends on
reconstitution of GAL4 activity via protein-protein interaction.
Colonies containing interacting polypeptides are detected with a
chromogenic substrate for .beta.-galactosidase. A complete kit
(MATCHMAKER.TM.) for identifying protein-protein interactions
between two specific proteins using the two-hybrid technique is
commercially available from Clontech. This system can also be
extended to map protein domains involved in specific protein
interactions as well as to pinpoint amino acid residues that are
crucial for these interactions.
[0681] 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.
[0682] 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.
[0683] As an alternative approach for receptor identification,
labeled TAT polypeptide can be photoaffinity-linked with cell
membrane or extract preparations that express the receptor
molecule. Cross-linked material is resolved by PAGE and exposed to
X-ray film. The labeled complex containing the receptor can be
excised, resolved into peptide fragments, and subjected to protein
micro-sequencing. The amino acid sequence obtained from
micro-sequencing would be used to design a set of degenerate
oligonucleotide probes to screen a cDNA library to identify the
gene encoding the putative receptor.
[0684] 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.
[0685] 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.
[0686] Another potential TAT polypeptide antagonist is an antisense
RNA or DNA construct prepared using antisense technology, where,
e.g., an antisense RNA or DNA molecule acts to block directly the
translation of mRNA by hybridizing to targeted mRNA and preventing
protein translation. Antisense technology can be used to control
gene expression through triple-helix formation or antisense DNA or
RNA, both of which methods are based on binding of a polynucleotide
to DNA or RNA. For example, the 5' coding portion of the
polynucleotide sequence, which encodes the mature TAT polypeptides
herein, is used to design an antisense RNA oligonucleotide of from
about 10 to 40 base pairs in length. A DNA oligonucleotide is
designed to be complementary to a region of the gene involved in
transcription (triple helix--see Lee et al., Nucl. Acids Res.,
6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan et
al., Science, 251:1360 (1991)), thereby preventing transcription
and the production of the TAT polypeptide. The antisense RNA
oligonucleotide hybridizes to the mRNA in vivo and blocks
translation of the mRNA molecule into the TAT polypeptide
(antisense--Okano, Neurochem., 56:560 (1991); Oligodeoxynucleotides
as Antisense Inhibitors of Gene Expression (CRC Press: Boca Raton,
Fla., 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.
[0687] 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.
[0688] 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).
[0689] 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.
[0690] 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.
[0691] 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.
[0692] 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.
[0693] 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).
[0694] 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.
[0695] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way.
[0696] All patent and literature references cited in the present
specification are hereby incorporated by reference in their
entirety.
EXAMPLES
[0697] 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.
[0698] 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 and
detectably upregulated in a particular human tumor tissue(s) of
interest as compared to other human tumor(s) and/or normal human
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 molecule(s) exhibit a tissue expression
profile showing high tissue expression and significant and
reproducibly detectable upregulation of expression in a specific
human tumor or tumors as compared to other human tumor(s) and/or
normal human tissues and optionally relatively low expression in
normal essential and/or normal proliferating human tissues.
[0699] Using the expression analysis described above, it was
determined that mRNA encoding the TAT10772 polypeptide is
significantly, reproducibly and detectably overexpressed in certain
types of human cancerous ovarian, breast and pancreatic tumors as
compared to the corresponding normal human ovarian, breast and
pancreatic tissues, respectively.
[0700] A. Ovary
[0701] In a first experiment, expression of TAT10772 was analyzed
in a group of 89 independent normal human ovarian tissue samples.
The results of these analyses demonstrated that the level of
TAT10772 mRNA expression in all of the normal human ovarian tissue
samples analyzed was remarkably consistent and fell within a very
tight distribution, with no normal human ovarian tissue sample
evidencing greater than a 6-fold increase in TAT10772 expression as
compared to the mean level of TAT10772 expression for the group of
samples as a whole.
[0702] For purposes of quantitative comparison, a variety of
independent and different types of cancerous human ovarian tissue
samples were also analyzed for TAT10772 expression. The results
obtained from these analyses demonstrated that the level of
expression of TAT10772 in the cancerous samples was quite variable,
with a significant number of the cancerous samples showing an at
least 6-fold (to as high as an about 580-fold) increase in TAT10772
expression when compared to the mean level of TAT10772 expression
for the group of normal ovarian tissue samples analyzed. More
specifically, detectable and reproducible TAT10772 overexpression
was observed for the following ovarian cancer types as compared to
normal ovarian (wherein the numbers shown in parentheses for each
cancer type represent the number of independent samples that
exhibited at least a 6-fold increase in TAT10772 expression when
compared to the mean level of TAT10772 expression for the group of
normal ovarian tissue samples analyzed/the total number of
independent tumor samples analyzed): endometrioid adenocarcinoma
(13/17), serous cystadenocarcinoma, including papillary (52/57),
and clear cell adenocarcinoma (7/10). Additional experiments were
conducted which confirmed these results.
B. Breast
[0703] In another experiment, expression of TAT10772 was analyzed
in a group of 22 independent normal human breast tissue samples.
The results of these analyses demonstrated that the level of
TAT10772 mRNA expression in all of the normal human breast tissue
samples analyzed was remarkably consistent and fell within a very
tight distribution, with no normal human breast tissue sample
evidencing greater than a 2-fold increase in TAT10772 expression as
compared to the mean level of TAT10772 expression for the group of
samples as a whole.
[0704] For purposes of quantitative comparison, 209 independent
human HER-2 negative infiltrating ductal carcinomas of the breast
tissue samples were also analyzed for TAT10772 expression. The
results obtained from these analyses demonstrated that the level of
expression of TAT10772 in the cancerous samples was quite variable,
with 76 of the 209 samples tested showing at least a 2-fold (to as
high as an about 15-fold) increase in TAT10772 expression when
compared to the mean level of TAT10772 expression for the group of
normal breast tissue samples analyzed.
[0705] C. Pancreas
[0706] In another experiment, expression of TAT10772 was analyzed
in a group of 51 independent normal human pancreas tissue samples.
The results of these analyses demonstrated that the level of
TAT10772 mRNA expression in all of the normal human pancreas tissue
samples analyzed was remarkably consistent and fell within a very
tight distribution, with no normal human pancreas tissue sample
evidencing greater than a 2-fold increase in TAT10772 expression as
compared to the mean level of TAT10772 expression for the group of
samples as a whole.
[0707] For purposes of quantitative comparison, 65 independent
human pancreatic adenocarcinoma tissue samples were also analyzed
for TAT10772 expression. The results obtained from these analyses
demonstrated that the level of expression of TAT10772 in the
cancerous samples was quite variable, with 33 of the 65 samples
tested showing at least a 2-fold (to as high as an about 21-fold)
increase in TAT10772 expression when compared to the mean level of
TAT10772 expression for the group of normal pancreas tissue samples
analyzed.
[0708] Given the above, the TAT10772 polypeptide, and the nucleic
acid encoding that polypeptide, are excellent targets which can be
exploited for quantitatively and qualitatively determining the
expression level of the TAT10772 polypeptide, and the mRNA encoding
it, in various mammalian tissue samples, thereby allowing one to
make quantitative and qualitative comparisons therebetween.
Therefore, the TAT10772 polypeptide, and the nucleic acid encoding
that polypeptide, are molecules whose unique expression profile can
be exploited for the diagnosis of certain types of cancerous tumors
in mammals as described above. Moreover, as this analysis
demonstrates that the TAT10772 polypeptide is significantly,
reproducibly and detectably overexpressed in certain human tumors
as compared to their corresponding normal human tissues, the
TAT10772 polypeptide serves as an excellent target that can be
exploited for the therapeutic treatment of such tumors in
mammals.
Example 2
Microarray Analysis to Detect Upregulation of TAT Polypeptides in
Cancerous Tumors
[0709] Nucleic acid microarrays, often containing thousands of gene
sequences, are useful for identifying differentially expressed
genes in diseased tissues as compared to their normal counterparts.
Using nucleic acid microarrays, test and control mRNA samples from
test and control tissue samples are reverse transcribed and labeled
to generate cDNA probes. The cDNA probes are then hybridized to an
array of nucleic acids immobilized on a solid support. The array is
configured such that the sequence and position of each member of
the array is known. For example, a selection of genes known to be
expressed in certain disease states may be arrayed on a solid
support. Hybridization of a labeled probe with a particular array
member indicates that the sample from which the probe was derived
expresses that gene. If the hybridization signal of a probe from a
test (disease tissue) sample is greater than hybridization signal
of a probe from a control (normal tissue) sample, the gene or genes
overexpressed in the disease tissue are identified. The implication
of this result is that an overexpressed protein in a diseased
tissue is useful not only as a diagnostic marker for the presence
of the disease condition, but also as a therapeutic target for
treatment of the disease condition.
[0710] The methodology of hybridization of nucleic acids and
microarray technology is well known in the art. In the present
example, the specific preparation of nucleic acids for
hybridization and probes, slides, and hybridization conditions are
all detailed in PCT Patent Application Serial No. PCT/US01/10482,
filed on Mar. 30, 2001 and which is herein incorporated by
reference.
[0711] In the present example, cancerous tumors derived from
various human tissues were studied for upregulated gene expression
relative to cancerous tumors from different tissue types and/or
non-cancerous human tissues in an attempt to identify those
polypeptides which are overexpressed in a particular cancerous
tumor(s). In certain experiments, cancerous human tumor tissue and
non-cancerous human tumor tissue of the same tissue type (often
from the same patient) were obtained and analyzed for TAT
polypeptide expression. Additionally, cancerous human tumor tissue
from any of a variety of different human tumors was obtained and
compared to a "universal" epithelial control sample which was
prepared by pooling non-cancerous human tissues of epithelial
origin, including liver, kidney, and lung. mRNA isolated from the
pooled epithelial tissues represents a mixture of expressed gene
products from various different epithelial tissues, thereby
providing an excellent negative control against which to
quantitatively compare gene expression levels in tumors of
epithelial origin. Microarray hybridization experiments using the
pooled control samples generated a linear plot in a 2-color
analysis. The slope of the line generated in a 2-color analysis was
then used to normalize the ratios of (test: control detection)
within each experiment. The normalized ratios from various
experiments were then compared and used to identify clustering of
gene expression. Thus, the pooled "universal control" sample not
only allowed effective relative gene expression determinations in a
simple 2-sample comparison, it also allowed multi-sample
comparisons across several experiments.
[0712] In the present experiments, nucleic acid probes derived from
the herein described TAT polypeptide-encoding nucleic acid
sequences were used in the creation of the microarray and RNA from
various tumor tissues were used for the hybridization thereto. A
value based upon the normalized ratio:experimental ratio was
designated as a "cutoff ratio". Only values that were above this
cutoff ratio were determined to be significant. Significance of
ratios were estimated from the amount of noise or scatter
associated with each experiment, but typically, a ratio cutoff of
1.8 fold-2 fold or greater was used to identify candidate genes
relatively overexpressed in tumor samples compared to the
corresponding normal tissue and/or the pooled normal epithelial
universal control. Ratios for genes identified in this way as being
relatively overexpressed in tumor samples varied from 2 fold to 40
fold, or even greater. By comparison, in a control experiment in
which the same RNA was labeled in each color and hybridized against
itself, for virtually all genes with signals above background, the
observed ratio is significantly less than 1.8 fold. This indicates
that experimental noise above a ratio of 1.8 fold is extremely low,
and that an observed fold change of 1.8 fold or greater is
significant and is expected to represent a real, detectably and
reproducible difference in expression between the samples analyzed
and compared.
[0713] The results of these experiments demonstrated that mRNA
encoding the TAT10772 polypeptide is significantly overexpressed
(i.e., at least 2-fold) in 8 of 10 independent human ovarian tumor
samples tested when compared to both normal human ovarian tissue
and the pooled epithelial control sample. These data also
demonstrate that the observed overexpression is significant,
detectable and reproducible across multiple human ovarian tumor
samples when compared to both normal counterpart human ovarian
samples as well as the pooled human epithelial control sample. As
described above, these data demonstrate that the TAT10772
polypeptide of the present invention, and the encoding nucleic
acid, are useful not only as diagnostic markers for the presence of
human ovarian tumors, but also serve as potential therapeutic
targets for the treatment of those tumors in humans.
EXAMPLE 3
Quantitative Analysis of TAT mRNA Expression
[0714] 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 and quantitative interpretation of the
data. This assay is well known and routinely used in the art to
quantitatively identify gene expression differences between two
different human tissue samples, see, e.g., Higuchi et al.,
Biotechnology 10:413-417 (1992); Livak et al., PCR Methods Appl.,
4:357-362 (1995); Heid et al., Genome Res. 6:986-994 (1996);
Pennica et al., Proc. Natl. Acad. Sci. USA 95(25):14717-14722
(1998); Pitti et al., Nature, 396(6712):699-703 (1998) and Bieche
et al., Int. J. Cancer, 78:661-666 (1998).
[0715] The 5' nuclease procedure is run on a real-time quantitative
PCR device such as the ABI Prism 7700.TM. Sequence Detection. The
system consists of a thermocycler, laser, charge-coupled device
(CCD) camera and computer. The system amplifies samples in a
96-well format on a thermocycler. During amplification,
laser-induced fluorescent signal is collected in real-time through
fiber optics cables for all 96 wells, and detected at the CCD. The
system includes software for running the instrument and for
analyzing the data.
[0716] 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. Frequently, tumor sample(s) are
directly compared to "matched" normal sample(s) of the same tissue
type, meaning that the tumor and normal sample(s) are obtained from
the same individual.
[0717] 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 and quantitatively measure the relative fold
increase in mRNA expression between two or more different tissues.
In this regard, it is well accepted in the art that this assay is
sufficiently technically sensitive to reproducibly detect an at
least 2-fold increase in mRNA expression in a human tumor sample
relative to a normal control.
[0718] Using this technique, it was determined that mRNA encoding
the TAT10772 polypeptide is significantly and reproducibly
overexpressed (i.e., at least 2-fold) in 9 of 10 independent human
ovarian tumor samples when compared to both normal human ovarian
samples from different human tissue donors as well as various
"matched" normal human ovarian tumor samples derived from the same
human tissue donor as from which the tumor sample(s) was derived.
As described above, therefore, these data demonstrate that the
TAT10772 polypeptide of the present invention, and the encoding
nucleic acid, are useful not only as diagnostic markers for the
presence of human ovarian tumors, but also serve as potential
therapeutic targets for the treatment of those tumors in
humans.
Example 4
In Situ Hybridization
[0719] 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.
[0720] 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 EC, 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 EC overnight. The slides were dipped in Kodak NTB2 nuclear
track emulsion and exposed for 4 weeks.
.sup.33P-Riboprobe Synthesis
[0721] 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:
[0722] 2.0 .mu.l 5.times. transcription buffer
[0723] 1.0 .mu.l DTT (100 mM)
[0724] 2.0 .mu.l NTP mix (2.5 mM: 10.mu.; each of 10 mM GTP, CTP
& ATP+10 .mu.l H.sub.2O)
[0725] 1.0 .mu.l UTP (50 .mu.M)
[0726] 1.0 .mu.l Rnasin
[0727] 1.0 .mu.l DNA template (1 .mu.g)
[0728] 1.0 .mu.l H.sub.2O
[0729] 1.0 .mu.l RNA polymerase (for PCR products T3=AS, T7=S,
usually)
[0730] The tubes were incubated at 37 EC for one hour. 1.0 .mu.l
RQ1 DNase were added, followed by incubation at 37 EC 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.
[0731] 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 EC 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 EC freezer one hour to overnight.
.sup.33P-Hybridization
[0732] A. Pretreatment of frozen sections
[0733] The slides were removed from the freezer, placed on aluminum
trays and thawed at room temperature for 5 minutes. The trays were
placed in 55 EC 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 0.5m/ml proteinase K for 10 minutes at 37 EC
(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.
[0734] B. Pretreatment of Paraffin-Embedded Sections
[0735] 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 EC, 15
minutes)--human embryo, or 8.times.proteinase K (100 .mu.l in 250
ml Rnase buffer, 37 EC, 30 minutes)--formalin tissues. Subsequent
rinsing in 0.5.times.SSC and dehydration were performed as
described above.
[0736] C. Prehybridization
[0737] The slides were laid out in a plastic box lined with Box
buffer (4.times.SSC, 50% formamide)--saturated filter paper.
[0738] D. Hybridization
[0739] 1.0.times.10.sup.6 cpm probe and 1.0 .mu.l tRNA (50 mg/ml
stock) per slide were heated at 95 EC for 3 minutes. The slides
were cooled on ice, and 48 .mu.l hybridization buffer were added
per slide. After vortexing, 50 .mu..sup.33P mix were added to 50
.mu.l prehybridization on slide. The slides were incubated
overnight at 55 EC.
[0740] E. Washes
[0741] Washing was done 2.times.10 minutes with 2.times.SSC, EDTA
at room temperature (400 ml 20.times.SSC+16 ml 0.25 M EDTA,
V.sub.f=4L), followed by RNaseA treatment at 37 EC for 30 minutes
(500 .mu.l of 10 mg/ml in 250 ml Rnase buffer=20 .mu.g/ml), The
slides were washed 2.times.10 minutes with 2.times.SSC, EDTA at
room temperature. The stringency wash conditions were as follows: 2
hours at 55 EC, 0.1.times.SSC, EDTA (20 ml 20.times.SSC+16 ml EDTA,
V.sub.f=4L).
[0742] F. Oligonucleotides
[0743] 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.
[0744] G. Results
[0745] With regard to expression of TAT10772 in normal human
tissues, strong expression is observed in bronchial mucosa and
submucous glands. However, all other normal human tissues tested
are negative for TAT10772 expression. In contrast, strong TAT10772
expression is observed in 13 of 15 human ovarian tumors
(adenocarcinoma and surface epithelial tumors) tested.
Additionally, strong TAT10772 expression is also observed in 8 of 9
human uterine adenocarcinomas.
Example 5
Preparation of Antibodies that Bind TAT10772
[0746] This example illustrates preparation of monoclonal
antibodies which can specifically bind TAT10772.
[0747] 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.
[0748] 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, Mt.) 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.
[0749] After a suitable antibody titer has been detected, the
animals "positive" for antibodies can be injected with a final
intravenous injection of TAT. Three to four days later, the mice
are sacrificed and the spleen cells are harvested. The spleen cells
are then fused (using 35% polyethylene glycol) to a selected murine
myeloma cell line such as P3X63AgU.1, available from ATCC, No. CRL
1597. The fusions generate hybridoma cells which can then be plated
in 96 well tissue culture plates containing HAT (hypoxanthine,
aminopterin, and thymidine) medium to inhibit proliferation of
non-fused cells, myeloma hybrids, and spleen cell hybrids.
[0750] 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.
[0751] 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.
[0752] Using the above described technique, 11 separate and
distinct hybridoma cell lines have been generated, each of which
produce monoclonal antibodies that bind to the TAT10772
polypeptide. These 11 hybridoma cell lines are herein referred to
as 16F7.1.15 (producing monoclonal antibody 16F7),17A8.1.3
(producing monoclonal antibody 17A8), 9F3.1.3 (producing monoclonal
antibody 9F3), 16E12.2.15 (producing monoclonal antibody 16E12),
16A7.1.3 (producing monoclonal antibody 16A7), 10G11.1.1 (producing
monoclonal antibody 10G11), 5B10 (producing monoclonal antibody
5B10), 11D10.1.14 (producing monoclonal antibody 11D10), 5F6.1.24
(producing monoclonal antibody 5F6), 7G6.2.6 (producing monoclonal
antibody 7G6), and 3A5.3 (producing monoclonal antibody 3A5.3). The
monoclonal antibodies produced by these 11 hybridoma lines have
been shown to bind to the TAT10772 polypeptide using well-known and
routinely employed techniques such as Western blot, ELISA analysis,
FACS sorting analysis of cells expressing the TAT10772 polypeptide
and/or immunohistochemistry analysis. Of the 11 hybridoma lines
that produce functional anti-TAT10772 monoclonal antibodies, two
(hybridoma clones 11D10.1.14 and 3A5.3) have been deposited under
the terms of the Budapest Treaty with the American Tissue Type
Collection, Manassas, Va. as described in further detail below.
Example 6
Competitive Binding Analyses and Epitope Mapping
[0753] The TAT10772 epitopes bound by the monoclonal antibodies
described were determined by standard competitive binding analysis
(Fendly et al., Cancer Research, 50:1550-1558 (1990)).
Cross-blocking studies were done on antibodies by direct
fluorescence on intact PC3 cells engineered to express TAT10772
using the PANDEX.TM. Screen Machine to quantitate fluorescence.
Each monoclonal antibody was conjugated with fluorescein
isothiocyanate (FITC), using established procedures (Wofsy et al.,
Selected Methods in Cellular Immunology, p. 287, Mishel and Schiigi
(eds.) San Francisco: W. J. Freeman Co. (1980)). Confluent
monolayers of TAT10772-expressing PC3 cells were trypsinized,
washed once, and resuspended at 1.75.times.10.sup.6 cell/ml in cold
PBS containing 0.5% bovine serum albumin (BSA) and 0.1% NaN.sub.3.
A final concentration of 1% latex particles (DC, Portland, Oreg.)
was added to reduce clogging of the PANDEX.TM. plate membranes.
Cells in suspension, 20 .mu.l, and 20 .mu.l of purified monoclonal
antibodies (100 .mu.g/ml to 0.1 .mu.g/ml) were added to the
PANDEX.TM. plate wells and incubated on ice for 30 minutes. A
predetermined dilution of FITC-labeled monoclonal antibodies in 20
.mu.l was added to each well, incubated for 30 minutes, washed, and
the fluorescence was quantitated by the PANDEX.TM. Screen Machine.
Monoclonal antibodies were considered to share an epitope if each
blocked binding of the other by 40% or greater in comparison to an
irrelevant monoclonal antibody control and at the same antibody
concentration. In this experiment, monoclonal antibodies 16F7,
17A8, 9F3, 16E12, 16A7, 10G11, 5B10, 11D10, 5F6, 7G6, and 3A5 were
assigned TAT10772 epitopes B, B, B, B, B, B, A, B, B, C, and D,
respectively. Using this assay, one of ordinary skill in the art
can identify other monoclonal antibodies that bind to the same
epitope as those described above.
[0754] Deletion analysis was also conducted to identify the
approximate location in the polypeptide sequence shown as SEQ ID
NO:2 of the above described antigenic epitopes. These analyses
demonstrated that TAT10772 antigenic epitope A is found between
amino acids 6471-6560 of SEQ ID NO:2, TAT10772 antigenic epitope B
is found between amino acids 6389-6470 of SEQ ID NO:2, TAT10772
antigenic epitope C is found between amino acids 6663-6806 of SEQ
ID NO:2, and TAT10772 antigenic epitope D is found between amino
acids 3765-6397 of SEQ ID NO:2 (which comprises approximately
seventeen 150 amino acid mucin-like repeat sequences and,
therefore, most likely comprises multiple similar antigenic epitope
sites). Polypeptides comprising any of these specifically
identified antigenic epitope sites (and nucleic acid molecules
encoding those polypeptides) are encompassed within the present
invention.
[0755] In a separate experiment, it was demonstrated that the
binding of monoclonal antibody to 3A5 to OVCAR-3, OVCA-432 and
SK-OV-3 cells as determined by standard flow cytometry analyses
parallels the expression level of TAT10772 mRNA expressed in each
of these three specific cell lines as determined by standard
quantitative PCR analyses. More specifically, as determined by
standard quantitative PCR analysis, OVCAR-3, OVCA-432 and SK-OV-3
cells express a high, moderate and low level of TAT10772 mRNA,
respectively. When monoclonal antibody 3A5 was employed in standard
flow cytometry analyses to quantitate the ability of 3A5 to bind to
these cells, it was observed that 3A5 binding quantitatively
parallels the relative amount of TAT10772 mRNA present in those
cell lines. These data suggest that the amount of TAT10772 mRNA in
any particular cell type is quantitatively determinative of the
amount of TAT10772 polypeptide expressed by that cell type and, in
turn, is determinative of the ability of any specific anti-TAT10772
antibody to bind to that cell type.
Example 7
Immunohistochemistry Analysis
[0756] Antibodies against TAT10772 were prepared as described above
and immunohistochemistry analysis was performed using the
monoclonal antibodies 3A5 and 11D10 as follows. Tissue sections
were first fixed for 5 minutes in acetone/ethanol (frozen or
paraffin-embedded). The sections were then washed in PBS and then
blocked with avidin and biotin (Vector kit) for 10 minutes each
followed by a wash in PBS. The sections were then blocked with 10%
serum for 20 minutes and then blotted to remove the excess. A
primary antibody was then added to the sections at a concentration
of 10 .mu.m/ml for 1 hour and then the sections were washed in PBS.
A biotinylated secondary antibody (anti-primary antibody) was then
added to the sections for 30 minutes and then the sections were
washed with PBS. The sections were then exposed to the reagents of
the Vector ABC kit for 30 minutes and then the sections were washed
in PBS. The sections were then exposed to Diaminobenzidine (Pierce)
for 5 minutes and then washed in PBS. The sections were then
counterstained with Mayers hematoxylin, covered with a coverslip
and visualized. Immunohistochemistry analysis can also be performed
as described in Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Press, 1989 and Ausubel et
al., Current Protocols of Molecular Biology, Unit 3.16, John Wiley
and Sons (1997).
[0757] The results from these analyses demonstrate that monoclonal
antibody 11D10 does not detectably bind to any of the following
normal human tissues: aorta, brain, colon, liver, kidney, small
intestine, stomach, lung (both alveolar and bronchial tissue),
testis, spleen thyroid, ovarian, uterine, urothelium and placenta.
However, 6 of 13 independent human ovarian adenocarcinoma samples
and 1 of 7 independent human endometrial adenocarcinoma samples
show strong binding to antibody 11D10. Moreover, in a separate
experiment, antibody 11D10 binds strongly to 1 of 9 human mucinous
adenocarcinoma tumor samples, 13 of 22 human endometrioid
adenocarcinoma tumor samples, 17 of 26 human serous
cystadenocarcinoma tumor samples and 3 of 8 human clear cell tumor
samples.
[0758] Moreover, the results from these analyses demonstrate that
monoclonal antibody 3A5, like monoclonal antibody 11D10, does not
detectably bind to any of the above listed normal human tissues.
However, antibody 3A5 binds strongly to 2 of 2 independent human
ovarian adenocarcinomas (membranous staining), 16 of 20 human
endometrioid adenocarcinoma tumor samples, 24 of 25 human serous
cystadenocarcinoma tumor samples and 5 of 10 human clear cell tumor
samples.
Example 8
Monoclonal Antibody 3A5 Is Internalized Upon Binding to TAT10772
Polypeptide on Cells
[0759] This experiment demonstrates that monoclonal antibody 3A5
becomes internalized into cells to which it binds TAT10772
polypeptide on the cell surface. Specifically, OVCAR-3 cells were
incubated for 18 hours with monoclonal antibody 3A5 and fluorescent
dextran and then cell-associated 3A5 was quantitatively detected
with a fluorescein-labeled anti-3A5 antibody. These analyses
demonstrated that antibody 3A5 co-localizes with dextran,
indicating trafficking of the 3A5 antibody into subcellular
components of the incubated cells, including the lysosomal
compartments of these cells.
