U.S. patent application number 15/220973 was filed with the patent office on 2017-03-23 for identification of tumor-associated antigens for diagnosis and therapy.
The applicant listed for this patent is BioNTech AG, Johannes Gutenberg-Universitat Mainz. Invention is credited to Michael Koslowski, Ugur Sahin, Ozlem Tureci, Dirk Usener.
Application Number | 20170080068 15/220973 |
Document ID | / |
Family ID | 35285406 |
Filed Date | 2017-03-23 |
United States Patent
Application |
20170080068 |
Kind Code |
A1 |
Sahin; Ugur ; et
al. |
March 23, 2017 |
Identification of Tumor-Associated Antigens for Diagnosis and
Therapy
Abstract
The invention relates to genetic products the expression of
which is associated with cancer diseases. The invention also
relates to the therapy and diagnosis of diseases in which the
genetic products are expressed or aberrantly expressed, in
particular cancer diseases.
Inventors: |
Sahin; Ugur; (Mainz, DE)
; Tureci; Ozlem; (Mainz, DE) ; Koslowski;
Michael; (Mainz, DE) ; Usener; Dirk;
(Wiesbaden, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BioNTech AG
Johannes Gutenberg-Universitat Mainz |
Mainz
Mainz |
|
DE
DE |
|
|
Family ID: |
35285406 |
Appl. No.: |
15/220973 |
Filed: |
July 27, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13946302 |
Jul 19, 2013 |
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15220973 |
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13086176 |
Apr 13, 2011 |
8975375 |
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13946302 |
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12066399 |
Jul 7, 2008 |
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PCT/EP2006/008695 |
Sep 6, 2006 |
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13086176 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
G01N 33/574 20130101; A61P 35/00 20180101; C12Q 1/6886 20130101;
C07K 16/2803 20130101; C12Q 2600/158 20130101; C07K 16/30 20130101;
C07K 2317/34 20130101; A61K 2039/57 20130101; C07K 14/4748
20130101; A61K 2039/54 20130101; A61P 43/00 20180101; A61K
2039/5256 20130101; A61K 2039/572 20130101; A61P 37/04 20180101;
A61K 39/0011 20130101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 45/06 20060101 A61K045/06; C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 12, 2005 |
EP |
05019786.2 |
Claims
1.-48. (canceled)
49. A method of treating a patient having prostate cancer
characterized by expression of or abnormal expression of a
tumor-associated antigen encoded by SEQ ID NO: 5, the method
comprising: administering to the patient a composition comprising a
nucleic acid molecule having a sequence that encodes a
tumor-associated antigen of SEQ ID NO: 6, or a fragment thereof
selected from the group consisting of SEQ ID NOs: 51, 52, 53, 54,
55, 56, and 57.
50. The method according to claim 49, further comprising a
therapeutic agent that is not the expressed tumor-associated
antigen.
51. The method of claim 50, wherein the therapeutic agent is
selected from the group consisting of anticancer agents,
radioactive iodine-labeled compounds, toxins, and cytostatic or
cytolytic drugs.
52. The method of claim 49, wherein the nucleic acid molecule is
present in a vector.
53. The method of claim 49, wherein the nucleic acid molecule is
present in a virus or host cell.
54. The method of claim 53, wherein the virus is selected from the
group consisting of adenoviruses, adeno-associated viruses, pox
viruses, vaccinia virus, attenuated pox viruses, Semliki Forest
virus, retroviruses, Sindbis virus, and Ty virus-like
particles.
55. The method of claim 49, wherein the composition further
comprises an adjuvant.
56. The method of claim 49, wherein the administering comprises
injection.
57. A method of treating a patient having prostate cancer that is
characterized by expression or abnormal expression of a
tumor-associated antigen encoded by SEQ ID NO: 5, which method
comprises the steps of: (i) providing a sample containing
immunoreactive cells, (ii) contacting said sample with a host cell
expressing said tumor-associated antigen of SEQ ID NO: 6, or a
fragment thereof selected from the group consisting of SEQ ID NOs:
51, 52, 53, 54, 55, 56, and 57, under conditions which favor
production of cytolytic or cytokine-releasing T cells against said
tumor-associated antigen or said fragment thereof, and (iii)
introducing the cytolytic or cytokine-releasing T cells into the
patient in an amount suitable for lysing prostate cancer cells
expressing the tumor-associated antigen or a fragment thereof.
58. The method of claim 57, in which the host cell recombinantly or
endogenously expresses an MHC molecule binding to the
tumor-associated antigen or to a fragment thereof.
59. The method of claim 57, in which the host cell endogenously
expresses an MHC molecule binding to the tumor-associated antigen
or to a fragment thereof.
60. A method of inducing an immune response in a patient suffering
from prostate cancer that is characterized by expression of or
abnormal expression of a tumor-associated antigen encoded by SEQ ID
NO: 5, the method comprising administering to the patient a
composition comprising a nucleic acid molecule having a sequence
that encodes a tumor-associated antigen of SEQ ID NO: 6, or a
fragment thereof selected from the group consisting of SEQ ID NOs:
51, 52, 53, 54, 55, 56, and 57.
61. The method of claim 60, wherein the method induces cytotoxic or
T helper cell response.
62. The method of claim 60, wherein the nucleic acid molecule is
present in a vector.
63. The method of claim 60, wherein the nucleic acid molecule is
present a virus or host cell.
64. The method of claim 63, wherein the virus is selected from the
group consisting of adenoviruses, adeno-associated viruses, pox
viruses, vaccinia virus, attenuated pox viruses, Semliki Forest
virus, retroviruses, Sindbis virus, and Ty virus-like
particles.
65. The method of claim 60, wherein the composition further
comprises an adjuvant.
66. The method of claim 60, wherein the administering comprises
injection.
67. A method of vaccinating a subject against prostate cancer that
is characterized by expression of or abnormal expression of a
tumor-associated antigen encoded by SEQ ID NO: 5, the method
comprising administering to the patient a vaccine composition
comprising a vector comprising a nucleic acid sequence which
comprises a sequence that encodes a tumor-associated antigen of SEQ
ID NO: 6, or a fragment thereof selected from the group consisting
of SEQ ID NOs: 51, 52, 53, 54, 55, 56, and 57.
68. The method of claim 67, wherein the vector comprises a virus or
host cell.
69. The method of claim 68, wherein the virus is selected from the
group consisting of adenoviruses, adeno-associated viruses, pox
viruses, vaccinia virus, attenuated pox viruses, Semliki Forest
virus, retroviruses, Sindbis virus, and Ty virus-like
particles.
70. The method of claim 67, wherein the composition further
comprises an adjuvant.
71. The method of claim 67, wherein the administering comprises
injection.
Description
[0001] Despite interdisciplinary approaches and exhaustive use of
classical therapeutic procedures, cancers are still among the
leading causes of death. More recent therapeutic concepts aim at
incorporating the patient's immune system into the overall
therapeutic concept by using recombinant tumor vaccines and other
specific measures such as antibody therapy. A prerequisite for the
success of such a strategy is the recognition of tumor-specific or
tumor-associated antigens or epitopes by the patient's immune
system whose effector functions are to be interventionally
enhanced. Tumor cells biologically differ substantially from their
nonmalignant cells of origin. These differences are due to genetic
alterations acquired during tumor development and result, inter
alia, also in the formation of qualitatively or quantitatively
altered molecular structures in the cancer cells. Tumor-associated
structures of this kind which are recognized by the specific immune
system of the tumor-harboring host are referred to as
tumor-associated antigens. The specific recognition of
tumor-associated antigens involves cellular and humoral mechanisms
which are two functionally interconnected units: CD4.sup.+ and
CD8.sup.+ T lymphocytes recognize the processed antigens presented
on the molecules of the MHC (major histocompatibility complex)
classes II and I, respectively, while B lymphocytes produce
circulating antibody molecules which bind directly to unprocessed
antigens. The potential clinical-therapeutical importance of
tumor-associated antigens results from the fact that the
recognition of antigens on neoplastic cells by the immune system
leads to the initiation of cytotoxic effector mechanisms and, in
the presence of T helper cells, can cause elimination of the cancer
cells (Pardoll, Nat. Med. 4:525-31, 1998). Accordingly, a central
aim of tumor immunology is to molecularly define these structures.
The molecular nature of these antigens has been enigmatic for a
long time. Only after development of appropriate cloning techniques
has it been possible to screen cDNA expression libraries of tumors
systematically for tumor-associated antigens by analyzing the
target structures of cytotoxic T lymphocytes (CTL) (van der Bruggen
et al., Science 254:1643-7, 1991) or by using circulating
autoantibodies (Sahin et al., Curr. Opin. Immunol. 9:709-16, 1997)
as probes. To this end, cDNA expression libraries were prepared
from fresh tumor tissue and recombinantly expressed as proteins in
suitable systems. Immunoeffectors isolated from patients, namely
CTL clones with tumor-specific lysis patterns, or circulating
autoantibodies were utilized for cloning the respective
antigens.
[0002] In recent years a multiplicity of antigens have been defined
in various neoplasias by these approaches.
[0003] However, the probes utilized for antigen identification in
the classical methods are immunoeffectors (circulating
autoantibodies or CTL clones) from patients usually having already
advanced cancer. A number of data indicate that tumors can lead,
for example, to tolerization and anergization of T cells and that,
during the course of the disease, especially those specificities
which could cause effective immune recognition are lost from the
immunoeffector repertoire. Current patient studies have not yet
produced any solid evidence of a real action of the previously
found and utilized tumor-associated antigens. Accordingly, it
cannot be ruled out that proteins evoking spontaneous immune
responses are the wrong target structures.
[0004] It was the object of the present invention to provide target
structures for a diagnosis and therapy of cancers.
[0005] This object is achieved by the subject matter of the
claims.
[0006] According to the invention, genes are identified which are
selectively or aberrantly expressed in tumor cells and thus,
provide tumor-associated antigens. These genes and/or their genetic
products and/or the derivatives and/or fragments thereof are useful
as target structures for therapeutic and diagnostic approaches.
[0007] The tumor-associated antigens identified according to the
invention have an amino acid sequence encoded by a nucleic acid
which is selected from the group consisting of (a) a nucleic acid
which comprises a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39,
41, 45, 49, 61, 62, and 64-67 of the sequence listing, a part or
derivative thereof, (b) a nucleic acid which hybridizes with the
nucleic acid of (a) under stringent conditions, (c) a nucleic acid
which is degenerate with respect to the nucleic acid of (a) or (b),
and (d) a nucleic acid which is complementary to the nucleic acid
of (a), (b) or (c). In a preferred embodiment, a tumor-associated
antigen identified according to the invention has an amino acid
sequence encoded by a nucleic acid which is selected from the group
consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39,
41, 45, 49, 61, 62, and 64-67 of the sequence listing. In a further
preferred embodiment, a tumor-associated antigen identified
according to the invention comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18,
22, 26, 29, 31, 36, 40, 42, 46, 50-60, 63, 68, and 69 of the
sequence listing, a part or derivative thereof.
[0008] The present invention generally relates to the use of
tumor-associated antigens identified according to the invention or
of parts or derivatives thereof, of nucleic acids coding for the
tumor-associated antigens identified according to the invention or
of parts or derivatives thereof or of nucleic acids directed
against said coding nucleic acids, of antibodies or T cells
directed against the tumor-associated antigens identified according
to the invention or parts or derivatives thereof and/or of host
cells expressing the tumor-associated antigens identified according
to the invention or parts or derivatives thereof for therapy,
prophylaxis, diagnosis and/or monitoring of neoplastic diseases.
This may also involve the use of a combination of two or more of
these antigens, nucleic acids, antibodies, T cells and/or host
cells, in one embodiment also in combination with tumor-associated
antigens other than those identified according to the invention,
nucleic acids coding therefor or nucleic acids directed against
said coding nucleic acids, antibodies or T cells directed against
said tumor-associated antigens and/or host cells expressing said
tumor associated antigens.
[0009] In those embodiments of the invention relating to the use of
antibodies directed against the tumor-associated antigens
identified according to the invention or parts or derivatives
thereof also T cell receptors directed against the tumor-associated
antigens identified according to the invention or parts or
derivatives thereof, optionally in a complex with MHC molecules,
may be used.
[0010] Especially suitable for therapy, prophylaxis, diagnosis
and/or monitoring is a part of the tumor-associated antigens
identified according to the invention which corresponds to the
non-transmembrane portion, in particular the extracellular portion
of the tumor-associated antigens or is comprised thereof.
Therefore, according to the invention, a part of the
tumor-associated antigens identified according to the invention
which corresponds to the non-transmembrane portion, in particular
the extracellular portion of the tumor-associated antigens or is
comprised thereof, or a corresponding part of the nucleic acids
coding for the tumor-associated antigens identified according to
the invention is preferred for therapy, prophylaxis, diagnosis
and/or monitoring. Similarly the use of antibodies is preferred
which are directed against a part of the tumor-associated antigens
identified according to the invention which corresponds to the
non-transmembrane portion, in particular the extracellular portion
of the tumor-associated antigens or is comprised thereof.
[0011] Preferred diseases for a therapy, prophylaxis and/or
diagnosis are those in which one or more of the tumor-associated
antigens identified according to the invention are selectively
expressed or abnormally expressed.
[0012] Furthermore, the invention relates to nucleic acids and
proteins or peptides, which result from altered splicing (splice
variants) of known genes or altered translation using alternative
open reading frames. In this aspect the invention relates to
nucleic acids which comprise a nucleic acid sequence selected from
the group consisting of SEQ ID NOs: 28 and 49 of the sequence
listing. Moreover, in this aspect, the invention relates to
proteins or peptides which comprise an amino acid sequence selected
from the group consisting of SEQ ID NOs: 29 and 50 of the sequence
listing.
[0013] Altered splicing of a gene results in an altered transcript
sequence (splice variant). Translation of a splice variant in the
region of its altered sequence results in an altered protein which
may be distinctly different in the structure and function from the
original protein. Tumor-associated splice variants may produce
tumor-associated transcripts and tumor-associated
proteins/antigens. These may be utilized as molecular markers both
for detecting tumor cells and for therapeutic targeting of tumors.
Detection of tumor cells in a sample from a patient may be carried
out according to the invention, for example, after extraction of
nucleic acids by PCR amplification with splice variant-specific
oligonucleotides.
[0014] According to the invention, all sequence-dependent detection
systems are suitable for detection. These are, apart from PCR, for
example gene chip/microarray systems, Northern blot, RNAse
protection assays (RDA) and others. All detection systems have in
common that detection is based on a specific hybridization with at
least one splice variant-specific nucleic acid sequence. However,
tumor cells may also be detected according to the invention by
antibodies which recognize a specific epitope encoded by the splice
variant. Said antibodies may be prepared by using for immunization
peptides which are specific for said splice variant. Suitable for
immunization are particularly the amino acid sequences which are
distinctly different from the variant(s) of the genetic product,
which is (are) preferably produced in healthy cells. Detection of
the tumor cells with antibodies may be carried out here on a sample
isolated from the patient or as imaging with intravenously
administered antibodies.
[0015] In addition to diagnostic usability, splice variants having
new or altered epitopes are attractive targets for immunotherapy as
these epitopes may be utilized for targeting antibodies or T
lymphocytes as described herein. In passive immunotherapy,
antibodies or T lymphocytes which recognize splice variant-specific
epitopes are adoptively transferred here. As in the case of other
antigens, antibodies may be generated also by using standard
technologies with utilization of polypeptides which include these
epitopes. Alternatively, it is possible to utilize for immunization
nucleic acids coding for peptides which contain said epitopes.
Various techniques for in vitro or in vivo generation of
epitope-specific T lymphocytes are known and have been described in
detail (for example Kessler J H, et al. 2001, Sahin et al., 1997)
and are likewise based on utilizing peptides which contain the
splice variant-specific epitopes or nucleic acids coding for said
peptides. Peptides which contain the splice variant-specific
epitopes or nucleic acids coding for said peptides may also be used
as pharmaceutically active substances in active immunotherapy (e.g.
vaccination, vaccine therapy).
[0016] In one aspect, the invention relates to a pharmaceutical
composition comprising an agent which recognizes a tumor-associated
antigen identified according to the invention or a nucleic acid
coding for the tumor-associated antigen and which is preferably
selective for cells which have expression or abnormal expression of
a tumor-associated antigen identified according to the invention.
In a further aspect, the invention relates to a pharmaceutical
composition comprising an agent which (I) inhibits expression or
activity of a tumor-associated antigen identified according to the
invention, and/or (II) has tumor-inhibiting or tumor-destroying
activity and is selective for cells expressing or abnormally
expressing a tumor-associated antigen identified according to the
invention, and/or (III) when administered, selectively increases
the amount of complexes between an MHC molecule and a
tumor-associated antigen identified according to the invention or a
part thereof, such as a peptide epitope. In particular embodiments,
said agent may cause induction of cell death, reduction in cell
growth, damage to the cell membrane or secretion of cytokines and
preferably have a tumor-inhibiting activity. In one embodiment, the
agent is an antisense nucleic acid which hybridizes selectively
with the nucleic acid coding for the tumor-associated antigen. In a
further embodiment, the agent is a siRNA preferably comprising a
sense RNA strand and an antisense RNA strand, wherein the sense and
antisense RNA strands form an RNA duplex, and wherein the sense RNA
strand comprises a nucleotide sequence substantially identical to a
target sequence of about 19 to about 25 contiguous nucleotides in a
nucleic acid coding for the tumor-associated antigen, preferably
mRNA coding for the tumor-associated antigen. In a further
embodiment, the agent is an antibody which binds selectively to the
tumor-associated antigen, in particular a complement-activated or
toxin conjugated antibody which binds selectively to the
tumor-associated antigen. In a preferred embodiment, the antibody
which binds selectively to the tumor-associated antigen is coupled
to a therapeutically useful substance and/or recruits natural or
artificial effector mechanisms to said cell expressing or
abnormally expressing said tumor-associated antigen. In a further
embodiment, the agent is a cytotoxic T lymphocyte which recognizes
the tumor-associated antigen or a part thereof bound by an MHC
molecule on a cell and lyses the cells labeled in this way. In a
further embodiment, the agent is a T helper lymphocyte which
enhances effector functions of other cells specifically recognizing
said tumor-associated antigen or a part thereof.
[0017] In a further embodiment, the agent comprises two or more
agents which each recognize different tumor-associated antigens
and/or inhibit expression or activity of different tumor-associated
antigens, and/or have tumor-inhibiting or tumor-destroying activity
and are selective for cells expressing or abnormally expressing
different tumor-associated antigens, and/or when administered,
selectively increase the amount of complexes between MHC molecules
and different tumor-associated antigens or parts thereof, wherein
at least one of said different tumor-associated antigens is a
tumor-associated antigen identified according to the invention.
Preferably, a tumor-associated antigen selectively limited to
tumors serves as a label for recruiting effector mechanisms to this
specific location. The invention includes embodiments wherein the
agent itself does not have an ability to inhibit activity of a
tumor-associated antigen or a tumor-inhibiting or tumor-destroying
activity but mediates such effect, in particular by recruiting
effector mechanisms, in particular those having cell damaging
potential, to a specific location, in particular a tumor or tumor
cells.
[0018] The activity of a tumor-associated antigen identified
according to the invention can be any activity of a protein or a
peptide. In one embodiment this activity is an enzymatic
activity.
[0019] According to the invention the phrase "inhibit expression or
activity" includes a complete or essentially complete inhibition of
expression or activity and a reduction in expression or
activity.
[0020] The agent which, when administered, selectively increases
the amount of complexes between an MHC molecule and a
tumor-associated antigen identified according to the invention or a
part thereof comprises one or more components selected from the
group consisting of (i) the tumor-associated antigen or a part
thereof, (ii) a nucleic acid which codes for said tumor-associated
antigen or a part thereof, (iii) a host cell which expresses said
tumor-associated antigen or a part thereof, and (iv) isolated
complexes between peptide epitopes from said tumor-associated
antigen and an MHC molecule.
[0021] The invention furthermore relates to a pharmaceutical
composition which comprises one or more components selected from
the group consisting of (i) a tumor-associated antigen identified
according to the invention or a part thereof, (ii) a nucleic acid
which codes for a tumor-associated antigen identified according to
the invention or a part thereof, (iii) an antibody which binds to a
tumor-associated antigen identified according to the invention or
to a part thereof, (iv) an antisense nucleic acid which hybridizes
specifically with a nucleic acid coding for a tumor-associated
antigen identified according to the invention, (v) an siRNA
directed against a nucleic acid coding for a tumor-associated
antigen identified according to the invention, (vi) a host cell
which expresses a tumor-associated antigen identified according to
the invention or a part thereof, and (vii) isolated complexes
between a tumor-associated antigen identified according to the
invention or a part thereof and an MHC molecule.
[0022] In one embodiment, a nucleic acid coding for a
tumor-associated antigen identified according to the invention or a
part thereof is present in the pharmaceutical composition in an
expression vector and functionally linked to a promoter. In a
further embodiment, a nucleic acid coding for a tumor-associated
antigen identified according to the invention or a part thereof is
present in the pharmaceutical composition in a virus as further
described below.
[0023] In a further embodiment, a host cell present in a
pharmaceutical composition of the invention secretes the
tumor-associated antigen or the part thereof, expresses it on the
surface and preferably additionally express an MHC molecule which
binds to said tumor-associated antigen or said part thereof. In one
embodiment, the host cell expresses the MHC molecule endogenously.
In a further embodiment, the host cell expresses the MHC molecule
and/or the tumor-associated antigen or the part thereof in a
recombinant manner. The host cell is preferably nonproliferative.
In a preferred embodiment, the host cell is an antigen-presenting
cell, in particular a dendritic cell, a monocyte or a
macrophage.
[0024] In a further embodiment, an antibody present in a
pharmaceutical composition of the invention is a monoclonal
antibody. In further embodiments, the antibody is a chimeric or
humanized antibody, a fragment of a natural antibody or a synthetic
antibody. The antibody may be coupled to a therapeutically or
diagnostically useful agent also termed therapeutic or diagnostic
agent herein.
[0025] An antisense nucleic acid present in a pharmaceutical
composition of the invention may comprise a sequence of 6-50, in
particular 10-30, 15-30 and 20-30, contiguous nucleotides of the
nucleic acid coding for the tumor-associated antigen identified
according to the invention.
[0026] In further embodiments, a tumor-associated antigen or a part
thereof, provided by a pharmaceutical composition of the invention
either directly or via expression of a nucleic acid, binds to MHC
molecules on the surface of cells, said binding preferably causing
a cytolytic response and/or inducing cytokine release.
[0027] In particular embodiments of the siRNA targeting the nucleic
acid according to SEQ ID NO: 1 the sense RNA strand has the
sequence of SEQ ID NO: 70 and the antisense RNA strand has the
sequence of SEQ ID NO: 71, or the sense RNA strand has the sequence
of SEQ ID NO: 72 and the antisense RNA strand has the sequence of
SEQ ID NO: 73.
[0028] A pharmaceutical composition of the invention may comprise a
pharmaceutically compatible carrier and/or an adjuvant.
[0029] A pharmaceutical composition of the invention is preferably
used for the treatment or prevention of a disease characterized by
selective expression or abnormal expression of a tumor-associated
antigen. In a preferred embodiment, the disease is a neoplastic
disease, preferably cancer.
[0030] In a preferred embodiment, the pharmaceutical composition of
the invention is in the form of a vaccine which may be used
therapeutically or prophylactically. Such vaccine preferably
comprises a tumor-associated antigen identified according to the
invention or a part thereof, and/or a nucleic acid which codes for
a tumor-associated antigen identified according to the invention or
a part thereof. In particular embodiments, the nucleic acid is
present in a virus or host cell.