Example 9
Humanization of Murine Monoclonal Antibodies
[0760] This example demonstrates the applicability of the method of
CDR-repair for humanization of murine antibodies 11D10 and 3A5
directed against TAT10772.
[0761] Three forms of TAT10772 were used during the humanization
process. The human TAT10772 shed antigen, CA125, encompasses of the
entire shed antigen and was purchased from US Biological C0050-10.
The TAT10772-stalk consists of the last, most C-terminal mucin
domain and the following C-terminal sequence leading to the
predicted transmembrane region (amino acids 6282-6979 of SEQ ID
NO:2). 5-domain TAT10772 (amino acids 4471-5171 of SEQ ID NO:2) is
a recombinant portion of the extracellular domain encoding 5 mucin
domains plus the C-terminal sequence leading to the predicted
transmembrane region. The MUC16-stalk and the 5-mucin domain were
expressed in CHO cells and purified by conventional means.
[0762] Residue numbers are according to Kabat (Kabat et al.,
Sequences of proteins of immunological interest, 5th Ed., Public
Health Service, National Institutes of Health, Bethesda, MD
(1991)). Single letter amino acid abbreviations are used. DNA
degeneracies are represented using the IUB code (N=A/C/G/T,
D=A/G/T, V=A/C/G, B=C/G/T, H=A/C/T, K=G/T, M=A/C, R=A/G, S=G/C,
W=A/T, Y=C/T).
Cloning of Murine 11D10 and 3A5 Variable Domains and Generation of
Chimeric 11D10 and 3A5 Antibodies
[0763] Total RNA was extracted from hybridoma cells producing 11D10
or 3A5 using standard methods. The variable light (VL) and variable
heavy (VH) domains were amplified using RT-PCR with degenerate
primers to the heavy and light chains. The forward primers were
specific for the N-terminal amino acid sequence of the VL and VH
regions. Respectively, the LC and HC reverse primers were designed
to anneal to a region in the constant light (CL) and constant heavy
domain 1 (CH1), which are highly conserved across species.
Amplified VL and VH were cloned into mammalian expression vectors.
The polynucleotide sequence of the inserts was determined using
routine sequencing methods. The 11D10 VL (mu11D10-L) and VH
(mu11D10-H) amino acid sequences are shown in FIGS. 3 and 4,
respectively (SEQ ID NOS:4 and 7, respectively); the 3A5 VL
(mu3A5-L) and VH (mu3A5-H) amino acid sequences are shown in FIGS.
5 and 6, respectively (SEQ ID NOS:9 and 11, respectively). HVR
regions according to Kabat numbering are shown in bold font in
FIGS. 3-6.
Direct Hypervariable Region Grafts onto the Acceptor Human
Consensus Framework
[0764] The phagemid used for this work is a monovalent Fab-g3
display vector and consists of 2 open reading frames under control
of the phoA promoter. The first open reading frame consists of the
stll signal sequence fused to the VL and CH1 domains of the
acceptor light chain and the second consists of the stII signal
sequence fused to the VH and CH1 domains of the acceptor heavy
chain followed by the minor phage coat protein P3.
[0765] The VL and VH domains from the murine 11D10 and 3A5
antibodies were aligned with the human VL kappa I (huKl; SEQ ID
NO:3) and human VH subgroup III (huIII; SEQ ID NO:6) consensus
sequences. To make the HVR grafts, hypervariable regions from the
murine antibodies were grafted into the huKI and huIII acceptor
frameworks. For 3A5, two acceptor VH frameworks were tested
(designated herein as 3A5.L and 3A5.F, respectively) differing only
at amino acid position 78 (see FIGS. 6A-B).
[0766] Hypervariable regions from murine 11D10 and 3A5 antibodies
were engineered into the acceptor human consensus framework to
generate the direct HVR-grafts, 11D10-graft, 3A5.L-graft and
3A5.F-graft. In the VL domain the following regions were grafted to
the human consensus acceptor: positions 24-34 (HVR-L1), 49-56
(HVR-L2) and 89-97 (HVR-L3). In the VH domain, positions 26-35A
(HVR-H1), 49-65 (HVR-H2) and 93-102 (HVR-H3) were grafted (FIGS. 3
through 6). MacCallum et al. (MacCallum et al. J. Mol. Biol. 262:
732-745 (1996)) have analyzed antibody and antigen complex crystal
structures and found position 49 of the light chain and positions
49, 93 and 94 of the heavy chain are part of the contact region
thus it seems reasonable to include these positions in the
definition of HVR-L2, HVR-H2 and HVR-H3 when humanizing
antibodies.
[0767] The direct-graft variants were generated by Kunkel
mutagenesis using a separate oligonucleotide for each hypervariable
region. Correct clones were assessed by DNA sequencing.
Randomization of the Hypervariable Regions
[0768] For each grafted antibody, sequence diversity was introduced
separately into each hypervariable region using a soft
randomization strategy (SR libraries) that maintains a bias towards
the murine hypervariable region sequence. This was accomplished
using a poisoned oligonucleotide synthesis strategy first described
by Gallop et al., J. Med. Chem. 37:1233-1251 (1994). For a given
position within a hypervariable region to be mutated, the codon
encoding the wild-type amino acid is poisoned with a 70-10-10-10
mixture of nucleotides resulting in an average 50 percent mutation
rate at each position.
[0769] Soft randomized oligonucleotides were patterned after the
murine hypervariable region sequences and encompassed the same
regions defined by the direct hypervariable region grafts. The
amino acid position at the beginning of H2 (position 49) in the VH
domain, was limited in sequence diversity to A, G, S or T by using
the codon RGC.
[0770] In addition to the soft randomization libraries outlined
above, each position in each hypervariable region of 3A5.L-graft
and 3A5.F-graft was fully randomized to all possible 20 amino acids
using oligonucleotides encoding NNS. This was accomplished in 2
types of libraries. In the first, multiple libraries were made each
consisting of 20 members having a single position located within
one of the hypervariable regions of 3A5 fully randomized. To cover
each position in the hypervariable regions, 63 libraries of this
type were generated and combined into a pooled "single position
library" (SP library) encompassing single mutations located
throughout each hypervariable position. The second library
introduced all 20 amino acids into all positions (FR library)
within a single hypervariable region at the same time. For both of
these library types there were 6 libraries each encompassing a
separate hypervariable region of the 3A5.L-graft or
3A5.F-graft.
[0771] To avoid reselecting the wild type CDR grafted sequence, a
stop codon (TAA) was introduced in the middle of each HVR by Kunkel
mutagenesis resulting in 6 different templates for each graft
(11D10-graft, 3A5.L-graft and 3A5.F-graft) each with a stop codon
introduced into a different HVR. When generating the SR, FR and SP
libraries, randomized oligonucleotides were used to introduce
diversity as well as to repair the stop codon in the corresponding
template. For 3A5 libraries, a mixture of 3A5.L and 3A5.F templates
was used during the construction of each library. All 3 types of
libraries were generated for humanization of 3A5, while only the SR
library was generated for humanization of 11D10.
Generation of Phage Libraries
[0772] Randomized oligonucleotide pools designed to introduce
diversity into each hypervariable region as outlined above, were
phosphorylated separately in 20 .mu.l reactions containing 660 ng
of oligonucleotide, 50 mM Tris pH 7.5, 10 mM MgCl.sub.2, 1 mM ATP,
20 mM DTT, and 5 U polynucleotide kinase for 1 h at 37.degree.
C.
[0773] To generate the SR and FR libraries each phosphorylated
oligonucleotide pool directed to introduce diversity into a single
HVR was combined with 20 .mu.g of Kunkel template containing the
corresponding stop codon. The reaction was performed in 50 mM Tris
pH 7.5, 10 mM MgCl.sub.2 in a final volume of 500 .mu.l resulting
in a oligonucleotide to template ratio of 3. The mixture was
annealed at 90.degree. C. for 4 min, 50.degree. C. for 5 min and
then cooled on ice. The annealed template (250 .mu.l) was then
filled in by adding 1 .mu.l 100 mM ATP, 10 .mu.l 25 mM dNTPs (25 mM
each of dATP, dCTP, dGTP and dTTP), 15 .mu.l 100 mM DTT, 25 .mu.l
10.times.TM buffer (0.5 M Tris pH 7.5, 0.1 M MgCl.sub.2), 2400 U T4
ligase, and 30 U T7 polymerase for 3 hours at room temperature. The
filled in product was then cleaned-up and electroporated into SS320
cells and propagated in the presence of M13/K07 helper phage as
described by Sidhu et al., Methods in Enzymology 328:333-363
(2000). Library sizes ranged from 1-2.times.10.sup.9 independent
clones. Random clones from the initial libraries were sequenced to
assess library quality.
[0774] Multiple (63) standard Kunkel mutagenesis reactions were
performed in a 96-well PCR plate to generate the 3A5 SP libraries.
From the phosphorylated oligonucleotides reactions (above), 2 .mu.l
was added to 300 ng Kunkel template containing the corresponding
stop codon in 50 mM Tris pH 7.5, 10 mM MgCl.sub.2 in a final volume
of 10 .mu.l. The mixture was annealed at 90.degree. C. for 2 min,
50.degree. C. for 5 min and then cooled on ice. The annealed
template was then filled in by adding 0.5 .mu.l 10 mM ATP, 0.5
.mu.l 10 mM dNTPs (10 mM each of dATP, dCTP, dGTP and dTTP), 1.mu.l
100 mM DTT, 1.mu.l 10.times.TM buffer (0.5 M Tris pH 7.5, 0.1 M
MgCl.sub.2), 80 U T4 ligase, and 4 U T7 polymerase in a total
volume of 20 .mu.l for 2 h at room temperature. These filled in and
ligated products were then each transformed into XL1-blue cells,
grown in 0.5 ml of 2YT containing 5 .mu.g/ml of tetracycline and
M13/K07 helper phage (MOI 10) for 2 hr at 37.degree. C. and then
pooled and transferred to 500 ml 2YT containing 50 .mu.g/ml
carbenacillin and grown 16 h at 37.degree. C.
Phage Selection
[0775] For the phage selections outlined below, TAT10772-stalk (2
.mu.g/ml), CA125 (17 .mu.g/ml), 5-domain TAT10772 (2 .mu.g/ml) or
neutravidin (2 .mu.g/ml) were immobilized in PBS on MaxiSorp
microtiter plates (Nunc) overnight at 4.degree. C. Plates were
blocked for at least 1 h using Casein Blocker (Pierce). Phage were
harvested from the culture supernatant and suspended in PBS
containing 1% BSA and 0.05% Tween 20 (PBSBT). Following phage
selection, as outlined below, microtiter wells were washed
extensively with PBS containing 0.05% Tween 20 (PBST) and bound
phage were eluted by incubating the wells with 100 mM HCl for 30
min. Phage were neutralized with 1 M Tris, pH 8 and amplified using
XL1-Blue cells and M13/K07 helper phage and grown overnight at
37.degree. C. in 2YT, 50 .mu.g/ml carbenacillin. The titers of
phage eluted from a target containing well were compared to titers
of phage recovered from a non-target containing well to assess
enrichment.
[0776] The solution sorting method has been described (Fuh et al.
J. Mol. Biol. (2004)) and enables the selection of faster on-rates
through a control of biotinylated target concentration and slower
off-rates resulting from competition with unlabeled target.
TAT10772-stalk and 5-domain TAT10772 were biotinylated using
Sulfo-NHS-LC-biotin (Pierce).
[0777] The TAT10772-stalk was used as a phage target for the
humanization of 11D10. The TAT10772-stalk was immobilized directly
on MaxiSorp microtiter plates (Nunc) at 2 ug/ml in PBS for the
first round of phage selection. Successive rounds of selection used
a soluble selection method (Fuh et al. J. Mol. Biol. (2004)).
Biotinylated-TAT10772-stalk was first incubated with the phage
library for 1 hr, followed by a 5 min capture of the bound phage on
a neutravidin-coated plate. Excess unlabeled TAT10772-stalk
(greater than 100 nM) was added prior to the capture step for
increasing lengths of time to increase selection stringency. The
following table summarizes the conditions that were used for
solution-panning the 11D10 libraries.
Selection Round [Biotinylated TAT10772-Stalk] Incubation with
Excess TAT10772-Stalk
TABLE-US-00006 2 10 nM 20 min at 25.degree. C. 3 10 nM 6.5 hr at
25.degree. C. 4 10 nM 88.5 hr at 25.degree. C. 5 1 nM 48 hr at
25.degree. C., then 52 hr at 37.degree. C.
CA125 and 5-domain TAT10772 were used as a phage targets for the
humanization of 3A5. Libraries were sorted individually for the
first round of selection against immobilized 5-domain TAT10772 (2
.mu.g/ml in PBS) or CA125 (17 .mu.g/ml in PBS) that was coated on
Nunc MaxiSorp microtiter plates. Following amplification, the
libraries were pooled according to their library type (FR/SR/SP)
and whether they were panned against CA125 or 5-domain TAT10772 and
sorted for an additional 2 rounds against their respective
immobilized targets. Three successive rounds of selection were
performed by continued panning against the immobilized targets or
by selection against soluble biotinylated 5-domain TAT10772 using a
solution sorting strategy (Fuh et al. J. Mol. Biol. (2004)). For
the solution sorting method, phage libraries were incubated with 1
nM biotinylated 5-domain TAT10772 for 1 hr followed by the addition
of an excess of unlabeled 5-domain TAT10772 (greater than 100 nM)
for up to 22 hrs to increase selection stringency. Phage bound to
the biotinylated 5-domain TAT10772 were captured briefly (5 min)
using a neutravidin-coated plate. TAT10772-stalk Phage ELISA
[0778] MaxiSorp microtiter plates were coated with TAT10772-stalk
at 2 .mu.g/ml in PBS over night and then blocked with Casein
Blocker. Phage from culture supernatants were incubated with
serially diluted TAT10772-stalk in PBST containing 1% BSA in a
tissue culture microtiter plate for 1 h after which 80 .mu.l of the
mixture was transferred to the target coated wells for 15 min to
capture unbound phage. The plate was washed with PBST and HRP
conjugated anti-M13 (Amersham Pharmacia Biotech) was added (1:5000
in PBSBT) for 40 min. The plate was washed with PBST and developed
by adding Tetramethylbenzidine substrate (Kirkegaard and Perry
Laboratories, Gaithersburg, Md.). The absorbance at 450 nm was
plotted as a function of target concentration in solution to
determine an IC50. This was used as an affinity estimate for the
Fab clone displayed on the surface of the phage.
Fab and IgG Production and Affinity Determination
[0779] To express Fab protein for affinity measurements, a stop
codon was introduced between the heavy chain and g3 in the phage
display vector. Clones were transformed into E. coli 34B8 cells and
grown in Complete C.R.A.P. media at 30.degree. C. (Presta et al.
Cancer Res. 57: 4593-4599 (1997)). Cells were harvested by
centrifugation, suspended in PBS, 100 uM PMSF, 100 uM benzamidine,
2.5 mM EDTA and broken open using a microfluidizer. Fab was
purified with Protein G affinity chromatography.
[0780] Affinity determinations were performed by surface plasmon
resonance using a BIAcore.TM.-2000. Either .about.500 RU of
5-domain TAT10772 or .about.300 RU IgG was immobilized in 10 mM
Sodium Acetate pH 4.8 on a CMS sensor chip and serial 2-fold
dilutions of the corresponding binding partner (1-1000nM) in PBST
were injected at a flow rate of 20 .mu.l/min. Each sample was
analyzed with 5-minute association and 10-minute dissociation.
After each injection the chip was regenerated using 10 mM Glycine
pH 1.5. Binding response was corrected by subtracting the RU from a
blank flow cell. A 1:1 Langmuir model of simultaneous fitting of
k.sub.on and k.sub.off was used for kinetics analysis.
Humanization of 11D10
[0781] The human acceptor framework used for humanization of 11D10
consists of the consensus human kappa I VL domain and a variant of
the human subgroup III consensus VH domain. The VL and VH domains
of murine 11D10 were each aligned with the human kappa I and
subgroup III domains; each complementarity determining region (CDR)
was identified and grafted into the human acceptor framework to
generate an HVR graft that could be displayed as an Fab on phage
(FIGS. 3 and 4). When phage displaying the 11D10 HVR graft were
tested for binding to immobilized CA125, phage binding was
observed. When the 11D10 HVR graft sequence was expressed as a Fab,
Biacore analysis also evidenced binding to CA125.
[0782] A SR library was generated for 11D10 in which each HVR was
soft randomized individually. The 6 SR libraries were each panned
separately against immobilized TAT10772-stalk for 5 rounds of
selection. Enrichment was observed beginning after round 3 and
following round 5, clones were picked for DNA sequence analysis.
Sequence changes targeting each of the HVRs were observed. Clones
were screened using the anti-TAT10772 phage ELISA. Select clones
were expressed as Fab for further analysis by Biacore. Several
clones were reformatted as IgG for Scatchard analysis. FACS
analysis using OVCAR-3 cells demonstrated that all 11D10 humanized
antibodies tested were capable of effectively FACS sorting said
cells. From these results it is clear that there are multiple
sequence changes that can repair the affinity of 11D10 grafted onto
a human framework and that this antibody can be humanized by
CDR-repair to generate affinities that meet or exceed that of the
initial murine antibody.
Humanization of 3A5
[0783] Two human acceptor frameworks, 3A5.L and 3A5.F, were used
for humanization of 3A5 and are based on the consensus human kappa
I VL domain and the human subgroup III consensus VH domain. The VL
and VH domains of murine 3A5 were each aligned with the human kappa
I and subgroup III domains; each complementarity determining region
(CDR) was identified and grafted into the human acceptor framework
to generate an HVR graft that could be displayed as an Fab on phage
(FIGS. 5 and 6). When phage displaying the 3A5 HVR grafts were
tested for binding to immobilized CA125, phage binding was observed
for both. When expressed as a Fab, Biacore analysis also evidenced
binding for both to 5-domain TAT10772.
[0784] SR, FR and SP libraries were generated in which diversity
was introduced separately into each HVR of the 3A5 HVR graft.
Libraries were panned against CA125 and 5-domain TAT10772 using
both solid phase and solution sorting strategies. The solution
sorting method allows high affinity clones to be selected through
manipulation of the biotinylated target concentration and phage
capture time while the addition of unlabeled target can be used to
eliminate clones with faster off rates (Fuh et al. J. Mol. Biol.
340, 1073-1093 (2004)). Enrichment was observed after the second
round in all libraries. FACS analysis using OVCAR-3 cells
demonstrated that all 3A5 humanized antibodies tested were capable
of effectively FACS sorting said cells.
[0785] Following round 5, clones were picked for DNA sequence
analysis from each library and revealed sequence changes targeted
at HVR-H3 suggesting that the redesign of this CDR was important to
the restoration of antigen binding.
Sequence Analysis of Humanized Clones
[0786] The amino acid sequences for all light chain and heavy chain
HVR regions of all of the humanized clones were obtained. For
humanized 11D10 antibodies, the obtained HVR sequences are shown in
FIGS. 7-12. For humanized 3A5 antibodies, the obtained HVR
sequences are shown in FIGS. 13-18. FIGS. 19 and 20 show exemplary
acceptor human consensus framework sequences for variable heavy and
variable light chains, respectively. The present invention
encompasses antibodies comprising at least one of the disclosed
acceptor human consensus framework sequences in combination with at
least one of the HVR sequences disclosed.
Binding Analyses for Selected Humanized 3A5 Antibody Clones
[0787] Several humanized 3A5 clones were selected to be expressed
as IgG and characterized for binding to TAT10772 by Biacore, a
competitive binding ELISA, and OVCAR-3 cell binding analyses.
Results from the standard ELISA analyses are shown in Table 7
below. Results from the standard Biacore analyses measuring binding
to 5'-domain TAT10772 to immobilized 3A5 variant IgG antibodies are
shown in Table 8 below. Note that all antibodies tested were IgG
and contained the variable light chain sequence shown herein as SEQ
ID NO:211. A back mutation of S49Y in VL was found to have no
affect on binding and was incorporated into the final humanized
variants as tyrosine is more commonly found at this position. The
variable heavy chain sequence of the antibody is referred to in
Tables 7 and 8. As shown in Tables land 8, several clones met or
exceeded the monomeric affinity of the chimeric antibody as
summarized.
TABLE-US-00007 TABLE 7 ELISA Kd (nM) 3A5 Antibody Version 5'-Domain
(VH Chain Sequence) Cells CA125 TAT10772 OVCAR-3 3A5 chimera 0.3
2.3 0.3 (mu3 A5-H; SEQ ID NO: 11) 3A5.L-graft (SEQ ID NO: 12) 7.1
3A5.F- graft (SEQ ID NO: 13) 51.4 90.3 0.6 3A5v1 (SEQ ID NO: 198)
0.6 3.0 0.5 3A5v2 (SEQ ID NO: 199) 0.8 3.7 0.7 3A5v3 (SEQ ID NO:
200) 0.5 1.6 0.2 3A5v4 (SEQ ID NO: 201) 0.3 2.4 0.8 3A5v5 (SEQ ID
NO: 202) 8.2 10.2 0.6 3A5v6 (SEQ ID NO: 203) 4.4 5.7 0.6 3A5v7 (SEQ
ID NO: 204) 1.2 3.3 0.8 3A5v8 (SEQ ID NO: 205) 0.4 2.6 0.5
TABLE-US-00008 TABLE 8 3A5 Antibody Version (VH Chain Sequence) ka
(1/Ms) Kd (1/s) Kd (nM) 3A5 chimera 4.48E+04 1.21E-04 2.7 (mu3A5-H;
SEQ ID NO: 11) 3A5.F- graft (SEQ ID NO: 13) 2.85E+04 2.92E-04 10
3A5v1 (SEQ ID NO: 198) 3.69E+04 1.78E-04 4.8 3A5v2 (SEQ ID NO: 199)
3.34E+04 1.21E-04 3.6 3A5v3 (SEQ ID NO: 200) 3.62E+04 1.30E-04 3.6
3A5v8 (SEQ ID NO: 205) 5.51E+04 1.27E-04 2.3
Several humanized 3A5 antibodies were also tested in competitive
binding ELISA (measuring binding to immobilized 5'-domain TAT10772
and CA125) and OVCAR-3 cell binding analyses, wherein the results
of these analyses are shown in FIGS. 23-25. As shown in FIGS.
23-25, all humanized 3A5 antibodies tested are capable of strongly
binding to the TAT10772 target polypeptide and effectively
competing for binding at antigenic sites on that target
polypeptide.
Removal of a Potential Glycosylation Site in CDR-H2 of Humanized
3A5 Variants
[0788] To avoid potential manufacturing issues, a potential
glycosylation site in CDR-H2 of the humanized 3A5 variants was
eliminated using phage selection methods to identify suitable
sequence changes. Separately both N52 and S54 were fully randomized
using the codon NNS to allow all possible amino acid substitutions.
These small 20-member phage libraries were selected for binding to
5'-domain TAT10772. Although both N52 and S54 were found, other
substitutions were frequently observed at both positions with the
changes N52S and S54A being the most abundant. Certain data from
standard scatchard analyses are shown in Table 9 below, where the
antibodies are expressed as IgGs having a variable light chain
sequence shown herein as SEQ ID NO:211 and the variable heavy chain
sequence shown in Table 9. When either of the described changes
were incorporated into the humanized variants 3A5.v1 or 3A5.v4 (see
SEQ ID NOS:206-209), they did not affect binding affinity for
TAT10772.
TABLE-US-00009 TABLE 9 3A5 Antibody Version (VH Chain Sequence) Kd
(nM) 3A5 chimera (mu3A5-H; SEQ ID NO: 11) 0.57 .+-. 0.3 3A5v1b 52
(SEQ ID NO: 206) 0.47 .+-. 0.1 3A5v1b 54 (SEQ ID NO: 207) 0.37 .+-.
0.4 3A5v4b 52 (SEQ ID NO: 208) 0.46 .+-. 0.5
Example 10
Preparation of Toxin-Conjugated Antibodies that Bind TAT10772
[0789] The use of antibody-drug conjugates (ADC), i.e.
immunoconjugates, for the local delivery of cytotoxic or cytostatic
agents, i.e. drugs to kill or inhibit tumor cells in the treatment
of cancer (Payne (2003) Cancer Cell, 3:207-212; Syrigos and
Epenetos (1999) Anticancer Research, 19:605-614; Niculescu-Duvaz
and Springer (1997) Adv. Drug Del. Rev., 26:151-172; U.S. Pat. No.
4,975,278) allows targeted delivery of the drug moiety to tumors,
and intracellular accumulation therein, where systemic
administration of these unconjugated drug agents may result in
unacceptable levels of toxicity to normal cells as well as the
tumor cells sought to be eliminated (Baldwin et al., (1986) Lancet,
(Mar. 15, 1986) pp. 603-05; Thorpe, (1985) "Antibody Carriers Of
Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal
Antibodies '84: Biological And Clinical Applications, Pinchera et
al. (eds.), pp. 475-506). Maximal efficacy with minimal toxicity is
sought thereby. Efforts to design and refine ADC have focused on
the selectivity of monoclonal antibodies (mAbs) as well as
drug-linking and drug-releasing properties. Both polyclonal
antibodies and monoclonal antibodies have been reported as useful
in these strategies (Rowland et al., (1986) Cancer Immunol.
Immunother., 21:183-87). Drugs used in these methods include
daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et
al., (1986) supra). Toxins used in antibody-toxin conjugates
include bacterial toxins such as diphtheria toxin, plant toxins
such as ricin, small molecule toxins such as geldanamycin (Mandler
et al. (2000) J. of the Nat. Cancer Inst., 92(19):1573-1581;
Mandler et al. (2000) Bioorganic & Med. Chem. Letters,
10:1025-1028; Mandler et al. (2002) Bioconjugate Chem.,
13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc.
Natl. Acad. Sci. USA, 93:8618-8623), and calicheamicin (Lode et al.
(1998) Cancer Res., 58:2928; Hinman et al. (1993) Cancer Res.,
53:3336-3342).
[0790] In the antibody drug conjugates (ADC) of the invention, an
antibody (Ab) is conjugated to one or more drug moieties (D), e.g.
about 1 to about 20 drug moieties per antibody, through a linker
(L). The ADC having the formula:
Ab-(L-D).sub.p
may be prepared by several routes, employing organic chemistry
reactions, conditions, and reagents known to those skilled in the
art, including: (1) reaction of a nucleophilic group of an antibody
with a bivalent linker reagent, to form Ab-L, via a covalent bond,
followed by reaction with a drug moiety D; and (2) reaction of a
nucleophilic group of a drug moiety with a bivalent linker reagent,
to form D-L, via a covalent bond, followed by reaction with the
nucleophilic group of an antibody. Additional methods for preparing
ADC are described herein.
[0791] The linker may be composed of one or more linker components.
Exemplary linker components include 6-maleimidocaproyl ("MC"),
maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"),
alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl
("PAB"), N-Succinimidyl 4-(2-pyridylthio) pentanoate ("SPP"),
N-Succinimidyl 4-(N-maleimidomethyl) cyclohexane-1 carboxylate
("SMCC`), and N-Succinimidyl (4-iodo-acetyl) aminobenzoate
("SIAB''). Additional linker components are known in the art and
some are described herein.
[0792] In some embodiments, the linker may comprise amino acid
residues. Exemplary amino acid linker components include a
dipeptide, a tripeptide, a tetrapeptide or a pentapeptide.
Exemplary dipeptides include: valine-citrulline (vc or val-cit),
alanine-phenylalanine (af or ala-phe). Exemplary tripeptides
include: glycine-valine-citrulline (gly-val-cit) and
glycine-glycine-glycine (gly-gly-gly). Amino acid residues which
comprise an amino acid linker component include those occurring
naturally, as well as minor amino acids and non-naturally occurring
amino acid analogs, such as citrulline. Amino acid linker
components can be designed and optimized in their selectivity for
enzymatic cleavage by a particular enzymes, for example, a
tumor-associated protease, cathepsin B, C and D, or a plasmin
protease.
[0793] Nucleophilic groups on antibodies include, but are not
limited to: (i) N-terminal amine groups, (ii) side chain amine
groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine,
and (iv) sugar hydroxyl or amino groups where the antibody is
glycosylated. Amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,
thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups
are nucleophilic and capable of reacting to form covalent bonds
with electrophilic groups on linker moieties and linker reagents
including: (i) active esters such as NHS esters, HOBt esters,
haloformates, and acid halides; (ii) alkyl and benzyl halides such
as haloacetamides; (iii) aldehydes, ketones, carboxyl, and
maleimide groups. Certain antibodies have reducible interchain
disulfides, i.e. cysteine bridges. Antibodies may be made reactive
for conjugation with linker reagents by treatment with a reducing
agent such as DTT (dithiothreitol). Each cysteine bridge will thus
form, theoretically, two reactive thiol nucleophiles. Additional
nucleophilic groups can be introduced into antibodies through the
reaction of lysines with 2-iminothiolane (Traut's reagent)
resulting in conversion of an amine into a thiol. Reactive thiol
groups may be introduced into the antibody (or fragment thereof) by
introducing one, two, three, four, or more cysteine residues (e.g.,
preparing mutant antibodies comprising one or more non-native
cysteine amino acid residues).
[0794] Antibody drug conjugates of the invention may also be
produced by modification of the antibody to introduce electrophilic
moieties, which can react with nucleophilic substituents on the
linker reagent or drug. The sugars of glycosylated antibodies may
be oxidized, e.g. with periodate oxidizing reagents, to form
aldehyde or ketone groups which may react with the amine group of
linker reagents or drug moieties. The resulting imine Schiff base
groups may form a stable linkage, or may be reduced, e.g. by
borohydride reagents to form stable amine linkages. In one
embodiment, reaction of the carbohydrate portion of a glycosylated
antibody with either galactose oxidase or sodium meta-periodate may
yield carbonyl (aldehyde and ketone) groups in the protein that can
react with appropriate groups on the drug (Hermanson, Bioconjugate
Techniques). In another embodiment, proteins containing N-terminal
serine or threonine residues can react with sodium meta-periodate,
resulting in production of an aldehyde in place of the first amino
acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146;
U.S. Pat. No. 5,362,852). Such aldehyde can be reacted with a drug
moiety or linker nucleophile.
[0795] Alternatively, a fusion protein comprising the 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.
[0796] 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).
[0797] Specific techniques for producing antibody-drug conjugates
by linking toxins to purified antibodies are well known and
routinely employed in the art. For example, conjugation of a
purified monoclonal antibody to the toxin DM1 may be accomplished
as follows. Purified antibody is derivatized with
N-succinimidyl-4-(2-pyridylthio)pentanoate to introduce
dithiopyridyl groups. Antibody (376.0 mg, 8 mg/mL) in 44.7 ml of 50
mM potassium phosphate buffer (pH 6.5) containing NaCl (50 mM) and
EDTA (1 mM) is treated with SPP (5.3 molar equivalents in 2.3 ml
ethanol). After incubation for 90 minutes under argon at ambient
temperature, the reaction mixture is gel filtered through a
Sephadex G25 column equilibrated with 35 mM sodium citrate, 154 mM
NaCl and 2 mM EDTA. Antibody containing fractions are then pooled
and assayed. Antibody-SPP-Py (337.0 mg with releasable
2-thiopyridine groups) is diluted with the above 35 mM sodium
citrate buffer, pH 6.5, to a final concentration of 2.5 mg/ml. DM1
(1.7 equivalents, 16.1 mols) in 3.0 mM dimethylacetamide (DMA, 3%
v/v in the final reaction mixture) is then added to the antibody
solution. The reaction is allowed to proceed at ambient temperature
under argon for 20 hours. The reaction is loaded on a Sephacryl
S300 gel filtration column (5.0 cm.times.90.0 cm, 1.77 L)
equilibrated with 35 mM sodium citrate, 154 mM NaCl, pH 6.5. The
flow rate is 5.0 ml/min and 65 fractions (20.0 ml each) are
collected. Fractions are pooled and assayed, wherein the number of
DM1 drug molecules linked per antibody molecule (p') is determined
by measuring the absorbance at 252 nm and 280 nm.
[0798] For illustrative purposes, conjugation of a purified
monoclonal antibody to the toxin DM1 may also be accomplished as
follows. Purified antibody is derivatized with (Succinimidyl
4-(N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC, Pierce
Biotechnology, Inc) to introduce the SMCC linker. The antibody is
treated at 20 mg/ml in 50 mM potassium phosphate/50 mM sodium
chloride/2 mM EDTA, pH 6.5 with 7.5 molar equivalents of SMCC (20
mM in DMSO, 6.7 mg/ml). After stirring for 2 hours under argon at
ambient temperature, the reaction mixture is filtered through a
Sephadex G25 column equilibrated with 50mM potassium phosphate/50
mM sodium chloride/2 mM EDTA, pH 6.5. Antibody containing fractions
are pooled and assayed. Antibody-SMCC is then diluted with 50 mM
potassium phosphate/50 mM sodium chloride/2 mM EDTA, pH 6.5, to a
final concentration of 10 mg/ml, and reacted with a 10 mM solution
of DM1 (1.7 equivalents assuming 5 SMCC/antibody, 7.37 mg/ml) in
dimethylacetamide. The reaction is stirred at ambient temperature
under argon 16.5 hours. The conjugation reaction mixture is then
filtered through a Sephadex G25 gel filtration column
(1.5.times.4.9 cm) with 1.times. PBS at pH 6.5. The DM1/antibody
ratio (p) is then measured by the absorbance at 252 nm and at 280
nm.
[0799] Moreover, a free cysteine on an antibody of choice may be
modified by the bis-maleimido reagent BM(PEO)4 (Pierce Chemical),
leaving an unreacted maleimido group on the surface of the
antibody. This may be accomplished by dissolving BM(PEO)4 in a 50%
ethanol/water mixture to a concentration of 10 mM and adding a
tenfold molar excess to a solution containing the antibody in
phosphate buffered saline at a concentration of approximately 1.6
mg/ml (10 micromolar) and allowing it to react for 1 hour. Excess
BM(PEO)4 is removed by gel filtration in 30 mM citrate, pH 6 with
150 mM NaCl buffer. An approximate 10 fold molar excess DM1 is
dissolved in dimethyl acetamide (DMA) and added to the
antibody-BMPEO intermediate. Dimethyl formamide (DMF) may also be
employed to dissolve the drug moiety reagent. The reaction mixture
is allowed to react overnight before gel filtration or dialysis
into PBS to remove unreacted drug. Gel filtration on 5200 columns
in PBS is used to remove high molecular weight aggregates and
furnish purified antibody-BMPEO-DM1 conjugate.
[0800] Cytotoxic drugs have typically been conjugated to antibodies
through the often numerous lysine residues of the antibody.
Conjugation through thiol groups present, or engineered into, the
antibody of interest has also been accomplished. For example,
cysteine residues have been introduced into proteins by genetic
engineering techniques to form covalent attachment sites for
ligands (Better et al. (1994) J. Biol. Chem., 13:9644-9650;
Bernhard et al. (1994) Bioconjugate Chem., 5:126-132; Greenwood et
al. (1994) Therapeutic Immunology 1:247-255; Tu et al. (1999) Proc.
Natl. Acad. Sci USA, 96:4862-4867; Kanno et al. (2000) J. of
Biotechnology, 76:207-214; Chmura et al. (2001) Proc. Nat. Acad.
Sci. USA 98(15):8480-8484; U.S. Pat. No. 6,248,564). Once a free
cysteine residue exists in the antibody of interest, toxins can be
linked to that site. As an example, the drug linker reagents,
maleimidocaproyl-monomethyl auristatin E (MMAE), i.e. MC-MMAE,
maleimidocaproyl-monomethyl auristatin F (MMAF), i.e. MC-MMAF,
MC-val-cit-PAB-MMAE or MC-val-cit-PAB-MMAF, dissolved in DMSO, is
diluted in acetonitrile and water at known concentration, and added
to chilled cysteine-derivatized antibody in phosphate buffered
saline (PBS). After about one hour, an excess of maleimide is added
to quench the reaction and cap any unreacted antibody thiol groups.
The reaction mixture is concentrated by centrifugal ultrafiltration
and the toxin conjugated antibody is purified and desalted by
elution through G25 resin in PBS, filtered through 0.2 m filters
under sterile conditions, and frozen for storage.
[0801] Additionally, anti-TAT antibodies of the present invention
may be conjugate to auristatin and dolastatin toxins (such as MMAE
and MMAF) using the following technique. Antibody, dissolved in
500mM sodium borate and 500 mM sodium chloride at pH 8.0 is treated
with an excess of 100mM dithiothreitol (DTT). After incubation at
37 .degree. C. for about 30 minutes, the buffer is exchanged by
elution over Sephadex G25 resin and eluted with PBS with 1 mM DTPA.
The thiol/Ab value is checked by determining the reduced antibody
concentration from the absorbance at 280 nm of the solution and the
thiol concentration by reaction with DTNB (Aldrich, Milwaukee,
Wis.) and determination of the absorbance at 412 nm. The reduced
antibody dissolved in PBS is chilled on ice.
[0802] The drug linker reagent, (1) maleimidocaproyl-monomethyl
auristatin E (MMAE), i.e. MC-MMAE, (2) MC-MMAF, (3)
MC-val-cit-PAB-MMAE, or (4) MC-val-cit-PAB-MMAF dissolved in DMSO,
is diluted in acetonitrile and water at known concentration, and
added to the chilled reduced antibody in PBS. After about one hour,
an excess of maleimide is added to quench the reaction and cap any
unreacted antibody thiol groups. The reaction mixture is
concentrated by centrifugal ultrafiltration and the conjugated
antibody is purified and desalted by elution through G25 resin in
PBS, filtered through 0.2 m filters under sterile conditions, and
frozen for storage.
Example 11
In Vitro Tumor Cell Killing Assay
[0803] 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.
[0804] 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.
[0805] In a first experiment and with specific regard to the
present invention, various concentrations of MMAF and MMAE
conjugates of the chimeric 3A5 and chimeric 11D10 antibodies were
tested for the ability to kill (1) the TAT10772
polypeptide-expressing cell line OVCAR-3, (2) a PC3-derived cell
line engineered to stably express TAT10772 polypeptide on its cell
surface (PC3/A5.3B2) and (3) a PC3 cell line that does not express
TAT10772 polypeptide (PC3/neo). The chimeric 3A5 antibodies
employed in these analyses contained the variable light chain amino
acid sequence shown herein as SEQ ID NO:211 and the variable heavy
chain amino acid sequence shown herein as SEQ ID NO:11. The
chimeric 11D10 antibodies employed in these analyses contained the
variable light chain amino acid sequence shown herein as SEQ ID
NO:4 and the variable heavy chain amino acid sequence shown herein
as SEQ ID NO:7. Results from these experiments are shown in FIGS.
26-31 and demonstrated that each of the toxin conjugated antibodies
caused significant levels of cell death in the OVCAR-3 and
PC3/A5.3B2 cells (i.e., cells that express TAT10772 polypeptide on
the cell surface), whereas no significant cell killing was observed
for any of the antibodies in the PC3/neo cells (which do not
express TAT10772 polypeptide on the cell surface). These data
demonstrate the tested antibodies are capable of binding to the
TAT10772 polypeptide on the surface of cells expressing that
polypeptide and causing the death of those cells in vitro.
Example 12
In Vivo Tumor Cell Killing Assay
[0806] Intraperitoneal tumor model
[0807] To test the efficacy of the chimeric 11D10 and 3A5
anti-TAT10772 polypeptide antibodies in vivo, 2.times.10.sup.7
OVCAR-3/luc cells per 110 SCID mouse were injected into the
peritoneal cavity and allowed to grow for 20 days post-injection.
At day 20 post-injection, the mice were segregated into 9 different
groups of from 9-10 mice per group and the tumor volume was
determined in each mouse. At days 23, 30, 37 and 44 post-injection,
mice were treated as follows: [0808] Group A--vehicle alone [0809]
Group B--2.5 mg/kg chimeric 11D10-MC-vc-PAB-MMAE [0810] Group
C--2.5 mg/kg chimeric 11D10-MC-vc-PAB-MMAF [0811] Group D--2.5
mg/kg chimeric 11D10-MC-MMAF [0812] Group E--2.5 mg/kg chimeric
3A5-MC-vc-PAB-MMAE [0813] Group F--2.5 mg/kg chimeric
3A5-MC-vc-PAB-MMAF [0814] Group G--2.5 mg/kg chimeric 3A5-MC-MMAF
[0815] Group H--2.5 mg/kg anti-ragweed-MC-vc-PAB-MMAE [0816] Group
I--2.5 mg/kg anti-ragweed-MC-vc-PAB-MMAF
[0817] Tumor volume was measured in each mouse on days 27, 34, 41,
48, 55 and 69 post-injection to determine the efficacy of each
treatment in reducing tumor volume. Additionally, % animal survival
was determined daily through 250 days post treatment.
[0818] The results of these in vivo analyses demonstrated that mice
which were treated with vehicle alone (Group A) or with the
non-TAT10772-specific anti-ragweed antibody (Groups H and I) showed
no observable reduction in tumor volume subsequent to treatment. In
fact, the tumors in these animals simply continue to increase in
size over time. These results demonstrate that antibodies that are
incapable of binding to TAT10772 polypeptide, even if
toxin-conjugated, provide no specific (or even non-specific)
therapeutic effect. In contrast, the majority of animals in Groups
B-G evidenced a significant and reproducible reduction in tumor
volume post-treatment, demonstrating that both chimeric 11D10 and
3A5 provide a specific in vivo therapeutic effect. In fact, many of
the animals in Groups B-G evidenced complete tumor necrosis. These
data clearly demonstrate that both chimeric antibodies 11D10 and
3A5 provide a specific, significant and reproducible in vivo
therapeutic effect for the treatment of tumors that express the
TAT10772 polypeptide.
[0819] With regard to percent survival, all of the animals in
Groups A, H and I had perished by day 125 post implantation. At the
same time point, however, 90% of the animals in Group B, 80% of the
animals in Groups E and F, and 55% of the animals in Groups C, D
and G remained alive evidencing that both chimeric antibodies 11D10
and 3A5 provide a specific, significant and reproducible in vivo
therapeutic effect for the treatment of tumors that express the
TAT10772 polypeptide. Results from a standard Cox proportional
hazard model is shown in Table 10 below, where a separate hazard
ratio (H.R.) for each of the eight non-vehicle subgroups was
determined (the vehicle only Group A was arbitrarily assigned a
hazard ratio of 1.0).
TABLE-US-00010 TABLE 10 95% confidence Group log H.R. S.E. of log
H.R. H.R. interval for H.R. B -2.86 0.55 0.057 (0.019, 0.168) C
-2.22 0.52 0.108 (0.039, 0.300) D -1.40 0.48 0.248 (0.096, 0.635) E
-4.51 0.71 0.011 (0.0027, 0.044) F -5.21 0.87 0.006 (0.001, 0.030)
G -3.12 0.59 0.044 (0.013, 0.140)
Subcutaneous Injection Model, Mammary Fat Pads Transplant Model,
and Xenograft Transplant Models
[0820] The results from additional in vivo experiments measuring
the therapeutic efficacy of 3A5 chimeric antibodies are shown in
FIGS. 32-37. More specifically, toxin-conjugated chimeric 3A5
antibodies were tested for their ability to decrease tumor size in
vivo in a variety of different in vivo formats including, tumor
formation by subcutaneous injection of PC3/C5.3B2 cells followed by
various antibody treatments (FIG. 32), OVCAR-3 cell transplantation
into the mammary fat pad of SCID beige mice followed by various
antibody treatments (FIGS. 33-35 and 37), and xenograft
transplantation of 10 million PC3/A5.3B2 cells per nude mouse
followed by various antibody treatments (FIG. 36). The results of
these experiments show that the various anti-TAT10772 antibodies
tested are effective in the therapeutic treatment of
TAT10772-expressing tumors in vivo.
Example 13
Use of TAT as a Hybridization Probe
[0821] 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.
[0822] 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.
[0823] 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.
[0824] 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 14
Expression of TAT in E. coli
[0825] This example illustrates preparation of an unglycosylated
form of TAT by recombinant expression in E. coli.
[0826] 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.
[0827] 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.
[0828] 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.
[0829] 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.
[0830] 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(laclq). Transformants are first grown in LB
containing 50 mg/ml carbenicillin at 30 EC 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 EC 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.
[0831] 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 EC. 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 EC. Protein
concentration is estimated by its absorbance at 280 nm using the
calculated extinction coefficient based on its amino acid
sequence.
[0832] 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 EC for 12-36 hours. The refolding
reaction is quenched by the addition of TFA to a final
concentration of 0.4% (pH of approximately 3). Before further
purification of the protein, the solution is filtered through a
0.22 micron filter and acetonitrile is added to 2-10% final
concentration. The refolded protein is chromatographed on a Poros
R1/H reversed phase column using a mobile buffer of 0.1% TFA with
elution with a gradient of acetonitrile from 10 to 80%. Aliquots of
fractions with A280 absorbance are analyzed on SDS polyacrylamide
gels and fractions containing homogeneous refolded protein are
pooled. Generally, the properly refolded species of most proteins
are eluted at the lowest concentrations of acetonitrile since those
species are the most compact with their hydrophobic interiors
shielded from interaction with the reversed phase resin. Aggregated
species are usually eluted at higher acetonitrile concentrations.
In addition to resolving misfolded forms of proteins from the
desired form, the reversed phase step also removes endotoxin from
the samples.
[0833] Fractions containing the desired folded TAT polypeptide are
pooled and the acetonitrile removed using a gentle stream of
nitrogen directed at the solution. Proteins are formulated into 20
mM Hepes, pH 6.8 with 0.14 M sodium chloride and 4% mannitol by
dialysis or by gel filtration using G25 Superfine (Pharmacia)
resins equilibrated in the formulation buffer and sterile
filtered.
[0834] Certain of the TAT polypeptides disclosed herein have been
successfully expressed and purified using this technique(s).
Example 15
Expression of TAT in Mammalian Cells
[0835] This example illustrates preparation of a potentially
glycosylated form of TAT by recombinant expression in mammalian
cells.
[0836] 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.
[0837] 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.
[0838] Approximately 24 hours after the transfections, the culture
medium is removed and replaced with culture medium (alone) or
culture medium containing 200 .mu.Ci/ml .sup.35S-cysteine and 200
.mu.Ci/ml .sup.35S-methionine. After a 12 hour incubation, the
conditioned medium is collected, concentrated on a spin filter, and
loaded onto a 15% SDS gel. The processed gel may be dried and
exposed to film for a selected period of time to reveal the
presence of TAT polypeptide. The cultures containing transfected
cells may undergo further incubation (in serum free medium) and the
medium is tested in selected bioassays.
[0839] 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.m/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.
[0840] 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.
[0841] 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.
[0842] 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.
[0843] 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.
[0844] 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
cDNAs. 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.
[0845] Twelve micrograms of the desired plasmid DNA is introduced
into approximately 10 million CHO cells using commercially
available transfection reagents SUPERFECT.RTM. (Qiagen),
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.
[0846] 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
.PHI.m filtered PS20 with 5% 0.2 .PHI.m diafiltered fetal bovine
serum). The cells are then aliquoted into a 100 mL spinner
containing 90 mL of selective media. After 1-2 days, the cells are
transferred into a 250 mL spinner filled with 150 mL selective
growth medium and incubated at 37.degree. C. After another 2-3
days, 250 mL, 500 mL and 2000 mL spinners are seeded with
3.times.10.sup.5 cells/mL. The cell media is exchanged with fresh
media by centrifugation and resuspension in production medium.
Although any suitable CHO media may be employed, a production
medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992
may actually be used. A 3L production spinner is seeded at
1.2.times.10.sup.6 cells/mL. On day 0, the cell number pH is
determined. On day 1, the spinner is sampled and sparging with
filtered air is commenced. On day 2, the spinner is sampled, the
temperature shifted to 33.degree. C., and 30 mL of 500 g/L glucose
and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane
emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout
the production, the pH is adjusted as necessary to keep it at
around 7.2. After 10 days, or until the viability dropped below
70%, the cell culture is harvested by centrifugation and filtering
through a 0.22 .PHI.m filter. The filtrate was either stored at
4.degree. C. or immediately loaded onto columns for
purification.
[0847] For the poly-His tagged constructs, the proteins are
purified using a Ni-NTA column (Qiagen). Before purification,
imidazole is added to the conditioned media to a concentration of 5
mM. The conditioned media is pumped onto a 6 ml Ni-NTA column
equilibrated in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl
and 5 mM imidazole at a flow rate of 4-5 ml/min. at 4.degree. C.
After loading, the column is washed with additional equilibration
buffer and the protein eluted with equilibration buffer containing
0.25 M imidazole. The highly purified protein is subsequently
desalted into a storage buffer containing 10 mM Hepes, 0.14 M NaCl
and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine (Pharmacia)
column and stored at -80.degree. C.
[0848] 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 .PHI.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 Edmann degradation.
[0849] Certain of the TAT polypeptides disclosed herein have been
successfully expressed and purified using this technique(s).
Example 16
Expression of TAT in Yeast
[0850] The following method describes recombinant expression of TAT
in yeast.
[0851] 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.
[0852] 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.
[0853] 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.
[0854] Certain of the TAT polypeptides disclosed herein have been
successfully expressed and purified using this technique(s).
Example 17
Expression of TAT in Baculovirus-Infected Insect Cells
[0855] The following method describes recombinant expression of TAT
in Baculovirus-infected insect cells.
[0856] 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.
[0857] Recombinant baculovirus is generated by co-transfecting the
above plasmid and BACULOGOLD.TM. virus DNA (Pharmingen) into
Spodoptera frugiperda ("Sf9") cells (ATCC CRL 1711) using
lipofectin (commercially available from GIBCO-BRL). After 4-5 days
of incubation at 28.degree. C., the released viruses are harvested
and used for further amplifications. Viral infection and protein
expression are performed as described by O'Reilley et al.,
Baculovirus expression vectors: A Laboratory Manual, Oxford: Oxford
University Press (1994).
[0858] 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 SD 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 .PHI.m filter. A
Ni.sup.2+-NTA agarose column (commercially available from Qiagen)
is prepared with a bed volume of 5 mL, washed with 25 mL of water
and equilibrated with 25 mL of loading buffer. The filtered cell
extract is loaded onto the column at 0.5 mL per minute. The column
is washed to baseline 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
.sub.A280 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.
[0859] 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.
[0860] Certain of the TAT polypeptides disclosed herein have been
successfully expressed and purified using this technique(s).
Example 18
Purification of TAT Polypeptides Using Specific Antibodies
[0861] 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.
[0862] 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.
[0863] Such an immunoaffinity column is utilized in the
purification of TAT polypeptide by preparing a fraction from cells
containing TAT polypeptide in a soluble form. This preparation is
derived by solubilization of the whole cell or of a subcellular
fraction obtained via differential centrifugation by the addition
of detergent or by other methods well known in the art.
Alternatively, soluble TAT polypeptide containing a signal sequence
may be secreted in useful quantity into the medium in which the
cells are grown.
[0864] A soluble TAT polypeptide-containing preparation is passed
over the immunoaffinity column, and the column is washed under
conditions that allow the preferential absorbance of TAT
polypeptide (e.g., high ionic strength buffers in the presence of
detergent). Then, the column is eluted under conditions that
disrupt antibody/TAT polypeptide binding (e.g., a low pH buffer
such as approximately pH 2-3, or a high concentration of a
chaotrope such as urea or thiocyanate ion), and TAT polypeptide is
collected.
Deposit of Material
[0865] The following materials have been deposited with the
American Type Culture Collection, 10801 University Blvd., Manassas,
Va. 20110-2209, USA (ATCC):
TABLE-US-00011 TABLE 11 Material ATCC Dep. No. Deposit Date
Hybridoma cell line 3A5.3 PTA-6695 May 4, 2005 Hybridoma cell line
11D10.1.14 PTA-6696 May 4, 2005
[0866] These deposits were made under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of a viable culture of the deposit for 30 years from the date of
deposit. The deposits will be made available by ATCC under the
terms of the Budapest Treaty, and subject to an agreement between
Genentech, Inc. and ATCC, which assures permanent and unrestricted
availability of the progeny of the culture of the deposit to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 USC .sctn. 122 and the
Commissioner's rules pursuant thereto (including 37 CFR .sctn. 1.14
with particular reference to 886 OG 638).