[0031] The invention furthermore relates to methods of treating,
preventing, diagnosing or monitoring, i.e. determining the
regression, progression, course and/or onset of, a disease
characterized by expression or abnormal expression of one of more
tumor-associated antigens identified according to the invention,
preferably a neoplastic disease, in particular cancer. In one
embodiment, the treatment or prevention comprises administering a
pharmaceutical composition of the invention.
[0032] Said methods of diagnosing and/or methods of monitoring
according to the invention generally concern the detection of
and/or determination of the quantity of one or more parameters
selected from the group consisting of (i) a nucleic acid, which
codes for a tumor-associated antigen identified according to the
invention, or a part thereof, (ii) a tumor-associated antigen
identified according to the invention, or a part thereof (iii) an
antibody against a tumor-associated antigen identified according to
the invention or a part thereof, and (iv) T lymphocytes, preferably
cytotoxic or T helper lymphocytes, which are specific for a
tumor-associated antigen identified according to the invention or a
part thereof and/or a complex between the tumor-associated antigen
or a part thereof and an MHC molecule, in a biological sample
isolated from a patient, preferably from a patient having said
disease, being suspected of having or falling ill with said disease
or having a potential for said disease. Means for accomplishing
said detection and/or determination of the quantity are described
herein and will be apparent to the skilled person.
[0033] Preferably, the presence of said nucleic acid, said
tumor-associated antigen or said part thereof, said antibody and/or
said T lymphocytes and/or a quantity of said nucleic acid, said
tumor-associated antigen or said part thereof, said antibody and/or
said T lymphocytes which is increased compared to a patient without
said disease is indicative for the presence of said disease or a
potential for a development of said disease.
[0034] The methods of diagnosing and/or monitoring of the invention
also include embodiments wherein by detection or determination of
the quantity of said nucleic acid, said tumor-associated antigen or
said part thereof, said antibody and/or said T lymphocytes it is
possible to assess and/or prognose the metastatic behavior of said
disease, wherein, preferably, the presence of said nucleic acid,
said tumor-associated antigen or said part thereof, said antibody
and/or said T lymphocytes and/or a quantity of said nucleic acid,
said tumor-associated antigen or said part thereof, said antibody
and/or said T lymphocytes which is increased compared to a patient
without said disease or without a metastasis of said disease is
indicative for a metastatic behavior of said disease or a potential
for a metastatic behavior of said disease.
[0035] In particular embodiments, said detection or determination
of the quantity comprises (i) contacting a biological sample with
an agent which binds specifically to said nucleic acid coding for
the tumor-associated antigen or said part thereof, to said
tumor-associated antigen or said part thereof, to said antibody or
said part thereof or to said T lymphocytes, and (ii) detecting the
formation of or determining the amount of a complex between the
agent and the nucleic acid or the part thereof, the
tumor-associated antigen or the part thereof, the antibody or the
part thereof, or the T lymphocytes. In one embodiment, the disease
is characterized by expression or abnormal expression of two or
more different tumor-associated antigens and a detection or
determination of the amount comprises a detection or determination
of the amount of two or more nucleic acids coding for said two or
more different tumor-associated antigens or of parts thereof, of
two or more different tumor-associated antigens or of parts
thereof, of two or more antibodies binding to said two or more
different tumor-associated antigens or to parts thereof and/or of
two or more T lymphocytes specific for said two or more different
tumor-associated antigens or parts thereof, or complexes thereof
with MHC molecules. In a further embodiment, the biological sample
isolated from the patient is compared to a comparable normal
biological sample.
[0036] The methods of monitoring according to the invention
preferably comprise a detection of and/or determination of the
quantity of one or more of the parameters mentioned above in a
first sample at a first point in time and in a further sample at a
second point in time, wherein the course of the disease is
determined by comparing the two samples.
[0037] According to the invention, detection of a nucleic acid or
of a part thereof or determining the quantity of a nucleic acid or
of a part thereof may be carried out using a oligo- or
polynucleotide probe which hybridizes specifically to said nucleic
acid or said part thereof or may be carried out by selective
amplification of said nucleic acid or said part thereof, e.g. by
means of PCR amplification. In one embodiment, the oligo- or
polynucleotide probe comprises a sequence of 6-50, in particular
10-30, 15-30 and 20-30, contiguous nucleotides of said nucleic
acid.
[0038] In particular embodiments, the tumor-associated antigen or
the part thereof which is to be detected or the amount of which is
to be determined in the methods of the present invention is present
intracellularly, on the cell surface or in a complex with an MHC
molecule. According to the invention, detection of a
tumor-associated antigen or of a part thereof or determining the
quantity of a tumor-associated antigen or of a part thereof may be
carried out using an antibody binding specifically to said
tumor-associated antigen or said part thereof.
[0039] According to the invention, detection of an antibody or
determining the quantity of an antibody may be carried out using a
protein or peptide binding specifically to said antibody.
[0040] According to the invention, detection of or determining the
quantity of T lymphocytes which are specific for a tumor-associated
antigen or a part thereof and/or a complex thereof with an MHC
molecule may be carried out using a cell presenting the complex
between said tumor-associated antigen or said part thereof and an
MHC molecule. T lymphocytes may additionally be detected by
detecting their proliferation, their cytokine production, and their
cytotoxic activity triggered by specific stimulation with a complex
of an MHC molecule and a tumor-associated antigen or a part
thereof. T lymphocytes may also be detected with aid of a
recombinant MHC molecule or a complex of two or more MHC molecules
loaded with immunogenic fragments of one or more tumor-associated
antigens.
[0041] An agent which is used for detection or determining the
quantity in the methods of the invention such as a oligo- or
polynucleotide probe, an antibody, a protein or peptide or a cell
is preferably labeled in a detectable manner, in particular by a
detectable marker such as a radioactive marker or an enzymic
marker.
[0042] In a particular aspect, the invention relates to a method of
treating, preventing, diagnosing or monitoring a disease
characterized by expression or abnormal expression of a
tumor-associated antigen identified according to the invention,
which method comprises administering an antibody which binds to
said tumor-associated antigen or to a part thereof and which is
coupled to a therapeutic or diagnostic agent. The antibody may be a
monoclonal antibody. In further embodiments, the antibody is a
chimeric or humanized antibody or a fragment of a natural
antibody.
[0043] In certain embodiments, the methods of the invention of
diagnosing or monitoring a disease characterized by expression or
abnormal expression of a tumor-associated antigen identified
according to the invention are performed with a biological sample
containing or suspected of containing disseminating tumor cells or
metastatic tumor cells. Such biological samples include, for
example, blood, serum, bone marrow, sputum, bronchial aspirate,
and/or bronchial lavage.
[0044] In one particular aspect, the invention relates to a method
of treating a patient having a disease characterized by expression
or abnormal expression of a tumor-associated antigen identified
according to the invention, which method comprises (i) providing a
sample containing immunoreactive cells, either obtained from said
patient or from another individual of the same species, in
particular a healthy individual, or an individual of a different
species, (ii) contacting said sample with a host cell expressing
said tumor-associated antigen or a part thereof, under conditions
which favor production of cytolytic T cells against said
tumor-associated antigen or a part thereof, and (iii) introducing
the cytolytic T cells into the patient in an amount suitable for
lysing cells expressing the tumor-associated antigen or a part
thereof. In one embodiment, the method includes cloning of the T
cell receptor of cytolytic T cells obtained and transferring the
nucleic acid coding for the T cell receptor to T cells, either
obtained from said patient or from another individual of the same
species, in particular a healthy individual, or an individual of a
different species, which T cells thus receive the desired
specificity and, as under (iii), may be introduced into the
patient.
[0045] In one embodiment, the host cell endogenously expresses an
MHC molecule. In a further embodiment, the host cell recombinantly
expresses an MHC molecule and/or the tumor-associated antigen or
the part thereof. Preferably, the host cell presents the
tumor-associated antigen or the part thereof by MHC molecules on
its surface. The host cell is preferably nonproliferative. In a
preferred embodiment, the host cell is an antigen-presenting cell,
in particular a dendritic cell, a monocyte or a macrophage.
[0046] The invention also relates to a method of treating a disease
characterized by expression or abnormal expression of a
tumor-associated antigen identified according to the invention,
which method comprises (i) identifying cells from the patient which
express abnormal amounts of the tumor-associated antigen, (ii)
isolating a sample of said cells, (iii) culturing said cells, and
(iv) introducing said cells into the patient in an amount suitable
for triggering an immune response to the cells.
[0047] The present invention furthermore relates to a nucleic acid
selected from the group consisting of (a) a nucleic acid which
comprises a nucleic acid sequence selected from the group
consisting of SEQ ID NOs: 1, 5, 9, 13, 17, 21, 25, 28, 30, 35, 39,
41, 45, 49, 61, 62, and 64-67, a part or derivative thereof, (b) a
nucleic acid which hybridizes with the nucleic acid of (a) under
stringent conditions, (c) a nucleic acid which is degenerate with
respect to the nucleic acid of (a) or (b), and (d) a nucleic acid
which is complementary to the nucleic acid of (a), (b) or (c). The
invention furthermore relates to a nucleic acid, which codes for a
protein or polypeptide comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26,
29, 31, 36, 40, 42, 46, 50-60, 63, 68, and 69, a part or derivative
thereof.
[0048] In a further aspect, the invention relates to a recombinant
nucleic acid molecule, in particular DNA or RNA molecule, which
comprises a nucleic acid of the invention.
[0049] The invention also relates to host cells which contain a
nucleic acid or recombinant nucleic acid molecule of the
invention.
[0050] The host cell may also comprise a nucleic acid coding for a
MHC molecule. In one embodiment, the host cell endogenously
expresses the MHC molecule. In a further embodiment, the host cell
recombinantly expresses the MHC molecule and/or the nucleic acid or
recombinant nucleic acid molecule of the invention or a part
thereof. Preferably, the host cell is nonproliferative. In a
preferred embodiment, the host cell is an antigen-presenting cell,
in particular a dendritic cell, a monocyte or a macrophage.
[0051] In a further embodiment, the invention relates to
oligonucleotides which hybridize with a nucleic acid identified
according to the invention and which may be used as genetic probes
or as "antisense" molecules. Nucleic acid molecules in the form of
oligonucleotide primers or competent probes, which hybridize with a
nucleic acid identified according to the invention or parts
thereof, may be used for finding nucleic acids which are homologous
to said nucleic acid identified according to the invention, e.g. by
PCR amplification, Southern and Northern hybridization.
Hybridization may be carried out under low stringency, more
preferably under medium stringency and most preferably under high
stringency conditions.
[0052] In a further aspect, the invention relates to a protein or
peptide which is encoded by a nucleic acid selected from the group
consisting of (a) a nucleic acid which comprises a nucleic acid
sequence selected from the group consisting of SEQ ID NOs: 1, 5, 9,
13, 17, 21, 25, 28, 30, 35, 39, 41, 45, 49, 61, 62, and 64-67, a
part or derivative thereof, (b) a nucleic acid which hybridizes
with the nucleic acid of (a) under stringent conditions, (c) a
nucleic acid which is degenerate with respect to the nucleic acid
of (a) or (b), and (d) a nucleic acid which is complementary to the
nucleic acid of (a), (b) or (c). In a preferred embodiment, the
invention relates to a protein or peptide which comprises an amino
acid sequence selected from the group consisting of SEQ ID NOs: 2,
6, 10, 14, 18, 22, 26, 29, 31, 36, 40, 42, 46, 50-60, 63, 68, and
69, a part or derivative thereof.
[0053] In a further aspect, the invention relates to an immunogenic
fragment of a tumor-associated antigen identified according to the
invention. Said fragment preferably binds to a MHC molecule or an
antibody, preferably to a human HLA receptor or a human antibody.
According to the invention, a fragment preferably comprises a
sequence of at least 6, in particular at least 8, at least 10, at
least 12, at least 15, at least 20, at least 30 or at least 50,
amino acids.
[0054] In this aspect the invention relates, in particular, to a
peptide which has or comprises a sequence selected from the group
consisting of SEQ ID NOs: 51-60, 68 and of the sequence listing, a
part or derivative thereof.
[0055] In a further aspect, the invention relates to an agent which
binds to a tumor-associated antigen identified according to the
invention or to a part thereof. In a preferred embodiment, the
agent is a protein or peptide, in particular an antibody, a T cell
receptor or an MHC molecule. In further embodiments, the antibody
is a monoclonal, chimeric, or humanized antibody, an antibody
produced by combinatory techniques, or a fragment of an antibody.
In one preferred embodiment, the invention relates to an antibody
which binds selectively to a complex of (i) a tumor-associated
antigen identified according to the invention or a part thereof and
(ii) an MHC molecule to which said tumor-associated antigen
identified according to the invention or said part thereof binds,
with said antibody not binding to (i) or (ii) alone.
[0056] In particular, the invention relates to such an agent, in
particular an antibody, which specifically binds to a peptide which
has or comprises a sequence selected from the group consisting of
SEQ ID NOs: 51-60, 68, and of the sequence listing, a part or
derivative thereof.
[0057] According to the invention, the term "binding" preferably
relates to a specific binding. "Specific binding" means that an
agent such as an antibody binds stronger to a target such as an
epitope for which it is specific compared to the binding to another
target. An agent binds stronger to a first target compared to a
second target if it binds to the first target with a dissociation
constant (K.sub.D) which is lower than the dissociation constant
for the second target. Preferably the dissociation constant
(K.sub.D) for the target to which the agent binds specifically is
more than 10-fold, preferably more than 20-fold, more preferably
more than 50-fold, even more preferably more than 100-fold,
200-fold, 500-fold or 1000-fold lower than the dissociation
constant (K.sub.D) for the target to which the agent does not bind
specifically.
[0058] Such specific antibodies may, for example, be obtained by
immunization using the aforementioned peptides.
[0059] The invention furthermore relates to a conjugate between an
agent of the invention which binds to a tumor-associated antigen
identified according to the invention or to a part thereof or an
antibody of the invention and a therapeutic or diagnostic agent. In
one embodiment, the therapeutic or diagnostic agent is a toxin.
[0060] In a further aspect, the invention relates to a kit for
detecting expression or abnormal expression of a tumor-associated
antigen identified according to the invention, which kit comprises
agents for detection or determining the quantity (i) of the nucleic
acid which codes for the tumor-associated antigen or of a part
thereof, (ii) of the tumor-associated antigen or of a part thereof,
(iii) of antibodies which bind to the tumor-associated antigen or
to a part thereof, and/or (iv) of T cells which are specific for
the tumor-associated antigen or a part thereof or a complex thereof
with an MHC molecule. In one embodiment, the agents for detection
of the nucleic acid or the part thereof are nucleic acid molecules
for selective amplification of said nucleic acid, which comprise,
in particular, a sequence of 6-50, in particular 10-30, 15-30 and
20-30, contiguous nucleotides of said nucleic acid.
DETAILED DESCRIPTION OF THE INVENTION
[0061] According to the invention, a "reference" such as a
reference sample or reference organism may be used to correlate and
compare the results obtained in the methods of the invention from a
test sample or test organism, i.e. a patient. Typically the
reference organism is a healthy organism, in particular an organism
which does not suffer from cancer.
[0062] A "reference value" can be determined from a reference
empirically by measuring a sufficiently large number of references.
Preferably the reference value is determined by measuring at least
2, preferably at least 3, preferably at least 5, preferably at
least 8, preferably at least 12, preferably at least 20, preferably
at least 30, preferably at least 50, or preferably at least 100
references.
[0063] "Derivative" of a nucleic acid means according to the
invention that single or multiple such as at least 2, at least 4,
or at least 6 and preferably up to 3, up to 4, up to 5, up to 6, up
to 10, up to 15, or up to 20 nucleotide substitutions, deletions
and/or additions are present in said nucleic acid. Furthermore, the
term "derivative" also comprises chemical derivatization of a
nucleic acid on a nucleotide base, on the sugar or on the
phosphate. The term "derivative" also comprises nucleic acids which
contain nucleotides and nucleotide analogs not occurring
naturally.
[0064] According to the invention, a nucleic acid is preferably
deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic
acids comprise according to the invention genomic DNA, cDNA, mRNA,
recombinantly produced and chemically synthesized molecules.
According to the invention, a nucleic acid may be present as a
single-stranded or double-stranded and linear or covalently
circularly closed molecule.
[0065] As used herein, the term "RNA" means a molecule comprising
at least one ribonucleotide residue. By "ribonucleotide" is meant a
nucleotide with a hydroxyl group at the 2'-position of a
beta-D-ribo-furanose moiety. The term includes double stranded RNA,
single stranded RNA, isolated RNA such as partially purified RNA,
essentially pure RNA, synthetic RNA, recombinantly produced RNA, as
well as altered RNA that differs from naturally occurring RNA by
the addition, deletion, substitution and/or alteration of one or
more nucleotides. Such alterations can include addition of
non-nucleotide material, such as to the end(s) of a RNA or
internally, for example at one or more nucleotides of the RNA.
Nucleotides in RNA molecules can also comprise non-standard
nucleotides, such as non-naturally occurring nucleotides or
chemically synthesized nucleotides or deoxynucleotides. These
altered RNAs can be referred to as analogs or analogs of
naturally-occurring RNA.
[0066] The nucleic acids described according to the invention have
preferably been isolated. The term "isolated nucleic acid" means
according to the invention that the nucleic acid was (i) amplified
in vitro, for example by polymerase chain reaction (PCR), (ii)
recombinantly produced by cloning, (iii) purified, for example by
cleavage and gel-electrophoretic fractionation, or (iv)
synthesized, for example by chemical synthesis. An isolated nucleic
acid is a nucleic acid which is available for manipulation by
recombinant DNA techniques.
[0067] A nucleic acid is "complementary" to another nucleic acid if
the two sequences are capable of hybridizing and forming a stable
duplex with one another, with hybridization preferably being
carried out under conditions which allow specific hybridization
between polynucleotides (stringent conditions). Stringent
conditions are described, for example, in Molecular Cloning: A
Laboratory Manual, J. Sambrook et al., Editors, 2nd Edition, Cold
Spring Harbor Laboratory press, Cold Spring Harbor, N.Y., 1989 or
Current Protocols in Molecular Biology, F. M. Ausubel et al.,
Editors, John Wiley & Sons, Inc., New York and refer, for
example, to hybridization at 65.degree. C. in hybridization buffer
(3.5.times.SSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02%
bovine serum albumin, 2.5 mM NaH.sub.2PO.sub.4 (pH 7), 0.5% SDS, 2
mM EDTA). SSC is 0.15 M sodium chloride/0.15 M sodium citrate, pH
7. After hybridization, the membrane to which the DNA has been
transferred is washed, for example, in 2.times.SSC at room
temperature and then in 0.1-0.5.times.SSC/0.1.times.SDS at
temperatures of up to 68.degree. C.
[0068] According to the invention, complementary nucleic acids have
at least 40%, in particular at least 50%, at least 60%, at least
70%, at least 80%, at least 90% and preferably at least 95%, at
least 98% or at least 99%, identical nucleotides.
[0069] The term "percentage identity" is intended to denote a
percentage of nucleotides or of amino acid residues which are
identical between the two sequences to be compared, obtained after
the best alignment, this percentage being purely statistical and
the differences between the two sequences being distributed
randomly and over their entire length. Sequence comparisons between
two nucleotide or amino acid sequences are conventionally carried
out by comparing these sequences after having aligned them
optimally, said comparison being carried out by segment or by
"window of comparison" in order to identify and compare local
regions of sequence similarity. The optimal alignment of the
sequences for comparison may be produced, besides manually, by
means of the local homology algorithm of Smith and Waterman, 1981,
Ads App. Math. 2, 482, by means of the local homology algorithm of
Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by means of the
similarity search method of Pearson and Lipman, 1988, Proc. Natl
Acad. Sci. USA 85, 2444, or by means of computer programs which use
these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA
in Wisconsin Genetics Software Package, Genetics Computer Group,
575 Science Drive, Madison, Wis.).
[0070] The percentage identity is calculated by determining the
number of identical positions between the two sequences being
compared, dividing this number by the number of positions compared
and multiplying the result obtained by 100 so as to obtain the
percentage identity between these two sequences.
[0071] Nucleic acids coding for tumor-associated antigens may,
according to the invention, be present alone or in combination with
other nucleic acids, in particular heterologous nucleic acids. In
preferred embodiments, a nucleic acid is functionally linked to
expression control sequences or regulatory sequences which may be
homologous or heterologous with respect to said nucleic acid. A
coding sequence and a regulatory sequence are "functionally" linked
to one another, if they are covalently linked to one another in
such a way that expression or transcription of said coding sequence
is under the control or under the influence of said regulatory
sequence. If the coding sequence is to be translated into a
functional protein, then, with a regulatory sequence functionally
linked to said coding sequence, induction of said regulatory
sequence results in transcription of said coding sequence, without
causing a frame shift in the coding sequence or said coding
sequence not being capable of being translated into the desired
protein or peptide.
[0072] The term "expression control sequence" or "regulatory
sequence" comprises according to the invention promoters, enhancers
and other control elements which regulate expression of a gene. In
particular embodiments of the invention, the expression control
sequences can be regulated. The exact structure of regulatory
sequences may vary as a function of the species or cell type, but
generally comprises 5'untranscribed and 5'untranslated sequences
which are involved in initiation of transcription and translation,
respectively, such as TATA box, capping sequence, CAAT sequence,
and the like. More specifically, 5'untranscribed regulatory
sequences comprise a promoter region which includes a promoter
sequence for transcriptional control of the functionally linked
gene. Regulatory sequences may also comprise enhancer sequences or
upstream activator sequences.
[0073] According to the invention, a nucleic acid may furthermore
be present in combination with another nucleic acid which codes for
a peptide controlling secretion of the protein or peptide encoded
by said nucleic acid from a host cell. According to the invention,
a nucleic acid may also be present in combination with another
nucleic acid which codes for a peptide causing the encoded protein
or peptide to be anchored on the cell membrane of the host cell or
compartmentalized into particular organelles of said cell.
Similarly, a combination with a nucleic acid is possible which
represents a reporter gene or any "tag".
[0074] In a preferred embodiment, a recombinant nucleic acid
molecule is according to the invention a vector, where appropriate
with a promoter, which controls expression of a nucleic acid, for
example a nucleic acid coding for a tumor-associated antigen
identified according to the invention. The term "vector" is used
here in its most general meaning and comprises any intermediary
vehicle for a nucleic acid which enables said nucleic acid, for
example, to be introduced into prokaryotic and/or eukaryotic cells
and, where appropriate, to be integrated into a genome. Vectors of
this kind are preferably replicated and/or expressed in the cells.
An intermediary vehicle may be adapted, for example, to the use in
electroporation, in bombardment with microprojectiles, in liposomal
administration, in the transfer with the aid of agrobacteria or in
insertion via DNA or RNA viruses. Vectors comprise plasmids,
phagemids, bacteriophages or viral genomes.
[0075] The nucleic acids coding for a tumor-associated antigen
identified according to the invention may be used for transfection
of host cells. Nucleic acids here mean both recombinant DNA and
RNA. Recombinant RNA may be prepared by in-vitro transcription of a
DNA template. Furthermore, it may be modified by stabilizing
sequences, capping and polyadenylation prior to application.
[0076] According to the invention, the term "host cell" relates to
any cell which can be transformed or transfected with an exogenous
nucleic acid. The term "host cells" comprises according to the
invention prokaryotic (e.g. E. coli) or eukaryotic cells (e.g.
dendritic cells, B cells, CHO cells, COS cells, K562 cells, yeast
cells and insect cells). Particular preference is given to
mammalian cells such as cells from humans, mice, hamsters, pigs,
goats, primates. The cells may be derived from a multiplicity of
tissue types and comprise primary cells and cell lines. Specific
examples comprise keratinocytes, peripheral blood leukocytes, stem
cells of the bone marrow and embryonic stem cells. In further
embodiments, the host cell is an antigen-presenting cell, in
particular a dendritic cell, monocyte or a macrophage. A nucleic
acid may be present in the host cell in the form of a single copy
or of two or more copies and, in one embodiment, is expressed in
the host cell.
[0077] According to the invention, the term "expression" is used in
its most general meaning and comprises the production of RNA or of
RNA and protein. It also comprises partial expression of nucleic
acids. Furthermore, expression may be carried out transiently or
stably. Preferred expression systems in mammalian cells comprise
pcDNA3.1 and pRc/CMV (Invitrogen, Carlsbad, Calif.), which contain
a selectable marker such as a gene imparting resistance to G418
(and thus enabling stably transfected cell lines to be selected)
and the enhancer-promoter sequences of cytomegalovirus (CMV).
[0078] In those cases of the invention in which a MHC molecule
presents a tumor-associated antigen or a part thereof, an
expression vector may also comprise a nucleic acid sequence coding
for said MHC molecule. The nucleic acid sequence coding for the MHC
molecule may be present on the same expression vector as the
nucleic acid coding for the tumor-associated antigen or the part
thereof, or both nucleic acids may be present on different
expression vectors. In the latter case, the two expression vectors
may be cotransfected into a cell. If a host cell expresses neither
the tumor-associated antigen or the part thereof nor the MHC
molecule, both nucleic acids coding therefor may be transfected
into the cell either on the same expression vector or on different
expression vectors. If the cell already expresses the MHC molecule,
only the nucleic acid sequence coding for the tumor-associated
antigen or the part thereof can be transfected into the cell.
[0079] The invention also comprises kits for amplification of a
nucleic acid coding for a tumor-associated antigen. Such kits
comprise, for example, a pair of amplification primers which
hybridize to the nucleic acid coding for the tumor-associated
antigen. The primers preferably comprise a sequence of 6-50, in
particular 10-30, 15-30 and 20-30 contiguous nucleotides of the
nucleic acid and are nonoverlapping, in order to avoid the
formation of primer dimers. One of the primers will hybridize to
one strand of the nucleic acid coding for the tumor-associated
antigen, and the other primer will hybridize to the complementary
strand in an arrangement which allows amplification of the nucleic
acid coding for the tumor-associated antigen.
[0080] "Antisense molecules" or "antisense nucleic acids" may be
used for regulating, in particular reducing, expression of a
nucleic acid. The term "antisense molecule" or "antisense nucleic
acid" refers according to the invention to an oligonucleotide which
is an oligoribonucleotide, oligodeoxyribonucleotide, modified
oligoribonucleotide or modified oligo-deoxyribonucleotide and which
hybridizes under physiological conditions to DNA comprising a
particular gene or to mRNA of said gene, thereby inhibiting
transcription of said gene and/or translation of said mRNA.
According to the invention, an "antisense molecule" also comprises
a construct which contains a nucleic acid or a part thereof in
reverse orientation with respect to its natural promoter. An
antisense transcript of a nucleic acid or of a part thereof may
form a duplex with the naturally occurring mRNA specifying the
enzyme and thus prevent accumulation of or translation of the mRNA
into the active enzyme. Another possibility is the use of ribozymes
for inactivating a nucleic acid. Antisense oligonucleotides
preferred according to the invention have a sequence of 6-50, in
particular 10-30, 15-30 and 20-30, contiguous nucleotides of the
target nucleic acid and preferably are fully complementary to the
target nucleic acid or to a part thereof.
[0081] In preferred embodiments, the antisense oligonucleotide
hybridizes with an N-terminal or 5' upstream site such as a
translation initiation site, transcription initiation site or
promoter site. In further embodiments, the antisense
oligonucleotide hybridizes with a 3'untranslated region or mRNA
splicing site.
[0082] In one embodiment, an oligonucleotide of the invention
consists of ribonucleotides, deoxyribonucleotides or a combination
thereof, with the 5' end of one nucleotide and the 3' end of
another nucleotide being linked to one another by a phosphodiester
bond. These oligonucleotides may be synthesized in the conventional
manner or produced recombinantly.
[0083] In preferred embodiments, an oligonucleotide of the
invention is a "modified" oligonucleotide. Here, the
oligonucleotide may be modified in very different ways, without
impairing its ability to bind its target, in order to increase, for
example, its stability or therapeutic efficacy. According to the
invention, the term "modified oligonucleotide" means an
oligonucleotide in which (i) at least two of its nucleotides are
linked to one another by a synthetic internucleoside bond (i.e. an
internucleoside bond which is not a phosphodiester bond) and/or
(ii) a chemical group which is usually not found in nucleic acids
is covalently linked to the oligonucleotide. Preferred synthetic
internucleoside bonds are phosphorothioates, alkyl phosphonates,
phosphorodithioates, phosphate esters, alkyl phosphonothioates,
phosphoramidates, carbamates, carbonates, phosphate triesters,
acetamidates, carboxymethyl esters and peptides.
[0084] The term "modified oligonucleotide" also comprises
oligonucleotides having a covalently modified base and/or sugar.
"Modified oligonucleotides" comprise, for example, oligonucleotides
with sugar residues which are covalently bound to low molecular
weight organic groups other than a hydroxyl group at the 3'
position and a phosphate group at the 5' position. Modified
oligonucleotides may comprise, for example, a 2'-O-alkylated ribose
residue or another sugar instead of ribose, such as arabinose.
[0085] It is to be understood that all embodiments described above
with respect to oligonucleotides may also apply to
polynucleotides.
[0086] By "small interfering RNA" or "siRNA" as used herein is
meant an isolated RNA molecule, preferably greater than 10
nucleotides in length, more preferably greater than 15 nucleotides
in length, and most preferably 18, 19, 20, 21, 22, 23, 24, 25, 26,
27, 28, 29, or 30 nucleotides in length that is used to identify a
target gene or mRNA to be degraded. A range of 19-25 nucleotides is
the most preferred size for siRNAs.
[0087] siRNA according to the invention can comprise partially
purified RNA, substantially pure RNA, synthetic RNA, or
recombinantly produced RNA, as well as altered RNA that differs
from naturally-occurring RNA by the addition, deletion,
substitution and/or alteration of one or more nucleotides. Such
alterations can include addition of non-nucleotide material, such
as to the end(s) of the siRNA or to one or more internal
nucleotides of the siRNA; modifications that make the siRNA
resistant to nuclease digestion (e. g., the use of 2'-substituted
ribonucleotides or modifications to the sugar-phosphate backbone);
or the substitution of one or more nucleotides in the siRNA with
deoxyribonucleotides. Furthermore, siRNA may be modified to
increase the stability thereof as described above for modified
oligonucleotides, in particular by introducing one or more
phosphorothioate linkages.
[0088] One or both strands of the siRNA can also comprise a
3'-overhang. As used herein, a "3'-overhang" refers to at least one
unpaired nucleotide extending from the 3'-end of an RNA strand.
Thus in one embodiment, the siRNA comprises at least one
3'-overhang of from 1 to about 6 nucleotides (which includes
ribonucleotides or deoxynucleotides) in length, preferably from 1
to about nucleotides in length, more preferably from 1 to about 4
nucleotides in length, and particularly preferably from about 2 to
about 4 nucleotides in length. In the embodiment in which both
strands of the siRNA molecule comprise a 3'-overhang, the length of
the overhangs can be the same or different for each strand. In a
most preferred embodiment, the 3'-overhang is present on both
strands of the siRNA, and is 2 nucleotides in length. For example,
each strand of the siRNA of the invention can comprise 3'-overhangs
of dideoxythymidylic acid ("TT") or diuridylic acid ("uu").
[0089] In order to enhance the stability of the siRNA, the
3'-overhangs can be also stabilized against degradation. In one
embodiment, the overhangs are stabilized by including purine
nucleotides, such as adenosine or guanosine nucleotides.
Alternatively, substitution of pyrimidine nucleotides by modified
analogues, e.g., substitution of uridine nucleotides in the
3'-overhangs with 2'-deoxythymidine, is tolerated and does not
affect the efficiency of RNAi degradation. In particular, the
absence of a 2'-hydroxyl in the 2'-deoxythymidine significantly
enhances the nuclease resistance of the 3'-overhang in tissue
culture medium.
[0090] The sense and antisense strands of the siRNA can comprise
two complementary, single-stranded RNA molecules or can comprise a
single molecule in which two complementary portions are base-paired
and are covalently linked by a single-stranded "hairpin" area. That
is, the sense region and antisense region can be covalently
connected via a linker molecule. The linker molecule can be a
polynucleotide or non-nucleotide linker. Without wishing to be
bound by any theory, it is believed that the hairpin area of the
latter type of siRNA molecule is cleaved intracellularly by the
"Dicer" protein (or its equivalent) to form a siRNA of two
individual base-paired RNA molecules.
[0091] As used herein, "target mRNA" refers to an RNA molecule that
is a target for downregulation.
[0092] siRNA can be expressed from pol III expression vectors
without a change in targeting site, as expression of RNAs from pol
III promoters is only believed to be efficient when the first
transcribed nucleotide is a purine.
[0093] siRNA according to the invention can be targeted to any
stretch of approximately 19-25 contiguous nucleotides in any of the
target mRNA sequences (the "target sequence"). Techniques for
selecting target sequences for siRNA are given, for example, in
Tuschl T. et al., "The siRNA User Guide", revised Oct. 11, 2002,
the entire disclosure of which is herein incorporated by reference.
"The siRNA User Guide" is available on the world wide web at a
website maintained by Dr. Thomas Tuschl, Laboratory of RNA
Molecular Biology, Rockefeller University, New York, USA, and can
be found by accessing the website of the Rockefeller University and
searching with the keyword "siRNA". Thus, the sense strand of the
present siRNA comprises a nucleotide sequence substantially
identical to any contiguous stretch of about 19 to about 25
nucleotides in the target mRNA.
[0094] Generally, a target sequence on the target mRNA can be
selected from a given cDNA sequence corresponding to the target
mRNA, preferably beginning 50 to 100 nt downstream (i.e., in the
3'-direction) from the start codon. The target sequence can,
however, be located in the 5'- or 3'-untranslated regions, or in
the region nearby the start codon.
[0095] siRNA can be obtained using a number of techniques known to
those of skill in the art. For example, siRNA can be chemically
synthesized or recombinantly produced using methods known in the
art, such as the Drosophila in vitro system described in U.S.
published application 2002/0086356 of Tuschl et al., the entire
disclosure of which is herein incorporated by reference.
[0096] Preferably, siRNA is chemically synthesized using
appropriately protected ribonucleoside phosphoramidites and a
conventional DNA/RNA synthesizer. siRNA can be synthesized as two
separate, complementary RNA molecules, or as a single RNA molecule
with two complementary regions.
[0097] Alternatively, siRNA can also be expressed from recombinant
circular or linear DNA plasmids using any suitable promoter. Such
embodiments are included according to the present invention when
reference is made herein to the administration of siRNA or the
incorporation of siRNA into pharmaceutical compositions. Suitable
promoters for expressing siRNA of the invention from a plasmid
include, for example, the U6 or H1 RNA pol III promoter sequences
and the cytomegalovirus promoter.
[0098] Selection of other suitable promoters is within the skill in
the art. The recombinant plasmids of the invention can also
comprise inducible or regulatable promoters for expression of the
siRNA in a particular tissue or in a particular intracellular
environment.
[0099] The siRNA expressed from recombinant plasmids can either be
isolated from cultured cell expression systems by standard
techniques, or can be expressed intracellularly. The use of
recombinant plasmids to deliver siRNA to cells in vivo is discussed
in more detail below. siRNA can be expressed from a recombinant
plasmid either as two separate, complementary RNA molecules, or as
a single RNA molecule with two complementary regions.
[0100] Selection of plasmids suitable for expressing siRNA, methods
for inserting nucleic acid sequences for expressing the siRNA into
the plasmid, and methods of delivering the recombinant plasmid to
the cells of interest are within the skill in the art.
[0101] siRNA can also be expressed from recombinant viral vectors
intracellularly in vivo. The recombinant viral vectors comprise
sequences encoding the siRNA and any suitable promoter for
expressing the siRNA sequences. The recombinant viral vectors can
also comprise inducible or regulatable promoters for expression of
the siRNA in a particular tissue or in a particular intracellular
environment. siRNA can be expressed from a recombinant viral vector
either as two separate, complementary RNA molecules, or as a single
RNA molecule with two complementary regions.
[0102] The term "peptide" comprises oligo- and polypeptides and
refers to substances comprising two or more, preferably 3 or more,
preferably 4 or more, preferably 6 or more, preferably 8 or more,
preferably 10 or more, preferably 13 or more, preferably 16 more,
preferably 21 or more and up to preferably 8, 10, 20, 30, 40 or 50,
in particular 100 amino acids joined covalently by peptide bonds.
The term "protein" refers to large peptides, preferably to peptides
with more than 100 amino acid residues, but in general the terms
"peptides" and "proteins" are synonyms and are used interchangeably
herein.
[0103] Preferably, the proteins and peptides described according to
the invention have been isolated. The terms "isolated protein" or
"isolated peptide" mean that the protein or peptide has been
separated from its natural environment. An isolated protein or
peptide may be in an essentially purified state. The term
"essentially purified" means that the protein or peptide is
essentially free of other substances with which it is associated in
nature or in vivo.
[0104] Such proteins and peptides may be used, for example, in
producing antibodies and in an immunological or diagnostic assay or
as therapeutics. Proteins and peptides described according to the
invention may be isolated from biological samples such as tissue or
cell homogenates and may also be expressed recombinantly in a
multiplicity of pro- or eukaryotic expression systems.
[0105] For the purposes of the present invention, "derivatives" of
a protein or peptide or of an amino acid sequence comprise amino
acid insertion variants, amino acid deletion variants and/or amino
acid substitution variants.
[0106] Amino acid insertion variants comprise amino- and/or
carboxy-terminal fusions and also insertions of single or two or
more amino acids in a particular amino acid sequence. In the case
of amino acid sequence variants having an insertion, one or more
amino acid residues are inserted into a particular site in an amino
acid sequence, although random insertion with appropriate screening
of the resulting product is also possible. Amino acid deletion
variants are characterized by the removal of one or more amino
acids from the sequence. Amino acid substitution variants are
characterized by at least one residue in the sequence being removed
and another residue being inserted in its place. Preference is
given to the modifications being in positions in the amino acid
sequence which are not conserved between homologous proteins or
peptides and/or to replacing amino acids with other ones having
similar properties such as hydrophobicity, hydrophilicity,
electronegativity, volume of the side chain and the like
(conservative substitution). Conservative substitutions, for
example, relate to the exchange of one amino acid with another
amino acid listed below in the same group as the amino acid to be
substituted: [0107] 1. small aliphatic, nonpolar or slightly polar
residues: Ala, Ser, Thr (Pro, Gly) [0108] 2. negatively charged
residues and their amides: Asn, Asp, Glu, Gln [0109] 3. positively
charged residues: His, Arg, Lys [0110] 4. large aliphatic, nonpolar
residues: Met, Leu, Ile, Val (Cys) [0111] 5. large aromatic
residues: Phe, Tyr, Trp.
[0112] Owing to their particular part in protein architecture,
three residues are shown in brackets. Gly is the only residue
without a side chain and thus imparts flexibility to the chain. Pro
has an unusual geometry which greatly restricts the chain. Cys can
form a disulfide bridge.
[0113] The amino acid variants described above may be readily
prepared with the aid of known peptide synthesis techniques such
as, for example, by solid phase synthesis (Merrifield, 1964) and
similar methods or by recombinant DNA manipulation. The
manipulation of DNA sequences for preparing proteins and peptides
having substitutions, insertions or deletions, is described in
detail in Sambrook et al. (1989), for example.
[0114] According to the invention, "derivatives" of proteins and
peptides also comprise single or multiple substitutions, deletions
and/or additions of any molecules associated with the protein or
peptide, such as carbohydrates, lipids and/or proteins or
peptides.
[0115] The term "derivative" also extends to all functional
chemical equivalents of said proteins and peptides.
[0116] According to the invention, a part or fragment of a
tumor-associated antigen preferably has a functional property of
the protein or peptide from which it has been derived. Such
functional properties comprise the interaction with antibodies, the
interaction with other peptides or proteins, the selective binding
of nucleic acids and an enzymatic activity. A particular property
is the ability to form a complex with MHC molecules and, where
appropriate, generate an immune response, preferably by stimulating
cytotoxic or T helper cells. A part or fragment of a
tumor-associated antigen of the invention preferably comprises a
sequence of at least 6, in particular at least 8, at least 10, at
least 12, at least 15, at least 20, at least 30 or at least 50,
consecutive amino acids of the tumor-associated antigen. A part or
fragment of a tumor-associated antigen of the invention preferably
comprises a sequence of up to 8, in particular up to 10, up to 12,
up to 15, up to 20, up to 30 or up to 55, consecutive amino acids
of the tumor-associated antigen. A part or fragment of a
tumor-associated antigen is preferably a part of the
tumor-associated antigen which corresponds to the non-transmembrane
portion, in particular the extracellular portion of the antigen, or
is comprised thereof.
[0117] Preferred parts or fragments of a tumor-associated antigen
according to the invention are in particular suitable for the
stimulation of cytotoxic T-lymphocytes in vivo but also for the
production of expanded and stimulated T-lymphocytes for the
therapeutic adoptive transfer ex vivo.
[0118] A part or a fragment of a nucleic acid coding for a
tumor-associated antigen relates according to the invention to the
part of the nucleic acid, which codes at least for the
tumor-associated antigen and/or for a part or a fragment of said
tumor-associated antigen, as defined above. A part or fragment of a
nucleic acid coding for a tumor-associated antigen is preferably
that part of the nucleic acid corresponding to the open reading
frame.
[0119] According to the invention, particular embodiments ought to
involve providing "dominant negative" proteins or peptides derived
from tumor-associated antigens. A dominant negative protein or
peptide is an inactive protein or peptide variant which, by way of
interacting with the cellular machinery, displaces an active
protein or peptide from its interaction with the cellular machinery
or which competes with the active protein or peptide, thereby
reducing the effect of said active protein.
[0120] Antisera which contain specific antibodies specifically
binding to the target protein can be prepared by various standard
processes; see, for example, "Monoclonal Antibodies: A Practical
Approach" by Philip Shepherd, Christopher Dean ISBN 0-19-963722-9;
"Antibodies: A Laboratory Manual" by Ed Harlow, David Lane, ISBN:
0879693142 and "Using Antibodies: A Laboratory Manual: Portable
Protocol NO" by Edward Harlow, David Lane, Ed Harlow ISBN
0879695447. Thereby it is also possible to generate affine and
specific antibodies which recognize complex membrane proteins in
their native form (Azorsa et al., J. Immunol. Methods 229: 35-48,
1999; Anderson et al., J. Immunol. 143: 1899-1904, 1989; Gardsvoll,
J. Immunol. Methods 234: 107-116, 2000). This is in particular
relevant for the preparation of antibodies which are to be used
therapeutically, but also for many diagnostic applications. In this
respect, it is possible to immunize with the whole protein, with
extracellular partial sequences as well as with cells which express
the target molecule in physiologically folded form.
[0121] Monoclonal antibodies are traditionally prepared using the
hybridoma technology. (for technical details see: "Monoclonal
Antibodies: A Practical Approach" by Philip Shepherd, Christopher
Dean ISBN 0-19-963722-9; "Antibodies: A Laboratory Manual" by Ed
Harlow, David Lane ISBN: 0879693142; "Using Antibodies: A
Laboratory Manual: Portable Protocol NO" by Edward Harlow, David
Lane, Ed Harlow ISBN: 0879695447).
[0122] It is known that only a small part of an antibody molecule,
the paratope, is involved in binding of the antibody to its epitope
(cf. Clark, W. R. (1986), The Experimental Foundations of Modern
Immunology, Wiley & Sons, Inc., New York; Roitt, I. (1991),
Essential Immunology, 7th Edition, Blackwell Scientific
Publications, Oxford). The pFc' and Fc regions are, for example,
effectors of the complement cascade but are not involved in antigen
binding. An antibody from which the pFc' region has been
enzymatically removed or which has been produced without the pFc'
region, referred to as F(ab').sub.2 fragment, carries both antigen
binding sites of a complete antibody. Similarly, an antibody from
which the Fc region has been enzymatically removed or which has
been produced without said Fc region, referred to as Fab fragment,
carries one antigen binding site of an intact antibody molecule.
Furthermore, Fab fragments consist of a covalently bound light
chain of an antibody and part of the heavy chain of said antibody,
referred to as Fd. The Fd fragments are the main determinants of
antibody specificity (a single Fd fragment can be associated with
up to ten different light chains, without altering the specificity
of the antibody) and Fd fragments, when isolated, retain the
ability to bind to an epitope.
[0123] Located within the antigen-binding part of an antibody are
complementary-determining regions (CDRs) which interact directly
with the antigen epitope and framework regions (FRs) which maintain
the tertiary structure of the paratope. Both the Fd fragment of the
heavy chain and the light chain of IgG immunoglobulins contain four
framework regions (FR1 to FR4) which are separated in each case by
three complementary-determining regions (CDR1 to CDR3). The CDRs
and, in particular, the CDR3 regions and, still more particularly,
the CDR3 region of the heavy chain are responsible to a large
extent for antibody specificity.
[0124] Non-CDR regions of a mammalian antibody are known to be able
to be replaced by similar regions of antibodies with the same or a
different specificity, with the specificity for the epitope of the
original antibody being retained. This made possible the
development of "humanized" antibodies in which nonhuman CDRs are
covalently linked to human FR and/or Fc/pFc' regions to produce a
functional antibody.
[0125] As another example, WO 92/04381 describes the production and
use of humanized murine RSV antibodies in which at least part of
the murine FR regions have been replaced with FR regions of a human
origin. Antibodies of this kind, including fragments of intact
antibodies with antigen-binding capability, are often referred to
as "chimeric" antibodies.
[0126] According to the invention, the term "antibody" also
includes F(ab').sub.2, Fab, Fv, and Fd fragments of antibodies,
chimeric antibodies, in which the Fc and/or FR and/or CDR1 and/or
CDR2 and/or light chain-CDR3 regions have been replaced with
homologous human or nonhuman sequences, chimeric
F(ab').sub.2-fragment antibodies in which the FR and/or CDR1 and/or
CDR2 and/or light chain-CDR3 regions have been replaced with
homologous human or nonhuman sequences, chimeric Fab-fragment
antibodies in which the FR and/or CDR1 and/or CDR2 and/or light
chain-CDR3 regions have been replaced with homologous human or
nonhuman sequences, and chimeric Fd-fragment antibodies in which
the FR and/or CDR1 and/or CDR2 regions have been replaced with
homologous human or nonhuman sequences. The term "antibody" also
comprises "single-chain" antibodies.
[0127] The invention also comprises proteins and peptides which
bind specifically to tumor-associated antigens. Binding substances
of this kind may be provided, for example, by degenerate peptide
libraries which may be prepared simply in solution in an
immobilized form or as phage-display libraries. It is likewise
possible to prepare combinatorial libraries of peptides with one or
more amino acids. Libraries of peptoids and nonpeptidic synthetic
residues may also be prepared.
[0128] Antibodies may also be coupled to specific diagnostic
substances for displaying cells and tissues expressing
tumor-associated antigens. They may also be coupled to
therapeutically useful substances.