[0867] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
[0868] 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
211121112DNAHomo sapiens 1cctgtgactt ctcttctcac ccctggcctg
gtgataacca cagacaggat 50gggcataagc agagaacctg gaaccagttc cacttcaaat
ttgagcagca 100cctcccatga gagactgacc actttggaag acactgtaga
tacagaagcc 150atgcagcctt ccacacacac agcagtgacc aacgtgagga
cctccatttc 200tggacatgaa tcacaatctt ctgtcctatc tgactcagag
acacccaaag 250ccacatctcc aatgggtacc acctacacca tgggggaaac
gagtgtttcc 300atatccactt ctgacttctt tgagaccagc agaattcaga
tagaaccaac 350atcctccctg acttctggat tgagggagac cagcagctct
gagaggatca 400gctcagccac agagggaagc actgtccttt ctgaagtgcc
cagtggtgct 450accactgagg tctccaggac agaagtgata tcctctaggg
gaacatccat 500gtcagggcct gatcagttca ccatatcacc agacatctct
actgaagcga 550tcaccaggct ttctacttcc cccattatga cagaatcagc
agaaagtgcc 600atcactattg agacaggttc tcctggggct acatcagagg
gtaccctcac 650cttggacacc tcaacaacaa ccttttggtc agggacccac
tcaactgcat 700ctccaggatt ttcacactca gagatgacca ctcttatgag
tagaactcct 750ggagatgtgc catggccgag ccttccctct gtggaagaag
ccagctctgt 800ctcttcctca ctgtcttcac ctgccatgac ctcaacttct
tttttctcca 850cattaccaga gagcatctcc tcctctcctc atcctgtgac
tgcacttctc 900acccttggcc cagtgaagac cacagacatg ttgcgcacaa
gctcagaacc 950tgaaaccagt tcacctccaa atttgagcag cacctcagct
gaaatattag 1000ccacgtctga agtcaccaaa gatagagaga aaattcatcc
ctcctcaaac 1050acacctgtag tcaatgtagg gactgtgatt tataaacatc
tatccccttc 1100ctctgttttg gctgacttag tgacaacaaa acccacatct
ccaatggcta 1150ccacctccac tctggggaat acaagtgttt ccacatcaac
tcctgccttc 1200ccagaaacta tgatgacaca gccaacttcc tccctgactt
ctggattaag 1250ggagatcagt acctctcaag agaccagctc agcaacagag
agaagtgctt 1300ctctttctgg aatgcccact ggtgctacta ctaaggtctc
cagaacagaa 1350gccctctcct taggcagaac atccacccca ggtcctgctc
aatccacaat 1400atcaccagaa atctccacgg aaaccatcac tagaatttct
actcccctca 1450ccacgacagg atcagcagaa atgaccatca cccccaaaac
aggtcattct 1500ggggcatcct cacaaggtac ctttaccttg gacacatcaa
gcagagcctc 1550ctggccagga actcactcag ctgcaactca cagatctcca
cactcaggga 1600tgaccactcc tatgagcaga ggtcctgagg atgtgtcatg
gccaagccgc 1650ccatcagtgg aaaaaactag ccctccatct tccctggtgt
ctttatctgc 1700agtaacctca ccttcgccac tttattccac accatctgag
agtagccact 1750cgtctcctct ccgggtgact tctcttttca cccctgtcat
gatgaagacc 1800acagacatgt tggacacaag cttggaacct gtgaccactt
cacctcccag 1850tatgaatatc acctcagatg agagtctggc cacttctaaa
gccaccatgg 1900agacagaggc aattcagctt tcagaaaaca cagctgtgac
tcagatgggc 1950accatcagtg ctagacaaga attctattcc tcttatccag
gcctcccaga 2000gccatccaaa gtgacatctc cagtggtcac ctcttccacc
ataaaagaca 2050ttgtttctac aaccatacct gcttcctctg agataacaag
aattgagatg 2100gagtcaacat ccaccctgac ccccacacca agggagacca
gcacctccca 2150ggagatccac tcagccacaa agccaagcac tgttccttac
aaggcactca 2200ctagtgccac gattgaggac tccatgacac aagtcatgtc
ctctagcaga 2250ggacctagcc ctgatcagtc cacaatgtca caagacatat
ccactgaagt 2300gatcaccagg ctctctacct cccccatcaa gacagaatct
acagaaatga 2350ccattaccac ccaaacaggt tctcctgggg ctacatcaag
gggtaccctt 2400accttggaca cttcaacaac ttttatgtca gggacccatt
caactgcatc 2450tcaaggattt tcacactcac agatgaccgc tcttatgagt
agaactcctg 2500gagaggtgcc atggctaagc catccctctg tggaagaagc
cagctctgcc 2550tctttctcac tgtcttcacc tgtcatgacc tcatcttctc
ccgtttcttc 2600cacattacca gacagcatcc actcttcttc gcttcctgtg
acatcacttc 2650tcacctcagg gctggtgaag accacagagc tgttgggcac
aagctcagaa 2700cctgaaacca gttcaccccc aaatttgagc agcacctcag
ctgaaatact 2750ggccaccact gaagtcacta cagatacaga gaaactggag
atgaccaatg 2800tggtaacctc aggttataca catgaatctc cttcctctgt
cctagctgac 2850tcagtgacaa caaaggccac atcttcaatg ggtatcacct
accccacagg 2900agatacaaat gttctcacat caacccctgc cttctctgac
accagtagga 2950ttcaaacaaa gtcaaagctc tcactgactc ctgggttgat
ggagaccagc 3000atctctgaag agaccagctc tgccacagaa aaaagcactg
tcctttctag 3050tgtgcccact ggtgctacta ctgaggtctc caggacagaa
gccatctctt 3100ctagcagaac atccatccca ggccctgctc aatccacaat
gtcatcagac 3150acctccatgg aaaccatcac tagaatttct acccccctca
caaggaaaga 3200atcaacagac atggccatca cccccaaaac aggtccttct
ggggctacct 3250cgcagggtac ctttaccttg gactcatcaa gcacagcctc
ctggccagga 3300actcactcag ctacaactca gagatttcca cggtcagtgg
tgacaactcc 3350tatgagcaga ggtcctgagg atgtgtcatg gccaagcccg
ctgtctgtgg 3400aaaaaaacag ccctccatct tccctggtat cttcatcttc
agtaacctca 3450ccttcgccac tttattccac accatctggg agtagccact
cctctcctgt 3500ccctgtcact tctcttttca cctctatcat gatgaaggcc
acagacatgt 3550tggatgcaag tttggaacct gagaccactt cagctcccaa
tatgaatatc 3600acctcagatg agagtctggc cgcttctaaa gccaccacgg
agacagaggc 3650aattcacgtt tttgaaaata cagcagcgtc ccatgtggaa
accaccagtg 3700ctacagagga actctattcc tcttccccag gcttctcaga
gccaacaaaa 3750gtgatatctc cagtggtcac ctcttcctct ataagagaca
acatggtttc 3800cacaacaatg cctggctcct ctggcattac aaggattgag
atagagtcaa 3850tgtcatctct gacccctgga ctgagggaga ccagaacctc
ccaggacatc 3900acctcatcca cagagacaag cactgtcctt tacaagatgc
cctctggtgc 3950cactcctgag gtctccagga cagaagttat gccctctagc
agaacatcca 4000ttcctggccc tgctcagtcc acaatgtcac tagacatctc
cgatgaagtt 4050gtcaccaggc tgtctacctc tcccatcatg acagaatctg
cagaaataac 4100catcaccacc caaacaggtt attctctggc tacatcccag
gttacccttc 4150ccttgggcac ctcaatgacc tttttgtcag ggacccactc
aactatgtct 4200caaggacttt cacactcaga gatgaccaat cttatgagca
ggggtcctga 4250aagtctgtca tggacgagcc ctcgctttgt ggaaacaact
agatcttcct 4300cttctctgac atcattacct ctcacgacct cactttctcc
tgtgtcctcc 4350acattactag acagtagccc ctcctctcct cttcctgtga
cttcacttat 4400cctcccaggc ctggtgaaga ctacagaagt gttggataca
agctcagagc 4450ctaaaaccag ttcatctcca aatttgagca gcacctcagt
tgaaataccg 4500gccacctctg aaatcatgac agatacagag aaaattcatc
cttcctcaaa 4550cacagcggtg gccaaagtga ggacctccag ttctgttcat
gaatctcatt 4600cctctgtcct agctgactca gaaacaacca taaccatacc
ttcaatgggt 4650atcacctccg ctgtggagga taccactgtt ttcacatcaa
atcctgcctt 4700ctctgagact aggaggattc cgacagagcc aacattctca
ttgactcctg 4750gattcaggga gactagcacc tctgaagaga ccacctcaat
cacagaaaca 4800agtgcagtcc tttttggagt gcccactagt gctactactg
aagtctccat 4850gacagaaata atgtcctcta atagaacaca catccctgac
tctgatcagt 4900ccacgatgtc tccagacatc atcactgaag tgatcaccag
gctctcttcc 4950tcatccatga tgtcagaatc aacacaaatg accatcacca
cccaaaaaag 5000ttctcctggg gctacagcac agagtactct taccttggcc
acaacaacag 5050cccccttggc aaggacccac tcaactgttc ctcctagatt
tttacactca 5100gagatgacaa ctcttatgag taggagtcct gaaaatccat
catggaagag 5150ctctcccttt gtggaaaaaa ctagctcttc atcttctctg
ttgtccttac 5200ctgtcacgac ctcaccttct gtttcttcca cattaccgca
gagtatccct 5250tcctcctctt tttctgtgac ttcactcctc accccaggca
tggtgaagac 5300tacagacaca agcacagaac ctggaaccag tttatctcca
aatctgagtg 5350gcacctcagt tgaaatactg gctgcctctg aagtcaccac
agatacagag 5400aaaattcatc cttcttcaag catggcagtg accaatgtgg
gaaccaccag 5450ttctggacat gaactatatt cctctgtttc aatccactcg
gagccatcca 5500aggctacata cccagtgggt actccctctt ccatggctga
aacctctatt 5550tccacatcaa tgcctgctaa ttttgagacc acaggatttg
aggctgagcc 5600attttctcat ttgacttctg gacttaggaa gaccaacatg
tccctggaca 5650ccagctcagt cacaccaaca aatacacctt cttctcctgg
gtccactcac 5700cttttacaga gttccaagac tgatttcacc tcttctgcaa
aaacatcatc 5750cccagactgg cctccagcct cacagtatac tgaaattcca
gtggacataa 5800tcaccccctt taatgcttct ccatctatta cggagtccac
tgggataacc 5850tccttcccag aatccaggtt tactatgtct gtaacagaaa
gtactcatca 5900tctgagtaca gatttgctgc cttcagctga gactatttcc
actggcacag 5950tgatgccttc tctatcagag gccatgactt catttgccac
cactggagtt 6000ccacgagcca tctcaggttc aggtagtcca ttctctagga
cagagtcagg 6050ccctggggat gctactctgt ccaccattgc agagagcctg
ccttcatcca 6100ctcctgtgcc attctcctct tcaaccttca ctaccactga
ttcttcaacc 6150atcccagccc tccatgagat aacttcctct tcagctaccc
catatagagt 6200ggacaccagt cttgggacag agagcagcac tactgaagga
cgcttggtta 6250tggtcagtac tttggacact tcaagccaac caggcaggac
atcttcatca 6300cccattttgg ataccagaat gacagagagc gttgagctgg
gaacagtgac 6350aagtgcttat caagttcctt cactctcaac acggttgaca
agaactgatg 6400gcattatgga acacatcaca aaaataccca atgaagcagc
acacagaggt 6450accataagac cagtcaaagg ccctcagaca tccacttcgc
ctgccagtcc 6500taaaggacta cacacaggag ggacaaaaag aatggagacc
accaccacag 6550ctctgaagac caccaccaca gctctgaaga ccacttccag
agccaccttg 6600accaccagtg tctatactcc cactttggga acactgactc
ccctcaatgc 6650atcaatgcaa atggccagca caatccccac agaaatgatg
atcacaaccc 6700catatgtttt ccctgatgtt ccagaaacga catcctcatt
ggctaccagc 6750ctgggagcag aaaccagcac agctcttccc aggacaaccc
catctgtttt 6800caatagagaa tcagagacca cagcctcact ggtctctcgt
tctggggcag 6850agagaagtcc ggttattcaa actctagatg tttcttctag
tgagccagat 6900acaacagctt catgggttat ccatcctgca gagaccatcc
caactgtttc 6950caagacaacc cccaattttt tccacagtga attagacact
gtatcttcca 7000cagccaccag tcatggggca gacgtcagct cagccattcc
aacaaatatc 7050tcacctagtg aactagatgc actgacccca ctggtcacta
tttcggggac 7100agatactagt acaacattcc caacactgac taagtcccca
catgaaacag 7150agacaagaac cacatggctc actcatcctg cagagaccag
ctcaactatt 7200cccagaacaa tccccaattt ttctcatcat gaatcagatg
ccacaccttc 7250aatagccacc agtcctgggg cagaaaccag ttcagctatt
ccaattatga 7300ctgtctcacc tggtgcagaa gatctggtga cctcacaggt
cactagttct 7350ggcacagaca gaaatatgac tattccaact ttgactcttt
ctcctggtga 7400accaaagacc atagcctcat tagtcaccca tcctgaagca
cagacaagtt 7450cggccattcc aacttcaact atctcgcctg ctgtatcacg
gttggtgacc 7500tcaatggtca ccagtttggc ggcaaagaca agtacaacta
atcgagctct 7550gacaaactcc cctggtgaac cagctacaac agtttcattg
gtcacgcatt 7600ctgcacagac cagcccaaca gttccctgga caacttccat
ttttttccat 7650agtaaatcag acaccacacc ttcaatgacc accagtcatg
gggcagaatc 7700cagttcagct gttccaactc caactgtttc aactgaggta
ccaggagtag 7750tgaccccttt ggtcaccagt tctagggcag tgatcagtac
aactattcca 7800attctgactc tttctcctgg tgaaccagag accacacctt
caatggccac 7850cagtcatggg gaagaagcca gttctgctat tccaactcca
actgtttcac 7900ctggggtacc aggagtggtg acctctctgg tcactagttc
tagggcagtg 7950actagtacaa ctattccaat tctgactttt tctcttggtg
aaccagagac 8000cacaccttca atggccacca gtcatgggac agaagctggc
tcagctgttc 8050caactgtttt acctgaggta ccaggaatgg tgacctctct
ggttgctagt 8100tctagggcag taaccagtac aactcttcca actctgactc
tttctcctgg 8150tgaaccagag accacacctt caatggccac cagtcatggg
gcagaagcca 8200gctcaactgt tccaactgtt tcacctgagg taccaggagt
ggtgacctct 8250ctggtcacta gttctagtgg agtaaacagt acaagtattc
caactctgat 8300tctttctcct ggtgaactag aaaccacacc ttcaatggcc
accagtcatg 8350gggcagaagc cagctcagct gttccaactc caactgtttc
acctggggta 8400tcaggagtgg tgacccctct ggtcactagt tccagggcag
tgaccagtac 8450aactattcca attctaactc tttcttctag tgagccagag
accacacctt 8500caatggccac cagtcatggg gtagaagcca gctcagctgt
tctaactgtt 8550tcacctgagg taccaggaat ggtgaccttt ctggtcacta
gttctagagc 8600agtaaccagt acaactattc caactctgac tatttcttct
gatgaaccag 8650agaccacaac ttcattggtc acccattctg aggcaaagat
gatttcagcc 8700attccaactt taggtgtctc ccctactgta caagggctgg
tgacttcact 8750ggtcactagt tctgggtcag agaccagtgc gttttcaaat
ctaactgttg 8800cctcaagtca accagagacc atagactcat gggtcgctca
tcctgggaca 8850gaagcaagtt ctgttgttcc aactttgact gtctccactg
gtgagccgtt 8900tacaaatatc tcattggtca cccatcctgc agagagtagc
tcaactcttc 8950ccaggacaac ctcaaggttt tcccacagtg aattagacac
tatgccttct 9000acagtcacca gtcctgaggc agaatccagc tcagccattt
caacaactat 9050ttcacctggt ataccaggtg tgctgacatc actggtcact
agctctggga 9100gagacatcag tgcaactttt ccaacagtgc ctgagtcccc
acatgaatca 9150gaggcaacag cctcatgggt tactcatcct gcagtcacca
gcacaacagt 9200tcccaggaca acccctaatt attctcatag tgaaccagac
accacaccat 9250caatagccac cagtcctggg gcagaagcca cttcagattt
tccaacaata 9300actgtctcac ctgatgtacc agatatggta acctcacagg
tcactagttc 9350tgggacagac accagtataa ctattccaac tctgactctt
tcttctggtg 9400agccagagac cacaacctca tttatcacct attctgagac
acatacaagt 9450tcagccattc caactctccc tgtctcccct gatgcatcaa
agatgctgac 9500ctcactggtc atcagttctg ggacagacag cactacaact
ttcccaacac 9550tgacggagac cccatatgaa ccagagacaa cagccataca
gctcattcat 9600cctgcagaga ccaacacaat ggttcccagg acaactccca
agttttccca 9650tagtaagtca gacaccacac tcccagtagc catcaccagt
cctgggccag 9700aagccagttc agctgtttca acgacaacta tctcacctga
tatgtcagat 9750ctggtgacct cactggtccc tagttctggg acagacacca
gtacaacctt 9800cccaacattg agtgagaccc catatgaacc agagactaca
gccacgtggc 9850tcactcatcc tgcagaaacc agcacaacgg tttctgggac
aattcccaac 9900ttttcccata ggggatcaga cactgcaccc tcaatggtca
ccagtcctgg 9950agtagacacg aggtcaggtg ttccaactac aaccatccca
cccagtatac 10000caggggtagt gacctcacag gtcactagtt ctgcaacaga
cactagtaca 10050gctattccaa ctttgactcc ttctcctggt gaaccagaga
ccacagcctc 10100atcagctacc catcctggga cacagactgg cttcactgtt
ccaattcgga 10150ctgttccctc tagtgagcca gatacaatgg cttcctgggt
cactcatcct 10200ccacagacca gcacacctgt ttccagaaca acctccagtt
tttcccatag 10250tagtccagat gccacacctg taatggccac cagtcctagg
acagaagcca 10300gttcagctgt actgacaaca atctcacctg gtgcaccaga
gatggtgact 10350tcacagatca ctagttctgg ggcagcaacc agtacaactg
ttccaacttt 10400gactcattct cctggtatgc cagagaccac agccttattg
agcacccatc 10450ccagaacaga gacaagtaaa acatttcctg cttcaactgt
gtttcctcaa 10500gtatcagaga ccacagcctc actcaccatt agacctggtg
cagagactag 10550cacagctctc ccaactcaga caacatcctc tctcttcacc
ctacttgtaa 10600ctggaaccag cagagttgat ctaagtccaa ctgcttcacc
tggtgtttct 10650gcaaaaacag ccccactttc cacccatcca gggacagaaa
ccagcacaat 10700gattccaact tcaactcttt cccttggttt actagagact
acaggcttac 10750tggccaccag ctcttcagca gagaccagca cgagtactct
aactctgact 10800gtttcccctg ctgtctctgg gctttccagt gcctctataa
caactgataa 10850gccccaaact gtgacctcct ggaacacaga aacctcacca
tctgtaactt 10900cagttggacc cccagaattt tccaggactg tcacaggcac
cactatgacc 10950ttgataccat cagagatgcc aacaccacct aaaaccagtc
atggagaagg 11000agtgagtcca accactatct tgagaactac aatggttgaa
gccactaatt 11050tagctaccac aggttccagt cccactgtgg ccaagacaac
aaccaccttc 11100aatacactgg ctggaagcct ctttactcct ctgaccacac
ctgggatgtc 11150caccttggcc tctgagagtg tgacctcaag aacaagttat
aaccatcggt 11200cctggatctc caccaccagc agttataacc gtcggtactg
gacccctgcc 11250accagcactc cagtgacttc tacattctcc ccagggattt
ccacatcctc 11300catccccagc tccacagcag ccacagtccc attcatggtg
ccattcaccc 11350tcaacttcac catcaccaac ctgcagtacg aggaggacat
gcggcaccct 11400ggttcaagga agttcaacgc cacagagaga gaactgcagg
gtctgctcaa 11450acccttgttc aggaatagca gtctggaata cctctattca
ggctgcagac 11500tagcctcact caggccagag aaggatagct cagccacggc
agtggatgcc 11550atctgcacac atcgccctga ccctgaagac ctcggactgg
acagagagcg 11600actgtactgg gagctgagca atctgacaaa tggcatccag
gagctgggcc 11650cttacaccct ggaccggaac agtctctatg tcaatggttt
cacccatcga 11700agctctatgc ccaccaccag cactcctggg acctccacag
tggatgtggg 11750aacctcaggg actccatcct ccagccccag ccccacgact
gctggccctc 11800tcctgatgcc gttcaccctc aacttcacca tcaccaacct
gcagtacgag 11850gaggacatgc gtcgcactgg ctccaggaag ttcaacacca
tggagagtgt 11900cctgcagggt ctgctcaagc cattgttcaa gaacaccagt
gttggccctt 11950tgtactctgg ctgcagattg accttgctca ggcccgagaa
agatggggca 12000gccactggag tggatgccat ctgcacccac cgccttgacc
ccaaaagccc 12050tggactcaac agggagcagc tgtactggga gctaagcaaa
ctgaccaatg 12100acattgaaga gctgggcccc tacaccctgg acaggaacag
tctctatgtc 12150aatggtttca cccatcagag ctctgtgtcc accaccagca
ctcctgggac 12200ctccacagtg gatctcagaa cctcagggac tccatcctcc
ctctccagcc 12250ccacaattat ggctgctggc cctctcctgg taccattcac
cctcaacttc 12300accatcacca acctgcagta tggggaggac atgggtcacc
ctggctccag 12350gaagttcaac accacagaga gggtcctgca gggtctgctt
ggtcccatat 12400tcaagaacac cagtgttggc cctctgtact ctggctgcag
actgacctct 12450ctcaggtccg agaaggatgg agcagccact ggagtggatg
ccatctgcat 12500ccatcatctt gaccccaaaa gccctggact caacagagag
cggctgtact 12550gggagctgag ccaactgacc aatggcatca aagagctggg
cccctacacc 12600ctggacagga acagtctcta tgtcaatggt ttcacccatc
ggacctctgt 12650gcccaccacc agcactcctg ggacctccac agtggacctt
ggaacctcag 12700ggactccatt ctccctccca agccccgcaa ctgctggccc
tctcctggtg 12750ctgttcaccc tcaacttcac catcaccaac ctgaagtatg
aggaggacat 12800gcatcgccct ggctccagga agttcaacac cactgagagg
gtcctgcaga 12850ccctggttgg tcctatgttc aagaacacca gtgttggcct
tctgtactct 12900ggctgcagac tgaccttgct caggtccgag aaggatggag
cagccactgg 12950agtggatgcc atctgcaccc accgtcttga ccccaaaagc
cctggagtgg 13000acagggagca gctatactgg gagctgagcc aactgaccaa
tggcatcaaa 13050gagctgggcc cctacaccct ggacaggaac agtctctatg
tcaatggttt 13100cacccattgg atccctgtgc ccaccagcag cacccctggg
acctccacag 13150tggaccttgg gtcagggact ccatcctccc tccccagccc
cacaagtgct 13200actgctggcc ctctcctggt gccgttcacc ctcaacttca
ccatcaccaa 13250cctgaagtac gaggaggaca tgcattgccc tggctccagg
aagttcaaca 13300ccacagagag agtcctgcag agtctgcttg gtcccatgtt
caagaacacc 13350agtgttggcc ctctgtactc tggctgcaga ctgaccttgc
tcaggtccga 13400gaaggatgga gcagccactg gagtggatgc catctgcacc
caccgtcttg 13450accccaaaag ccctggagtg gacagggagc agctatactg
ggagctgagc 13500cagctgacca atggcatcaa agagctgggt ccctacaccc
tggacagaaa 13550cagtctctat gtcaatggtt tcacccatca gacctctgcg
cccaacacca 13600gcactcctgg gacctccaca gtggaccttg ggacctcagg
gactccatcc 13650tccctcccca gccctacatc tgctggccct ctcctggtgc
cattcaccct 13700caacttcacc atcaccaacc tgcagtacga ggaggacatg
catcacccag 13750gctccaggaa gttcaacacc acggagcggg tcctgcaggg
tctgcttggt 13800cccatgttca agaacaccag tgtcggcctt ctgtactctg
gctgcagact 13850gaccttgctc aggcctgaga agaatggggc agccactgga
atggatgcca 13900tctgcagcca ccgtcttgac cccaaaagcc ctggactcaa
cagagagcag 13950ctgtactggg agctgagcca gctgacccat ggcatcaaag
agctgggccc 14000ctacaccctg gacaggaaca gtctctatgt caatggtttc
acccatcgga 14050gctctgtggc ccccaccagc actcctggga cctccacagt
ggaccttggg 14100acctcaggga ctccatcctc cctccccagc cccacaacag
ctgttcctct 14150cctggtgccg ttcaccctca actttaccat caccaatctg
cagtatgggg 14200aggacatgcg tcaccctggc tccaggaagt tcaacaccac
agagagggtc 14250ctgcagggtc tgcttggtcc cttgttcaag aactccagtg
tcggccctct 14300gtactctggc tgcagactga tctctctcag gtctgagaag
gatggggcag 14350ccactggagt ggatgccatc tgcacccacc accttaaccc
tcaaagccct 14400ggactggaca gggagcagct gtactggcag ctgagccaga
tgaccaatgg 14450catcaaagag ctgggcccct acaccctgga ccggaacagt
ctctacgtca 14500atggtttcac ccatcggagc tctgggctca ccaccagcac
tccttggact 14550tccacagttg accttggaac ctcagggact ccatcccccg
tccccagccc 14600cacaactgct ggccctctcc tggtgccatt caccctaaac
ttcaccatca 14650ccaacctgca gtatgaggag gacatgcatc gccctggatc
taggaagttc 14700aacgccacag agagggtcct gcagggtctg cttagtccca
tattcaagaa 14750ctccagtgtt ggccctctgt actctggctg cagactgacc
tctctcaggc 14800ccgagaagga tggggcagca actggaatgg atgctgtctg
cctctaccac 14850cctaatccca aaagacctgg gctggacaga gagcagctgt
actgggagct 14900aagccagctg acccacaaca tcactgagct gggcccctac
agcctggaca 14950gggacagtct ctatgtcaat ggtttcaccc atcagaactc
tgtgcccacc 15000accagtactc ctgggacctc cacagtgtac tgggcaacca