[0129] Diagnostic substances include any label that functions to:
(i) provide a detectable signal; (ii) interact with a second label
to modify the detectable signal provided by the first or second
label, e.g. FRET (Fluorescence Resonance Energy Transfer); (iii)
affect mobility, e.g. electrophoretic mobility, by charge,
hydrophobicity, shape, or other physical parameters, or (iv)
provide a capture moiety, e.g., affinity, antibody/antigen, or
ionic complexation. Suitable as label are structures, such as
fluorescent labels, luminescent labels, chromophore labels,
radioisotopic labels, isotopic labels, preferably stable isotopic
labels, isobaric labels, enzyme labels, particle labels, in
particular metal particle labels, magnetic particle labels, polymer
particle labels, small organic molecules such as biotin, ligands of
receptors or binding molecules such as cell adhesion proteins or
lectins, label-sequences comprising nucleic acids and/or amino acid
residues which can be detected by use of binding agents, etc.
Diagnostic substances comprise, in a nonlimiting manner, barium
sulfate, iocetamic acid, iopanoic acid, calcium ipodate, sodium
diatrizoate, meglumine diatrizoate, metrizamide, sodium tyropanoate
and radio diagnostic, including positron emitters such as
fluorine-18 and carbon-11, gamma emitters such as iodine-123,
technetium-99m, iodine-131 and indium-111, nuclides for nuclear
magnetic resonance, such as fluorine and gadolinium.
[0130] According to the invention, the term "therapeutically useful
substance" means any molecule which may exert a therapeutic effect.
According to the invention, a therapeutically useful substance is
preferably selectively guided to a cell which expresses one or more
tumor-associated antigens and includes anticancer agents,
radioactive iodine-labeled compounds, toxins, cytostatic or
cytolytic drugs, etc. Anticancer agents comprise, for example,
aminoglutethimide, azathioprine, bleomycin sulfate, busulfan,
carmustine, chlorambucil, cisplatin, cyclophosphamide,
cyclosporine, cytarabidine, dacarbazine, dactinomycin, daunorubin,
doxorubicin, taxol, etoposide, fluorouracil, interferon-.alpha.,
lomustine, mercaptopurine, methotrexate, mitotane, procarbazine
HCl, thioguanine, vinblastine sulfate and vincristine sulfate.
Other anticancer agents are described, for example, in Goodman and
Gilman, "The Pharmacological Basis of Therapeutics", 8th Edition,
1990, McGraw-Hill, Inc., in particular Chapter 52 (Antineoplastic
Agents (Paul Calabresi and Bruce A. Chabner). Toxins may be
proteins such as pokeweed antiviral protein, cholera toxin,
pertussis toxin, ricin, gelonin, abrin, diphtheria exotoxin or
Pseudomonas exotoxin. Toxin residues may also be high
energy-emitting radionuclides such as cobalt-60.
[0131] The term "major histocompatibility complex" or "MHC" relates
to a complex of genes present in all vertebrates. MHC proteins or
molecules are involved in signaling between lymphocytes and antigen
presenting cells in normal immune reactions by binding peptides and
presenting them for recognition by T cell receptors (TCR). MHC
molecules bind peptides within an intracellular processing
compartment and present these peptides on the surface of antigen
presenting cells for recognition by T cells. The human MHC region
also termed HLA is located on chromosome 6 and includes the class I
and class II region. In one preferred embodiment of all aspects of
the invention an MHC molecule is an HLA molecule.
[0132] "Reduce" or "inhibit" as used herein means the ability to
cause an overall decrease, preferably of 20% or greater, more
preferably of 50% or greater, and most preferably of 75% or
greater, in the level, e.g. in the level of protein or mRNA as
compared to a reference sample (e.g., a sample not treated with
siRNA). This reduction or inhibition of RNA or protein expression
can occur through targeted mRNA cleavage or degradation. Assays for
protein expression or nucleic acid expression are known in the art
and include, for example, ELISA, western blot analysis for protein
expression, and northern blotting or RNase protection assays for
RNA.
[0133] The term "patient" means according to the invention a human
being, a nonhuman primate or another animal, in particular a mammal
such as a cow, horse, pig, sheep, goat, dog, cat or a rodent such
as a mouse and rat. In a particularly preferred embodiment, the
patient is a human being.
[0134] "Abnormal expression" means according to the invention that
expression is altered, preferably increased, compared to the state
in a healthy individual.
[0135] According to the invention the term "increased" or
"increased amount" preferably refers to an increase by at least
10%, in particular at least 20%, at least 50% or at least 100%. The
amount of a substance is also increased in a test sample such as a
biological sample compared to a reference sample if it is
detectable in the test sample but absent or not detectable in the
reference sample.
[0136] According to the invention, the term "disease" refers to any
pathological state in which tumor-associated antigens are expressed
or abnormally expressed. "Abnormal expression" means according to
the invention that expression is altered, preferably increased,
compared to the state in a healthy individual. An increase in
expression refers to an increase by at least 10%, in particular at
least 20%, at least 50% or at least 100%. In one embodiment, the
tumor-associated antigen is expressed only in tissue of a diseased
individual, while expression in a healthy individual is repressed.
One example of such a disease is cancer, wherein the term "cancer"
according to the invention comprises leukemias, seminomas,
melanomas, teratomas, lymphomas, neuroblastomas, gliomas, rectal
cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid
cancer, blood cancer, skin cancer, cancer of the brain, cervical
cancer, intestinal cancer, liver cancer, colon cancer, stomach
cancer, intestine cancer, head and neck cancer, gastrointestinal
cancer, lymph node cancer, esophagus cancer, colorectal cancer,
pancreas cancer, ear, nose and throat (ENT) cancer, breast cancer,
prostate cancer, cancer of the uterus, ovarian cancer and lung
cancer and the matastases thereof. Examples thereof are lung
carcinomas, mamma carcinomas, prostate carcinomas, colon
carcinomas, renal cell carcinomas, cervical carcinomas, or
metastases of the cancer types or tumors described above. The term
cancer according to the invention also comprises cancer
metastases.
[0137] By "tumor" is meant an abnormal group of cells or tissue
that grows by a rapid, uncontrolled cellular proliferation and
continues to grow after the stimuli that initiated the new growth
cease. Tumors show partial or complete lack of structural
organization and functional coordination with the normal tissue,
and usually form a distinct mass of tissue, which may be either
benign or malignant.
[0138] By "metastasis" is meant the spread of cancer cells from its
original site to another part of the body. The formation of
metastasis is a very complex process and depends on detachment of
malignant cells from the primary tumor, invasion of the
extracellular matrix, penetration of the endothelial basement
membranes to enter the body cavity and vessels, and then, after
being transported by the blood, infiltration of target organs.
Finally, the growth of a new tumor at the target site depends on
angiogenesis. Tumor metastasis often occurs even after the removal
of the primary tumor because tumor cells or components may remain
and develop metastatic potential. In one embodiment, the term
"metastasis" according to the invention relates to "distant
metastasis" which relates to a metastasis which is remote from the
primary tumor and the regional lymph node system.
[0139] According to the invention, a biological sample may be a
tissue sample, including bodily fluids, and/or a cellular sample
and may be obtained in the conventional manner such as by tissue
biopsy, including punch biopsy, and by taking blood, bronchial
aspirate, sputum, urine, feces or other body fluids. According to
the invention, the term "biological sample" also includes fractions
of biological samples.
[0140] According to the invention, the term "immunoreactive cell"
means a cell which can mature into an immune cell (such as B cell,
T helper cell, or cytolytic T cell) with suitable stimulation.
Immunoreactive cells comprise CD34.sup.+ hematopoietic stem cells,
immature and mature T cells and immature and mature B cells. If
production of cytolytic or T helper cells recognizing a
tumor-associated antigen is desired, the immunoreactive cell is
contacted with a cell expressing a tumor-associated antigen under
conditions which favor production, differentiation and/or selection
of cytolytic T cells and of T helper cells. The differentiation of
T cell precursors into a cytolytic T cell, when exposed to an
antigen, is similar to clonal selection of the immune system.
[0141] The terms "T cell" and "T lymphocyte" are used
interchangeably herein and include T helper cells and cytotoxic T
cells which comprise cytolytic T cells.
[0142] Some therapeutic methods are based on a reaction of the
immune system of a patient, which results in a lysis of
antigen-presenting cells such as cancer cells which present one or
more tumor-associated antigens. In this connection, for example
autologous cytotoxic lymphocytes specific for a complex of a
tumor-associated antigen and an MHC molecule are administered to a
patient having a cellular abnormality. The production of such
cytotoxic T lymphocytes in vitro is known. An example of a method
of differentiating T cells can be found in WO-A-9633265. Generally,
a sample containing cells such as blood cells is taken from the
patient and the cells are contacted with a cell which presents the
complex and which can cause propagation of cytotoxic T lymphocytes
(e.g. dendritic cells). The target cell may be a transfected cell
such as a COS cell. These transfected cells present the desired
complex on their surface and, when contacted with cytotoxic T
lymphocytes, stimulate propagation of the latter. The clonally
expanded autologous cytotoxic T lymphocytes are then administered
to the patient.
[0143] In another method of selecting antigen-specific cytotoxic T
lymphocytes, fluorogenic tetramers of MHC class I molecule/peptide
complexes are used for obtaining specific clones of cytotoxic T
lymphocytes (Altman et al., Science 274:94-96, 1996; Dunbar et al.,
Curr. Biol. 8:413-416, 1998).
[0144] The present invention also includes therapeutic methods
referred to as adoptive transfer (Greenberg, J. Immunol.
136(5):1917, 1986; Riddel et al., Science 257:238, 1992; Lynch et
al., Eur. J. Immunol. 21:1403-1410, 1991; Kast et al., Cell
59:603-614, 1989), wherein cells presenting the desired complex
(e.g. dendritic cells) are combined with cytotoxic T lymphocytes of
the patient to be treated, resulting in a propagation of specific
cytotoxic T lymphocytes. The propagated cytotoxic T lymphocytes are
then administered to a patient having a cellular anomaly
characterized by particular abnormal cells presenting the specific
complex. The cytotoxic T lymphocytes then lyse the abnormal cells,
thereby achieving a desired therapeutic effect.
[0145] Furthermore, cells presenting the desired complex (e.g.
dendritic cells) may be combined with cytotoxic T lymphocytes of
healthy individuals or another species (e.g. mouse) which may
result in propagation of specific cytotoxic T lymphocytes with high
affinity. The high affinity T cell receptor of these propagated
specific T lymphocytes may be cloned and optionally humanized to a
different extent, and the T cell receptors thus obtained then
transduced via gene transfer, for example using retroviral vectors,
into T cells of patients. Adoptive transfer may then be carried out
using these genetically altered T lymphocytes (Stanislawski et al.,
Nat Immunol. 2:962-70, 2001; Kessels et al., Nat Immunol. 2:957-61,
2001).
[0146] Adoptive transfer is not the only form of therapy which can
be applied according to the invention. Cytotoxic T lymphocytes may
also be generated in vivo in a manner known per se. One method uses
nonproliferative cells expressing the complex. The cells used here
will be those which usually express the complex, such as irradiated
tumor cells or cells transfected with one or both genes necessary
for presentation of the complex (i.e. the antigenic peptide and the
presenting MHC molecule). Another preferred form is the
introduction of the tumor-associated antigen in the form of
recombinant RNA which may be introduced into cells by liposomal
transfer or by electroporation, for example. The resulting cells
present the complex of interest and are recognized by autologous
cytotoxic T lymphocytes which then propagate.
[0147] A similar effect can be achieved by combining the
tumor-associated antigen or a fragment thereof with an adjuvant in
order to make incorporation into antigen-presenting cells in vivo
possible. The tumor-associated antigen or a fragment thereof may be
represented as protein, as DNA (e.g. within a vector) or as RNA.
The tumor-associated antigen is processed to produce a peptide
partner for the MHC molecule, while a fragment thereof may be
presented without the need for further processing. The latter is
the case in particular, if these can bind to MHC molecules.
Preference is given to administration forms in which the complete
antigen is processed in vivo by a dendritic cell, since this may
also produce T helper cell responses which are needed for an
effective immune response (Ossendorp et al., Immunol Lett. 74:75-9,
2000; Ossendorp et al., J. Exp. Med. 187:693-702, 1998). In
general, it is possible to administer an effective amount of the
tumor-associated antigen to a patient by intradermal injection, for
example. However, injection may also be carried out intranodally
into a lymph node (Maloy et al., Proc Natl Acad Sci USA
98:3299-303, 2001).
[0148] The pharmaceutical compositions and methods of treatment
described according to the invention may also be used for
immunization or vaccination to therapeutically treat or prevent a
disease described herein. According to the invention, the terms
"immunization" or "vaccination" preferably relate to an increase in
or activation of an immune response to an antigen. It is possible
to use animal models for testing an immunizing effect on cancer by
using a tumor-associated antigen or a nucleic acid coding therefor.
For example, human cancer cells may be introduced into a mouse to
generate a tumor, and one or more nucleic acids coding for
tumor-associated antigens may be administered. The effect on the
cancer cells (for example reduction in tumor size) may be measured
as a measure for the effectiveness of an immunization by the
nucleic acid.
[0149] As part of the composition for an immunization or a
vaccination, preferably one or more tumor-associated antigens or
stimulating fragments thereof are administered together with one or
more adjuvants for inducing an immune response or for increasing an
immune response. An adjuvant is a substance which is incorporated
into the antigen or administered together with the latter and which
enhances the immune response. Adjuvants may enhance the immune
response by providing an antigen reservoir (extracellularly or in
macrophages), activating macrophages and/or stimulating particular
lymphocytes. Adjuvants are known and comprise in a nonlimiting way
monophosphoryl lipid A (MPL, SmithKline Beecham), saponins such as
QS21 (SmithKline Beecham), DQS21 (SmithKline Beecham; WO 96/33739),
QS7, QS17, QS18 and QS-L1 (So et al., Mol. Cells 7:178-186, 1997),
incomplete Freund's adjuvant, complete Freund's adjuvant, vitamin
E, montanide, alum, CpG oligonucleotides (cf. Kreig et al., Nature
374:546-9, 1995) and various water-in-oil emulsions prepared from
biologically degradable oils such as squalene and/or tocopherol.
Preferably, the peptides are administered in a mixture with
DQS21/MPL. The ratio of DQS21 to MPL is typically about 1:10 to
10:1, preferably about 1:5 to 5:1 and in particular about 1:1. For
administration to humans, a vaccine formulation typically contains
DQS21 and MPL in a range from about 1 .mu.g to about 100 .mu.g.
[0150] Other substances which stimulate an immune response of the
patient may also be administered. It is possible, for example, to
use cytokines in a vaccination, owing to their regulatory
properties on lymphocytes. Such cytokines comprise, for example,
interleukin-12 (IL-12) which was shown to increase the protective
actions of vaccines (cf. Science 268:1432-1434, 1995), GM-CSF and
IL-18.
[0151] There are a number of compounds which enhance an immune
response and which therefore may be used in a vaccination. Said
compounds comprise costimulating molecules provided in the form of
proteins or nucleic acids such as B7-1 and B7-2 (CD80 and CD86,
respectively).
[0152] The invention also provides for administration of nucleic
acids, proteins or peptides. Proteins and peptides may be
administered in a manner known per se. In one embodiment, nucleic
acids are administered by ex vivo methods, i.e. by removing cells
from a patient, genetic modification of said cells in order to
incorporate a tumor-associated antigen and reintroduction of the
altered cells into the patient. This generally comprises
introducing a functional copy of a gene into the cells of a patient
in vitro and reintroducing the genetically altered cells into the
patient. The functional copy of the gene is under the functional
control of regulatory elements which allow the gene to be expressed
in the genetically altered cells. Transfection and transduction
methods are known to the skilled worker. The invention also
provides for administering nucleic acids in vivo by using vectors
such as viruses and target-controlled liposomes. If according to
the invention reference is made to the administration or
incorporation into pharmaceutical compositions of nucleic acids
this includes embodiments wherein the nucleic acid is present in
such vectors.
[0153] In a preferred embodiment, a virus or viral vector for
administering a nucleic acid coding for a tumor-associated antigen
is selected from the group consisting of adenoviruses,
adeno-associated viruses, pox viruses, including vaccinia virus and
attenuated pox viruses, Semliki Forest virus, retroviruses, Sindbis
virus and Ty virus-like particles. Particular preference is given
to adenoviruses and retroviruses. The retroviruses are typically
replication-deficient (i.e. they are incapable of generating
infectious particles).
[0154] Methods of introducing nucleic acids into cells in vitro or
in vivo comprise transfection of nucleic acid calcium phosphate
precipitates, transfection of nucleic acids associated with DEAE,
transfection or infection with the above viruses carrying the
nucleic acids of interest, liposome-mediated transfection, and the
like. In particular embodiments, preference is given to directing
the nucleic acid to particular cells. In such embodiments, a
carrier used for administering a nucleic acid to a cell (e.g. a
retrovirus or a liposome) may have a bound target control molecule.
For example, a molecule such as an antibody specific for a surface
membrane protein on the target cell or a ligand for a receptor on
the target cell may be incorporated into or attached to the nucleic
acid carrier. Preferred antibodies comprise antibodies which bind
selectively a tumor-associated antigen. If administration of a
nucleic acid via liposomes is desired, proteins binding to a
surface membrane protein associated with endocytosis may be
incorporated into the liposome formulation in order to make target
control and/or uptake possible. Such proteins comprise capsid
proteins or fragments thereof which are specific for a particular
cell type, antibodies to proteins which are internalized, proteins
addressing an intracellular site, and the like.
[0155] The therapeutic compositions of the invention may be
administered in pharmaceutically compatible preparations. Such
preparations may usually contain pharmaceutically compatible
concentrations of salts, buffer substances, preservatives,
carriers, supplementing immunity-enhancing substances such as
adjuvants, e.g. CpG oligonucleotides, cytokines, chemokines,
saponin, GM-CSF and/or RNA and, where appropriate, other
therapeutically active compounds.
[0156] The therapeutically active compounds of the invention may be
administered via any conventional route, including by injection or
infusion. The administration may be carried out, for example,
orally, intravenously, intraperitonealy, intramuscularly,
subcutaneously or transdermally. Preferably, antibodies are
therapeutically administered by way of a lung aerosol. Antisense
nucleic acids are preferably administered by slow intravenous
administration.
[0157] The compositions of the invention are administered in
effective amounts. An "effective amount" refers to the amount which
achieves a desired reaction or a desired effect alone or together
with further doses. In the case of treatment of a particular
disease or of a particular condition characterized by expression of
one or more tumor-associated antigens, the desired reaction
preferably relates to inhibition of the course of the disease. This
comprises slowing down the progress of the disease and, in
particular, interrupting or reversing the progress of the disease.
The desired reaction in a treatment of a disease or of a condition
may also be delay of the onset or a prevention of the onset of said
disease or said condition. According to the invention, a diagnosis
or treatment of cancer may also include the diagnosis or treatment
of cancer metastases which have already formed or will form.
According to the invention, the term "treatment" comprises
therapeutic and prophylactic treatment, i.e. prevention.
[0158] An effective amount of a composition of the invention will
depend on the condition to be treated, the severeness of the
disease, the individual parameters of the patient, including age,
physiological condition, size and weight, the duration of
treatment, the type of an accompanying therapy (if present), the
specific route of administration and similar factors.
[0159] The pharmaceutical compositions of the invention are
preferably sterile and contain an effective amount of the
therapeutically active substance to generate the desired reaction
or the desired effect.
[0160] The doses administered of the compositions of the invention
may depend on various parameters such as the type of
administration, the condition of the patient, the desired period of
administration, etc. In the case that a reaction in a patient is
insufficient with an initial dose, higher doses (or effectively
higher doses achieved by a different, more localized route of
administration) may be used.
[0161] Generally, doses of the tumor-associated antigen of from 1
ng to 1 mg, preferably from 10 ng to 100 .mu.g, are formulated and
administered for a treatment or for generating or increasing an
immune response. If the administration of nucleic acids (DNA and
RNA) coding for tumor-associated antigens is desired, doses of from
1 ng to 0.1 mg are formulated and administered.
[0162] The pharmaceutical compositions of the invention are
generally administered in pharmaceutically compatible amounts and
in pharmaceutically compatible compositions. The term
"pharmaceutically compatible" refers to a nontoxic material which
does not interact with the action of the active component of the
pharmaceutical composition. Preparations of this kind may usually
contain salts, buffer substances, preservatives, carriers and,
where appropriate, other therapeutically active compounds. When
used in medicine, the salts should be pharmaceutically compatible.
However, salts which are not pharmaceutically compatible may used
for preparing pharmaceutically compatible salts and are included in
the invention. Pharmacologically and pharmaceutically compatible
salts of this kind comprise in a nonlimiting way those prepared
from the following acids: hydrochloric, hydrobromic, sulfuric,
nitric, phosphoric, maleic, acetic, salicylic, citric, formic,
malonic, succinic acids, and the like. Pharmaceutically compatible
salts may also be prepared as alkali metal salts or alkaline earth
metal salts, such as sodium salts, potassium salts or calcium
salts.
[0163] A pharmaceutical composition of the invention may comprise a
pharmaceutically compatible carrier. According to the invention,
the term "pharmaceutically compatible carrier" refers to one or
more compatible solid or liquid fillers, diluents or encapsulating
substances, which are suitable for administration to humans. The
term "carrier" refers to an organic or inorganic component, of a
natural or synthetic nature, in which the active component is
combined in order to facilitate application. The components of the
pharmaceutical composition of the invention are usually such that
no interaction occurs which substantially impairs the desired
pharmaceutical efficacy.
[0164] The pharmaceutical compositions of the invention may contain
suitable buffer substances such as acetic acid in a salt, citric
acid in a salt, boric acid in a salt and phosphoric acid in a
salt.
[0165] The pharmaceutical compositions may, where appropriate, also
contain suitable preservatives such as benzalkonium chloride,
chlorobutanol, paraben and thimerosal.
[0166] The pharmaceutical compositions are usually provided in a
uniform dosage form and may be prepared in a manner known per se.
Pharmaceutical compositions of the invention may be in the form of
capsules, tablets, lozenges, solutions, suspensions, syrups,
elixirs or in the form of an emulsion, for example.
[0167] Compositions suitable for parenteral administration usually
comprise a sterile aqueous or nonaqueous preparation of the active
compound, which is preferably isotonic to the blood of the
recipient. Examples of compatible carriers and solvents are Ringer
solution and isotonic sodium chloride solution. In addition,
usually sterile, fixed oils are used as solution or suspension
medium.
[0168] The present invention is described in detail by the figures
and examples below, which are used only for illustration purposes
and are not meant to be limiting. Owing to the description and the
examples, further embodiments which are likewise included in the
invention are accessible to the skilled worker.
FIGURES
[0169] FIG. 1. ISC-468 mRNA expression [0170] A. RT-PCR
investigations with ISC-468-specific primers showed no significant
expression within all tested normal tissues except placenta. [0171]
B. ISC-468 mRNA expression in head and neck, liver, kidney and
colon carcinomas. [0172] C. ISC-468 mRNA expression in breast,
ovarian and stomach carcinomas.
[0173] FIG. 2. Quantitative PCR analysis of ISC-468 mRNA expression
in normal control tissues and breast cancers
[0174] Real-time PCR investigation with ISC-468-specific primers
showed selective mRNA expression in normal testis, placenta,
stomach and PBMC, and in all breast carcinoma biopsies.
[0175] FIG. 3. Specific ISC-507 expression in normal testis and
prostate carcinoma
[0176] RT-PCR analysis with gene-specific ISC-507 primers shows
cDNA amplification exclusively in normal testis (A) and in prostate
carcinoma biopsies (B).
[0177] FIG. 4. Quantitative expression of ISC-507
[0178] Quantitative RT-PCR with ISC-507-specific primers showed
selective expression in testis, lymph node and prostate samples and
prostate cancer samples.
[0179] FIG. 5. ISC-466 expression in normal testis and various
tumor samples
[0180] RT-PCR analysis with ISC-466-specific primers showed no
expression within normal tissues except placenta (A), but
expression in head and neck carcinoma biopsies and in kidney
carcinoma biopsies (B). Distinct expression was also detected in
breast and lung carcinoma cell lines, as well as in ovarian
carcinoma cell lines (C and D).
[0181] FIG. 6. ISC-518 mRNA expression
[0182] RT-PCR analysis with ISC-518-specific primers showed no
expression within normal tissues except testis.
[0183] FIG. 7. Quantitative expression of ISC-518
[0184] Quantitative RT-PCR showed high and selective expression in
normal testis and in one liver carcinoma-pool.
[0185] FIG. 8. ISC-477 expression in normal and tumor tissues
[0186] RT-PCR investigations with ISC-477-specific primers showed
selective expression in placenta and ovary normal tissue (A) and
high expression in investigated stomach carcinomas (B), breast,
colon and lung carcinomas (C), as well as in ovarian and pancreas
carcinoma samples (D).
[0187] FIG. 9. ISC-489 mRNA expression
[0188] RT-PCR investigations with ISC-489-specific primers showed
selective expression in placenta control tissue and additionally
various levels of expression in lung carcinoma samples (A, C),
stomach carcinomas (B, C), head and neck tumors (C) and liver
carcinoma samples (C).
[0189] FIG. 10. ISC-461 expression in normal testis and various
tumor samples
[0190] RT-PCR investigations with ISC-461-specific primers showed
selective expression in placenta control tissue and additionally
various levels of expression in breast carcinomas and melanomas
(B), as well as in breast carcinoma, lung carcinoma and melanoma
cell lines (C) and ovarian carcinoma cell lines (D).
[0191] FIG. 11. ISC-465 mRNA expression in placenta and cancer
derived samples
[0192] RT-PCR investigations with ISC-465-specific primers showed
selective expression in placenta (A) and in some cell lines derived
from breast cancer, melanoma, lung cancer or stomach cancer
(B).
[0193] FIG. 12. Quantitative expression of Mem-030 [0194] A.
Quantitative RT-PCR with Mem-030-specific primers showed a
significant overexpression in all investigated head and neck
carcinoma samples. The following normal tissues were analyzed:
bladder, brain, bone marrow, cervix, colon, duodenum, heart, lung,
lymph node, breast, muscle, ovary, PBMC, PBMC-activated, placenta,
prostate, retina, spleen, stomach, testis, thymus and tonsil.
[0195] B. Prevalence of Mem-030 in esophageal, liver, uterus
carcimomas and melanoma derived tissues.
[0196] FIG. 13. Quantitative expression of Mem-055
[0197] Quantitative RT-PCR with Mem-055-specific primers show high
and selective expression in normal control tissues and a
significant overexpression in stomach and lung cancer derived
tissues (A). Mem-055 is also overexpressed in liver carcinomas,
ovarian carcinomas and breast cancer samples (B).
[0198] FIG. 14. Mem-062 mRNA expression
[0199] RT-PCR analysis with Mem-064-specific primers showed
selective expression in testis and weak expression in lung cancer
derived tissues (A). Strong, significant expression levels of
Mem-064 transcripts were detectable in various ovarian tumors
(B).
[0200] FIG. 15. Specific Mem-068 expression in normal testis and
renal cell carcinomas
[0201] RT-PCR analysis with gene-specific Mem-068 primers shows
cDNA amplification in normal testis, weak in placenta (A), in renal
cell carcinomas and in stomach cancers (B).
[0202] FIG. 16. Mem-071 expression in normal testis and various
tumor samples
[0203] RT-PCR analysis with Mem-071-specific primers showed no
expression within normal tissues except testis (A). Distinct
expression was also detected in renal cell carcinoma samples and in
stomach cancers (B).
[0204] FIG. 17. Mem-072 mRNA expression
[0205] RT-PCR analysis with Mem-072-specific primers showed no
expression within normal tissues (A) and significant expression in
various lung cancer samples (A+B).
[0206] FIG. 18. Mem-106 expression in normal and tumor tissues
[0207] RT-PCR investigations with Mem-106-specific primers showed
no expression within normal tissues except in testis (A) and high
expression were investigated in ovarian- and prostate carcinomas,
as well as in melanomas and colon cancer cellines (B).
[0208] FIG. 19. Mem-131 mRNA expression
[0209] RT-PCR investigations with Mem-131-specfic primers showed no
significant expression within all tested normal tissues except
activated PBMC.Mem-131 mRNA expression in breast- and
lung-carcinomas.Mem-131 mRNA expression in lung- and ovarian
carcinomas.
[0210] FIG. 20. ISC-468 mRNA expression
[0211] (A) RT-PCR and B) Real-time PCR (investigation with
ISC-468-specific primers showed selective mRNA expression in normal
testis, placenta, and in 80% of breast carcinoma biopsies.
[0212] FIG. 21. Immunofluorescence analysis of ISC-468
expression
[0213] (A) Specificity of anti-ISC-468 antibodies were confirmed by
staining of ISC-468-eGFP transfected cells. (B) Staining of
MeOH-fixed cells either transfected with ISC-468-specific RNAi
duplexes, or non-silencing control duplexes. (C) Staining of
non-fixed cells either transfected with ISC-468-specific RNAi
duplexes, or non-silencing control duplexes.
[0214] FIG. 22. Immunochistochmical analysis of ISC-468
expression
[0215] No expression was detectable in normal breast tissue (A)
100.times., (B) 200.times.. In contrast, strong and homogeneous
membrane-staining was observed in breast carcinoma specimens (C)
100.times., (D) 200.times..
[0216] FIG. 23. RNAi-induced knock-down of ISC-468 mRNA
expression
[0217] Transfection of cells with ISC-468-specific siRNA duplexes
resulted in distinct knock-down of ISC-468 mRNA expression compared
to control cells.
[0218] FIG. 24. Cell proliferation analysis
[0219] Transfection of cells with ISC-468-specific siRNA duplexes
resulted in distinct impairment of cell proliferation compared to
control cells.
[0220] FIG. 25. Cell cycle analysis
[0221] Transfection of cells with ISC-468-specific siRNA duplexes
resulted in Gl/S arrest in (A) MCF-7 and (B) BT-549 breast
carcinoma cells compared to control cells.
[0222] FIG. 26. AKT phosphorylierung
[0223] Transfection of cells with ISC-468-specific siRNA duplexes
resulted in distinct impairment of AKT phosphorylation compared to
control cells.
[0224] FIG. 27. Antibody-mediated proliferation inhibition
[0225] Incubation of MCF-7 breast carcinoma cells with ISC-468
specific antibodies resulted in reduced proliferation compared to
cells incubated with an irrelevant control antibody.
[0226] FIG. 28. Cell proliferation analysis
[0227] Transfection of cells with ISC-468-specific siRNA duplexes
resulted in distinct impairment of (A) chemotaxis, (B)
chemokinesis, and (C) invasion compared to control cells.
[0228] FIG. 29. Estrogen receptor correlation
[0229] Expression levels of ISC-468 mRNA in breast carcinoma
samples correlates with the estrogen receptor state. Shown are the
median, 10.sup.th, and 90.sup.th percentiles with error bars.
[0230] FIG. 30. 17.beta.-estradiol treatment
[0231] ISC-468 mRNA expression was induced by treatment of estrogen
receptor positive breast carcinoma cell line MCF-7 with 100 nM
17.beta.-estradiol. No induction was seen in estrogen receptor
negative cell line MDA-MB-231.
[0232] FIG. 31. Sequences
[0233] The sequences to which reference is made herein are
shown.
EXAMPLES
Material and Methods
[0234] The techniques and methods mentioned herein are carried out
in a manner known per se and are described, for example, in
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd
Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. All methods including the use of kits and reagents are
carried out according to the manufacturers' information.
RNA Extraction, Preparation of Poly-d(T) Primed cDNA and
Conventional RT-PCR Analysis
[0235] Total RNA was extracted from native tissue material by using
guanidium isothiocyanate as chaotropic agent (Chomczynski &
Sacchi, Anal. Biochem. 162:156-9, 1987). After extraction with
acidic phenol and precipitation with isopropanol, said RNA was
dissolved in DEPC-treated water.
[0236] First strand cDNA synthesis from 4 .mu.g of total RNA was
carried out in a 20 .mu.l reaction mixture by means of Superscript
II (Invitrogen), according to the manufacturer's information. The
primer used was a dT(18) oligonucleotide. Integrity and quality of
the cDNA were checked by amplification of p53 in a 30 cycle PCR
((SEQ ID NO:33,34)), hybridization temperature 67.degree. C.)
[0237] An archive of first strand cDNA was prepared from a number
of normal tissues and tumor entities. For expression studies, 0.5
.mu.l of these cDNAs was amplified in a 30 .mu.l reaction mixture,
using GOI-specific primers (see below) and 1 U of HotStarTaq DNA
polymerase (Qiagen). Each reaction mixture contained 150 .mu.M
dNTPs, 0.3 .mu.M of each primer and 3 .mu.l of 10.times.reaction
buffer. The primers were selected so as to be located in two
different exons, and elimination of the interference by
contaminating genomic DNA as the reason for false-positive results
was confirmed by testing nonreverse-transcribed DNA as template.
After 15 minutes at 95.degree. C. to activate the HotStarTaq DNA
polymerase, 35 cycles of PCR were carried out (0.5 min at
94.degree. C., 0.5 min at the particular hybridization temperature,
0.5 min at 72.degree. C. and final elongation at 72.degree. C. for
6 min).
[0238] 20 .mu.l of this reaction were fractionated and analyzed on
an ethidium bromide-stained agarose gel.
Preparation of Random Hexamer-Primed cDNA and Quantitative
Real-Time PCR
[0239] The expression of several genes was quantified by real-time
PCR. The PCR products were detected using SYBR Green as
intercalating reporter dye. The reporter fluorescence of SYBR Green
is suppressed in solution and the dye is active only after binding
to double-stranded DNA fragments. The increase in the SYBR Green
fluorescence as a result of the specific amplification using
GOI-specific primers after each PCR cycle is utilized for
quantification. Expression of the target gene is quantified
absolutely or relative to the expression of a control gene with
constant expression in the tissues to be investigated. Expression
was measured after standardization of the samples against 18s RNA
as so-called housekeeping gene using the .DELTA..DELTA.-C.sub.t
method (PE Biosystems, USA). The reactions were carried out in
duplicates and determined in triplicates. The QuantiTect SYBR Green
PCR kit (Qiagen, Hilden) was used in accordance with the
manufacturer's instructions. The cDNA was synthesized with random
primers (Invitrogen) using the protocol described above. Each 5
.mu.l portions of the diluted cDNA were employed in a total volume
of 30 .mu.l for the PCR: sense primer 300 nM, antisense primer 300
nM; initial denaturation 95.degree. C. for 15 min; 95.degree. C.
for 30 sec; annealing for 30 sec; 72.degree. C. for 30 sec; 40
cycles. The sequences of the primers used are indicated in the
respective examples.
Cloning and Sequence Analysis
[0240] Cloning of full-lengths and gene fragments took place by
conventional methods. To ascertain the sequence, corresponding
antigenes were amplified using the proofreading polymerase pfu
(Stratagene). After completion of the PCR, adenosine was ligated by
means of HotStarTaq DNA polymerase to the ends of the amplicon in
order to clone the fragments in accordance with the manufacturer's
instructions into the TOPO-TA vector. The sequencing was carried
out by a commercial service. The sequences were analysed using
conventional prediction programs and algorithms.
Cell Proliferation Analysis
[0241] 24 h after transfection with siRNA duplexes 1.times.10.sup.4
cells were cultured in medium supplemented with varying
concentrations of FCS for 48 h. Proliferation was analyzed by
measuring the incorporation of BrdU into newly synthesized DNA
strands using the DELFIA cell proliferation Kit (Perkin Elmer)
according to the manufacturer's instructions on a Wallac Victor2
multi-label counter (Perkin Elmer).
Cell Cycle Analysis and Apoptosis
[0242] Cells were cultured in medium supplemented with FCS in
varying concentrations, harvested after 48 h and stained with
propidiumiodide prior to flowcytometric DNA content analysis.
Apoptotic cells and cells in S/G2/M phases of the cell cycle were
quantified using CellQuest-Software (Becton Dickinson).
Cell Migration
[0243] Cell migration assays were conducted in transwell chambers
with 8.0 .mu.m pore membranes (BD Biosciences) with cells cultured
in serum-free medium for 12 h prior to the experiments. For siRNA
experiments cells were transferred to serum-free conditions 24 h
after transfection with siRNA duplexes as described above.
4.times.10.sup.4 cells in 400 .mu.l serum-free culture medium were
added to the upper chamber. The bottom chambers contained 800 .mu.l
culture medium supplemented with either FCS, PDGF-BB
(Sigma-Aldrich) or SDF-1.alpha./CXCL12 (R&D Systems) as
chemoattractants. After 24 hours cells that had migrated to the
bottom side of the membrane were fixed in ice-cold methanol;
membranes were excised, placed on microscope slides and mounted
with Hoechst (Dako) for fluorescence microscopy. Cells in five
random visual fields (100.times. magnification) were counted for
each membrane. All experiments were done in triplicates. Effects on
chemokinesis of cells was analyzed using the same experimental
setup with (i) no chemoattractant added to the upper and lower
chamber and (ii) with chemoattractant added to both the upper and
lower chamber.
In Vitro Invasion Assay
[0244] In vivo invasion assays were conducted in transwell chambers
with 8.0 .mu.m pore membranes (BD Biosciences) with cells cultured
in serum-free medium for 12 h prior to the experiments. Upper
chambers were prepared with 100 .mu.l of Matrigel (BD Biosciences)
diluted to 1 mg/ml in serum free medium. Chambers were incubated at
37.degree. C. for 5 h for gelling. For siRNA experiments cells were
transferred to serum-free conditions 24 h after transfection with
siRNA duplexes as described above. 1.times.10.sup.5 cells in 400
.mu.l serum-free culture medium were added to the upper chamber.
The bottom chambers contained 800 .mu.l culture medium supplemented
with FCS as chemoattractant. After 24 hours invaded cells at the
bottom side of the membrane were fixed in ice-cold methanol;
membranes were excised, placed on microscope slides and mounted
with Hoechst (Dako) for fluorescence microscopy. Cells in five
random visual fields (100.times. magnification) were counted for
each membrane. All experiments were done in triplicates.
Example 1
Identification of ISC-468 as Therapeutic and Diagnostic Cancer
Target
[0245] ISC-468 (SEQ ID NO:1) encodes a protein of 212 amino acids
(SEQ ID NO:2) and with a molecular weight of 23.6 kDa.
[0246] It has been previously described as placenta-specific
protein expressed during pregnancy (Fant et al., Mol Reprod Dev.
63:430-6, 2002)
[0247] The protein is predicted to have a cleavable signal peptide
from aa 1-23, followed by a short putative transmembrane domain (aa
25-47) as analysed by bioinformatics tools (TMpred, SOUSI). The
remaining protein is predicted to be extracellular and can
therefore be used according to the invention as target structure
for monoclonal antibodies.
[0248] According to the invention, a gene-specific primer pair (SEQ
ID NO:3, 4) for ISC-468 was used in RT-PCR analyses to amplify cDNA
derived from a comprehensive panel of normal and tumor tissues. As
expected, placenta was confirmed as the only healthy tissue
expressing this gene (FIG. 1). No significant expression,
whatsoever, was detected in any other normal organ tissue. Most
surprisingly, when cancer specimen were investigated, we found high
and significant levels of expression in a number of different tumor
types, including colon, pancreatic, esophageal, stomach, lung,
breast, ovrian, head&neck, kidney, prostate and liver
carcinomas (FIGS. 1 and 2 as well as tab.1). Quantitative real-time
RT-PCR analysis of ISC-468 expression in 60 breast carcinoma
samples revealed that 80% of all samples expressed significant
levels of ISC-468 (FIG. 20A,B).
TABLE-US-00001 TABLE 1 ISC-468 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Brain -
Colon carcinoma + Myocardium - Pancreatic + Skeletal muscle -
carcinoma Myocardium - Esophageal + Stomach - carcinoma Colon -
Stomach carcinoma + Pancreas - Lung cancer + Kidney - Breast cancer
+++ Liver - Ovarian carcinoma + Testis - Head & Neck Cancer +
Thymus - Kidney cancer + Breast - Prostate carcinoma + Ovary -
Liver carcinoma ++ Uterus - Skin - Lung - Placenta +++ Lymph nodes
- Spleen - PBMC - Prostate -
[0249] The selective and high expression of ISC-468 transcripts in
tumors was not previously known and can be utilized according to
the invention for molecular diagnostic methods such as RT-PCR for
detecting disseminating tumor cells in the serum and bone marrow
and for detecting metastases in other tissues. This molecule can be
further used as specific target for therapeutic approaches.
[0250] The following peptides, inter alia, were selected for
producing ISC-468 specific antibodies according to the invention:
SEQ ID NO:58, 59, 60, 68, 69, 2. Specificity of the antibodies was
confirmed by immunofluorescence analysis of ISC-468-eGFP
transfected cells (FIG. 21A).
[0251] The subcellular localization of ISC-468 in endogenously
expressing breast carcinoma cell lines MCF-7 and BT-549 was
analyzed by immunofluorescence analyses. Staining of either
MeOH-fixed (FIG. 21B) or non-fixed (FIG. 21C) cells revealed that
ISC-468 is localized at the plasma membranes of the expressing
cells. Specificity of the staining was confirmed by RNAi-induced
knock-down of ISC-468 expression, resulting in the loss of plasma
membrane staining.
[0252] Furthermore, ISC-468 specific antibodies were used for
immunohistochemical analysis of ISC-468 expression in clinical
samples of normal breast and breast carcinomas. Expression of
ISC-468 was not detectable in normal breast specimens (FIG. 22A,B).
In contrast, breast carcinoma specimens showed strong and
homogeneous expression of ISC-468 (FIG. 22C,D). Signals were
accentuated at the plasmamembrane of the expressing cancer cells,
confirming that ISC-468 is a membrane protein selectively expressed
in cancer cells.
[0253] The extracellular domains of ISC-468 can be used according
to the invention as target structure for immunodiagnosis and
therapy by means of monoclonal antibodies. In addition, ISC-468 can
be employed according to the invention as vaccine (RNA, DNA,
protein, peptides) for inducing tumor-specific immune responses (T
and B cell-mediated immune responses).
[0254] RNAi-induced knock-down of ISC-468 expression was achieved
by transfection of cells with siRNA duplexes specifically targeting
ISC-468 mRNA (SEQ ID NOs: 70-73). Transfection of endogenously
expressing breast carcinoma cell lines MCF-7 and BT-549 resulted in
stable and specific reduction of ISC-468 mRNA expression (FIG.
23).
[0255] To gain insight into the physiological role of ISC-468
expression several RNAi-based in vitro cell assays were performed.
Transfection of breast carcionoma cell lines MCF-7 and BT-549 with
siRNA duplexes resulted in a distinct reduction of cell
proliferation compared to the respective controls, as analyzed in a
BrdU-based proliferation assay (FIG. 24). FACS-based cell cycle
analysis showed that the abrogation of cell proliferation resulted
from a Gl/S arrest (FIG. 25A,B). Additionally, it could be shown
that RNAi-induced knock-down of ISC-468 profoundly affects the AKT
signaling pathway in endogenously expressing cancer cells by
inhibition of AKT phosphorylation (FIG. 26). Furthermore,
proliferation of MCF-7 cells was attenuated when cells were
incubated with ISC-468 specific antibodies generated against
ISC-468 specific peptides (SEQ ID NO:68,69) compared to an
irrelevant control antibody (FIG. 27). These results indicate that
ISC-468 is a critical factor for the proliferation of cancer cells
presumably by mediating growth factor-induced activation of the AKT
signaling pathway and others. ISC-468 itself might represent a
receptor, co-receptor or membrane-bound chaperone for
growth-factors, chemokines or other substances.
[0256] Furthermore, the impact of ISC-468 expression on the
migratory ability of cancer cells was analyzed. RNAi-induced
knock-down of ISC-468 expression in breastcarcinoma cell lines
MCF-7 and BT-549 resulted in distinct impairment of chemotaxis,
chemokinesis and invasion of the cells, as assessed in transwell
migration assays (FIG. 28A,B,C). Chemotaxis, chemokinesis and
invasion are critical factors for the metastasis of cancer cells to
other organs. Therefore, expression of ISC-468 in cancer cells
might be a positive factor for cancer cell metastasis.
[0257] In breast carcinomas, it could be shown that expression of
ISC-468 is correlated with the estrogen-receptor state of the
tumor. Quantitative real-time RT-PCR analysis of ISC-468 expression
in 60 breast carcinoma samples revealed that estrogen-receptor
positive breast carcinomas showed significantly higher levels of
ISC-468 expression than receptor-negative tumors (FIG. 29).
Accordingly, expression of ISC-468 could be induced in
estrogen-receptor positive breast carcinoma cell line MCF-7 by
treatment with 178-estradiol (FIG. 30).
Example 2
Identification of ISC-507 as Therapeutic and Diagnostic Cancer
Target
[0258] ISC-507 (SEQ ID NO:5) encodes a 754 aa protein (SEQ ID NO:6)
with a molecular weight of 85.6 kDa. ISC-507 is member of a family
of zinc-binding proteins with disintegrin and metalloprotease
activities that can function as adhesion proteins and/or
endopeptidases. Members of this family have been described as
involved in a number of biologic processes, including
fertilization, neurogenesis, muscle development, and immune
response (Seals et al., Genes Dev. 17(1):7-30, 2003)
[0259] ISC-507 has one transmembrane domain (aa 671-687), a large
N-terminal extracellular and a shorter C-terminal cytoplasmatic
region.
[0260] ISC-507 expression has been reported to be specifically
restricted to mammalian epididymis, the small gland adjacent to the
testicle, which is critically involved in maturation of sperm.
According to literature, ISC-507 is transferred from the epididymis
to the sperm surface and redistributed in the sperm head during
acrosome reaction (Adachi et al., Mol Reprod Dev. 64:414-21,
2003).
[0261] RT-PCR investigations with ISC-507 specific primers (SEQ ID
NO:7, 8) confirmed selective expression in the testis and absence
of ISC-507 from any other normal tissue (tab.2, FIG. 3), except
weak expression in prostate and lymph node derived tissues (tab.2,
FIG. 4).
[0262] However and most surprisingly, we observed expression of
ISC-507 in a significant number of prostate cancers (FIG. 3,4).
This protein had not been reported before to be involved in
cancer.
TABLE-US-00002 TABLE 2 ISC-507 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Brain -
Colon carcinoma - Cerebellum - Pancreatic - carcinoma Myocardium -
Esophageal - carcinoma Skeletal muscle - Stomach carcinoma -
Myocardium Lung cancer - Stomach - Breast cancer - Colon - Ovarian
carcinoma - Pancreas - Uterus carcinoma - Kidney - Head & Neck
Cancer - Liver - Kidney cancer - Testis +++ Prostate carcinoma +++
Thymus - Liver carcinoma - Breast - Ovary - Uterus - Skin - Lung -
Placenta - Lymph nodes + Spleen - PBMC - Prostate +
[0263] The absence from toxicity relevant normal tissues and the
frequent and significant expression of ISC-507 in prostate cancers
make this protein according to the invention a valuable diagnostic
and therapeutic marker. This includes according to the invention
the detection of disseminated tumor cells in serum, bone marrow,
urine, and the detection of metastases in other organs by means of
RT-PCR. In addition, the extracellular domains of ISC-507 can be
used according to the invention as target structure for
immunodiagnosis and therapy by means of monoclonal antibodies. In
addition, ISC-507 can be employed according to the invention as
vaccine (RNA, DNA, protein, peptides) for inducing tumor-specific
immune responses (T and B cell-mediated immune responses).