ctgggactcc 15050atcctccttc cccggccaca cagagcctgg ccctctcctg
ataccattca 15100ctttcaactt taccatcacc aacctgcatt atgaggaaaa
catgcaacac 15150cctggttcca ggaagttcaa caccacggag agggttctgc
agggtctgct 15200caagcccttg ttcaagaaca ccagtgttgg ccctctgtac
tctggctgca 15250gactgacctt gctcagacct gagaagcagg aggcagccac
tggagtggac 15300accatctgta cccaccgcgt tgatcccatc ggacctggac
tggacagaga 15350gcggctatac tgggagctga gccagctgac caacagcatc
acagagctgg 15400gaccctacac cctggatagg gacagtctct atgtcaatgg
cttcaaccct 15450tggagctctg tgccaaccac cagcactcct gggacctcca
cagtgcacct 15500ggcaacctct gggactccat cctccctgcc tggccacaca
gcccctgtcc 15550ctctcttgat accattcacc ctcaacttta ccatcaccaa
cctgcattat 15600gaagaaaaca tgcaacaccc tggttccagg aagttcaaca
ccacggagag 15650ggttctgcag ggtctgctca agcccttgtt caagagcacc
agcgttggcc 15700ctctgtactc tggctgcaga ctgaccttgc tcagacctga
gaaacatggg 15750gcagccactg gagtggacgc catctgcacc ctccgccttg
atcccactgg 15800tcctggactg gacagagagc ggctatactg ggagctgagc
cagctgacca 15850acagcgttac agagctgggc ccctacaccc tggacaggga
cagtctctat 15900gtcaatggct tcacccatcg gagctctgtg ccaaccacca
gtattcctgg 15950gacctctgca gtgcacctgg aaacctctgg gactccagcc
tccctccctg 16000gccacacagc ccctggccct ctcctggtgc cattcaccct
caacttcact 16050atcaccaacc tgcagtatga ggaggacatg cgtcaccctg
gttccaggaa 16100gttcaacacc acggagagag tcctgcaggg tctgctcaag
cccttgttca 16150agagcaccag tgttggccct ctgtactctg gctgcagact
gaccttgctc 16200aggcctgaaa aacgtggggc agccaccggc gtggacacca
tctgcactca 16250ccgccttgac cctctaaacc ctggactgga cagagagcag
ctatactggg 16300agctgagcaa actgacccgt ggcatcatcg agctgggccc
ctacctcctg 16350gacagaggca gtctctatgt caatggtttc acccatcgga
actttgtgcc 16400catcaccagc actcctggga cctccacagt acacctagga
acctctgaaa 16450ctccatcctc cctacctaga cccatagtgc ctggccctct
cctggtgcca 16500ttcaccctca acttcaccat caccaacttg cagtatgagg
aggccatgcg 16550acaccctggc tccaggaagt tcaataccac ggagagggtc
ctacagggtc 16600tgctcaggcc cttgttcaag aataccagta tcggccctct
gtactccagc 16650tgcagactga ccttgctcag gccagagaag gacaaggcag
ccaccagagt 16700ggatgccatc tgtacccacc accctgaccc tcaaagccct
ggactgaaca 16750gagagcagct gtactgggag ctgagccagc tgacccacgg
catcactgag 16800ctgggcccct acaccctgga cagggacagt ctctatgtcg
atggtttcac 16850tcattggagc cccataccaa ccaccagcac tcctgggacc
tccatagtga 16900acctgggaac ctctgggatc ccaccttccc tccctgaaac
tacagccacc 16950ggccctctcc tggtgccatt cacactcaac ttcaccatca
ctaacctaca 17000gtatgaggag aacatgggtc accctggctc caggaagttc
aacatcacgg 17050agagtgttct gcagggtctg ctcaagccct tgttcaagag
caccagtgtt 17100ggccctctgt attctggctg cagactgacc ttgctcaggc
ctgagaagga 17150cggagtagcc accagagtgg acgccatctg cacccaccgc
cctgacccca 17200aaatccctgg gctagacaga cagcagctat actgggagct
gagccagctg 17250acccacagca tcactgagct gggaccctac accctggata
gggacagtct 17300ctatgtcaat ggtttcaccc agcggagctc tgtgcccacc
accagcactc 17350ctgggacttt cacagtacag ccggaaacct ctgagactcc
atcatccctc 17400cctggcccca cagccactgg ccctgtcctg ctgccattca
ccctcaattt 17450taccatcatt aacctgcagt atgaggagga catgcatcgc
cctggctcca 17500ggaagttcaa caccacggag agggtccttc agggtctgct
tatgcccttg 17550ttcaagaaca ccagtgtcag ctctctgtac tctggttgca
gactgacctt 17600gctcaggcct gagaaggatg gggcagccac cagagtggat
gctgtctgca 17650cccatcgtcc tgaccccaaa agccctggac tggacagaga
gcggctgtac 17700tggaagctga gccagctgac ccacggcatc actgagctgg
gcccctacac 17750cctggacagg cacagtctct atgtcaatgg tttcacccat
cagagctcta 17800tgacgaccac cagaactcct gatacctcca caatgcacct
ggcaacctcg 17850agaactccag cctccctgtc tggacctacg accgccagcc
ctctcctggt 17900gctattcaca attaacttca ccatcactaa cctgcggtat
gaggagaaca 17950tgcatcaccc tggctctaga aagtttaaca ccacggagag
agtccttcag 18000ggtctgctca ggcctgtgtt caagaacacc agtgttggcc
ctctgtactc 18050tggctgcaga ctgaccttgc tcaggcccaa gaaggatggg
gcagccacca 18100aagtggatgc catctgcacc taccgccctg atcccaaaag
ccctggactg 18150gacagagagc agctatactg ggagctgagc cagctaaccc
acagcatcac 18200tgagctgggc ccctacaccc tggacaggga cagtctctat
gtcaatggtt 18250tcacacagcg gagctctgtg cccaccacta gcattcctgg
gacccccaca 18300gtggacctgg gaacatctgg gactccagtt tctaaacctg
gtccctcggc 18350tgccagccct ctcctggtgc tattcactct caacttcacc
atcaccaacc 18400tgcggtatga ggagaacatg cagcaccctg gctccaggaa
gttcaacacc 18450acggagaggg tccttcaggg cctgctcagg tccctgttca
agagcaccag 18500tgttggccct ctgtactctg gctgcagact gactttgctc
aggcctgaaa 18550aggatgggac agccactgga gtggatgcca tctgcaccca
ccaccctgac 18600cccaaaagcc ctaggctgga cagagagcag ctgtattggg
agctgagcca 18650gctgacccac aatatcactg agctgggccc ctatgccctg
gacaacgaca 18700gcctctttgt caatggtttc actcatcgga gctctgtgtc
caccaccagc 18750actcctggga cccccacagt gtatctggga gcatctaaga
ctccagcctc 18800gatatttggc ccttcagctg ccagccatct cctgatacta
ttcaccctca 18850acttcaccat cactaacctg cggtatgagg agaacatgtg
gcctggctcc 18900aggaagttca acactacaga gagggtcctt cagggcctgc
taaggccctt 18950gttcaagaac accagtgttg gccctctgta ctctggctgc
aggctgacct 19000tgctcaggcc agagaaagat ggggaagcca ccggagtgga
tgccatctgc 19050acccaccgcc ctgaccccac aggccctggg ctggacagag
agcagctgta 19100tttggagctg agccagctga cccacagcat cactgagctg
ggcccctaca 19150cactggacag ggacagtctc tatgtcaatg gtttcaccca
tcggagctct 19200gtacccacca ccagcaccgg ggtggtcagc gaggagccat
tcacactgaa 19250cttcaccatc aacaacctgc gctacatggc ggacatgggc
caacccggct 19300ccctcaagtt caacatcaca gacaacgtca tgcagcacct
gctcagtcct 19350ttgttccaga ggagcagcct gggtgcacgg tacacaggct
gcagggtcat 19400cgcactaagg tctgtgaaga acggtgctga gacacgggtg
gacctcctct 19450gcacctacct gcagcccctc agcggcccag gtctgcctat
caagcaggtg 19500ttccatgagc tgagccagca gacccatggc atcacccggc
tgggccccta 19550ctctctggac aaagacagcc tctaccttaa cggttacaat
gaacctggtc 19600cagatgagcc tcctacaact cccaagccag ccaccacatt
cctgcctcct 19650ctgtcagaag ccacaacagc catggggtac cacctgaaga
ccctcacact 19700caacttcacc atctccaatc tccagtattc accagatatg
ggcaagggct 19750cagctacatt caactccacc gagggggtcc ttcagcacct
gctcagaccc 19800ttgttccaga agagcagcat gggccccttc tacttgggtt
gccaactgat 19850ctccctcagg cctgagaagg atggggcagc cactggtgtg
gacaccacct 19900gcacctacca ccctgaccct gtgggccccg ggctggacat
acagcagctt 19950tactgggagc tgagtcagct gacccatggt gtcacccaac
tgggcttcta 20000tgtcctggac agggatagcc tcttcatcaa tggctatgca
ccccagaatt 20050tatcaatccg gggcgagtac cagataaatt tccacattgt
caactggaac 20100ctcagtaatc cagaccccac atcctcagag tacatcaccc
tgctgaggga 20150catccaggac aaggtcacca cactctacaa aggcagtcaa
ctacatgaca 20200cattccgctt ctgcctggtc accaacttga cgatggactc
cgtgttggtc 20250actgtcaagg cattgttctc ctccaatttg gaccccagcc
tggtggagca 20300agtctttcta gataagaccc tgaatgcctc attccattgg
ctgggctcca 20350cctaccagtt ggtggacatc catgtgacag aaatggagtc
atcagtttat 20400caaccaacaa gcagctccag cacccagcac ttctacctga
atttcaccat 20450caccaaccta ccatattccc aggacaaagc ccagccaggc
accaccaatt 20500accagaggaa caaaaggaat attgaggatg cgctcaacca
actcttccga 20550aacagcagca tcaagagtta tttttctgac tgtcaagttt
caacattcag 20600gtctgtcccc aacaggcacc acaccggggt ggactccctg
tgtaacttct 20650cgccactggc tcggagagta gacagagttg ccatctatga
ggaatttctg 20700cggatgaccc ggaatggtac ccagctgcag aacttcaccc
tggacaggag 20750cagtgtcctt gtggatgggt attctcccaa cagaaatgag
cccttaactg 20800ggaattctga ccttcccttc tgggctgtca tcctcatcgg
cttggcagga 20850ctcctgggac tcatcacatg cctgatctgc ggtgtcctgg
tgaccacccg 20900ccggcggaag aaggaaggag aatacaacgt ccagcaacag
tgcccaggct 20950actaccagtc acacctagac ctggaggatc tgcaatgact
ggaacttgcc 21000ggtgcctggg gtgcctttcc cccagccagg gtccaaagaa
gcttggctgg 21050ggcagaaata aaccatattg gtcggaaaaa aaaaaaaaaa
aaaaaaaaaa 21100aaaaaaaaaa aa 2111226995PRTHomo sapiens 2Pro Val
Thr Ser Leu Leu Thr Pro Gly Leu Val Ile Thr Thr Asp 1 5 10 15Arg
Met Gly Ile Ser Arg Glu Pro Gly Thr Ser Ser Thr Ser Asn 20 25 30Leu
Ser Ser Thr Ser His Glu Arg Leu Thr Thr Leu Glu Asp Thr 35 40 45Val
Asp Thr Glu Ala Met Gln Pro Ser Thr His Thr Ala Val Thr 50 55 60Asn
Val Arg Thr Ser Ile Ser Gly His Glu Ser Gln Ser Ser Val 65 70 75Leu
Ser Asp Ser Glu Thr Pro Lys Ala Thr Ser Pro Met Gly Thr 80 85 90Thr
Tyr Thr Met Gly Glu Thr Ser Val Ser Ile Ser Thr Ser Asp 95 100
105Phe Phe Glu Thr Ser Arg Ile Gln Ile Glu Pro Thr Ser Ser Leu 110
115 120Thr Ser Gly Leu Arg Glu Thr Ser Ser Ser Glu Arg Ile Ser Ser
125 130 135Ala Thr Glu Gly Ser Thr Val Leu Ser Glu Val Pro Ser Gly
Ala 140 145 150Thr Thr Glu Val Ser Arg Thr Glu Val Ile Ser Ser Arg
Gly Thr 155 160 165Ser Met Ser Gly Pro Asp Gln Phe Thr Ile Ser Pro
Asp Ile Ser 170 175 180Thr Glu Ala Ile Thr Arg Leu Ser Thr Ser Pro
Ile Met Thr Glu 185 190 195Ser Ala Glu Ser Ala Ile Thr Ile Glu Thr
Gly Ser Pro Gly Ala 200 205 210Thr Ser Glu Gly Thr Leu Thr Leu Asp
Thr Ser Thr Thr Thr Phe 215 220 225Trp Ser Gly Thr His Ser Thr Ala
Ser Pro Gly Phe Ser His Ser 230 235 240Glu Met Thr Thr Leu Met Ser
Arg Thr Pro Gly Asp Val Pro Trp 245 250 255Pro Ser Leu Pro Ser Val
Glu Glu Ala Ser Ser Val Ser Ser Ser 260 265 270Leu Ser Ser Pro Ala
Met Thr Ser Thr Ser Phe Phe Ser Thr Leu 275 280 285Pro Glu Ser Ile
Ser Ser Ser Pro His Pro Val Thr Ala Leu Leu 290 295 300Thr Leu Gly
Pro Val Lys Thr Thr Asp Met Leu Arg Thr Ser Ser 305 310 315Glu Pro
Glu Thr Ser Ser Pro Pro Asn Leu Ser Ser Thr Ser Ala 320 325 330Glu
Ile Leu Ala Thr Ser Glu Val Thr Lys Asp Arg Glu Lys Ile 335 340
345His Pro Ser Ser Asn Thr Pro Val Val Asn Val Gly Thr Val Ile 350
355 360Tyr Lys His Leu Ser Pro Ser Ser Val Leu Ala Asp Leu Val Thr
365 370 375Thr Lys Pro Thr Ser Pro Met Ala Thr Thr Ser Thr Leu Gly
Asn 380 385 390Thr Ser Val Ser Thr Ser Thr Pro Ala Phe Pro Glu Thr
Met Met 395 400 405Thr Gln Pro Thr Ser Ser Leu Thr Ser Gly Leu Arg
Glu Ile Ser 410 415 420Thr Ser Gln Glu Thr Ser Ser Ala Thr Glu Arg
Ser Ala Ser Leu 425 430 435Ser Gly Met Pro Thr Gly Ala Thr Thr Lys
Val Ser Arg Thr Glu 440 445 450Ala Leu Ser Leu Gly Arg Thr Ser Thr
Pro Gly Pro Ala Gln Ser 455 460 465Thr Ile Ser Pro Glu Ile Ser Thr
Glu Thr Ile Thr Arg Ile Ser 470 475 480Thr Pro Leu Thr Thr Thr Gly
Ser Ala Glu Met Thr Ile Thr Pro 485 490 495Lys Thr Gly His Ser Gly
Ala Ser Ser Gln Gly Thr Phe Thr Leu 500 505 510Asp Thr Ser Ser Arg
Ala Ser Trp Pro Gly Thr His Ser Ala Ala 515 520 525Thr His Arg Ser
Pro His Ser Gly Met Thr Thr Pro Met Ser Arg 530 535 540Gly Pro Glu
Asp Val Ser Trp Pro Ser Arg Pro Ser Val Glu Lys 545 550 555Thr Ser
Pro Pro Ser Ser Leu Val Ser Leu Ser Ala Val Thr Ser 560 565 570Pro
Ser Pro Leu Tyr Ser Thr Pro Ser Glu Ser Ser His Ser Ser 575 580
585Pro Leu Arg Val Thr Ser Leu Phe Thr Pro Val Met Met Lys Thr 590
595 600Thr Asp Met Leu Asp Thr Ser Leu Glu Pro Val Thr Thr Ser Pro
605 610 615Pro Ser Met Asn Ile Thr Ser Asp Glu Ser Leu Ala Thr Ser
Lys 620 625 630Ala Thr Met Glu Thr Glu Ala Ile Gln Leu Ser Glu Asn
Thr Ala 635 640 645Val Thr Gln Met Gly Thr Ile Ser Ala Arg Gln Glu
Phe Tyr Ser 650 655 660Ser Tyr Pro Gly Leu Pro Glu Pro Ser Lys Val
Thr Ser Pro Val 665 670 675Val Thr Ser Ser Thr Ile Lys Asp Ile Val
Ser Thr Thr Ile Pro 680 685 690Ala Ser Ser Glu Ile Thr Arg Ile Glu
Met Glu Ser Thr Ser Thr 695 700 705Leu Thr Pro Thr Pro Arg Glu Thr
Ser Thr Ser Gln Glu Ile His 710 715 720Ser Ala Thr Lys Pro Ser
Thr
Val Pro Tyr Lys Ala Leu Thr Ser 725 730 735Ala Thr Ile Glu Asp Ser
Met Thr Gln Val Met Ser Ser Ser Arg 740 745 750Gly Pro Ser Pro Asp
Gln Ser Thr Met Ser Gln Asp Ile Ser Thr 755 760 765Glu Val Ile Thr
Arg Leu Ser Thr Ser Pro Ile Lys Thr Glu Ser 770 775 780Thr Glu Met
Thr Ile Thr Thr Gln Thr Gly Ser Pro Gly Ala Thr 785 790 795Ser Arg
Gly Thr Leu Thr Leu Asp Thr Ser Thr Thr Phe Met Ser 800 805 810Gly
Thr His Ser Thr Ala Ser Gln Gly Phe Ser His Ser Gln Met 815 820
825Thr Ala Leu Met Ser Arg Thr Pro Gly Glu Val Pro Trp Leu Ser 830
835 840His Pro Ser Val Glu Glu Ala Ser Ser Ala Ser Phe Ser Leu Ser
845 850 855Ser Pro Val Met Thr Ser Ser Ser Pro Val Ser Ser Thr Leu
Pro 860 865 870Asp Ser Ile His Ser Ser Ser Leu Pro Val Thr Ser Leu
Leu Thr 875 880 885Ser Gly Leu Val Lys Thr Thr Glu Leu Leu Gly Thr
Ser Ser Glu 890 895 900Pro Glu Thr Ser Ser Pro Pro Asn Leu Ser Ser
Thr Ser Ala Glu 905 910 915Ile Leu Ala Thr Thr Glu Val Thr Thr Asp
Thr Glu Lys Leu Glu 920 925 930Met Thr Asn Val Val Thr Ser Gly Tyr
Thr His Glu Ser Pro Ser 935 940 945Ser Val Leu Ala Asp Ser Val Thr
Thr Lys Ala Thr Ser Ser Met 950 955 960Gly Ile Thr Tyr Pro Thr Gly
Asp Thr Asn Val Leu Thr Ser Thr 965 970 975Pro Ala Phe Ser Asp Thr
Ser Arg Ile Gln Thr Lys Ser Lys Leu 980 985 990Ser Leu Thr Pro Gly
Leu Met Glu Thr Ser Ile Ser Glu Glu Thr 995 1000 1005Ser Ser Ala
Thr Glu Lys Ser Thr Val Leu Ser Ser Val Pro Thr 1010 1015 1020Gly
Ala Thr Thr Glu Val Ser Arg Thr Glu Ala Ile Ser Ser Ser 1025 1030
1035Arg Thr Ser Ile Pro Gly Pro Ala Gln Ser Thr Met Ser Ser Asp
1040 1045 1050Thr Ser Met Glu Thr Ile Thr Arg Ile Ser Thr Pro Leu
Thr Arg 1055 1060 1065Lys Glu Ser Thr Asp Met Ala Ile Thr Pro Lys
Thr Gly Pro Ser 1070 1075 1080Gly Ala Thr Ser Gln Gly Thr Phe Thr
Leu Asp Ser Ser Ser Thr 1085 1090 1095Ala Ser Trp Pro Gly Thr His
Ser Ala Thr Thr Gln Arg Phe Pro 1100 1105 1110Arg Ser Val Val Thr
Thr Pro Met Ser Arg Gly Pro Glu Asp Val 1115 1120 1125Ser Trp Pro
Ser Pro Leu Ser Val Glu Lys Asn Ser Pro Pro Ser 1130 1135 1140Ser
Leu Val Ser Ser Ser Ser Val Thr Ser Pro Ser Pro Leu Tyr 1145 1150
1155Ser Thr Pro Ser Gly Ser Ser His Ser Ser Pro Val Pro Val Thr
1160 1165 1170Ser Leu Phe Thr Ser Ile Met Met Lys Ala Thr Asp Met
Leu Asp 1175 1180 1185Ala Ser Leu Glu Pro Glu Thr Thr Ser Ala Pro
Asn Met Asn Ile 1190 1195 1200Thr Ser Asp Glu Ser Leu Ala Ala Ser
Lys Ala Thr Thr Glu Thr 1205 1210 1215Glu Ala Ile His Val Phe Glu
Asn Thr Ala Ala Ser His Val Glu 1220 1225 1230Thr Thr Ser Ala Thr
Glu Glu Leu Tyr Ser Ser Ser Pro Gly Phe 1235 1240 1245Ser Glu Pro
Thr Lys Val Ile Ser Pro Val Val Thr Ser Ser Ser 1250 1255 1260Ile
Arg Asp Asn Met Val Ser Thr Thr Met Pro Gly Ser Ser Gly 1265 1270
1275Ile Thr Arg Ile Glu Ile Glu Ser Met Ser Ser Leu Thr Pro Gly
1280 1285 1290Leu Arg Glu Thr Arg Thr Ser Gln Asp Ile Thr Ser Ser
Thr Glu 1295 1300 1305Thr Ser Thr Val Leu Tyr Lys Met Pro Ser Gly
Ala Thr Pro Glu 1310 1315 1320Val Ser Arg Thr Glu Val Met Pro Ser
Ser Arg Thr Ser Ile Pro 1325 1330 1335Gly Pro Ala Gln Ser Thr Met
Ser Leu Asp Ile Ser Asp Glu Val 1340 1345 1350Val Thr Arg Leu Ser
Thr Ser Pro Ile Met Thr Glu Ser Ala Glu 1355 1360 1365Ile Thr Ile
Thr Thr Gln Thr Gly Tyr Ser Leu Ala Thr Ser Gln 1370 1375 1380Val
Thr Leu Pro Leu Gly Thr Ser Met Thr Phe Leu Ser Gly Thr 1385 1390
1395His Ser Thr Met Ser Gln Gly Leu Ser His Ser Glu Met Thr Asn
1400 1405 1410Leu Met Ser Arg Gly Pro Glu Ser Leu Ser Trp Thr Ser
Pro Arg 1415 1420 1425Phe Val Glu Thr Thr Arg Ser Ser Ser Ser Leu
Thr Ser Leu Pro 1430 1435 1440Leu Thr Thr Ser Leu Ser Pro Val Ser
Ser Thr Leu Leu Asp Ser 1445 1450 1455Ser Pro Ser Ser Pro Leu Pro
Val Thr Ser Leu Ile Leu Pro Gly 1460 1465 1470Leu Val Lys Thr Thr
Glu Val Leu Asp Thr Ser Ser Glu Pro Lys 1475 1480 1485Thr Ser Ser
Ser Pro Asn Leu Ser Ser Thr Ser Val Glu Ile Pro 1490 1495 1500Ala
Thr Ser Glu Ile Met Thr Asp Thr Glu Lys Ile His Pro Ser 1505 1510
1515Ser Asn Thr Ala Val Ala Lys Val Arg Thr Ser Ser Ser Val His
1520 1525 1530Glu Ser His Ser Ser Val Leu Ala Asp Ser Glu Thr Thr
Ile Thr 1535 1540 1545Ile Pro Ser Met Gly Ile Thr Ser Ala Val Glu
Asp Thr Thr Val 1550 1555 1560Phe Thr Ser Asn Pro Ala Phe Ser Glu
Thr Arg Arg Ile Pro Thr 1565 1570 1575Glu Pro Thr Phe Ser Leu Thr
Pro Gly Phe Arg Glu Thr Ser Thr 1580 1585 1590Ser Glu Glu Thr Thr
Ser Ile Thr Glu Thr Ser Ala Val Leu Phe 1595 1600 1605Gly Val Pro
Thr Ser Ala Thr Thr Glu Val Ser Met Thr Glu Ile 1610 1615 1620Met
Ser Ser Asn Arg Thr His Ile Pro Asp Ser Asp Gln Ser Thr 1625 1630
1635Met Ser Pro Asp Ile Ile Thr Glu Val Ile Thr Arg Leu Ser Ser
1640 1645 1650Ser Ser Met Met Ser Glu Ser Thr Gln Met Thr Ile Thr
Thr Gln 1655 1660 1665Lys Ser Ser Pro Gly Ala Thr Ala Gln Ser Thr
Leu Thr Leu Ala 1670 1675 1680Thr Thr Thr Ala Pro Leu Ala Arg Thr
His Ser Thr Val Pro Pro 1685 1690 1695Arg Phe Leu His Ser Glu Met
Thr Thr Leu Met Ser Arg Ser Pro 1700 1705 1710Glu Asn Pro Ser Trp
Lys Ser Ser Pro Phe Val Glu Lys Thr Ser 1715 1720 1725Ser Ser Ser
Ser Leu Leu Ser Leu Pro Val Thr Thr Ser Pro Ser 1730 1735 1740Val
Ser Ser Thr Leu Pro Gln Ser Ile Pro Ser Ser Ser Phe Ser 1745 1750
1755Val Thr Ser Leu Leu Thr Pro Gly Met Val Lys Thr Thr Asp Thr
1760 1765 1770Ser Thr Glu Pro Gly Thr Ser Leu Ser Pro Asn Leu Ser
Gly Thr 1775 1780 1785Ser Val Glu Ile Leu Ala Ala Ser Glu Val Thr
Thr Asp Thr Glu 1790 1795 1800Lys Ile His Pro Ser Ser Ser Met Ala
Val Thr Asn Val Gly Thr 1805 1810 1815Thr Ser Ser Gly His Glu Leu
Tyr Ser Ser Val Ser Ile His Ser 1820 1825 1830Glu Pro Ser Lys Ala
Thr Tyr Pro Val Gly Thr Pro Ser Ser Met 1835 1840 1845Ala Glu Thr
Ser Ile Ser Thr Ser Met Pro Ala Asn Phe Glu Thr 1850 1855 1860Thr
Gly Phe Glu Ala Glu Pro Phe Ser His Leu Thr Ser Gly Leu 1865 1870
1875Arg Lys Thr Asn Met Ser Leu Asp Thr Ser Ser Val Thr Pro Thr
1880 1885 1890Asn Thr Pro Ser Ser Pro Gly Ser Thr His Leu Leu Gln
Ser Ser 1895 1900 1905Lys Thr Asp Phe Thr Ser Ser Ala Lys Thr Ser
Ser Pro Asp Trp 1910 1915 1920Pro Pro Ala Ser Gln Tyr Thr Glu Ile
Pro Val Asp Ile Ile Thr 1925 1930 1935Pro Phe Asn Ala Ser Pro Ser
Ile Thr Glu Ser Thr Gly Ile Thr 1940 1945 1950Ser Phe Pro Glu Ser
Arg Phe Thr Met Ser Val Thr Glu Ser Thr 1955 1960 1965His His Leu
Ser Thr Asp Leu Leu Pro Ser Ala Glu Thr Ile Ser 1970 1975 1980Thr
Gly Thr Val Met Pro Ser Leu Ser Glu Ala Met Thr Ser Phe 1985 1990
1995Ala Thr Thr Gly Val Pro Arg Ala Ile Ser Gly Ser Gly Ser Pro
2000 2005 2010Phe Ser Arg Thr Glu Ser Gly Pro Gly Asp Ala Thr Leu
Ser Thr 2015 2020 2025Ile Ala Glu Ser Leu Pro Ser Ser Thr Pro Val
Pro Phe Ser Ser 2030 2035 2040Ser Thr Phe Thr Thr Thr Asp Ser Ser
Thr Ile Pro Ala Leu His 2045 2050 2055Glu Ile Thr Ser Ser Ser Ala
Thr Pro Tyr Arg Val Asp Thr Ser 2060 2065 2070Leu Gly Thr Glu Ser
Ser Thr Thr Glu Gly Arg Leu Val Met Val 2075 2080 2085Ser Thr Leu
Asp Thr Ser Ser Gln Pro Gly Arg Thr Ser Ser Ser 2090 2095 2100Pro
Ile Leu Asp Thr Arg Met Thr Glu Ser Val Glu Leu Gly Thr 2105 2110
2115Val Thr Ser Ala Tyr Gln Val Pro Ser Leu Ser Thr Arg Leu Thr
2120 2125 2130Arg Thr Asp Gly Ile Met Glu His Ile Thr Lys Ile Pro
Asn Glu 2135 2140 2145Ala Ala His Arg Gly Thr Ile Arg Pro Val Lys
Gly Pro Gln Thr 2150 2155 2160Ser Thr Ser Pro Ala Ser Pro Lys Gly
Leu His Thr Gly Gly Thr 2165 2170 2175Lys Arg Met Glu Thr Thr Thr
Thr Ala Leu Lys Thr Thr Thr Thr 2180 2185 2190Ala Leu Lys Thr Thr
Ser Arg Ala Thr Leu Thr Thr Ser Val Tyr 2195 2200 2205Thr Pro Thr