[0264] Antibodies for detecting ISC-507 could be produced with
following peptides and proteins: SEQ ID NO:51, 52, 53, 54, 55, 6,
56 and 57.
[0265] According to the invention an antibody which binds to
ISC-507 might be useful for therapeutic or diagnostic purposes.
Example 3
Identification of ISC-466 as Therapeutic and Diagnostic Cancer
Target
[0266] ISC-466 (SEQ ID NO:9) encodes a 426 aa protein (SEQ ID
NO:10) with a molecular weight of 48.2 kDA.
[0267] It belongs to the family of pregnancy-specific
glycoproteins. The human pregnancy-specific glycoproteins (PSGs)
are a group of molecules that are mainly produced by the placental
syncytiotrophoblasts during pregnancy and are part of the
immunoglobulin superfamily (Beauchemin et al., Exp Cell Res.
252(2):243-9, 1999)
[0268] As other PSGs, ISC-466 as well has been reported to be
restricted to placenta.
[0269] According to the invention, a gene-specific primer pair (SEQ
ID NO:11, 12) for ISC-466 was used in RT-PCR analyses to amplify
cDNA derived from a comprehensive panel of normal and tumor
tissues. The RT-PCR analysis reveals expression of ISC-466
transcripts in normal placenta, and weak expression in thymus and
ovary (tab.3, FIG. 5A). No significant expression was detected in
any other normal organ tissue. Most surprisingly, when cancer cell
lines were investigated, we found high and significant levels of
expression in a number of tumor types, including breast cancer
(FIG. 5C), lung cancer (FIG. 5C), ovarian carcinoma (FIG. 5D) and
head and neck- and kidney carcinomas (FIG. 5B).
TABLE-US-00003 TABLE 3 ISC-466 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Brain -
Colon carcinoma + Thymus + Pancreatic + carcinoma Myocardium -
Esophageal + carcinoma Skeletal muscle - Stomach carcinoma -
Myocardium Lung cancer ++ Stomach - Breast cancer +++ Colon -
Ovarian carcinoma ++ Pancreas - Cervix carcinoma - Kidney - Head
& Neck Cancer +++ Liver - Kidney cancer ++ Testis +++ Prostate
carcinoma + Thymus - Liver carcinoma - Breast - Melanoma + Ovary +
Uterus - Skin - Lung - Placenta - Lymph nodes - Spleen - PBMC -
Prostate -
[0270] In contrast to the observation, that ISC-466 is involved by
colorectal carcinomas (Salahshor et al., BMC Cancer. 5:66, 2005),
our investigations reveal ISC-466 according to the invention as
diagnostic and therapeutic marker for head & neck, breast,
ovarian, prostate cancer and melanoma.
Example 4
Identification of ISC-518 as Therapeutic and Diagnostic Cancer
Target
[0271] ISC-518 (SEQ ID NO:13) encodes a 237 aa translation product
(SEQ ID NO:14). However, no data with regard to tissue distribution
and no connection to cancer is available so far.
[0272] ISC-518 is a hypothetical, bioinformaticly predicted
gene/protein. Sequence analyses revealed that the protein has a
transmembrane domain (aa 102-118). The extracellular C-terminus
features a functional domain, which occurs in cell-surface
glycoproteins.
[0273] According to the invention, a gene-specific primer pair (SEQ
ID NO:15, 16) for ISC-518 was used in RT-PCR analyses to amplify
cDNA derived from a comprehensive panel of normal and tumor
tissues. The only normal tissue we found to express this gene was
testis, whereas no significant expression of ISC-518 was detectable
in any other normal organ (FIG. 6). Most surprisingly, when cancer
specimen were investigated, we found high and significant levels of
expression in hepatocarcinomas (FIG. 7)
TABLE-US-00004 TABLE 4 ISC-518 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Brain -
Colon carcinoma - Cerebellum - Pancreatic - carcinoma Myocardium -
Esophageal - carcinoma Skeletal muscle - Stomach carcinoma +
Myocardium Lung cancer + Stomach - Breast cancer + Colon - Ovarian
carcinoma + Pancreas - Uterus carcinoma - Kidney - Head & Neck
Cancer - Liver - Kidney cancer - Testis +++ Prostate carcinoma +
Thymus - Liver carcinoma ++ Breast - Ovary - Uterus - Skin - Lung -
Placenta - Lymph nodes - Spleen - PBMC - Prostate -
[0274] Bioinformatic investigations showed that the protein encoded
by ISC-518 represents a cell surface molecule. The previous unknown
selective expression of this surface molecule makes it a target for
therapeutic purposes and for developing diagnostic methods for the
detection of tumor cells and therapeutic methods for the
elimination of tumor cells.
Example 5
Identification of ISC-477 as Therapeutic and Diagnostic Cancer
Target
[0275] ISC-477 (SEQ ID NO:17) encodes a 130 aa translation product
(SEQ ID NO:18). ISC-477 is a hypothetical protein. No data with
regard to tissue distribution and no connection to cancer was
publicly available.
[0276] Structural analysis reveals a hydrophobic region, which
might be a transmembrane region or signal peptide.
[0277] According to the invention, a gene-specific primer pair (SEQ
ID NO: 19, 20) for ISC-477 was used in RT-PCR analyses to amplify
cDNA derived from a comprehensive panel of normal and tumor
tissues. The only normal tissues we found to express this gene were
placenta and ovary. In contrast, no significant expression of
ISC-477 was detectable in any other normal organ (FIG. 8A). Most
surprisingly, when cancer specimens were investigated, we found
high and significant levels of expression in lung, ovarian, colon
and stomach cancer (FIG. 8A-D). Expression levels are clearly
higher than expression in normal ovary.
TABLE-US-00005 TABLE 5 ISC-477 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Brain -
Colon carcinoma ++ Cerebellum - Pancreatic + carcinoma Myocardium -
Esophageal - carcinoma Skeletal muscle - Stomach carcinoma ++
Myocardium Lung cancer +++ Stomach - Breast cancer ++ Colon -
Ovarian carcinoma ++ Pancreas - Kidney cancer - Kidney - Prostate
carcinoma - Liver - Liver carcinoma - Testis - Thymus - Breast -
Ovary ++ Uterus - Skin - Lung - Placenta +++ Lymph nodes - Spleen -
PBMC - Prostate -
Example 6
Identification of ISC-489 as Therapeutic and Diagnostic Cancer
Target
[0278] ISC-489 (SEQ ID NO:21) encodes a 363 aa translation product
(SEQ ID NO:22). The protein is a newly described member of the
family of G-protein coupled receptors. However, no data with regard
to tissue distribution and no connection to cancer was publically
available.
[0279] According to the invention, a gene-specific primer pair (SEQ
ID NO:23,24) for ISC-489 was used in RT-PCR analyses to amplify
cDNA derived from a comprehensive panel of normal and tumor
tissues. The only normal tissues we found to express this gene were
placenta and esophagus (weak expression). In contrast, no
significant expression of ISC-489 was detectable in any other
normal organ (FIG. 9A). Most surprisingly, when cancer specimens
were investigated, we found high and significant levels of
expression in head and neck, and stomach cancers (FIG. 9B, 9C).
[0280] As member of the G-protein coupled receptor family, ISC-489
is an integral membrane protein with 7 transmembrane domains and
several extracellular loops, which can be targeted on the cell
surface.
TABLE-US-00006 TABLE 6 ISC-489 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Brain -
Colon carcinoma + Esophagus + Stomach carcinoma ++ Myocardium -
Lung cancer + Skeletal muscle - Breast cancer - Myocardium Ovarian
carcinoma - Stomach - Head & Neck Cancer +++ Colon - Kidney
cancer + Pancreas - Prostate carcinoma - Kidney - Liver carcinoma +
Liver - Testis - Thymus - Breast - Ovary - Uterus - Skin - Lung -
Placenta +++ Lymph nodes - Spleen - PBMC - Prostate -
[0281] The pronounced expression and unexpected high incidence of
ISC-489 in head and neck carcinomas make this protein according to
the invention a highly interesting diagnostic and therapeutic
marker.
Example 7
Identification of ISC-461 as Therapeutic and Diagnostic Cancer
Target
[0282] ISC-461 (SEQ ID NO:25) encodes a 419 aa protein (SEQ ID
NO:26) with a molecular weight of 47.1 kDA.
[0283] It belongs to the family of pregnancy-specific
glycoproteins. The human pregnancy-specific glycoproteins (PSGs)
are a group of molecules that are mainly produced by the placental
syncytiotrophoblasts during pregnancy and are part of the
immunoglobulin superfamily (Beauchemin et al., Exp Cell Res.
252(2):243-9, 1999).
[0284] As other PSGs, ISC-461 as well has been reported to be
restricted to placenta.
[0285] According to the invention, a gene-specific primer pair (SEQ
ID NO:11, 27) for ISC-461 was used in RT-PCR analyses to amplify
cDNA derived from a comprehensive panel of normal and tumor
tissues. As expected, placenta was confirmed as expressing this
gene, besides weak expression in testis and ovary (FIGS. 10A and
10B). No significant expression, whatsoever, was detected in any
other normal organ tissue. Most surprisingly, when cancer derived
tissues and cancer cell lines were investigated, we found high and
significant levels of expression in a number of tumor types,
including breast cancer (FIG. 10C), ovarian carcinoma (FIG. 10D)
and melanoma (FIG. 10B, 10C).
TABLE-US-00007 TABLE 7 ISC-461 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Brain -
Colon carcinoma - Cerebellum - Pancreatic + carcinoma Myocardium -
Esophageal + carcinoma Skeletal muscle - Stomach carcinoma +
Myocardium Lung cancer + Stomach - Breast cancer ++ Colon - Ovarian
carcinoma ++ Pancreas - Kidney cancer - Kidney - Prostate carcinoma
- Liver - Liver carcinoma - Testis + Melanoma ++ Thymus - Breast -
Ovary + Uterus - Skin - Lung - Placenta +++ Lymph nodes - Spleen -
PBMC - Prostate -
[0286] A further aim according to the invention was to identify
splice variants for ISC-461 which can be utilized both for
diagnosis and for therapy.
[0287] On investigation of splice variants we could identify a
splice form (SEQ ID NO:28) and the protein encoded thereby (SEQ ID
NO:29).
Example 8
Identification of ISC-465 as Therapeutic and Diagnostic Cancer
Target
[0288] ISC-465 (SEQ ID NO:30) encodes a 419 aa protein (SEQ ID
NO:31) with a molecular weight of 47.0 kDA.
[0289] It belongs to the family of pregnancy-specific
glycoproteins. The human pregnancy-specific glycoproteins (PSGs)
are a group of molecules that are mainly produced by the placental
syncytiotrophoblasts during pregnancy and are part of the
immunoglobulin superfamily (Beauchemin et al., Exp Cell Res.
252(2):243-9, 1999).
[0290] As other PSGs, ISC-465 as well has been reported to be
restricted to placenta.
[0291] According to the invention, a gene-specific primer pair (SEQ
ID NO:11, 32) for ISC-465 was used in RT-PCR analyses to amplify
cDNA derived from a comprehensive panel of normal and tumor
tissues. As expected, placenta was confirmed as expressing this
gene, besides weak expression in normal ovary (FIG. 11A). No
significant expression, whatsoever, was detected in any other
normal organ tissue. Most surprisingly, when cancer derived tissues
and cancer cell lines were investigated, we found high and
significant levels of expression in a number of tumor types (FIG.
11A, 11B), especially breast cancer (FIG. 11B).
TABLE-US-00008 TABLE 8 ISC-461 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Brain -
Colon carcinoma - Cerebellum - Pancreatic + carcinoma Myocardium -
Esophageal + carcinoma Skeletal muscle - Stomach carcinoma +
Myocardium Lung cancer + Stomach - Breast cancer ++ Colon - Ovarian
carcinoma ++ Pancreas - Kidney cancer - Kidney - Prostate carcinoma
- Liver - Liver carcinoma - Testis + Melanoma + Thymus - Breast -
Ovary + Uterus - Skin - Lung - Placenta +++ Lymph nodes - Spleen -
PBMC - Prostate -
[0292] The selective and high expression of ISC-465 transcripts in
tumors was not previously known and can be utilized according to
the invention for molecular diagnostic methods such as RT-PCR for
detecting disseminating tumor cells in the serum and bone marrow
and for detecting metastases in other tissues. This molecule can be
further used as specific target for therapeutic approaches.
Example 9
Identification of Mem-030 as Therapeutic and Diagnostic Cancer
Target
[0293] Mem-030 (SEQ ID NO:35) encodes a 592 aa protein (SEQ ID
NO:36) with a molecular weight of 67.9 kDA. Mem-030 belongs to the
GBP-proteins, which are large GTPases being able to bind GTP, GDP,
and GMP and to catalyze the hydrolysis of GTP to GDP, as well as
GMP (Cheng et al., J Biol. Chem. 260:15834-9, 1985). GTPases play
an important role in cell proliferation, differentiation, signal
transduction, and intracellular protein transportation and are
interferon inducible (Boehm et al., J Immunol. 161(12):6715-23,
1998).
[0294] Also, Mem-030 counteracts the proliferative effect of
inflammatory cytokines like IFN-g, interleukin 1-b (IL-1b), and
tumor necrosis factor-a (TNF-.alpha.) 1 on endothelial cells
(Guenzi et al., EMBO J. 20(20):5568-77, 2001).
[0295] According to the invention, a gene-specific primer pair (SEQ
ID NO:37, 38) for Mem-030 was used in real time RT-PCR analyses to
amplify cDNA derived from a comprehensive panel of normal and tumor
tissues. Mem-030 show an ubiquitous expression pattern (FIG. 12A,
tab.9).
[0296] Most surprisingly, when cancer derived tissues and cancer
cell lines were investigated, we found high and significant levels
of overexpression in a number of tumor types (FIG. 12A, 12B),
especially head and neck carcinomas.
TABLE-US-00009 TABLE 9 Mem-030 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Brain +
Colon carcinoma + Myocardium + Pancreatic + carcinoma Skeletal
muscle + Esophageal ++ carcinoma Myocardium + Stomach carcinoma +
Stomach + Lung cancer + Colon + Breast cancer + Pancreas + Ovarian
carcinoma + Kidney + Uterus carcinoma ++ Liver + Head & Neck
Cancer +++ Testis + Kidney cancer + Thymus + Prostate carcinoma +
Breast + Liver carcinoma ++ Ovary + Melanoma ++ Uterus + Skin +
Lung + Placenta + Lymph nodes + Spleen + PBMC + Prostate +
[0297] Due to bioinformatics and literature analysis, a homologous
gene of Mem-030 might be also an attractive therapeutic target (SEQ
ID NO:39) and encodes a 586 aa protein (SEQ ID NO:40) with a
molecular weight of 66.6 kDA.
[0298] Bioinformatic investigations showed that both proteins
represent cell surface molecules. The previously unknown selective
overexpression of this surface molecule makes it a target for
therapeutic purposes and for developing diagnostic methods for the
detection of tumor cells and therapeutic methods for the
elimination of tumor cells.
Example 10
Identification of Mem-055 as Therapeutic and Diagnostic Cancer
Target
[0299] Mem-055 (SEQ ID NO:41) encodes a 250 aa protein (SEQ ID
NO:42) with a molecular weight of 27.9 kDA. The protein encoded by
this gene is a lysosomal thiol reductase that at low pH can reduce
protein disulfide bonds. The enzyme is expressed constitutively in
antigen-presenting cells and induced by gamma-interferon in other
cell types. This enzyme has an important role in MHC class
II-restricted antigen processing (Arunachalam et al. Proc Natl Acad
Sci USA. 97(2):745-50, 2000).
[0300] The localization of Mem-055 and the protein topology was
predicted by analysis of the putative signal sequences and
transmembrane domains with bioinformatic tools (TMPRED, SOUSI).
Mem-055 might have an extracellular C-terminus.
[0301] According to the invention, a gene-specific primer pair (SEQ
ID NO:43, 44) for Mem-055 was used in real time RT-PCR analyses to
amplify cDNA derived from a comprehensive panel of normal and tumor
tissues. Mem-055 show an ubiquitous expression pattern (FIG. 13A,
tab.10).
[0302] Most surprisingly, when Mem-055 expression within cancer
derived tissues was investigated, we found high and significant
levels of overexpression in a number of tumor types (FIG. 13A,
13B), especially stomach cancers.
TABLE-US-00010 TABLE 10 Mem-055 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Brain +
Colon carcinoma + Myocardium + Pancreatic + carcinoma Skeletal
muscle + Esophageal + carcinoma Myocardium + Stomach carcinoma +++
Stomach + Lung cancer ++ Colon + Breast cancer ++ Pancreas +
Ovarian carcinoma ++ Kidney + Uterus carcinoma + Liver + Head &
Neck Cancer + Testis + Kidney cancer + Thymus + Prostate carcinoma
+ Breast + Liver carcinoma ++ Ovary + Melanoma + Uterus + Skin +
Lung + Placenta + Lymph nodes + Spleen + PBMC + Prostate +
[0303] Mem-055 is a target structure for therapeutic and diagnostic
purposes, because of the putative extracellular domain and the
unexpected overexpression in different carcinoma types.
Example 11
Identification of Mem-062 as Therapeutic and Diagnostic Cancer
Target
[0304] Mem-062 (SEQ ID NO:45) encodes a 271 aa protein (SEQ ID
NO:46) with a molecular weight of 30.7 kDA.
[0305] By a computer-based screening method Mem-062 could
previously be identified and was described as testis, prostate and
placenta specifically expressed (Bera et al., Biochem Biophys Res
Commun. 312(4):1209-15, 2003)
[0306] According to the invention, a gene-specific primer pair (SEQ
ID NO:47, 48) for Mem-062 was used in RT-PCR analyses. Mem-062
surprisingly showed a cancer-testis specific expression pattern
(FIG. 14A, tab.11). No expression was detected in any other normal
organ tissue. Most surprisingly, when cancer derived tissues were
investigated, we found significant levels of Mem-expression (FIG.
14B), especially in ovarian carcinomas.
TABLE-US-00011 TABLE 11 Mem-062 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Brain -
Colon carcinoma + Myocardium - Pancreatic - carcinoma Skeletal
muscle - Esophageal - carcinoma Myocardium - Stomach carcinoma -
Stomach - Lung cancer - Colon - Breast cancer - Pancreas - Ovarian
carcinoma ++ Kidney - Uterus carcinoma - Liver - Head & Neck
Cancer - Testis - Kidney cancer - Thymus - Prostate carcinoma -
Breast - Liver carcinoma - Ovary - Melanoma - Uterus - Skin - Lung
- Placenta - Lymph nodes - Spleen - PBMC - Prostate -
[0307] Alternative splicing results in an alternative transcript
(SEQ ID NO:49) and its corresponding translation product (SEQ ID
NO:50).
Example 12
Identification of Mem-068 as Therapeutic and Diagnostic Cancer
Target
[0308] Mem-068 (SEQ ID NO:61) is a newly identified cDNA clone.
[0309] By a bioinformatic prediction approach (Genscan) Mem-068
could be described as multiple exon gene on chromosome 9 (SEQ ID
NO:62). The deduced protein sequence (SEQ ID NO:63) has 751 aa and
forms a protein with a molecular weight of 82.4 kDA.
[0310] According to the invention, a gene-specific primer pair for
Mem-068 was used in RT-PCR analyses. Mem-068 show surprisingly a
cancer-testis specific expression pattern (FIG. 15A, tab.12). No
expression was detected in any other normal organ tissue except
placenta (weak expression). Most surprisingly, when cancer derived
tissues were investigated, we found significant levels of Mem-068
expressed (FIG. 15B), especially in renal cell carcinomas and in
stomach cancers.
TABLE-US-00012 TABLE 12 Mem-068 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Brain -
Colon carcinoma + Breast Renal cell carcinoma ++ Colon - Stomach
carcinoma + Kidney - Lung cancer + Liver - Breast cancer - Lung -
Ovarian carcinoma - Lymph nodes - Melanoma - Ovary - Prostate
carcinoma - Pancreas - Placenta + PBMC - PBMC activated - Prostate
- Skeletal muscle - Skin - Stomach - Spleen - Testis + Uterus -
[0311] According to the transmembrane prediction programme TMpred
Mem-068 might be expressed at the cell surface, which makes it an
interesting target for therapeutic or diagnostic purposes.
Example 13
Identification of Mem-071 as Therapeutic and Diagnostic Cancer
Target
[0312] Mem-071 (SEQ ID NO:64) is a new cDNA clone, which is encoded
in 2 exons on chromosome 1.
[0313] According to the invention, a gene-specific primer pair for
Mem-071 was used in RT-PCR analyses to amplify cDNA derived from a
comprehensive panel of normal and tumor tissues. The only normal
tissues we found to express this gene was testis (FIG. 16A). In
contrast, when cancer specimen were investigated, we found high and
significant levels of expression in renal cell carcinomas and
stomach cancers (FIG. 16B).
TABLE-US-00013 TABLE 13 Mem-071 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Brain -
Colon carcinoma - Breast - Renal cell carcinoma ++ Colon - Stomach
carcinoma + Kidney - Lung cancer - Liver - Breast cancer - Lung -
Ovarian carcinoma - Lymph nodes - Melanoma - Ovary - Prostate
carcinoma - Pancreas - Placenta - PBMC - PBMC activated - Prostate
- Skeletal muscle - Skin - Stomach - Spleen - Testis + Uterus -
[0314] The unexpected high incidence of Mem-071 in renal cell
carcinomas make this protein according to the invention a highly
interesting diagnostic and therapeutic marker.
Example 14
Identification of Mem-072 as Therapeutic and Diagnostic Cancer
Target
[0315] Mem-072 (SEQ ID NO:65) is a newly identified gene, which is
encoded in 3 exons on chromosome 16.
[0316] According to the invention, a gene-specific primer pair for
Mem-072 was used in RT-PCR analyses to amplify cDNA derived from a
comprehensive panel of normal and tumor tissues. No expression
within all tested normal tissues could be found (FIG. 17A, tab.14).
When cancer derived tissues and cancer cell lines were
investigated, we found high and significant levels of expression in
lung cancer samples (FIG. 17B).
TABLE-US-00014 TABLE 14 Mem-072 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Brain -
Colon carcinoma - Breast - Renal cell carcinoma - Colon - Stomach
carcinoma - Kidney - Lung cancer ++ Liver - Breast cancer - Lung -
Ovarian carcinoma - Lymph nodes - Melanoma - Ovary - Prostate
carcinoma - Pancreas - Placenta - PBMC - PBMC activated - Prostate
- Skeletal muscle - Skin - Stomach - Spleen - Testis - Uterus -
[0317] The selective and high expression of Mem-072 in lung tumors
was not previously known and can be utilized according to the
invention for molecular diagnostic methods such as RT-PCR for
detecting disseminating tumor cells in the serum and bone marrow
and for detecting metastases in other tissues. This molecule can be
further used as specific target for therapeutic approaches.
Example 15
Identification of Mem-106 as Therapeutic and Diagnostic Cancer
Target
[0318] Mem-106 (SEQ ID NO:66) is a newly identified cDNA, which is
intronless encoded on chromosome 2.
[0319] According to the invention, a gene-specific primer pair for
Mem-106 was used in RT-PCR analyses. Mem-106 surprisingly showed a
cancer-testis specific expression pattern (FIG. 18A, tab.15). No
expression was detected in any other normal organ tissue. Most
surprisingly, when cancer derived tissues were investigated, we
found significant levels of Mem-106 expression (FIG. 18B),
especially in ovarian carcinomas.
TABLE-US-00015 TABLE 15 Mem-106 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Breast -
Colon carcinoma + Colon - Renal cell carcinoma - Kidney - Stomach
carcinoma - Liver - Lung cancer - Lung - Breast cancer - Lymph
nodes - Ovarian carcinoma ++ Ovary - Melanoma ++ Pancreas -
Prostate carcinoma - Placenta - PBMC - PBMC activated - Prostate -
Skeletal muscle - Skin - Stomach - Spleen - Testis ++ Uterus -
[0320] Mem-106 is a target structure for therapeutic and diagnostic
purposes, because of the unexpected overexpression in different
carcinoma types.
Example 16
Identification of Mem-131 as Therapeutic and Diagnostic Cancer
Target
[0321] Mem-131 (SEQ ID NO:67) is a newly identified cDNA clone.
Mem-131 is a 2 exone gene on chromosome 15.
[0322] According to the invention, a gene-specific primer pair for
Mem-131 was used in RT-PCR analyses to amplify cDNA derived from a
comprehensive panel of normal and tumor tissues. The RT-PCR
analysis reveals expression of Mem-131 transcripts only in normal
activated PBMCs (tab.16, FIG. 19A). No significant expression was
detected in any other normal organ tissue. Most surprisingly, when
cancer samples were investigated, we found high and significant
levels of expression in a number of tumor types, including breast
cancer (FIG. 19B), lung cancer (FIG. 19B+C) and ovarian carcinoma
(FIG. 19C).
TABLE-US-00016 TABLE 16 Mem-131 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Breast -
Lung cancer ++ Duodenum - Breast cancer ++ Bladder - Ovarian
carcinoma ++ Skin - Brain - Bone marrow - Colon - Liver - Lung -
Lymph node - Stomach - Spleen - Myocard - Kidney - Esophagus -
Ovary - Pancreas - PBMC - PBMC activated ++ Placenta - Muscle -
Testis - Thymus -
[0323] Our investigations reveals Mem-131 according to the
invention as diagnostic and therapeutic marker for lung, breast and
ovarian cancers.
Sequence CWU 1
1
7311126DNAHomo sapiens 1atatatcaga ccatcagaag gatttgtata aagagtgact
ctcctatgaa ggtaaaggcc 60acccctcttc agttccagtg actgagatac atttttccaa
tcctgggggc aaatacagac 120acagcaagtt ccttcttccc tttggaaatt
tggcagctgc cttcaccagt gagcacaaag 180ccacatttca aaggaaactg
acaaattatc cccagctgcc agaagaagaa atcctcactg 240gacggcttcc
tgtttcctgt ggttcattat ctgattggct gcagggatga aagtttttaa
300gttcatagga ctgatgatcc tcctcacctc tgcgttttca gccggttcag
gacaaagtcc 360aatgactgtg ctgtgctcca tagactggtt catggtcaca
gtgcacccct tcatgctaaa 420caacgatgtg tgtgtacact ttcatgaact
acacttgggc ctgggttgcc ccccaaacca 480tgttcagcca cacgcctacc
agttcaccta ccgtgttact gaatgtggca tcagggccaa 540agctgtctct
caggacatgg ttatctacag cactgagata cactactctt ctaagggcac
600gccatctaag tttgtgatcc cagtgtcatg tgctgccccc caaaagtccc
catggctcac 660caagccctgc tccatgagag tagccagcaa gagcagggcc
acagcccaga aggatgagaa 720atgctacgag gtgttcagct tgtcacagtc
cagtcaaagg cccaactgcg attgtccacc 780ttgtgtcttc agtgaagaag
agcataccca ggtcccttgt caccaagcag gggctcagga 840ggctcaacct
ctgcagccat ctcactttct tgatatttct gaggattggt ctcttcacac
900agatgatatg attgggtcca tgtgatcctc aggtttgggg tctcctgaag
atgctatttc 960tagaattagt atatagtgta caaatgtctg acaaataagt
gctcttgtga ccctcatgtg 1020agcacttttg agaaagagaa acctatagca
acttcatgaa ttaagccttt ttctatattt 1080ttatattcat gtgtaaacaa
aaaataaaat aaaattctga tcgcat 11262212PRTHomo sapiens 2Met Lys Val
Phe Lys Phe Ile Gly Leu Met Ile Leu Leu Thr Ser Ala 1 5 10 15 Phe
Ser Ala Gly Ser Gly Gln Ser Pro Met Thr Val Leu Cys Ser Ile 20 25
30 Asp Trp Phe Met Val Thr Val His Pro Phe Met Leu Asn Asn Asp Val
35 40 45 Cys Val His Phe His Glu Leu His Leu Gly Leu Gly Cys Pro
Pro Asn 50 55 60 His Val Gln Pro His Ala Tyr Gln Phe Thr Tyr Arg
Val Thr Glu Cys 65 70 75 80 Gly Ile Arg Ala Lys Ala Val Ser Gln Asp
Met Val Ile Tyr Ser Thr 85 90 95 Glu Ile His Tyr Ser Ser Lys Gly
Thr Pro Ser Lys Phe Val Ile Pro 100 105 110 Val Ser Cys Ala Ala Pro
Gln Lys Ser Pro Trp Leu Thr Lys Pro Cys 115 120 125 Ser Met Arg Val
Ala Ser Lys Ser Arg Ala Thr Ala Gln Lys Asp Glu 130 135 140 Lys Cys
Tyr Glu Val Phe Ser Leu Ser Gln Ser Ser Gln Arg Pro Asn 145 150 155
160 Cys Asp Cys Pro Pro Cys Val Phe Ser Glu Glu Glu His Thr Gln Val
165 170 175 Pro Cys His Gln Ala Gly Ala Gln Glu Ala Gln Pro Leu Gln
Pro Ser 180 185 190 His Phe Leu Asp Ile Ser Glu Asp Trp Ser Leu His
Thr Asp Asp Met 195 200 205 Ile Gly Ser Met 210 321DNAArtificial
SequenceDescription of artificial sequence Oligonucleotide
3aaatttggca gctgccttca c 21421DNAArtificial SequenceDescription of
artificial sequence Oligonucleotide 4tgatgccaca ttcagtaaca c
2152582DNAHomo sapiens 5atccctgcag tggaagtgag gaggaagaaa ggtgaactcc
ttttctcaag cacttctgct 60ctcctctacc agaatcactc agaatgcttc ccgggtgtat
attcttgatg attttactca 120ttcctcaggt taaagaaaag ttcatccttg
gagtagaggg tcaacaactg gttcgtccta 180aaaagcttcc tctgatacag
aagcgagata ctggacacac ccatgatgat gacatactga 240aaacgtatga
agaagaattg ttgtatgaaa taaaactaaa tagaaaaacc ttagtccttc
300atcttctaag atccagggag ttcctaggct caaattacag tgaaacattc
tactccatga 360aaggagaagc gttcaccagg catcctcaga tcatggatca
ttgtttttac caaggatcca 420tagtacacga atatgattca gctgccagta
tcagtacgtg taatggtcta aggggattct 480tcagaataaa cgaccaaaga
tacctcattg aaccagtgaa atactcagat gagggagaac 540atttggtgtt
caaatataac ctgagggtgc cgtatggtgc caattattcc tgtacagagc
600ttaattttac cagaaaaact gttccagggg ataatgaatc tgaagaagac
tccaaaataa 660aaggcatcca tgatgaaaag tatgttgaat tgttcattgt
tgctgatgat actgtgtatc 720gcagaaatgg tcatcctcac aataaactaa
ggaaccgaat ttggggaatg gtcaattttg 780tcaacatgat ttataaaacc
ttaaacatcc atgtgacgtt ggttggcatt gaaatatgga 840cacatgaaga
taaaatagaa ctatattcaa atatagaaac taccttattg cgtttttcat
900tttggcaaga aaagatcctt aaaacacgga aggattttga tcatgttgta
ttactcagtg 960ggaagtggct ctactcacat gtgcaaggaa tttcttatcc
agggggtatg tgcctgccct 1020attattccac cagtatcatt aaggatcttt
tacctgacac aaacataatt gcaaacagaa 1080tggcacatca actggggcat
aaccttggga tgcagcatga cgagttccca tgcacctgtc 1140cttcaggaaa
atgcgtgatg gacagtgatg gaagcattcc tgcactgaaa ttcagtaaat
1200gcagccaaaa ccaataccac cagtacttga aggattataa gccaacatgc
atgctcaaca 1260ttccatttcc ttacaatttt catgatttcc aattttgtgg
aaacaagaag ttggatgagg 1320gtgaagagtg tgactgtggc cctgctcagg
agtgtactaa tccttgctgt gatgcacaca 1380catgtgtact gaagccagga
tttacttgtg cagaaggaga atgctgtgaa tcttgtcaga 1440taaaaaaagc
agggtccata tgcagaccgg cgaaagatga atgtgatttt cctgagatgt
1500gcactggcca ctcgcctgcc tgtcctaagg accagttcag ggtcaatgga
tttccttgca 1560agaactcaga aggctactgt ttcatgggga aatgtccaac
tcgtgaggat cagtgctctg 1620aactatttga tgatgatgca atagagagtc
atgatatctg ctacaagatg aatacaaaag 1680gaaataaatt tggatactgc
aaaaacaagg aaaacagatt tcttccctgt gaggagaaag 1740atgtcagatg
tggaaagatc tactgcactg gaggggagct ttcctctctc cttggagaag
1800acaagactta tcaccttaag gatccccaga agaatgctac tgtcaaatgc
aaaactattt 1860ttttatacca tgattctaca gacattggcc tggtggcgtc
aggaacaaaa tgtggagagg 1920gaatggtgtg caacaatggt gaatgtctaa
acatggaaaa ggtctatatc tcaaccaatt 1980gcccctctca gtgcaatgaa
aatcctgtgg atggccacgg actccagtgc cactgtgagg 2040aaggacaggc
acctgtagcc tgtgaagaaa ccttacatgt taccaatatc accatcttgg
2100ttgttgtgct tgtcctggtt attgtcggta tcggagttct tatactatta
gttcgttacc 2160gaaaatgtat caagttgaag caagttcaga gcccacctac
agaaaccctg ggagtggaga 2220acaaaggata ctttggtgat gagcagcaga
taaggactga gccaatcctg ccagaaattc 2280atttcctaaa taaacctgca
agtaaagatt caagaggaat cgcagatccc aatcaaagtg 2340ccaagtgagc
ttgaagttgg atatccaaaa tggccgtgca agcttaggct ggggattctg
2400gatgcaacgt ctttacaacc ttacctagat atctgctact cacatttttg
gtagtgtttc 2460aaacgttctt tatccagaca gacaatgttt aagagaaaca
acttatttct gttaatattt 2520accggtagaa ttcacaccct ctatcataaa
catatgctgc agaaaaaaaa aaaaaaaaaa 2580aa 25826754PRTHomo sapiens
6Met Leu Pro Gly Cys Ile Phe Leu Met Ile Leu Leu Ile Pro Gln Val 1
5 10 15 Lys Glu Lys Phe Ile Leu Gly Val Glu Gly Gln Gln Leu Val Arg
Pro 20 25 30 Lys Lys Leu Pro Leu Ile Gln Lys Arg Asp Thr Gly His
Thr His Asp 35 40 45 Asp Asp Ile Leu Lys Thr Tyr Glu Glu Glu Leu
Leu Tyr Glu Ile Lys 50 55 60 Leu Asn Arg Lys Thr Leu Val Leu His
Leu Leu Arg Ser Arg Glu Phe 65 70 75 80 Leu Gly Ser Asn Tyr Ser Glu
Thr Phe Tyr Ser Met Lys Gly Glu Ala 85 90 95 Phe Thr Arg His Pro
Gln Ile Met Asp His Cys Phe Tyr Gln Gly Ser 100 105 110 Ile Val His
Glu Tyr Asp Ser Ala Ala Ser Ile Ser Thr Cys Asn Gly 115 120 125 Leu
Arg Gly Phe Phe Arg Ile Asn Asp Gln Arg Tyr Leu Ile Glu Pro 130 135
140 Val Lys Tyr Ser Asp Glu Gly Glu His Leu Val Phe Lys Tyr Asn Leu
145 150 155 160 Arg Val Pro Tyr Gly Ala Asn Tyr Ser Cys Thr Glu Leu
Asn Phe Thr 165 170 175 Arg Lys Thr Val Pro Gly Asp Asn Glu Ser Glu
Glu Asp Ser Lys Ile 180 185 190 Lys Gly Ile His Asp Glu Lys Tyr Val
Glu Leu Phe Ile Val Ala Asp 195 200 205 Asp Thr Val Tyr Arg Arg Asn
Gly His Pro His Asn Lys Leu Arg Asn 210 215 220 Arg Ile Trp Gly Met
Val Asn Phe Val Asn Met Ile Tyr Lys Thr Leu 225 230 235 240 Asn Ile
His Val Thr Leu Val Gly Ile Glu Ile Trp Thr His Glu Asp 245 250 255
Lys Ile Glu Leu Tyr Ser Asn Ile Glu Thr Thr Leu Leu Arg Phe Ser 260
265 270 Phe Trp Gln Glu Lys Ile Leu Lys Thr Arg Lys Asp Phe Asp His
Val 275 280 285 Val Leu Leu Ser Gly Lys Trp Leu Tyr Ser His Val Gln
Gly Ile Ser 290 295 300 Tyr Pro Gly Gly Met Cys Leu Pro Tyr Tyr Ser
Thr Ser Ile Ile Lys 305 310 315 320 Asp Leu Leu Pro Asp Thr Asn Ile
Ile Ala Asn Arg Met Ala His Gln 325 330 335 Leu Gly His Asn Leu Gly
Met Gln His Asp Glu Phe Pro Cys Thr Cys 340 345 350 Pro Ser Gly Lys
Cys Val Met Asp Ser Asp Gly Ser Ile Pro Ala Leu 355 360 365 Lys Phe
Ser Lys Cys Ser Gln Asn Gln Tyr His Gln Tyr Leu Lys Asp 370 375 380
Tyr Lys Pro Thr Cys Met Leu Asn Ile Pro Phe Pro Tyr Asn Phe His 385
390 395 400 Asp Phe Gln Phe Cys Gly Asn Lys Lys Leu Asp Glu Gly Glu
Glu Cys 405 410 415 Asp Cys Gly Pro Ala Gln Glu Cys Thr Asn Pro Cys
Cys Asp Ala His 420 425 430 Thr Cys Val Leu Lys Pro Gly Phe Thr Cys
Ala Glu Gly Glu Cys Cys 435 440 445 Glu Ser Cys Gln Ile Lys Lys Ala
Gly Ser Ile Cys Arg Pro Ala Lys 450 455 460 Asp Glu Cys Asp Phe Pro
Glu Met Cys Thr Gly His Ser Pro Ala Cys 465 470 475 480 Pro Lys Asp
Gln Phe Arg Val Asn Gly Phe Pro Cys Lys Asn Ser Glu 485 490 495 Gly
Tyr Cys Phe Met Gly Lys Cys Pro Thr Arg Glu Asp Gln Cys Ser 500 505
510 Glu Leu Phe Asp Asp Asp Ala Ile Glu Ser His Asp Ile Cys Tyr Lys
515 520 525 Met Asn Thr Lys Gly Asn Lys Phe Gly Tyr Cys Lys Asn Lys
Glu Asn 530 535 540 Arg Phe Leu Pro Cys Glu Glu Lys Asp Val Arg Cys
Gly Lys Ile Tyr 545 550 555 560 Cys Thr Gly Gly Glu Leu Ser Ser Leu
Leu Gly Glu Asp Lys Thr Tyr 565 570 575 His Leu Lys Asp Pro Gln Lys
Asn Ala Thr Val Lys Cys Lys Thr Ile 580 585 590 Phe Leu Tyr His Asp
Ser Thr Asp Ile Gly Leu Val Ala Ser Gly Thr 595 600 605 Lys Cys Gly
Glu Gly Met Val Cys Asn Asn Gly Glu Cys Leu Asn Met 610 615 620 Glu
Lys Val Tyr Ile Ser Thr Asn Cys Pro Ser Gln Cys Asn Glu Asn 625 630
635 640 Pro Val Asp Gly His Gly Leu Gln Cys His Cys Glu Glu Gly Gln
Ala 645 650 655 Pro Val Ala Cys Glu Glu Thr Leu His Val Thr Asn Ile
Thr Ile Leu 660 665 670 Val Val Val Leu Val Leu Val Ile Val Gly Ile
Gly Val Leu Ile Leu 675 680 685 Leu Val Arg Tyr Arg Lys Cys Ile Lys
Leu Lys Gln Val Gln Ser Pro 690 695 700 Pro Thr Glu Thr Leu Gly Val
Glu Asn Lys Gly Tyr Phe Gly Asp Glu 705 710 715 720 Gln Gln Ile Arg
Thr Glu Pro Ile Leu Pro Glu Ile His Phe Leu Asn 725 730 735 Lys Pro
Ala Ser Lys Asp Ser Arg Gly Ile Ala Asp Pro Asn Gln Ser 740 745 750
Ala Lys 721DNAArtificial SequenceDescription of artificial sequence
Oligonucleotide 7gtgcagaagg agaatgctgt g 21821DNAArtificial
SequenceDescription of artificial sequence Oligonucleotide
8tccacatctg acatctttct c 2191731DNAHomo sapiens 9agaaggagga
aggacagcac agctgacagc cgtgctcaga cagcttctgg atcccaggct 60catctccaca
gaggagaaca cacaggcagc agagaccatg gggcccctcc cagccccttc
120ctgcacacag cgcatcacct ggaaggggct cctgctcaca gcatcacttt
taaacttctg 180gaacccgccc accactgccg aagtcacgat tgaagcccag
ccacccaaag tttctgaggg 240gaaggatgtt cttctacttg tccacaattt
gccccagaat cttcctggct acttctggta 300caaaggggaa atgacggacc
tctaccatta cattatatcg tatatagttg atggtaaaat 360aattatatat
gggcctgcat acagtggaag agaaacagta tattccaacg catccctgct
420gatccagaat gtcacccgga aggatgcagg aacctacacc ttacacatca
taaagcgagg 480tgatgagact agagaagaaa ttcgacattt caccttcacc
ttatacttgg agactcccaa 540gccctacatc tccagcagca acttaaaccc
cagggaggcc atggaggctg tgcgcttaat 600ctgtgatcct gagactctgg
acgcaagcta cctatggtgg atgaatggtc agagcctccc 660tgtgactcac
aggttgcagc tgtccaaaac caacaggacc ctctatctat ttggtgtcac
720aaagtatatt gcaggaccct atgaatgtga aatacggaac ccagtgagtg
ccagtcgcag 780tgacccagtc accctgaatc tcctcccgaa gctgcccatc
ccctacatca ccatcaacaa 840cttaaacccc agggagaata aggatgtctt
agccttcacc tgtgaaccta agagtgagaa 900ctacacctac atttggtggc
taaacggtca gagcctcccc gtcagtcccg gggtaaagcg 960acccattgaa
aacaggatac tcattctacc cagtgtcacg agaaatgaaa caggacccta
1020tcaatgtgaa atacaggacc gatatggtgg cctccgcagt aacccagtca
tcctaaatgt 1080cctctatggt ccagacctcc ccagaattta cccttcattc
acctattacc gttcaggaga 1140aaacctcgac ttgtcctgct tcacggaatc
taacccaccg gcagagtatt tttggacaat 1200taatgggaag tttcagcaat
caggacaaaa gctctttatc ccccaaatta ctagaaatca 1260tagcgggctc
tatgcttgct ctgttcataa ctcagccact ggcaaggaaa tctccaaatc
1320catgacagtc aaagtctctg gtccctgcca tggagacctg acagagtctc
agtcatgact 1380gcaacaactg agacactgag aaaaagaaca ggctgatacc
ttcatgaaat tcaagacaaa 1440gaagaaaaaa actcaatgtt attggactaa
ataatcaaaa ggataatgtt ttcataattt 1500tttattggaa aatgtgctga
ttctttgaat gttttattct ccagatttat gaactttttt 1560tcttcagcaa
ttggtaaagt atacttttat aaacaaaaat tgaaatattt gcttttgctg
1620tctatctgaa tgccccagaa ttgtgaaact attcatgagt attcataggt
ttatggtaat 1680aaagttattt gcacatgttc caaaaaaaaa aaaaaaaaaa
aaaaaaaaaa a 173110426PRTHomo sapiens 10Met Gly Pro Leu Pro Ala Pro
Ser Cys Thr Gln Arg Ile Thr Trp Lys 1 5 10 15 Gly Leu Leu Leu Thr
Ala Ser Leu Leu Asn Phe Trp Asn Pro Pro Thr 20 25 30 Thr Ala Glu
Val Thr Ile Glu Ala Gln Pro Pro Lys Val Ser Glu Gly 35 40 45 Lys
Asp Val Leu Leu Leu Val His Asn Leu Pro Gln Asn Leu Pro Gly 50 55
60 Tyr Phe Trp Tyr Lys Gly Glu Met Thr Asp Leu Tyr His Tyr Ile Ile
65 70 75 80 Ser Tyr Ile Val Asp Gly Lys Ile Ile Ile Tyr Gly Pro Ala
Tyr Ser 85 90 95 Gly Arg Glu Thr Val Tyr Ser Asn Ala Ser Leu Leu
Ile Gln Asn Val 100 105 110 Thr Arg Lys Asp Ala Gly Thr Tyr Thr Leu
His Ile Ile Lys Arg Gly 115 120 125 Asp Glu Thr Arg Glu Glu Ile Arg
His Phe Thr Phe Thr Leu Tyr Leu 130 135 140 Glu Thr Pro Lys Pro Tyr
Ile Ser Ser Ser Asn Leu Asn Pro Arg Glu 145 150 155 160 Ala Met Glu
Ala Val Arg Leu Ile Cys Asp Pro Glu Thr Leu Asp Ala 165 170 175 Ser
Tyr Leu Trp Trp Met Asn Gly Gln Ser Leu Pro Val Thr His Arg 180 185
190 Leu Gln Leu Ser Lys Thr Asn Arg Thr Leu Tyr Leu Phe Gly Val Thr
195 200 205 Lys Tyr Ile Ala Gly Pro Tyr Glu Cys Glu Ile Arg Asn Pro
Val Ser 210 215 220 Ala Ser Arg Ser Asp Pro Val Thr Leu Asn Leu Leu
Pro Lys Leu Pro 225 230 235 240 Ile Pro Tyr Ile Thr Ile Asn Asn Leu
Asn Pro Arg Glu Asn Lys Asp 245 250 255 Val Leu Ala Phe Thr Cys Glu
Pro Lys Ser Glu Asn Tyr Thr Tyr Ile 260 265 270 Trp Trp Leu Asn Gly