Leu Gly Thr Leu Thr Pro Leu Asn Ala Ser Met Gln 2210 2215 2220Met
Ala Ser Thr Ile Pro Thr Glu Met Met Ile Thr Thr Pro Tyr 2225 2230
2235Val Phe Pro Asp Val Pro Glu Thr Thr Ser Ser Leu Ala Thr Ser
2240 2245 2250Leu Gly Ala Glu Thr Ser Thr Ala Leu Pro Arg Thr Thr
Pro Ser 2255 2260 2265Val Phe Asn Arg Glu Ser Glu Thr Thr Ala Ser
Leu Val Ser Arg 2270 2275 2280Ser Gly Ala Glu Arg Ser Pro Val Ile
Gln Thr Leu Asp Val Ser 2285 2290 2295Ser Ser Glu Pro Asp Thr Thr
Ala Ser Trp Val Ile His Pro Ala 2300 2305 2310Glu Thr Ile Pro Thr
Val Ser Lys Thr Thr Pro Asn Phe Phe His 2315 2320 2325Ser Glu Leu
Asp Thr Val Ser Ser Thr Ala Thr Ser His Gly Ala 2330 2335 2340Asp
Val Ser Ser Ala Ile Pro Thr Asn Ile Ser Pro Ser Glu Leu 2345 2350
2355Asp Ala Leu Thr Pro Leu Val Thr Ile Ser Gly Thr Asp Thr Ser
2360 2365 2370Thr Thr Phe Pro Thr Leu Thr Lys Ser Pro His Glu Thr
Glu Thr 2375 2380 2385Arg Thr Thr Trp Leu Thr His Pro Ala Glu Thr
Ser Ser Thr Ile 2390 2395 2400Pro Arg Thr Ile Pro Asn Phe Ser His
His Glu Ser Asp Ala Thr 2405 2410 2415Pro Ser Ile Ala Thr Ser Pro
Gly Ala Glu Thr Ser Ser Ala Ile 2420 2425 2430Pro Ile Met Thr Val
Ser Pro Gly Ala Glu Asp Leu Val Thr Ser 2435 2440 2445Gln Val Thr
Ser Ser Gly Thr Asp Arg Asn Met Thr Ile Pro Thr 2450 2455 2460Leu
Thr Leu Ser Pro Gly Glu Pro Lys Thr Ile Ala Ser Leu Val 2465 2470
2475Thr His Pro Glu Ala Gln Thr Ser Ser Ala Ile Pro Thr Ser Thr
2480 2485 2490Ile Ser Pro Ala Val Ser Arg Leu Val Thr Ser Met Val
Thr Ser 2495 2500 2505Leu Ala Ala Lys Thr Ser Thr Thr Asn Arg Ala
Leu Thr Asn Ser 2510 2515 2520Pro Gly Glu Pro Ala Thr Thr Val Ser
Leu Val Thr His Ser Ala 2525 2530 2535Gln Thr Ser Pro Thr Val Pro
Trp Thr Thr Ser Ile Phe Phe His 2540 2545 2550Ser Lys Ser Asp Thr
Thr Pro Ser Met Thr Thr Ser His Gly Ala 2555 2560 2565Glu Ser Ser
Ser Ala Val Pro Thr Pro Thr Val Ser Thr Glu Val 2570 2575 2580Pro
Gly Val Val Thr Pro Leu Val Thr Ser Ser Arg Ala Val Ile 2585 2590
2595Ser Thr Thr Ile Pro Ile Leu Thr Leu Ser Pro Gly Glu Pro Glu
2600 2605 2610Thr Thr Pro Ser Met Ala Thr Ser His Gly Glu Glu Ala
Ser Ser 2615 2620 2625Ala Ile Pro Thr Pro Thr Val Ser Pro Gly Val
Pro Gly Val Val 2630 2635 2640Thr Ser Leu Val Thr Ser Ser Arg Ala
Val Thr Ser Thr Thr Ile 2645 2650 2655Pro Ile Leu Thr Phe Ser Leu
Gly Glu Pro Glu Thr Thr Pro Ser 2660 2665 2670Met Ala Thr Ser His
Gly Thr Glu Ala Gly Ser Ala Val Pro Thr 2675 2680 2685Val Leu Pro
Glu Val Pro Gly Met Val Thr Ser Leu Val Ala Ser 2690 2695 2700Ser
Arg Ala Val Thr Ser Thr Thr Leu Pro Thr Leu Thr Leu Ser 2705 2710
2715Pro Gly Glu Pro Glu Thr Thr Pro Ser Met Ala Thr Ser His Gly
2720 2725 2730Ala Glu Ala Ser Ser Thr Val Pro Thr Val Ser Pro Glu
Val Pro 2735 2740 2745Gly Val Val Thr Ser Leu Val Thr Ser Ser Ser
Gly Val Asn Ser 2750 2755 2760Thr Ser Ile Pro Thr Leu Ile Leu Ser
Pro Gly Glu Leu Glu Thr 2765 2770 2775Thr Pro Ser Met Ala Thr Ser
His Gly Ala Glu Ala Ser Ser Ala 2780 2785 2790Val Pro Thr Pro Thr
Val Ser Pro Gly Val Ser Gly Val Val Thr 2795 2800 2805Pro Leu Val
Thr Ser Ser Arg Ala Val Thr Ser Thr Thr Ile Pro 2810 2815 2820Ile
Leu Thr Leu Ser Ser Ser Glu Pro Glu Thr Thr Pro Ser Met 2825 2830
2835Ala Thr Ser His Gly Val Glu Ala Ser Ser Ala Val Leu Thr Val
2840 2845 2850Ser Pro Glu Val Pro Gly Met Val Thr Phe Leu Val Thr
Ser Ser 2855 2860 2865Arg Ala Val Thr Ser Thr Thr Ile Pro Thr Leu
Thr Ile Ser Ser 2870 2875 2880Asp Glu Pro Glu Thr Thr Thr Ser Leu
Val Thr His Ser Glu Ala 2885 2890 2895Lys Met Ile Ser Ala Ile Pro
Thr Leu Gly Val Ser Pro Thr Val 2900 2905 2910Gln Gly Leu Val Thr
Ser Leu Val Thr Ser Ser Gly Ser Glu Thr 2915 2920 2925Ser Ala Phe
Ser Asn Leu Thr Val Ala Ser Ser Gln Pro Glu Thr 2930 2935 2940Ile
Asp Ser Trp Val Ala His Pro Gly Thr Glu Ala Ser Ser Val 2945 2950
2955Val Pro Thr Leu Thr Val Ser Thr Gly Glu Pro Phe Thr Asn Ile
2960 2965 2970Ser Leu Val Thr His Pro Ala Glu Ser Ser Ser Thr Leu
Pro Arg 2975 2980 2985Thr Thr Ser Arg Phe Ser His Ser Glu Leu Asp
Thr
Met Pro Ser 2990 2995 3000Thr Val Thr Ser Pro Glu Ala Glu Ser Ser
Ser Ala Ile Ser Thr 3005 3010 3015Thr Ile Ser Pro Gly Ile Pro Gly
Val Leu Thr Ser Leu Val Thr 3020 3025 3030Ser Ser Gly Arg Asp Ile
Ser Ala Thr Phe Pro Thr Val Pro Glu 3035 3040 3045Ser Pro His Glu
Ser Glu Ala Thr Ala Ser Trp Val Thr His Pro 3050 3055 3060Ala Val
Thr Ser Thr Thr Val Pro Arg Thr Thr Pro Asn Tyr Ser 3065 3070
3075His Ser Glu Pro Asp Thr Thr Pro Ser Ile Ala Thr Ser Pro Gly
3080 3085 3090Ala Glu Ala Thr Ser Asp Phe Pro Thr Ile Thr Val Ser
Pro Asp 3095 3100 3105Val Pro Asp Met Val Thr Ser Gln Val Thr Ser
Ser Gly Thr Asp 3110 3115 3120Thr Ser Ile Thr Ile Pro Thr Leu Thr
Leu Ser Ser Gly Glu Pro 3125 3130 3135Glu Thr Thr Thr Ser Phe Ile
Thr Tyr Ser Glu Thr His Thr Ser 3140 3145 3150Ser Ala Ile Pro Thr
Leu Pro Val Ser Pro Asp Ala Ser Lys Met 3155 3160 3165Leu Thr Ser
Leu Val Ile Ser Ser Gly Thr Asp Ser Thr Thr Thr 3170 3175 3180Phe
Pro Thr Leu Thr Glu Thr Pro Tyr Glu Pro Glu Thr Thr Ala 3185 3190
3195Ile Gln Leu Ile His Pro Ala Glu Thr Asn Thr Met Val Pro Arg
3200 3205 3210Thr Thr Pro Lys Phe Ser His Ser Lys Ser Asp Thr Thr
Leu Pro 3215 3220 3225Val Ala Ile Thr Ser Pro Gly Pro Glu Ala Ser
Ser Ala Val Ser 3230 3235 3240Thr Thr Thr Ile Ser Pro Asp Met Ser
Asp Leu Val Thr Ser Leu 3245 3250 3255Val Pro Ser Ser Gly Thr Asp
Thr Ser Thr Thr Phe Pro Thr Leu 3260 3265 3270Ser Glu Thr Pro Tyr
Glu Pro Glu Thr Thr Ala Thr Trp Leu Thr 3275 3280 3285His Pro Ala
Glu Thr Ser Thr Thr Val Ser Gly Thr Ile Pro Asn 3290 3295 3300Phe
Ser His Arg Gly Ser Asp Thr Ala Pro Ser Met Val Thr Ser 3305 3310
3315Pro Gly Val Asp Thr Arg Ser Gly Val Pro Thr Thr Thr Ile Pro
3320 3325 3330Pro Ser Ile Pro Gly Val Val Thr Ser Gln Val Thr Ser
Ser Ala 3335 3340 3345Thr Asp Thr Ser Thr Ala Ile Pro Thr Leu Thr
Pro Ser Pro Gly 3350 3355 3360Glu Pro Glu Thr Thr Ala Ser Ser Ala
Thr His Pro Gly Thr Gln 3365 3370 3375Thr Gly Phe Thr Val Pro Ile
Arg Thr Val Pro Ser Ser Glu Pro 3380 3385 3390Asp Thr Met Ala Ser
Trp Val Thr His Pro Pro Gln Thr Ser Thr 3395 3400 3405Pro Val Ser
Arg Thr Thr Ser Ser Phe Ser His Ser Ser Pro Asp 3410 3415 3420Ala
Thr Pro Val Met Ala Thr Ser Pro Arg Thr Glu Ala Ser Ser 3425 3430
3435Ala Val Leu Thr Thr Ile Ser Pro Gly Ala Pro Glu Met Val Thr
3440 3445 3450Ser Gln Ile Thr Ser Ser Gly Ala Ala Thr Ser Thr Thr
Val Pro 3455 3460 3465Thr Leu Thr His Ser Pro Gly Met Pro Glu Thr
Thr Ala Leu Leu 3470 3475 3480Ser Thr His Pro Arg Thr Glu Thr Ser
Lys Thr Phe Pro Ala Ser 3485 3490 3495Thr Val Phe Pro Gln Val Ser
Glu Thr Thr Ala Ser Leu Thr Ile 3500 3505 3510Arg Pro Gly Ala Glu
Thr Ser Thr Ala Leu Pro Thr Gln Thr Thr 3515 3520 3525Ser Ser Leu
Phe Thr Leu Leu Val Thr Gly Thr Ser Arg Val Asp 3530 3535 3540Leu
Ser Pro Thr Ala Ser Pro Gly Val Ser Ala Lys Thr Ala Pro 3545 3550
3555Leu Ser Thr His Pro Gly Thr Glu Thr Ser Thr Met Ile Pro Thr
3560 3565 3570Ser Thr Leu Ser Leu Gly Leu Leu Glu Thr Thr Gly Leu
Leu Ala 3575 3580 3585Thr Ser Ser Ser Ala Glu Thr Ser Thr Ser Thr
Leu Thr Leu Thr 3590 3595 3600Val Ser Pro Ala Val Ser Gly Leu Ser
Ser Ala Ser Ile Thr Thr 3605 3610 3615Asp Lys Pro Gln Thr Val Thr
Ser Trp Asn Thr Glu Thr Ser Pro 3620 3625 3630Ser Val Thr Ser Val
Gly Pro Pro Glu Phe Ser Arg Thr Val Thr 3635 3640 3645Gly Thr Thr
Met Thr Leu Ile Pro Ser Glu Met Pro Thr Pro Pro 3650 3655 3660Lys
Thr Ser His Gly Glu Gly Val Ser Pro Thr Thr Ile Leu Arg 3665 3670
3675Thr Thr Met Val Glu Ala Thr Asn Leu Ala Thr Thr Gly Ser Ser
3680 3685 3690Pro Thr Val Ala Lys Thr Thr Thr Thr Phe Asn Thr Leu
Ala Gly 3695 3700 3705Ser Leu Phe Thr Pro Leu Thr Thr Pro Gly Met
Ser Thr Leu Ala 3710 3715 3720Ser Glu Ser Val Thr Ser Arg Thr Ser
Tyr Asn His Arg Ser Trp 3725 3730 3735Ile Ser Thr Thr Ser Ser Tyr
Asn Arg Arg Tyr Trp Thr Pro Ala 3740 3745 3750Thr Ser Thr Pro Val
Thr Ser Thr Phe Ser Pro Gly Ile Ser Thr 3755 3760 3765Ser Ser Ile
Pro Ser Ser Thr Ala Ala Thr Val Pro Phe Met Val 3770 3775 3780Pro
Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr Glu Glu 3785 3790
3795Asp Met Arg His Pro Gly Ser Arg Lys Phe Asn Ala Thr Glu Arg
3800 3805 3810Glu Leu Gln Gly Leu Leu Lys Pro Leu Phe Arg Asn Ser
Ser Leu 3815 3820 3825Glu Tyr Leu Tyr Ser Gly Cys Arg Leu Ala Ser
Leu Arg Pro Glu 3830 3835 3840Lys Asp Ser Ser Ala Thr Ala Val Asp
Ala Ile Cys Thr His Arg 3845 3850 3855Pro Asp Pro Glu Asp Leu Gly
Leu Asp Arg Glu Arg Leu Tyr Trp 3860 3865 3870Glu Leu Ser Asn Leu
Thr Asn Gly Ile Gln Glu Leu Gly Pro Tyr 3875 3880 3885Thr Leu Asp
Arg Asn Ser Leu Tyr Val Asn Gly Phe Thr His Arg 3890 3895 3900Ser
Ser Met Pro Thr Thr Ser Thr Pro Gly Thr Ser Thr Val Asp 3905 3910
3915Val Gly Thr Ser Gly Thr Pro Ser Ser Ser Pro Ser Pro Thr Thr
3920 3925 3930Ala Gly Pro Leu Leu Met Pro Phe Thr Leu Asn Phe Thr
Ile Thr 3935 3940 3945Asn Leu Gln Tyr Glu Glu Asp Met Arg Arg Thr
Gly Ser Arg Lys 3950 3955 3960Phe Asn Thr Met Glu Ser Val Leu Gln
Gly Leu Leu Lys Pro Leu 3965 3970 3975Phe Lys Asn Thr Ser Val Gly
Pro Leu Tyr Ser Gly Cys Arg Leu 3980 3985 3990Thr Leu Leu Arg Pro
Glu Lys Asp Gly Ala Ala Thr Gly Val Asp 3995 4000 4005Ala Ile Cys
Thr His Arg Leu Asp Pro Lys Ser Pro Gly Leu Asn 4010 4015 4020Arg
Glu Gln Leu Tyr Trp Glu Leu Ser Lys Leu Thr Asn Asp Ile 4025 4030
4035Glu Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asn Ser Leu Tyr Val
4040 4045 4050Asn Gly Phe Thr His Gln Ser Ser Val Ser Thr Thr Ser
Thr Pro 4055 4060 4065Gly Thr Ser Thr Val Asp Leu Arg Thr Ser Gly
Thr Pro Ser Ser 4070 4075 4080Leu Ser Ser Pro Thr Ile Met Ala Ala
Gly Pro Leu Leu Val Pro 4085 4090 4095Phe Thr Leu Asn Phe Thr Ile
Thr Asn Leu Gln Tyr Gly Glu Asp 4100 4105 4110Met Gly His Pro Gly
Ser Arg Lys Phe Asn Thr Thr Glu Arg Val 4115 4120 4125Leu Gln Gly
Leu Leu Gly Pro Ile Phe Lys Asn Thr Ser Val Gly 4130 4135 4140Pro
Leu Tyr Ser Gly Cys Arg Leu Thr Ser Leu Arg Ser Glu Lys 4145 4150
4155Asp Gly Ala Ala Thr Gly Val Asp Ala Ile Cys Ile His His Leu
4160 4165 4170Asp Pro Lys Ser Pro Gly Leu Asn Arg Glu Arg Leu Tyr
Trp Glu 4175 4180 4185Leu Ser Gln Leu Thr Asn Gly Ile Lys Glu Leu
Gly Pro Tyr Thr 4190 4195 4200Leu Asp Arg Asn Ser Leu Tyr Val Asn
Gly Phe Thr His Arg Thr 4205 4210 4215Ser Val Pro Thr Thr Ser Thr
Pro Gly Thr Ser Thr Val Asp Leu 4220 4225 4230Gly Thr Ser Gly Thr
Pro Phe Ser Leu Pro Ser Pro Ala Thr Ala 4235 4240 4245Gly Pro Leu
Leu Val Leu Phe Thr Leu Asn Phe Thr Ile Thr Asn 4250 4255 4260Leu
Lys Tyr Glu Glu Asp Met His Arg Pro Gly Ser Arg Lys Phe 4265 4270
4275Asn Thr Thr Glu Arg Val Leu Gln Thr Leu Val Gly Pro Met Phe
4280 4285 4290Lys Asn Thr Ser Val Gly Leu Leu Tyr Ser Gly Cys Arg
Leu Thr 4295 4300 4305Leu Leu Arg Ser Glu Lys Asp Gly Ala Ala Thr
Gly Val Asp Ala 4310 4315 4320Ile Cys Thr His Arg Leu Asp Pro Lys
Ser Pro Gly Val Asp Arg 4325 4330 4335Glu Gln Leu Tyr Trp Glu Leu
Ser Gln Leu Thr Asn Gly Ile Lys 4340 4345 4350Glu Leu Gly Pro Tyr
Thr Leu Asp Arg Asn Ser Leu Tyr Val Asn 4355 4360 4365Gly Phe Thr
His Trp Ile Pro Val Pro Thr Ser Ser Thr Pro Gly 4370 4375 4380Thr
Ser Thr Val Asp Leu Gly Ser Gly Thr Pro Ser Ser Leu Pro 4385 4390
4395Ser Pro Thr Ser Ala Thr Ala Gly Pro Leu Leu Val Pro Phe Thr
4400 4405 4410Leu Asn Phe Thr Ile Thr Asn Leu Lys Tyr Glu Glu Asp
Met His 4415 4420 4425Cys Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu
Arg Val Leu Gln 4430 4435 4440Ser Leu Leu Gly Pro Met Phe Lys Asn
Thr Ser Val Gly Pro Leu 4445 4450 4455Tyr Ser Gly Cys Arg Leu Thr
Leu Leu Arg Ser Glu Lys Asp Gly 4460 4465 4470Ala Ala Thr Gly Val
Asp Ala Ile Cys Thr His Arg Leu Asp Pro 4475 4480 4485Lys Ser Pro
Gly Val Asp Arg Glu Gln Leu Tyr Trp Glu Leu Ser 4490 4495 4500Gln
Leu Thr Asn Gly Ile Lys Glu Leu Gly Pro Tyr Thr Leu Asp 4505 4510
4515Arg Asn Ser Leu Tyr Val Asn Gly Phe Thr His Gln Thr Ser Ala
4520 4525 4530Pro Asn Thr Ser Thr Pro Gly Thr Ser Thr Val Asp Leu
Gly Thr 4535 4540 4545Ser Gly Thr Pro Ser Ser Leu Pro Ser Pro Thr
Ser Ala Gly Pro 4550 4555 4560Leu Leu Val Pro Phe Thr Leu Asn Phe
Thr Ile Thr Asn Leu Gln 4565 4570 4575Tyr Glu Glu Asp Met His His
Pro Gly Ser Arg Lys Phe Asn Thr 4580 4585 4590Thr Glu Arg Val Leu
Gln Gly Leu Leu Gly Pro Met Phe Lys Asn 4595 4600 4605Thr Ser Val
Gly Leu Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu 4610 4615 4620Arg
Pro Glu Lys Asn Gly Ala Ala Thr Gly Met Asp Ala Ile Cys 4625 4630
4635Ser His Arg Leu Asp Pro Lys Ser Pro Gly Leu Asn Arg Glu Gln
4640 4645 4650Leu Tyr Trp Glu Leu Ser Gln Leu Thr His Gly Ile Lys
Glu Leu 4655 4660 4665Gly Pro Tyr Thr Leu Asp Arg Asn Ser Leu Tyr
Val Asn Gly Phe 4670 4675 4680Thr His Arg Ser Ser Val Ala Pro Thr
Ser Thr Pro Gly Thr Ser 4685 4690 4695Thr Val Asp Leu Gly Thr Ser
Gly Thr Pro Ser Ser Leu Pro Ser 4700 4705 4710Pro Thr Thr Ala Val
Pro Leu Leu Val Pro Phe Thr Leu Asn Phe 4715 4720 4725Thr Ile Thr
Asn Leu Gln Tyr Gly Glu Asp Met Arg His Pro Gly 4730 4735 4740Ser
Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln Gly Leu Leu 4745 4750
4755Gly Pro Leu Phe Lys Asn Ser Ser Val Gly Pro Leu Tyr Ser Gly
4760 4765 4770Cys Arg Leu Ile Ser Leu Arg Ser Glu Lys Asp Gly Ala
Ala Thr 4775 4780 4785Gly Val Asp Ala Ile Cys Thr His His Leu Asn
Pro Gln Ser Pro 4790 4795 4800Gly Leu Asp Arg Glu Gln Leu Tyr Trp
Gln Leu Ser Gln Met Thr 4805 4810 4815Asn Gly Ile Lys Glu Leu Gly
Pro Tyr Thr Leu Asp Arg Asn Ser 4820 4825 4830Leu Tyr Val Asn Gly
Phe Thr His Arg Ser Ser Gly Leu Thr Thr 4835 4840 4845Ser Thr Pro
Trp Thr Ser Thr Val Asp Leu Gly Thr Ser Gly Thr 4850 4855 4860Pro
Ser Pro Val Pro Ser Pro Thr Thr Ala Gly Pro Leu Leu Val 4865 4870
4875Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln Tyr Glu Glu
4880 4885 4890Asp Met His Arg Pro Gly Ser Arg Lys Phe Asn Ala Thr
Glu Arg 4895 4900 4905Val Leu Gln Gly Leu Leu Ser Pro Ile Phe Lys
Asn Ser Ser Val 4910 4915 4920Gly Pro Leu Tyr Ser Gly Cys Arg Leu
Thr Ser Leu Arg Pro Glu 4925 4930 4935Lys Asp Gly Ala Ala Thr Gly
Met Asp Ala Val Cys Leu Tyr His 4940 4945 4950Pro Asn Pro Lys Arg
Pro Gly Leu Asp Arg Glu Gln Leu Tyr Trp 4955 4960 4965Glu Leu Ser
Gln Leu Thr His Asn Ile Thr Glu Leu Gly Pro Tyr 4970 4975 4980Ser
Leu Asp Arg Asp Ser Leu Tyr Val Asn Gly Phe Thr His Gln 4985 4990
4995Asn Ser Val Pro Thr Thr Ser Thr Pro Gly Thr Ser Thr Val Tyr
5000 5005 5010Trp Ala Thr Thr Gly Thr Pro Ser Ser Phe Pro Gly His
Thr Glu 5015 5020 5025Pro Gly Pro Leu Leu Ile Pro Phe Thr Phe Asn
Phe Thr Ile Thr 5030 5035 5040Asn Leu His Tyr Glu Glu Asn Met Gln
His Pro Gly Ser Arg Lys 5045 5050 5055Phe Asn Thr Thr Glu Arg Val
Leu Gln Gly Leu Leu Lys Pro Leu 5060 5065 5070Phe Lys Asn Thr Ser
Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu 5075 5080 5085Thr Leu Leu
Arg Pro Glu Lys Gln Glu Ala Ala Thr Gly Val Asp 5090 5095 5100Thr
Ile Cys Thr His Arg Val Asp Pro Ile Gly Pro Gly Leu Asp 5105 5110
5115Arg Glu Arg Leu Tyr Trp Glu Leu Ser Gln Leu Thr Asn Ser Ile
5120 5125 5130Thr Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asp Ser Leu
Tyr Val 5135 5140 5145Asn Gly Phe Asn Pro Trp Ser Ser Val Pro Thr
Thr Ser Thr Pro 5150 5155 5160Gly Thr Ser Thr Val His Leu Ala Thr
Ser Gly Thr Pro Ser Ser 5165 5170 5175Leu Pro Gly His Thr Ala Pro
Val Pro Leu Leu Ile Pro Phe Thr 5180 5185 5190Leu Asn Phe Thr Ile
Thr Asn Leu His Tyr Glu Glu Asn Met Gln 5195 5200 5205His Pro Gly
Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu Gln 5210 5215 5220Gly
Leu Leu Lys Pro Leu Phe Lys Ser Thr Ser Val Gly Pro Leu 5225 5230
5235Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu Lys His Gly
5240 5245
5250Ala Ala Thr Gly Val Asp Ala Ile Cys Thr Leu Arg Leu Asp Pro
5255 5260 5265Thr Gly Pro Gly Leu Asp Arg Glu Arg Leu Tyr Trp Glu
Leu Ser 5270 5275 5280Gln Leu Thr Asn Ser Val Thr Glu Leu Gly Pro
Tyr Thr Leu Asp 5285 5290 5295Arg Asp Ser Leu Tyr Val Asn Gly Phe
Thr His Arg Ser Ser Val 5300 5305 5310Pro Thr Thr Ser Ile Pro Gly
Thr Ser Ala Val His Leu Glu Thr 5315 5320 5325Ser Gly Thr Pro Ala
Ser Leu Pro Gly His Thr Ala Pro Gly Pro 5330 5335 5340Leu Leu Val
Pro Phe Thr Leu Asn Phe Thr Ile Thr Asn Leu Gln 5345 5350 5355Tyr
Glu Glu Asp Met Arg His Pro Gly Ser Arg Lys Phe Asn Thr 5360 5365
5370Thr Glu Arg Val Leu Gln Gly Leu Leu Lys Pro Leu Phe Lys Ser
5375 5380 5385Thr Ser Val Gly Pro Leu Tyr Ser Gly Cys Arg Leu Thr
Leu Leu 5390 5395 5400Arg Pro Glu Lys Arg Gly Ala Ala Thr Gly Val
Asp Thr Ile Cys 5405 5410 5415Thr His Arg Leu Asp Pro Leu Asn Pro
Gly Leu Asp Arg Glu Gln 5420 5425 5430Leu Tyr Trp Glu Leu Ser Lys
Leu Thr Arg Gly Ile Ile Glu Leu 5435 5440 5445Gly Pro Tyr Leu Leu
Asp Arg Gly Ser Leu Tyr Val Asn Gly Phe 5450 5455 5460Thr His Arg
Asn Phe Val Pro Ile Thr Ser Thr Pro Gly Thr Ser 5465 5470 5475Thr
Val His Leu Gly Thr Ser Glu Thr Pro Ser Ser Leu Pro Arg 5480 5485
5490Pro Ile Val Pro Gly Pro Leu Leu Val Pro Phe Thr Leu Asn Phe
5495 5500 5505Thr Ile Thr Asn Leu Gln Tyr Glu Glu Ala Met Arg His
Pro Gly 5510 5515 5520Ser Arg Lys Phe Asn Thr Thr Glu Arg Val Leu
Gln Gly Leu Leu 5525 5530 5535Arg Pro Leu Phe Lys Asn Thr Ser Ile
Gly Pro Leu Tyr Ser Ser 5540 5545 5550Cys Arg Leu Thr Leu Leu Arg
Pro Glu Lys Asp Lys Ala Ala Thr 5555 5560 5565Arg Val Asp Ala Ile
Cys Thr His His Pro Asp Pro Gln Ser Pro 5570 5575 5580Gly Leu Asn
Arg Glu Gln Leu Tyr Trp Glu Leu Ser Gln Leu Thr 5585 5590 5595His
Gly Ile Thr Glu Leu Gly Pro Tyr Thr Leu Asp Arg Asp Ser 5600 5605
5610Leu Tyr Val Asp Gly Phe Thr His Trp Ser Pro Ile Pro Thr Thr
5615 5620 5625Ser Thr Pro Gly Thr Ser Ile Val Asn Leu Gly Thr Ser
Gly Ile 5630 5635 5640Pro Pro Ser Leu Pro Glu Thr Thr Ala Thr Gly
Pro Leu Leu Val 5645 5650 5655Pro Phe Thr Leu Asn Phe Thr Ile Thr
Asn Leu Gln Tyr Glu Glu 5660 5665 5670Asn Met Gly His Pro Gly Ser
Arg Lys Phe Asn Ile Thr Glu Ser 5675 5680 5685Val Leu Gln Gly Leu
Leu Lys Pro Leu Phe Lys Ser Thr Ser Val 5690 5695 5700Gly Pro Leu
Tyr Ser Gly Cys Arg Leu Thr Leu Leu Arg Pro Glu 5705 5710 5715Lys
Asp Gly Val Ala Thr Arg Val Asp Ala Ile Cys Thr His Arg 5720 5725
5730Pro Asp Pro Lys Ile Pro Gly Leu Asp Arg Gln Gln Leu Tyr Trp
5735 5740 5745Glu Leu Ser Gln Leu Thr His Ser Ile Thr Glu Leu Gly
Pro Tyr 5750 5755 5760Thr Leu Asp Arg Asp Ser Leu Tyr Val Asn Gly
Phe Thr Gln Arg 5765 5770 5775Ser Ser Val Pro Thr Thr Ser Thr Pro
Gly Thr Phe Thr Val Gln 5780 5785 5790Pro Glu Thr Ser Glu Thr Pro
Ser Ser Leu Pro Gly Pro Thr Ala 5795 5800 5805Thr Gly Pro Val Leu
Leu Pro Phe Thr Leu Asn Phe Thr Ile Ile 5810 5815 5820Asn Leu Gln
Tyr Glu Glu Asp Met His Arg Pro Gly Ser Arg Lys 5825 5830 5835Phe
Asn Thr Thr Glu Arg Val Leu Gln Gly Leu Leu Met Pro Leu 5840 5845
5850Phe Lys Asn Thr Ser Val Ser Ser Leu Tyr Ser Gly Cys Arg Leu
5855 5860 5865Thr Leu Leu Arg Pro Glu Lys Asp Gly Ala Ala Thr Arg
Val Asp 5870 5875 5880Ala Val Cys Thr His Arg Pro Asp Pro Lys Ser
Pro Gly Leu Asp 5885 5890 5895Arg Glu Arg Leu Tyr Trp Lys Leu Ser
Gln Leu Thr His Gly Ile 5900 5905 5910Thr Glu Leu Gly Pro Tyr Thr
Leu Asp Arg His Ser Leu Tyr Val 5915 5920 5925Asn Gly Phe Thr His
Gln Ser Ser Met Thr Thr Thr Arg Thr Pro 5930 5935 5940Asp Thr Ser
Thr Met His Leu Ala Thr Ser Arg Thr Pro Ala Ser 5945 5950 5955Leu
Ser Gly Pro Thr Thr Ala Ser Pro Leu Leu Val Leu Phe Thr 5960 5965
5970Ile Asn Phe Thr Ile Thr Asn Leu Arg Tyr Glu Glu Asn Met His
5975 5980 5985His Pro Gly Ser Arg Lys Phe Asn Thr Thr Glu Arg Val
Leu Gln 5990 5995 6000Gly Leu Leu Arg Pro Val Phe Lys Asn Thr Ser
Val Gly Pro Leu 6005 6010 6015Tyr Ser Gly Cys Arg Leu Thr Leu Leu
Arg Pro Lys Lys Asp Gly 6020 6025 6030Ala Ala Thr Lys Val Asp Ala
Ile Cys Thr Tyr Arg Pro Asp Pro 6035 6040 6045Lys Ser Pro Gly Leu
Asp Arg Glu Gln Leu Tyr Trp Glu Leu Ser 6050 6055 6060Gln Leu Thr
His Ser Ile Thr Glu Leu Gly Pro Tyr Thr Leu Asp 6065 6070 6075Arg
Asp Ser Leu Tyr Val Asn Gly Phe Thr Gln Arg Ser Ser Val 6080 6085
6090Pro Thr Thr Ser Ile Pro Gly Thr Pro Thr Val Asp Leu Gly Thr
6095 6100 6105Ser Gly Thr Pro Val Ser Lys Pro Gly Pro Ser Ala Ala
Ser Pro 6110 6115 6120Leu Leu Val Leu Phe Thr Leu Asn Phe Thr Ile
Thr Asn Leu Arg 6125 6130 6135Tyr Glu Glu Asn Met Gln His Pro Gly
Ser Arg Lys Phe Asn Thr 6140 6145 6150Thr Glu Arg Val Leu Gln Gly
Leu Leu Arg Ser Leu Phe Lys Ser 6155 6160 6165Thr Ser Val Gly Pro
Leu Tyr Ser Gly Cys Arg Leu Thr Leu Leu 6170 6175 6180Arg Pro Glu
Lys Asp Gly Thr Ala Thr Gly Val Asp Ala Ile Cys 6185 6190 6195Thr
His His Pro Asp Pro Lys Ser Pro Arg Leu Asp Arg Glu Gln 6200 6205
6210Leu Tyr Trp Glu Leu Ser Gln Leu Thr His Asn Ile Thr Glu Leu
6215 6220 6225Gly Pro Tyr Ala Leu Asp Asn Asp Ser Leu Phe Val Asn
Gly Phe 6230 6235 6240Thr His Arg Ser Ser Val Ser Thr Thr Ser Thr
Pro Gly Thr Pro 6245 6250 6255Thr Val Tyr Leu Gly Ala Ser Lys Thr
Pro Ala Ser Ile Phe Gly 6260 6265 6270Pro Ser Ala Ala Ser His Leu
Leu Ile Leu Phe Thr Leu Asn Phe 6275 6280 6285Thr Ile Thr Asn Leu
Arg Tyr Glu Glu Asn Met Trp Pro Gly Ser 6290 6295 6300Arg Lys Phe
Asn Thr Thr Glu Arg Val Leu Gln Gly Leu Leu Arg 6305 6310 6315Pro
Leu Phe Lys Asn Thr Ser Val Gly Pro Leu Tyr Ser Gly Cys 6320 6325
6330Arg Leu Thr Leu Leu Arg Pro Glu Lys Asp Gly Glu Ala Thr Gly
6335 6340 6345Val Asp Ala Ile Cys Thr His Arg Pro Asp Pro Thr Gly
Pro Gly 6350 6355 6360Leu Asp Arg Glu Gln Leu Tyr Leu Glu Leu Ser
Gln Leu Thr His 6365 6370 6375Ser Ile Thr Glu Leu Gly Pro Tyr Thr
Leu Asp Arg Asp Ser Leu 6380 6385 6390Tyr Val Asn Gly Phe Thr His
Arg Ser Ser Val Pro Thr Thr Ser 6395 6400 6405Thr Gly Val Val Ser
Glu Glu Pro Phe Thr Leu Asn Phe Thr Ile 6410 6415 6420Asn Asn Leu
Arg Tyr Met Ala Asp Met Gly Gln Pro Gly Ser Leu 6425 6430 6435Lys
Phe Asn Ile Thr Asp Asn Val Met Gln His Leu Leu Ser Pro 6440 6445
6450Leu Phe Gln Arg Ser Ser Leu Gly Ala Arg Tyr Thr Gly Cys Arg
6455 6460 6465Val Ile Ala Leu Arg Ser Val Lys Asn Gly Ala Glu Thr
Arg Val 6470 6475 6480Asp Leu Leu Cys Thr Tyr Leu Gln Pro Leu Ser
Gly Pro Gly Leu 6485 6490 6495Pro Ile Lys Gln Val Phe His Glu Leu
Ser Gln Gln Thr His Gly 6500 6505 6510Ile Thr Arg Leu Gly Pro Tyr
Ser Leu Asp Lys Asp Ser Leu Tyr 6515 6520 6525Leu Asn Gly Tyr Asn
Glu Pro Gly Pro Asp Glu Pro Pro Thr Thr 6530 6535 6540Pro Lys Pro
Ala Thr Thr Phe Leu Pro Pro Leu Ser Glu Ala Thr 6545 6550 6555Thr
Ala Met Gly Tyr His Leu Lys Thr Leu Thr Leu Asn Phe Thr 6560 6565
6570Ile Ser Asn Leu Gln Tyr Ser Pro Asp Met Gly Lys Gly Ser Ala
6575 6580 6585Thr Phe Asn Ser Thr Glu Gly Val Leu Gln His Leu Leu
Arg Pro 6590 6595 6600Leu Phe Gln Lys Ser Ser Met Gly Pro Phe Tyr
Leu Gly Cys Gln 6605 6610 6615Leu Ile Ser Leu Arg Pro Glu Lys Asp
Gly Ala Ala Thr Gly Val 6620 6625 6630Asp Thr Thr Cys Thr Tyr His
Pro Asp Pro Val Gly Pro Gly Leu 6635 6640 6645Asp Ile Gln Gln Leu
Tyr Trp Glu Leu Ser Gln Leu Thr His Gly 6650 6655 6660Val Thr Gln
Leu Gly Phe Tyr Val Leu Asp Arg Asp Ser Leu Phe 6665 6670 6675Ile
Asn Gly Tyr Ala Pro Gln Asn Leu Ser Ile Arg Gly Glu Tyr 6680 6685
6690Gln Ile Asn Phe His Ile Val Asn Trp Asn Leu Ser Asn Pro Asp
6695 6700 6705Pro Thr Ser Ser Glu Tyr Ile Thr Leu Leu Arg Asp Ile
Gln Asp 6710 6715 6720Lys Val Thr Thr Leu Tyr Lys Gly Ser Gln Leu
His Asp Thr Phe 6725 6730 6735Arg Phe Cys Leu Val Thr Asn Leu Thr
Met Asp Ser Val Leu Val 6740 6745 6750Thr Val Lys Ala Leu Phe Ser
Ser Asn Leu Asp Pro Ser Leu Val 6755 6760 6765Glu Gln Val Phe Leu
Asp Lys Thr Leu Asn Ala Ser Phe His Trp 6770 6775 6780Leu Gly Ser
Thr Tyr Gln Leu Val Asp Ile His Val Thr Glu Met 6785 6790 6795Glu
Ser Ser Val Tyr Gln Pro Thr Ser Ser Ser Ser Thr Gln His 6800 6805
6810Phe Tyr Leu Asn Phe Thr Ile Thr Asn Leu Pro Tyr Ser Gln Asp
6815 6820 6825Lys Ala Gln Pro Gly Thr Thr Asn Tyr Gln Arg Asn Lys
Arg Asn 6830 6835 6840Ile Glu Asp Ala Leu Asn Gln Leu Phe Arg Asn
Ser Ser Ile Lys 6845 6850 6855Ser Tyr Phe Ser Asp Cys Gln Val Ser
Thr Phe Arg Ser Val Pro 6860 6865 6870Asn Arg His His Thr Gly Val
Asp Ser Leu Cys Asn Phe Ser Pro 6875 6880 6885Leu Ala Arg Arg Val
Asp Arg Val Ala Ile Tyr Glu Glu Phe Leu 6890 6895 6900Arg Met Thr
Arg Asn Gly Thr Gln Leu Gln Asn Phe Thr Leu Asp 6905 6910 6915Arg
Ser Ser Val Leu Val Asp Gly Tyr Ser Pro Asn Arg Asn Glu 6920 6925
6930Pro Leu Thr Gly Asn Ser Asp Leu Pro Phe Trp Ala Val Ile Leu
6935 6940 6945Ile Gly Leu Ala Gly Leu Leu Gly Leu Ile Thr Cys Leu
Ile Cys 6950 6955 6960Gly Val Leu Val Thr Thr Arg Arg Arg Lys Lys
Glu Gly Glu Tyr 6965 6970 6975Asn Val Gln Gln Gln Cys Pro Gly Tyr
Tyr Gln Ser His Leu Asp 6980 6985 6990Leu Glu Asp Leu Gln
69953108PRTHomo sapiens 3Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val 1 5 10 15Gly Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser 20 25 30Asn Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile Tyr Ala Ala Ser Ser Leu
Glu Ser Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu Gln Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln 80 85 90Tyr Asn Ser Leu Pro Trp Thr Phe Gly
Gln Gly Thr Lys Val Glu 95 100 105Ile Lys Arg4109PRTHomo sapiens
4Asp Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Leu 1 5 10
15Gly Glu Arg Val Thr Met Thr Cys Thr Ala Ser Ser Ser Val Ser 20 25
30Ser Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Ser Ser Pro 35 40
45Lys Leu Trp Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro 50 55
60Gly Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr 65 70
75Ile Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys His 80 85
90Gln Tyr His Arg Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val 95
100 105Glu Ile Lys Arg5109PRTHomo sapiens 5Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15Gly Asp Arg Val Thr
Ile Thr Cys Thr Ala Ser Ser Ser Val Ser 20 25 30Ser Ser Tyr Leu His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro 35 40 45Lys Leu Leu Ile Tyr
Ser Thr Ser Asn Leu Ala Ser Gly Val Pro 50 55 60Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75Ile Ser Ser Leu Gln
Pro Glu Asp Phe Ala Thr Tyr Tyr Cys His 80 85 90Gln Tyr His Arg Ser
Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val 95 100 105Glu Ile Lys
Arg6113PRTHomo sapiens 6Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser 20 25 30Ser Tyr Ala Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Ser Val Ile Ser Gly Asp Gly
Gly Ser Thr Tyr Tyr 50 55 60Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser 65 70 75Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Gly Phe
Asp Tyr Trp Gly Gln 95 100 105Gly Thr Leu Val Thr Val Ser Ser 110
7120PRTHomo sapiens 7Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Val Lys Pro Gly 1 5 10 15Ala Ser Val Lys Leu Ser Cys Thr Ala Ser
Gly Phe Asn Ile Lys 20 25 30Asp Thr Tyr Met His Trp Val Lys Gln Arg
Pro Glu Gln Gly Leu 35 40 45Glu Trp Ile Gly Arg Val Asp Pro Ala Asn
Gly Asn Thr Lys Tyr 50 55 60Asp Pro Lys Phe Gln Gly Lys Ala Thr Leu
Thr Ala Asp Thr Ser 65 70 75Ser Asn Thr Ala Tyr Leu Gln Leu Ser Ser
Leu Thr Ser Glu Asp
80 85 90Thr Ala Val Tyr Phe Cys Val Arg Asp Tyr Tyr Gly His Thr Tyr
95 100 105Gly Phe Ala Phe Cys Asp Gln Gly Thr Thr Leu Thr Val Ser
Ala 110 115 1208120PRTHomo sapiens 8Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Asn Ile Lys 20 25 30Asp Thr Tyr Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu 35 40 45Glu Trp Val Gly Arg Val Asp
Pro Ala Asn Gly Asn Thr Lys Tyr 50 55 60Asp Pro Lys Phe Gln Gly Arg
Phe Thr Ile Ser Ala Asp Thr Ser 65 70 75Lys Asn Thr Ala Tyr Leu Gln
Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Val
Arg Asp Tyr Tyr Gly His Thr Tyr 95 100 105Gly Phe Ala Phe Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 110 115 1209107PRTHomo sapiens
9Asp Ile Gln Met Thr Gln Ser Ser Ser Phe Leu Ser Val Ser Leu 1 5 10
15Gly Gly Arg Val Thr Ile Thr Cys Lys Ala Ser Asp Leu Ile His 20 25
30Asn Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg 35 40
45Leu Leu Ile Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser 50 55
60Arg Phe Ser Gly Ser Gly Ser Gly Asn Asp Tyr Thr Leu Ser Ile 65 70
75Ala Ser Leu Gln Thr Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln 80 85
90Tyr Trp Thr Thr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu 95
100 105Ile Lys10108PRTHomo sapiens 10Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15Gly Asp Arg Val Thr Ile
Thr Cys Lys Ala Ser Asp Leu Ile His 20 25 30Asn Trp Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile Ser Gly Ala
Thr Ser Leu Glu Thr Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu Gln Pro Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Tyr Trp Thr Thr Pro Phe
Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys
Arg11116PRTHomo sapiens 11Asp Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Asn Pro Ser 1 5 10 15Gln Ser Leu Ser Leu Thr Cys Thr Val
Thr Gly Tyr Ser Ile Thr 20 25 30Asn Asp Tyr Ala Trp Asn Trp Ile Arg
Gln Phe Pro Gly Asn Lys 35 40 45Leu Glu Trp Met Gly Tyr Ile Asn Tyr
Ser Gly Tyr Thr Thr Tyr 50 55 60Asn Pro Ser Leu Lys Ser Arg Ile Ser
Ile Thr Arg Asp Thr Ser 65 70 75Lys Asn Gln Phe Phe Leu His Leu Asn
Ser Val Thr Thr Glu Asp 80 85 90Thr Ala Thr Tyr Tyr Cys Ala Arg Trp
Asp Gly Gly Leu Thr Tyr 95 100 105Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ala 110 115 12116PRTHomo sapiens 12Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr 20 25 30Asn Asp Tyr Ala Trp
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 35 40 45Leu Glu Trp Val Gly
Tyr Ile Asn Tyr Ser Gly Tyr Thr Thr Tyr 50 55 60Asn Pro Ser Leu Lys
Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser 65 70 75Lys Asn Thr Leu Tyr
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr
Cys Ala Arg Trp Asp Gly Gly Leu Thr Tyr 95 100 105Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 110 115 13116PRTHomo sapiens 13Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15Gly
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr 20 25 30Asn
Asp Tyr Ala Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 35 40 45Leu
Glu Trp Val Gly Tyr Ile Asn Tyr Ser Gly Tyr Thr Thr Tyr 50 55 60Asn
Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Arg Asp Asn Ser 65 70 75Lys
Asn Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr
Ala Val Tyr Tyr Cys Ala Arg Trp Asp Gly Gly Leu Thr Tyr 95 100
105Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115 1412PRTHomo
sapiens 14Thr Ala Ser Ser Ser Val Ser Ser Ser Tyr Leu His 5 10
1512PRTHomo sapiens 15Thr Gln Arg Thr Ser Val Lys Arg Ser Tyr Ile
Ser 5 10 1612PRTHomo sapiens 16Thr Pro Arg Gly Arg Val Arg Ser Ser
Tyr Leu Ser 5 10 1712PRTHomo sapiensUnsure4Unknown amino acid 17Pro
Glu Cys Xaa Ser Leu Gly Thr Ile Tyr Leu His 5 10 1812PRTHomo
sapiens 18Ser Ala Ser Ser Ser Val Asn Ser Thr Tyr Leu His 5 10
1912PRTHomo sapiens 19Thr Ala Ser Thr Ala Val Gly Ser Ser Tyr Leu
His 5 10 2011PRTHomo sapiens 20Asn Ser Arg Ser Val Ser Thr Arg Tyr
Leu His 5 10 2112PRTHomo sapiens 21Asn Thr Thr Arg Ser Val Ser Thr
Gly Tyr Leu His 5 10 2212PRTHomo sapiens 22Thr Ala Ser Ser Arg Val
Thr Ser Thr Tyr Leu His 5 10 2312PRTHomo sapiens 23Asn Thr Pro Thr
Gly Val Asn Pro Val Tyr Leu His 5 10 2412PRTHomo sapiens 24Ala Ala
Ser Ser Asp Val Ile Gly Ser Tyr Val His 5 10 2512PRTHomo sapiens
25Gly Leu Ser Thr Ser Val Asn Ser Ser Tyr Met His 5 10 2612PRTHomo
sapiensUnsure10Unknown amino acid 26Asn Ala Lys Ser Gly Val Arg Ser
Ser Xaa Val His 5 10 2712PRTHomo sapiens 27Asn Ser Asn Gly Ser Val
Ser Ser Lys Tyr Ile His 5 10 2812PRTHomo sapiens 28Thr Pro Ser Arg
Ile Val Ser Gly Ser Tyr Leu Ser 5 10 2912PRTHomo sapiens 29Asn Pro
Ser Arg Arg Val Thr Gly His Tyr Val Ser 5 10 3012PRTHomo sapiens
30Thr Ser Ser Ser Ala Val Ser Gly Ser Tyr Val Ser 5 10 3112PRTHomo
sapiens 31Thr Ser Thr Thr Ile Val Arg Gly Arg Tyr Val Ser 5 10
3212PRTHomo sapiens 32Thr Ala Ser Ser Thr Leu Ser Ser Asn Tyr Leu
Thr 5 10 3312PRTHomo sapiens 33Thr Pro Thr Gly Ser Ile Ser Arg Arg
Tyr Leu Ser 5 10 3412PRTHomo sapiens 34Thr Ala Gly Ser Lys Ala Asn
Ser Ser Tyr Ile His 5 10 358PRTHomo sapiens 35Tyr Ser Thr Ser Asn
Leu Ala Ser 5 368PRTHomo sapiens 36Tyr Ser Thr Ser His Phe Ala Ser
5 378PRTHomo sapiens 37Tyr Ser Ala Ser Asn Val Pro Ser 5 388PRTHomo
sapiens 38Tyr Ser Thr Ile Asn Leu Ala Thr 5 398PRTHomo sapiens
39Tyr Ser Thr Ser Lys Val Ala Asn 5 408PRTHomo sapiens 40Tyr Ser
Thr Thr Asn Leu Ala Ser 5 418PRTHomo sapiens 41Tyr Ser Thr Asn His
Leu Ala Ser 5 428PRTHomo sapiens 42Tyr Ser Thr Asn Asn Leu Ala Ser
5 438PRTHomo sapiens 43Tyr Ser Thr Ile His Pro Ala Ser 5 448PRTHomo
sapiens 44Tyr Ser Thr Ser His Leu Ser Tyr 5 458PRTHomo sapiens
45Tyr Ser Thr Arg Thr Met Ala Ser 5 468PRTHomo
sapiensUnsure5Unknown amino acid 46Tyr Ser Thr Ser Xaa Leu Phe Ser
5 478PRTHomo sapiens 47Tyr Asn Thr Ser Asn Arg Ala Ser 5 488PRTRana
catesbeiana 48Tyr Gly Thr Ser His Leu Ala Ser 5 498PRTHomo sapiens
49Tyr Gly Thr Gly Ser Pro Ala Ser 5 508PRTHomo sapiens 50Tyr Ser
Thr Asn Lys Leu Ala Arg 5 518PRTHomo sapiens 51Tyr Ser Thr Ser Gln
Leu Gly Arg 5 528PRTHomo sapiens 52Tyr Ser Thr Ser Asn Val Pro Gln
5 538PRTHomo sapiens 53Tyr Gly Thr Tyr Asn Leu Pro Ile 5 548PRTHomo
sapiens 54Tyr Gly Ser Asn Asn Arg Ala Tyr 5 558PRTHomo
sapiensUnsure7Unknown amino acid 55Tyr Ser Ser Ser Asn Thr Xaa Ser
5 568PRTHomo sapiens 56Tyr Ser Ala Asn Lys Leu Ala Ser 5 578PRTHomo
sapiens 57Tyr Ser Ala Thr Arg Arg Ala Ser 5 588PRTHomo sapiens
58Tyr Ser Ala Ser Asn Arg Ala Arg 5 599PRTHomo sapiens 59His Gln
Tyr His Arg Ser Pro Tyr Thr 5 609PRTHomo sapiens 60His Gln Tyr His
Arg Ser Pro Tyr Lys 5 619PRTHomo sapiens 61His Gln Tyr His Arg Thr
Pro Tyr Lys 5 629PRTHomo sapiens 62His Gln Tyr His Arg Ser Pro Tyr
Gly 5 639PRTHomo sapiens 63His Gln Tyr His Arg Ser Pro Tyr Asn 5
649PRTHomo sapiens 64His Gln Tyr His Arg Ser Pro Tyr Ser 5
659PRTHomo sapiens 65His Gln Tyr Tyr Arg Ser Pro Tyr Thr 5
669PRTHomo sapiens 66His Gln Tyr Tyr Arg Thr Pro Tyr Ser 5
679PRTHomo sapiens 67His Gln Tyr Gln Arg Ser Pro Tyr Thr 5
689PRTHomo sapiens 68His Gln Tyr Gln Arg Ser Pro Tyr Arg 5
699PRTHomo sapiens 69His Gln Tyr Asn Arg Ser Pro Tyr Ala 5
709PRTHomo sapiens 70His Gln Tyr His Arg Thr Pro Tyr Thr 5
719PRTHomo sapiens 71His Gln Tyr His Arg Ser Pro Tyr Ile 5
729PRTHomo sapiens 72His Gln Tyr His Arg Arg Pro Tyr Arg 5
739PRTHomo sapiens 73His Gln Tyr His Arg Asn Pro Tyr Ile 5
7410PRTHomo sapiens 74Gly Phe Asn Ile Lys Asp Thr Tyr Met His 5
107510PRTHomo sapiens 75Ala Phe Asn Ile Ala Asp Thr Tyr Ile His 5
107610PRTHomo sapiens 76Arg Phe Arg Ile Lys Asp Thr Tyr Val His 5
107710PRTHomo sapiens 77Arg Phe Asn Ile Lys Asp Thr Tyr Ile His 5
107810PRTHomo sapiens 78Ser Phe Gln Ile Asn Asp Thr Tyr Ile His 5
107910PRTHomo sapiens 79Ser Phe Gln Met Ser Asp Thr Tyr Val His 5
108010PRTHomo sapiens 80Asp Phe Asn Ile Lys Asp Thr Tyr Ile His 5
108110PRTHomo sapiens 81Gly Phe Asn Ile Ile Asp Thr Tyr Ile His 5
108210PRTHomo sapiens 82Gly Leu Gln Ile Val Asp Thr Tyr Ile His 5
108310PRTHomo sapiens 83Gly Phe Asn Ile Lys Asp Thr Tyr Leu His 5
108410PRTHomo sapiens 84Gly Phe Asn Ile Gln Asp Leu Tyr Leu His 5
108510PRTHomo sapiens 85Gly Phe Asn Ile Ile Asp Thr Tyr Met His 5
108610PRTHomo sapiens 86Gly Trp Lys Met Thr Asp Thr Tyr Met His 5
108710PRTHomo sapiens 87Glu Phe Lys Ile Lys Asp Thr Tyr Val His 5
108810PRTHomo sapiens 88Gly Phe Asn Ile Lys Asp Thr Tyr Val His 5
108910PRTHomo sapiens 89Gly Phe Tyr Ile Ser Asn Thr Tyr Ile His 5
109010PRTHomo sapiens 90Gly Phe Asn Ile Lys Asn Thr Tyr Leu His 5
109110PRTHomo sapiens 91Gly Phe Ser Ile Glu Asn Thr Tyr Met His 5
109210PRTHomo sapiens 92Gly Phe Asn Ile Lys Asn Thr Tyr Met His 5
109310PRTHomo sapiens 93Asp Phe Lys Ile Glu Asn Thr Tyr Val His 5
109418PRTHomo sapiens 94Gly Arg Val Asp Pro Ala Asn Gly Asn Thr Lys
Tyr Asp Pro Lys 1 5 10 15Phe Gln Gly9518PRTHomo sapiens 95Gly Arg
Val Asp Pro Ala Asn Gly Asn Thr Lys Ser Asp Pro Lys 1 5 10 15Val
Arg Gly9618PRTHomo sapiens 96Gly Arg Val Asp Pro Ala Asn Gly Leu
Thr Lys Tyr Asp Pro Lys 1 5 10 15Phe Gln Gly9718PRTHomo sapiens
97Gly Arg Val Asp Pro Ala Asn Gly Glu Ile Lys Ser His Pro Ile 1 5
10 15Phe Gln Gly9818PRTHomo sapiens 98Gly Arg Val Asp Pro Ala Asn
Gly Asn Thr Lys Glu Asp Arg Gln 1 5 10 15Phe Gln Gly9918PRTHomo
sapiens 99Gly Arg Val Asp Pro Glu Tyr Gly Asn Thr Lys Tyr Asp Pro
Lys 1 5 10 15Phe Gln Gly10018PRTHomo sapiens 100Gly Arg Leu Asp Pro
Ala Asn Gly Asn Thr Lys Tyr Asp Pro Lys 1 5 10 15Phe Gln
Gly10118PRTHomo sapiens 101Gly Arg Val Asp Pro Ala Asn Gly Asp Thr
Lys Tyr Asp Pro Lys 1 5 10 15Phe Gln Gly10218PRTHomo sapiens 102Gly
Arg Val Asp Pro Ala Asn Gly Lys Thr Lys Tyr Asp Pro Lys 1 5 10
15Phe Gln Gly10318PRTHomo sapiens 103Gly Arg Val Asp Pro Ala Asn
Gly Leu Thr Lys Tyr Asn Pro Lys 1 5 10 15Phe Gln Gly10418PRTHomo
sapiens 104Gly Arg Val Asp Pro Ala Asn Gly Tyr Thr Lys Tyr Asn Pro
Lys 1 5 10 15Phe Gln Gly10518PRTHomo sapiens 105Gly Arg Val Asp Pro
Ala Asn Gly Tyr Thr Lys Tyr Asp Pro Lys 1 5 10 15Phe Gln
Gly10618PRTHomo sapiens 106Gly Arg Val Asp Pro Ala Asn Gly Asn Tyr
Lys Tyr Asp Pro Lys 1 5 10 15Phe Gln Gly10718PRTHomo sapiens 107Gly
Arg Val Asp Pro Ala Asn Gly Asn Ser Lys Tyr Asp Pro Lys 1 5 10
15Phe Gln Gly10818PRTHomo sapiens 108Gly Arg Val Asp Pro Ala Asn
Gly Asn Thr Lys Tyr Asp His Arg 1 5 10 15Phe Gln Gly10918PRTHomo
sapiens 109Gly Arg Val Asp Pro Ala Asn Gly Asn Thr Lys Tyr Asp Pro
Lys 1 5 10 15Phe Arg Gly11018PRTHomo sapiens 110Gly Arg Val Asp Pro
Ser Asn Gly Asn Thr Lys Ser Asp Gly Lys 1 5 10 15Phe Asn
Gly11118PRTHomo sapiens 111Gly Arg Val Asp Pro Val Asp Gly Lys Thr
Lys Tyr Asn Pro Gln 1 5 10 15Ile Gln Gly11218PRTHomo sapiens 112Gly
Arg Val Asp Pro Ala His Gly Asn Ile Lys Tyr Asp Pro Gln 1 5 10
15Ile Met Gly11313PRTHomo sapiens 113Val Arg Asp Tyr Tyr Gly His
Thr Tyr Gly Phe Ala Phe 5 10 11413PRTHomo sapiens 114Val Arg Asp
Tyr Tyr Gly His Thr Tyr Gly Phe Gln Pro 5 10 11513PRTHomo sapiens
115Ala Arg Asp Asn Tyr Gly His Thr Tyr Gly Phe Gly Phe 5 10
11613PRTHomo sapiens 116Val Arg Asp Thr Tyr Gly His Thr Tyr Gly Phe
Ala Tyr 5 10 11713PRTHomo sapiens 117Val Arg Asp Tyr Tyr Gly His
Thr Tyr Gly Phe Gly Tyr 5 10 11813PRTHomo sapiens 118Val Arg Asp
Tyr Tyr Gly His Thr Tyr Gly Phe Gly Val 5 10 11911PRTHomo sapiens
119Lys Ala Ser Asp Leu Ile His Asn Trp Leu Ala 5 10 1208PRTHomo
sapiens 120Ser Gly Ala Thr Ser Leu Glu Thr 5 1218PRTHomo sapiens
121Tyr Gly Ala Thr Ser Leu Glu Thr 5 1229PRTHomo sapiens 122Gln Gln
Tyr Trp Thr Thr Pro Phe Thr 5 12311PRTHomo sapiens 123Gly Tyr Ser
Ile Thr Asn Asp Tyr Ala Trp Asn 5 10 12417PRTHomo sapiens 124Gly
Tyr Ile Asn Tyr Ser Gly Tyr Thr Thr Tyr Asn Pro Ser Leu 1 5 10
15Lys Ser12517PRTHomo sapiens 125Gly Tyr Ile Ser Tyr Ser Gly Tyr
Thr Thr Tyr Asn Pro Ser Leu 1 5 10 15Lys Ser12617PRTHomo sapiens
126Gly Tyr Ile Asn Tyr Ala Gly Tyr Thr Thr
Tyr Asn Pro Ser Leu 1 5 10 15Lys Ser12717PRTHomo sapiens 127Gly Tyr
Ile Ser Tyr Ala Gly Tyr Thr Thr Tyr Asn Pro Ser Leu 1 5 10 15Lys
Ser1289PRTHomo sapiens 128Ala Arg Trp Asp Gly Gly Leu Thr Tyr 5
1299PRTHomo sapiens 129Ala Arg Trp Ala Ala Gly Leu Thr Asn 5
1309PRTHomo sapiens 130Ala Arg Trp Asp Ala Gly Leu Ser Tyr 5
1319PRTHomo sapiens 131Ala Arg Trp Asp Ala Gly Leu Thr Tyr 5
1329PRTHomo sapiens 132Ala Arg Trp Glu Ala Gly Leu Asn His 5
1339PRTHomo sapiens 133Ala Arg Trp Glu Ala Gly Leu Asn Tyr 5
1349PRTHomo sapiens 134Ala Arg Trp Met Ala Gly Leu Ser Asp 5
1359PRTHomo sapiens 135Ala Arg Trp Ser Ala Gly Leu Asp His 5
1369PRTHomo sapiens 136Ala Arg Trp Thr Ala Gly Leu Asp Tyr 5
1379PRTHomo sapiens 137Ala Arg Trp Thr Ala Gly Leu Thr His 5
1389PRTHomo sapiens 138Ala Arg Trp Val Ala Gly Leu Thr Asn 5
1399PRTHomo sapiens 139Ala Arg Trp Ala Gly Gly Leu Glu Asn 5
1409PRTHomo sapiens 140Ala Arg Trp Asp Gly Gly Leu Ser Tyr 5
1419PRTHomo sapiens 141Ala Arg Trp Asp Arg Gly Leu Thr Tyr 5
1429PRTHomo sapiens 142Ala Arg Trp Ala Ser Gly Leu Ser His 5
1439PRTHomo sapiens 143Ala Arg Trp Ala Ser Gly Leu Ser Asn 5
1449PRTHomo sapiens 144Ala Arg Trp Ala Ser Gly Leu Ser Tyr 5
1459PRTHomo sapiens 145Ala Arg Trp Ala Ser Gly Leu Thr His 5
1469PRTHomo sapiens 146Ala Arg Trp Ala Ser Gly Leu Thr Asn 5
1479PRTHomo sapiens 147Ala Arg Trp Asp Ser Gly Leu Lys Tyr 5
1489PRTHomo sapiens 148Ala Arg Trp Asp Ser Gly Leu Asn Tyr 5
1499PRTHomo sapiens 149Ala Arg Trp Asp Ser Gly Leu Ser Ser 5
1509PRTHomo sapiens 150Ala Arg Trp Asp Ser Gly Leu Ser Val 5
1519PRTHomo sapiens 151Ala Arg Trp Asp Ser Gly Leu Ser Tyr 5
1529PRTHomo sapiens 152Ala Arg Trp Asp Ser Gly Leu Thr Tyr 5
1539PRTHomo sapiens 153Ala Arg Trp Glu Ser Gly Leu Ser His 5
1549PRTHomo sapiens 154Ala Arg Trp Glu Ser Gly Leu Ser Val 5
1559PRTHomo sapiens 155Ala Arg Trp Lys Ser Gly Leu Asp Ser 5
1569PRTHomo sapiens 156Ala Arg Trp Lys Ser Gly Leu Glu Tyr 5
1579PRTRana catesbeiana 157Ala Arg Trp Leu Ser Gly Leu Asp Phe 5
1589PRTHomo sapiens 158Ala Arg Trp Leu Ser Gly Leu Asp Ser 5
1599PRTHomo sapiens 159Ala Arg Trp Leu Ser Gly Leu Glu Ser 5
1609PRTHomo sapiens 160Ala Arg Trp Leu Ser Gly Leu Ser Asp 5
1619PRTHomo sapiens 161Ala Arg Trp Arg Ser Gly Leu Glu His 5
1629PRTHomo sapiens 162Ala Arg Trp Ser Ser Gly Leu Asn Tyr 5
1639PRTHomo sapiens 163Ala Arg Trp Ser Ser Gly Leu Thr Tyr 5
1649PRTHomo sapiens 164Ala Arg Trp Thr Ser Gly Met Asp Ser 5
1659PRTHomo sapiens 165Ala Arg Trp Thr Ser Gly Leu Thr Tyr 5
1669PRTHomo sapiens 166Ala Arg Trp Asp Thr Gly Leu Thr Tyr 5
1679PRTHomo sapiens 167Ala Arg Trp Ala Ala Gly Leu Asp His 5
1689PRTHomo sapiens 168Ala Arg Trp Ala Ala Gly Leu Asp Ser 5
1699PRTHomo sapiens 169Ala Arg Trp Leu Ala Gly Leu Ser Asn 5
1709PRTHomo sapiens 170Ala Arg Trp Thr Ala Gly Leu Asp Gln 5
1719PRTHomo sapiens 171Ala Arg Trp Ala Ser Gly Leu Asp His 5
1729PRTHomo sapiens 172Ala Arg Trp Ala Ser Gly Leu Asp Asn 5
1739PRTHomo sapiens 173Ala Arg Trp Ala Ser Gly Leu Asp Ser 5
1749PRTHomo sapiens 174Ala Arg Trp Ala Ser Gly Leu Asp Tyr 5
1759PRTHomo sapiens 175Ala Arg Trp Lys Ser Gly Leu Asp Thr 5
1769PRTRana catesbeiana 176Ala Arg Trp Lys Ser Gly Leu Gly Pro 5
1779PRTHomo sapiens 177Ala Arg Trp Met Ser Gly Leu Asp Ser 5
1789PRTHomo sapiens 178Ala Arg Trp Arg Ser Gly Leu Glu Ser 5
1799PRTHomo sapiens 179Ala Arg Trp Arg Ser Gly Leu Glu Tyr 5
1809PRTHomo sapiens 180Ala Arg Trp Thr Ser Gly Leu Asp Ser 5
1819PRTHomo sapiens 181Ala Arg Trp Thr Ser Gly Leu Asp Thr 5
1829PRTHomo sapiens 182Ala Arg Trp Thr Ser Gly Leu Asp Val 5
1839PRTHomo sapiens 183Ala Arg Trp Thr Ser Gly Leu Asp Tyr 5
18487PRTHomo sapiens 184Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly 1 5 10 15Ala Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr 20 25 30Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met Gly Arg 35 40 45Val Thr Ile Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr Met Glu 50 55 60Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys Ala 65 70 75Arg Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 80 85 18581PRTHomo sapiens 185Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly 1 5 10 15Ala Ser Val Lys Val Ser
Cys Lys Ala Ser Trp Val Arg Gln Ala 20 25 30Pro Gly Gln Gly Leu Glu
Trp Met Arg Val Thr Ile Thr Ala Asp 35 40 45Thr Ser Thr Ser Thr Ala
Tyr Met Glu Leu Ser Ser Leu Arg Ser 50 55 60Glu Asp Thr Ala Val Tyr
Tyr Cys Ala Arg Trp Gly Gln Gly Thr 65 70 75Leu Val Thr Val Ser Ser
80 18680PRTHomo sapiens 186Gln Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly 1 5 10 15Ala Ser Val Lys Val Ser Cys Lys Ala
Ser Trp Val Arg Gln Ala 20 25 30Pro Gly Gln Gly Leu Glu Trp Met Arg
Val Thr Ile Thr Ala Asp 35 40 45Thr Ser Thr Ser Thr Ala Tyr Met Glu
Leu Ser Ser Leu Arg Ser 50 55 60Glu Asp Thr Ala Val Tyr Tyr Cys Ala
Trp Gly Gln Gly Thr Leu 65 70 75Val Thr Val Ser Ser 8018779PRTHomo
sapiens 187Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly 1 5 10 15Ala Ser Val Lys Val Ser Cys Lys Ala Ser Trp Val Arg
Gln Ala 20 25 30Pro Gly Gln Gly Leu Glu Trp Met Arg Val Thr Ile Thr
Ala Asp 35 40 45Thr Ser Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu
Arg Ser 50 55 60Glu Asp Thr Ala Val Tyr Tyr Cys Trp Gly Gln Gly Thr
Leu Val 65 70 75Thr Val Ser Ser18887PRTHomo sapiens 188Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15Gln Thr
Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser 20 25 30Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly Arg 35 40 45Val Thr
Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Lys 50 55 60Leu Ser
Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 65 70 75Arg Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 80 85 18981PRTHomo sapiens
189Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5
10 15Gln Thr Leu Ser Leu Thr Cys Thr Val Ser Trp Ile Arg Gln Pro 20
25 30Pro Gly Lys Gly Leu Glu Trp Ile Arg Val Thr Ile Ser Val Asp 35
40 45Thr Ser Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala 50
55 60Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Trp Gly Gln Gly Thr 65
70 75Leu Val Thr Val Ser Ser 80 19080PRTHomo sapiens 190Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15Gln Thr
Leu Ser Leu Thr Cys Thr Val Ser Trp Ile Arg Gln Pro 20 25 30Pro Gly
Lys Gly Leu Glu Trp Ile Arg Val Thr Ile Ser Val Asp 35 40 45Thr Ser
Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser Val Thr Ala 50 55 60Ala Asp
Thr Ala Val Tyr Tyr Cys Ala Trp Gly Gln Gly Thr Leu 65 70 75Val Thr
Val Ser Ser 8019179PRTHomo sapiens 191Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu Val Lys Pro Ser 1 5 10 15Gln Thr Leu Ser Leu Thr
Cys Thr Val Ser Trp Ile Arg Gln Pro 20 25 30Pro Gly Lys Gly Leu Glu
Trp Ile Arg Val Thr Ile Ser Val Asp 35 40 45Thr Ser Lys Asn Gln Phe
Ser Leu Lys Leu Ser Ser Val Thr Ala 50 55 60Ala Asp Thr Ala Val Tyr
Tyr Cys Trp Gly Gln Gly Thr Leu Val 65 70 75Thr Val Ser
Ser19279PRTHomo sapiens 192Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Trp Val Arg Gln Ala 20 25 30Pro Gly Lys Gly Leu Glu Trp Val Arg
Phe Thr Ile Ser Arg Asp 35 40 45Asn Ser Lys Asn Thr Leu Tyr Leu Gln
Met Asn Ser Leu Arg Ala 50 55 60Glu Asp Thr Ala Val Tyr Tyr Cys Trp
Gly Gln Gly Thr Leu Val 65 70 75Thr Val Ser Ser19379PRTHomo sapiens
193Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5
10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Trp Val Arg Gln Ala 20
25 30Pro Gly Lys Gly Leu Glu Trp Val Arg Phe Thr Ile Ser Arg Asp 35
40 45Asn Ser Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala 50
55 60Glu Asp Thr Ala Val Tyr Tyr Cys Trp Gly Gln Gly Thr Leu Val 65
70 75Thr Val Ser Ser19480PRTHomo sapiens 194Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15Gly Asp Arg Val Thr
Ile Thr Cys Trp Tyr Gln Gln Lys Pro Gly 20 25 30Lys Ala Pro Lys Leu
Leu Ile Gly Val Pro Ser Arg Phe Ser Gly 35 40 45Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 50 55 60Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Phe Gly Gln Gly Thr Lys 65 70 75Val Glu Ile Lys Arg
8019580PRTHomo sapiens 195Asp Ile Val Met Thr Gln Ser Pro Leu Ser
Leu Pro Val Thr Pro 1 5 10 15Gly Glu Pro Ala Ser Ile Ser Cys Trp
Tyr Leu Gln Lys Pro Gly 20 25 30Gln Ser Pro Gln Leu Leu Ile Tyr Gly
Val Pro Asp Arg Phe Ser 35 40 45Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile Ser Arg Val 50 55 60Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Phe Gly Gln Gly Thr 65 70 75Lys Val Glu Ile Lys 8019680PRTHomo
sapiens 196Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
Pro 1 5 10 15Gly Glu Arg Ala Thr Leu Ser Cys Trp Tyr Gln Gln Lys
Pro Gly 20 25 30Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ile Pro Asp Arg
Phe Ser 35 40 45Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Arg Leu 50 55 60Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Phe Gly Gln
Gly Thr 65 70 75Lys Val Glu Ile Lys 8019780PRTHomo sapiens 197Asp
Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu 1 5 10
15Gly Glu Arg Ala Thr Ile Asn Cys Trp Tyr Gln Gln Lys Pro Gly 20 25
30Gln Pro Pro Lys Leu Leu Ile Tyr Gly Val Pro Asp Arg Phe Ser 35 40
45Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 50 55
60Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Phe Gly Gln Gly Thr 65 70
75Lys Val Glu Ile Lys 80198116PRTHomo sapiens 198Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr 20 25 30Asn Asp Tyr
Ala Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 35 40 45Leu Glu Trp
Val Gly Tyr Ile Asn Tyr Ser Gly Tyr Thr Thr Tyr 50 55 60Asn Pro Ser
Leu Lys Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser 65 70 75Lys Asn Thr
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val
Tyr Tyr Cys Ala Arg Trp Ala Ser Gly Leu Asp Tyr 95 100 105Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 110 115 199116PRTHomo sapiens
199Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5
10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr 20
25 30Asn Asp Tyr Ala Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 35
40 45Leu Glu Trp Val Gly Tyr Ile Asn Tyr Ser Gly Tyr Thr Thr Tyr 50
55 60Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser 65
70 75Lys Asn Thr Phe Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80
85 90Thr Ala Val Tyr Tyr Cys Ala Arg Trp Ala Ser Gly Leu Ser His 95
100 105Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115
200116PRTHomo sapiens 200Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Tyr Ser Ile Thr 20 25 30Asn Asp Tyr Ala Trp Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly 35 40 45Leu Glu Trp Val Gly Tyr Ile Asn Tyr
Ser Gly Tyr Thr Thr Tyr 50 55 60Asn Pro Ser Leu Lys Ser Arg Phe Thr
Ile Ser Arg Asp Thr Ser 65 70 75Lys Asn Thr Phe Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Trp
Ala Ser Gly Leu Ser Tyr 95 100 105Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 110 115 201116PRTHomo sapiens 201Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr 20 25 30Asn Asp Tyr Ala
Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 35 40 45Leu Glu Trp Val
Gly Tyr Ile Asn Tyr Ser Gly Tyr Thr Thr Tyr 50 55 60Asn Pro Ser Leu
Lys Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser 65 70 75Lys Asn Thr Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr
Tyr Cys Ala Arg Trp Thr Ser Gly Leu Asp Tyr 95
100 105Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115
202116PRTHomo sapiens 202Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Tyr Ser Ile Thr 20 25 30Asn Asp Tyr Ala Trp Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly 35 40 45Leu Glu Trp Val Gly Tyr Ile Asn Tyr
Ser Gly Tyr Thr Thr Tyr 50 55 60Asn Pro Ser Leu Lys Ser Arg Phe Thr
Ile Ser Arg Asp Thr Ser 65 70 75Lys Asn Thr Phe Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Trp
Asp Ala Gly Leu Thr Tyr 95 100 105Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 110 115 203116PRTHomo sapiens 203Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr 20 25 30Asn Asp Tyr Ala
Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 35 40 45Leu Glu Trp Val
Gly Tyr Ile Asn Tyr Ser Gly Tyr Thr Thr Tyr 50 55 60Asn Pro Ser Leu
Lys Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser 65 70 75Lys Asn Thr Phe
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr
Tyr Cys Ala Arg Trp Asp Ser Gly Leu Thr Tyr 95 100 105Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 110 115 204116PRTHomo sapiens
204Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5
10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr 20
25 30Asn Asp Tyr Ala Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 35
40 45Leu Glu Trp Val Gly Tyr Ile Asn Tyr Ser Gly Tyr Thr Thr Tyr 50
55 60Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser 65
70 75Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80
85 90Thr Ala Val Tyr Tyr Cys Ala Arg Trp Lys Ser Gly Leu Asp Ser 95
100 105Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115
205116PRTHomo sapiens 205Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Tyr Ser Ile Thr 20 25 30Asn Asp Tyr Ala Trp Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly 35 40 45Leu Glu Trp Val Gly Tyr Ile Asn Tyr
Ser Gly Tyr Thr Thr Tyr 50 55 60Asn Pro Ser Leu Lys Ser Arg Phe Thr
Ile Ser Arg Asp Thr Ser 65 70 75Lys Asn Thr Leu Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Trp
Thr Ser Gly Leu Asp Ser 95 100 105Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 110 115 206116PRTHomo sapiens 206Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr 20 25 30Asn Asp Tyr Ala
Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 35 40 45Leu Glu Trp Val
Gly Tyr Ile Ser Tyr Ser Gly Tyr Thr Thr Tyr 50 55 60Asn Pro Ser Leu
Lys Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser 65 70 75Lys Asn Thr Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr
Tyr Cys Ala Arg Trp Ala Ser Gly Leu Asp Tyr 95 100 105Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 110 115 207116PRTHomo sapiens
207Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5
10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr 20
25 30Asn Asp Tyr Ala Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 35
40 45Leu Glu Trp Val Gly Tyr Ile Asn Tyr Ala Gly Tyr Thr Thr Tyr 50
55 60Asn Pro Ser Leu Lys Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser 65
70 75Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80
85 90Thr Ala Val Tyr Tyr Cys Ala Arg Trp Ala Ser Gly Leu Asp Tyr 95
100 105Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 110 115
208116PRTHomo sapiens 208Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Tyr Ser Ile Thr 20 25 30Asn Asp Tyr Ala Trp Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly 35 40 45Leu Glu Trp Val Gly Tyr Ile Ser Tyr
Ser Gly Tyr Thr Thr Tyr 50 55 60Asn Pro Ser Leu Lys Ser Arg Phe Thr
Ile Ser Arg Asp Thr Ser 65 70 75Lys Asn Thr Leu Tyr Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr Tyr Cys Ala Arg Trp
Thr Ser Gly Leu Asp Tyr 95 100 105Trp Gly Gln Gly Thr Leu Val Thr
Val Ser Ser 110 115 209116PRTHomo sapiens 209Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 1 5 10 15Gly Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Tyr Ser Ile Thr 20 25 30Asn Asp Tyr Ala
Trp Asn Trp Val Arg Gln Ala Pro Gly Lys Gly 35 40 45Leu Glu Trp Val
Gly Tyr Ile Asn Tyr Ala Gly Tyr Thr Thr Tyr 50 55 60Asn Pro Ser Leu
Lys Ser Arg Phe Thr Ile Ser Arg Asp Thr Ser 65 70 75Lys Asn Thr Leu
Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 80 85 90Thr Ala Val Tyr
Tyr Cys Ala Arg Trp Thr Ser Gly Leu Asp Tyr 95 100 105Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 110 115 210108PRTHomo sapiens
210Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5
10 15Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Asp Leu Ile His 20
25 30Asn Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35
40 45Leu Leu Ile Ser Gly Ala Thr Ser Leu Glu Thr Gly Val Pro Ser 50
55 60Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65
70 75Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80
85 90Tyr Trp Thr Thr Pro Phe Thr Phe Gly Gln Gly Thr Lys Val Glu 95
100 105Ile Lys Arg211108PRTHomo sapiens 211Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15Gly Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Asp Leu Ile His 20 25 30Asn Trp Leu Ala Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys 35 40 45Leu Leu Ile Tyr Gly
Ala Thr Ser Leu Glu Thr Gly Val Pro Ser 50 55 60Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 65 70 75Ser Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 80 85 90Tyr Trp Thr Thr Pro
Phe Thr Phe Gly Gln Gly Thr Lys Val Glu 95 100 105Ile Lys Arg
* * * * *