Gln Ser Leu Pro Val Ser Pro Gly Val Lys Arg 275 280 285 Pro Ile Glu
Asn Arg Ile Leu Ile Leu Pro Ser Val Thr Arg Asn Glu 290 295 300 Thr
Gly Pro Tyr Gln Cys Glu Ile Gln Asp Arg Tyr Gly Gly Leu Arg 305 310
315 320 Ser Asn Pro Val Ile Leu Asn Val Leu Tyr Gly Pro Asp Leu Pro
Arg 325 330 335 Ile Tyr Pro Ser Phe Thr Tyr Tyr Arg Ser Gly Glu Asn
Leu Asp Leu 340 345 350 Ser Cys Phe Thr Glu Ser Asn Pro Pro Ala Glu
Tyr Phe Trp Thr Ile 355 360 365 Asn Gly Lys Phe Gln Gln Ser Gly Gln
Lys Leu Phe Ile Pro Gln Ile 370 375 380 Thr Arg Asn His Ser Gly Leu
Tyr Ala Cys Ser Val His Asn Ser Ala 385 390
395 400 Thr Gly Lys Glu Ile Ser Lys Ser Met Thr Val Lys Val Ser Gly
Pro 405 410 415 Cys His Gly Asp Leu Thr Glu Ser Gln Ser 420 425
1121DNAArtificial SequenceDescription of artificial sequence
Oligonucleotide 11ctcctcyatg gtccagacct c 211222DNAArtificial
SequenceDescription of artificial sequence Oligonucleotide
12gttgcagtca tgactgagac tc 22131634DNAHomo sapiens 13atgacagtga
ctccacagta tctaccagaa tacaagggca agcatccaaa atgtgactca 60ctggtggtgt
tccgcaatgt gtgcgtctgt gtgtccaccg cgacaggcat cagtacattg
120gatcagagtg tcgctttcag ttgtaacgga cttcatcaca tcacaaattg
tactcgttct 180catcctttta agaaagttca gacccaggaa aatttccata
gtaccttaat gaaaaagata 240gaaatcagtg ggacgtgtct ttcctttcat
ctccttttcg gcttggaaat cagaatgaga 300aggattgttt ttgctggtgt
tatcttattc cgcctcttag gtgttatctt attccgcctc 360ttaggtgtta
tcttattcgg ccgcttaggt gacctgggaa cctgccagac aaaacctggt
420cagtactgga aagaagaggt ccacattcaa gatgttggag gtttgatttg
cagagcatgc 480aatctttcac tgcccttcca tggatgtctt ttagacctgg
gaacctgcca ggcagaacct 540ggtcagtact gtaaagaaga ggtccacatt
caaggtggca ttcaatggta ttcagtcaaa 600ggctgcacaa agaacacatc
agagtgcttc aagagtactc tcgtcaagag aattctgcaa 660ctgcatgaac
ttgtaactac tcactgctgc aatcattctt tgtgcaattt ctgagtcagt
720ggcccatatc taaaatgctt ggcagatcaa tcagtctcga agcctgacct
ggctatcaca 780aaatgatggc tattgtcaat tagcccactt cagaaacctc
agacccttgt aggtagaagg 840aattttgatc tgaaattgac tttggttttc
aatattccca atatctcccc caccacctcc 900aactcatctg agaaatagcc
ctttcaacac catttctctc ctctcctcct tctgcttaat 960ttaccttcct
accacaaggc tacaaagaag gaaaaatgtt agtgattctc caagtcaaac
1020taggcatgtc acctctaact actttcattt ccctcaataa ttccatactc
caaaatatgg 1080ttacaaatgt ttcacaagac agcaagtgac ctgagaatat
tcatttggtt tccaaagcaa 1140actgccttgc tcctttgggg tgatttatgg
tatagaagaa actgacttaa catatactat 1200agggaaaaaa taagccatga
atcagcaagc caggcctgcg ggaaaagtat gaacccaaac 1260aggaaagggc
tgaggcaggt ggtagggctg gcacttattt cttccatctg cctcagagtt
1320tatccaaatt ttgaattttc cgtaccttaa ccatgcctaa atgctttggc
ttgttcaatt 1380ttggcagatt aagcagttca aggtaagcag agaagtaagt
tcccaaccac aaggaattta 1440aaaggagtag gaacgtactt tgaactacat
ttcccatttt gcgatcatta cgtcttctat 1500tacaatgccc tactttggca
gcatgaagag tactgcatta atttaattta atttaaaatt 1560taatttaaaa
ctgtctttct ctgtattttc aggagtttga aaattcaaaa aataaataat
1620aaatgtcaat aaaa 163414237PRTHomo sapiens 14Met Thr Val Thr Pro
Gln Tyr Leu Pro Glu Tyr Lys Gly Lys His Pro 1 5 10 15 Lys Cys Asp
Ser Leu Val Val Phe Arg Asn Val Cys Val Cys Val Ser 20 25 30 Thr
Ala Thr Gly Ile Ser Thr Leu Asp Gln Ser Val Ala Phe Ser Cys 35 40
45 Asn Gly Leu His His Ile Thr Asn Cys Thr Arg Ser His Pro Phe Lys
50 55 60 Lys Val Gln Thr Gln Glu Asn Phe His Ser Thr Leu Met Lys
Lys Ile 65 70 75 80 Glu Ile Ser Gly Thr Cys Leu Ser Phe His Leu Leu
Phe Gly Leu Glu 85 90 95 Ile Arg Met Arg Arg Ile Val Phe Ala Gly
Val Ile Leu Phe Arg Leu 100 105 110 Leu Gly Val Ile Leu Phe Arg Leu
Leu Gly Val Ile Leu Phe Gly Arg 115 120 125 Leu Gly Asp Leu Gly Thr
Cys Gln Thr Lys Pro Gly Gln Tyr Trp Lys 130 135 140 Glu Glu Val His
Ile Gln Asp Val Gly Gly Leu Ile Cys Arg Ala Cys 145 150 155 160 Asn
Leu Ser Leu Pro Phe His Gly Cys Leu Leu Asp Leu Gly Thr Cys 165 170
175 Gln Ala Glu Pro Gly Gln Tyr Cys Lys Glu Glu Val His Ile Gln Gly
180 185 190 Gly Ile Gln Trp Tyr Ser Val Lys Gly Cys Thr Lys Asn Thr
Ser Glu 195 200 205 Cys Phe Lys Ser Thr Leu Val Lys Arg Ile Leu Gln
Leu His Glu Leu 210 215 220 Val Thr Thr His Cys Cys Asn His Ser Leu
Cys Asn Phe 225 230 235 1521DNAArtificial SequenceDescription of
artificial sequence Oligonucleotide 15gacaaaacct ggtcagtact g
211623DNAArtificial SequenceDescription of artificial sequence
Oligonucleotide 16cccattgaat gccaccttga atg 2317744DNAHomo sapiens
17atcaggttca acgcagtgac tgctcagtag aagccatggc tcgcagacac tgcttctcct
60actggttact ggtatgctgg ttggtggtaa ctgtggcaga aggacaagaa gaggtattta
120cgcttcctgg agattcacaa aataatgcgg acgctaccga ctgccagatc
tttacactca 180cccctccacc tgccccgagg agtccggtca caagggccca
gcccatcaca aagacaccca 240ggtgtccctt ccattttttt ccacgaaggc
ccagaatcca ttttaggttt ccaaacagac 300ctttcgtccc ttcaaggtgt
aaccaccgtt ttccattcca gccattttat tggccacacc 360gttaccttac
ttataggtat ttccccagaa gaagactcca gagaggaagc tcatctgagg
420aaagctgaga gggaagagaa acccaaacat actgaagcaa aaaaaagcct
atccttcaga 480aaaaagcaac aaaaagattt ctgttttatc tttcgaaact
aaaactattg gatttgaaga 540ttaagtatcc taaacatcac tgactagaaa
ctgttctctt tgtcagcagt gaagatattg 600gatcataggt tattgatggt
tgcaaaattg gacaataacc acgttatttt tatcctcaac 660ctcttatggt
cacaggatat ttatgcaaat aaaatcttta aatgggaaaa aaaaaaaaaa
720aaaaaaaaaa aaaaaaaaaa aaaa 74418130PRTHomo sapiens 18Met Ala Arg
Arg His Cys Phe Ser Tyr Trp Leu Leu Val Cys Trp Leu 1 5 10 15 Val
Val Thr Val Ala Glu Gly Gln Glu Glu Val Phe Thr Leu Pro Gly 20 25
30 Asp Ser Gln Asn Asn Ala Asp Ala Thr Asp Cys Gln Ile Phe Thr Leu
35 40 45 Thr Pro Pro Pro Ala Pro Arg Ser Pro Val Thr Arg Ala Gln
Pro Ile 50 55 60 Thr Lys Thr Pro Arg Cys Pro Phe His Phe Phe Pro
Arg Arg Pro Arg 65 70 75 80 Ile His Phe Arg Phe Pro Asn Arg Pro Phe
Val Pro Ser Arg Cys Asn 85 90 95 His Arg Phe Pro Phe Gln Pro Phe
Tyr Trp Pro His Arg Tyr Leu Thr 100 105 110 Tyr Arg Tyr Phe Pro Arg
Arg Arg Leu Gln Arg Gly Ser Ser Ser Glu 115 120 125 Glu Ser 130
1921DNAArtificial SequenceDescription of artificial sequence
Oligonucleotide 19taactgtggc agaaggacaa g 212021DNAArtificial
SequenceDescription of artificial sequence Oligonucleotide
20agatgagctt cctctctgga g 21211955DNAHomo sapiens 21acagaagcgc
gcagagtccc atcctgccac gccacgagga gagaagaagg aaagatacag 60tgttaggaaa
gagacctccc tcgcccctac gccccgcgcc cctgcgcctc gcttcagcct
120caggacagtc ctgccgggac ggtgagcgca ttcagcaccc tggacagcac
cgcggttgcg 180ctgcctccag ggcggccccg ggctgctcct gctccgcaga
gctacgccct ccccccgggt 240gccccggacc ctgcacttgc cgccgctttc
ctcgcgctgc tctggacctt gctagccggc 300tctgcacctc ccagaagccg
tgggcgcgcc gctcagctgc tccatcgcct cactttccca 360ggctcgcgcc
cgaagcagag ccatgagaac cccagggtgc ctggcgagcc gctagcgcca
420tgggccccgg cgaggcgctg ctggcgggtc tcctggtgat ggtactggcc
gtggcgctgc 480tatccaacgc actggtgctg ctttgttgcg cctacagcgc
tgagctccgc actcgagcct 540caggcgtcct cctggtgaat ctgtctctgg
gccacctgct gctggcggcg ctggacatgc 600ccttcacgct gctcggtgtg
atgcgcgggc ggacaccgtc ggcgcccggc gcatgccaag 660tcattggctt
cctggacacc ttcctggcgt ccaacgcggc gctgagcgtg gcggcgctga
720gcgcagacca gtggctggca gtgggcttcc cactgcgcta cgccggacgc
ctgcgaccgc 780gctatgccgg cctgctgctg ggctgtgcct ggggacagtc
gctggccttc tcaggcgctg 840cacttggctg ctcgtggctt ggctacagca
gcgccttcgc gtcctgttcg ctgcgcctgc 900cgcccgagcc tgagcgtccg
cgcttcgcag ccttcaccgc cacgctccat gccgtgggct 960tcgtgctgcc
gctggcggtg ctctgcctca cctcgctcca ggtgcaccgg gtggcacgca
1020gacactgcca gcgcatggac accgtcacca tgaaggcgct cgcgctgctc
gccgacctgc 1080accccagtgt gcggcagcgc tgcctcatcc agcagaagcg
gcgccgccac cgcgccacca 1140ggaagattgg cattgctatt gcgaccttcc
tcatctgctt tgccccgtat gtcatgacca 1200ggctggcgga gctcgtgccc
ttcgtcaccg tgaacgccca gtggggcatc ctcagcaagt 1260gcctgaccta
cagcaaggcg gtggccgacc cgttcacgta ctctctgctc cgccggccgt
1320tccgccaagt cctggccggc atggtgcacc ggctgctgaa gagaaccccg
cgcccagcat 1380ccacccatga cagctctctg gatgtggccg gcatggtgca
ccagctgctg aagagaaccc 1440cgcgcccagc gtccacccac aacggctctg
tggacacaga gaatgattcc tgcctgcagc 1500agacacactg agggcctggc
agggctcatc gcccccacct tctaagaagc cctgtggaaa 1560gggcactggc
cctgccacag agatgccact ggggaccccc agacaccagt ggcttgactt
1620tgagctaagg ctgaagtaca ggaggaggag gaggagaggg ccggatgtgg
gtgtggacag 1680cagtagtggc ggaggagagc tcggggctgg gctgcctggc
tgctgggtgg ccccgggaca 1740gtggcttttc ctctctgaac cttagcttcc
tcacccttgt tctggggtca tggcgatgct 1800tcgagacagt gggtagggaa
gtgccctgtg tggcatatgg tactcgtggg cgtgctataa 1860gtgactgctg
ttcatgtggg tgaggtggtc actcttgctc agggtctgtt gtgcagccca
1920gatggacacc tgtttctcca aaaaaaaaaa aaaaa 195522363PRTHomo sapiens
22Met Gly Pro Gly Glu Ala Leu Leu Ala Gly Leu Leu Val Met Val Leu 1
5 10 15 Ala Val Ala Leu Leu Ser Asn Ala Leu Val Leu Leu Cys Cys Ala
Tyr 20 25 30 Ser Ala Glu Leu Arg Thr Arg Ala Ser Gly Val Leu Leu
Val Asn Leu 35 40 45 Ser Leu Gly His Leu Leu Leu Ala Ala Leu Asp
Met Pro Phe Thr Leu 50 55 60 Leu Gly Val Met Arg Gly Arg Thr Pro
Ser Ala Pro Gly Ala Cys Gln 65 70 75 80 Val Ile Gly Phe Leu Asp Thr
Phe Leu Ala Ser Asn Ala Ala Leu Ser 85 90 95 Val Ala Ala Leu Ser
Ala Asp Gln Trp Leu Ala Val Gly Phe Pro Leu 100 105 110 Arg Tyr Ala
Gly Arg Leu Arg Pro Arg Tyr Ala Gly Leu Leu Leu Gly 115 120 125 Cys
Ala Trp Gly Gln Ser Leu Ala Phe Ser Gly Ala Ala Leu Gly Cys 130 135
140 Ser Trp Leu Gly Tyr Ser Ser Ala Phe Ala Ser Cys Ser Leu Arg Leu
145 150 155 160 Pro Pro Glu Pro Glu Arg Pro Arg Phe Ala Ala Phe Thr
Ala Thr Leu 165 170 175 His Ala Val Gly Phe Val Leu Pro Leu Ala Val
Leu Cys Leu Thr Ser 180 185 190 Leu Gln Val His Arg Val Ala Arg Arg
His Cys Gln Arg Met Asp Thr 195 200 205 Val Thr Met Lys Ala Leu Ala
Leu Leu Ala Asp Leu His Pro Ser Val 210 215 220 Arg Gln Arg Cys Leu
Ile Gln Gln Lys Arg Arg Arg His Arg Ala Thr 225 230 235 240 Arg Lys
Ile Gly Ile Ala Ile Ala Thr Phe Leu Ile Cys Phe Ala Pro 245 250 255
Tyr Val Met Thr Arg Leu Ala Glu Leu Val Pro Phe Val Thr Val Asn 260
265 270 Ala Gln Trp Gly Ile Leu Ser Lys Cys Leu Thr Tyr Ser Lys Ala
Val 275 280 285 Ala Asp Pro Phe Thr Tyr Ser Leu Leu Arg Arg Pro Phe
Arg Gln Val 290 295 300 Leu Ala Gly Met Val His Arg Leu Leu Lys Arg
Thr Pro Arg Pro Ala 305 310 315 320 Ser Thr His Asp Ser Ser Leu Asp
Val Ala Gly Met Val His Gln Leu 325 330 335 Leu Lys Arg Thr Pro Arg
Pro Ala Ser Thr His Asn Gly Ser Val Asp 340 345 350 Thr Glu Asn Asp
Ser Cys Leu Gln Gln Thr His 355 360 2321DNAArtificial
SequenceDescription of artificial sequence Oligonucleotide
23ccgtatgtca tgaccaggct g 212421DNAArtificial SequenceDescription
of artificial sequence Oligonucleotide 24aagtcaagcc actggtgtct g
21252059DNAHomo sapiens 25agcacagaag gaggaaggac agcacagctg
acagccgtac tcaggaagct tctggatcct 60aggcttatct ccacagagga gaacacacaa
gcagcagaga ccatggggcc cctctcagcc 120cctccctgca cacagcgcat
cacctggaag ggggtcctgc tcacagcatc acttttaaac 180ttctggaatc
cgcccacaac tgcccaagtc acgattgaag cccagccacc caaagtttct
240gaggggaagg atgttcttct acttgtccac aatttgcccc agaatcttgc
tggctacatt 300tggtacaaag ggcaaatgac atacctctac cattacatta
catcatatgt agtagacggt 360caaagaatta tatatgggcc tgcatacagt
ggaagagaaa gagtatattc caatgcatcc 420ctgctgatcc agaatgtcac
gcaggaggat gcaggatcct acaccttaca catcataaag 480cgacgcgatg
ggactggagg agtaactgga catttcacct tcaccttaca cctggagact
540cccaagccct ccatctccag cagcaactta aatcccaggg aggccatgga
ggctgtgatc 600ttaacctgtg atcctgcgac tccagccgca agctaccagt
ggtggatgaa tggtcagagc 660ctccctatga ctcacaggtt gcagctgtcc
aaaaccaaca ggaccctctt tatatttggt 720gtcacaaagt atattgcagg
accctatgaa tgtgaaatac ggaacccagt gagtgccagc 780cgcagtgacc
cagtcaccct gaatctcctc ccaaagctgt ccaagcccta catcacaatc
840aacaacttaa accccagaga gaataaggat gtcttaacct tcacctgtga
acctaagagt 900aagaactaca cctacatttg gtggctaaat ggtcagagcc
tccctgtcag tcccagggta 960aagcgaccca ttgaaaacag gatcctcatt
ctacccaatg tcacgagaaa tgaaacagga 1020ccttatcaat gtgaaatacg
ggaccgatat ggtggcatcc gcagtgaccc agtcaccctg 1080aatgtcctct
atggtccaga cctccccagc atttaccctt cattcaccta ttaccgttca
1140ggagaaaacc tctacttgtc ctgcttcgcc gagtctaacc cacgggcaca
atattcttgg 1200acaattaatg ggaagtttca gctatcagga caaaagctct
ctatccccca aataactaca 1260aagcatagtg ggctctatgc ttgctctgtt
cgtaactcag ccactggcaa ggaaagctcc 1320aaatccatca cagtcaaagt
ctctgactgg atattaccct gaattctact agttcctcca 1380attccatttt
ctcccatgga atcacgaaga gcaagaccca ctctgttcca gaagccctat
1440aagctggagg tggacaactc gatgtaaatt tcatgggaaa acccttgtac
ctgacatgtg 1500agccactcag aactcaccaa aatgttcgac accataacaa
cagctactca aactgtaaac 1560caggataaca agttgatgac ttcacactgt
ggacagtttt tccaaagatg tcagaacaag 1620actccccatc atgataaggc
tcccacccct cttaaccgtc cttgctcatg cctgcctctt 1680tcacttggca
ggataatgca gtcattagaa tttcacatgt agtagcttct gagggtaaca
1740acagagtgtc agatatgtca tctcaacctc aaacttttac gtaacatctc
aggggaaatg 1800tggctctctc catcttgcat acagggctcc caatagaaat
gaacacagag atattgcctg 1860tgtgtttgca gagaagatgg tttctataaa
gagtaggaaa gctgaaatta tagtagagtc 1920tcctttaaat gcacattgtg
tggatggctc tcaccatttc ctaagagata cagtgtaaaa 1980cgtgacagta
atactgattc tagcagaata aaacatgtac cacatttgct aaaaaaaaaa
2040aaaaaaaaaa aaaaaaaaa 205926419PRTHomo sapiens 26Met Gly Pro Leu
Ser Ala Pro Pro Cys Thr Gln Arg Ile Thr Trp Lys 1 5 10 15 Gly Val
Leu Leu Thr Ala Ser Leu Leu Asn Phe Trp Asn Pro Pro Thr 20 25 30
Thr Ala Gln Val Thr Ile Glu Ala Gln Pro Pro Lys Val Ser Glu Gly 35
40 45 Lys Asp Val Leu Leu Leu Val His Asn Leu Pro Gln Asn Leu Ala
Gly 50 55 60 Tyr Ile Trp Tyr Lys Gly Gln Met Thr Tyr Leu Tyr His
Tyr Ile Thr 65 70 75 80 Ser Tyr Val Val Asp Gly Gln Arg Ile Ile Tyr
Gly Pro Ala Tyr Ser 85 90 95 Gly Arg Glu Arg Val Tyr Ser Asn Ala
Ser Leu Leu Ile Gln Asn Val 100 105 110 Thr Gln Glu Asp Ala Gly Ser
Tyr Thr Leu His Ile Ile Lys Arg Arg 115 120 125 Asp Gly Thr Gly Gly
Val Thr Gly His Phe Thr Phe Thr Leu His Leu 130 135 140 Glu Thr Pro
Lys Pro Ser Ile Ser Ser Ser Asn Leu Asn Pro Arg Glu 145 150 155 160
Ala Met Glu Ala Val Ile Leu Thr Cys Asp Pro Ala Thr Pro Ala Ala 165
170 175 Ser Tyr Gln Trp Trp Met Asn Gly Gln Ser Leu Pro Met Thr His
Arg 180 185 190 Leu Gln Leu Ser Lys Thr Asn Arg Thr Leu Phe Ile Phe
Gly Val Thr 195 200 205 Lys Tyr Ile Ala Gly Pro Tyr Glu Cys Glu Ile
Arg Asn Pro Val Ser 210 215 220 Ala Ser Arg Ser Asp Pro Val Thr Leu
Asn Leu Leu Pro Lys Leu Ser 225 230 235 240 Lys Pro Tyr Ile Thr Ile
Asn Asn Leu Asn Pro Arg Glu Asn Lys Asp 245 250 255 Val Leu Thr Phe
Thr Cys Glu Pro Lys Ser Lys Asn Tyr Thr Tyr Ile 260 265 270 Trp Trp
Leu Asn Gly Gln Ser Leu Pro Val Ser Pro Arg Val Lys Arg 275 280 285
Pro Ile Glu Asn Arg Ile Leu Ile Leu Pro Asn Val Thr Arg Asn Glu 290
295 300 Thr Gly Pro Tyr Gln Cys Glu Ile Arg Asp Arg Tyr Gly Gly Ile
Arg 305 310 315 320 Ser Asp Pro Val Thr Leu Asn Val Leu Tyr Gly Pro
Asp Leu Pro Ser 325 330 335 Ile Tyr Pro Ser Phe Thr Tyr Tyr Arg Ser
Gly Glu Asn Leu Tyr Leu 340 345 350 Ser Cys Phe Ala Glu Ser Asn Pro
Arg
Ala Gln Tyr Ser Trp Thr Ile 355 360 365 Asn Gly Lys Phe Gln Leu Ser
Gly Gln Lys Leu Ser Ile Pro Gln Ile 370 375 380 Thr Thr Lys His Ser
Gly Leu Tyr Ala Cys Ser Val Arg Asn Ser Ala 385 390 395 400 Thr Gly
Lys Glu Ser Ser Lys Ser Ile Thr Val Lys Val Ser Asp Trp 405 410 415
Ile Leu Pro 2721DNAArtificial SequenceDescription of artificial
sequence Oligonucleotide 27gttgttaccc tcagaagcta c 21281780DNAHomo
sapiens 28agcacagaag gaggaaggac agcacagctg acagccgtac tcaggaagct
tctggatcct 60aggcttatct ccacagagga gaacacacaa gcagcagaga ccatggggcc
cctctcagcc 120cctccctgca cacagcgcat cacctggaag ggggtcctgc
tcacagcatc acttttaaac 180ttctggaatc cgcccacaac tgcccaagtc
acgattgaag cccagccacc caaagtttct 240gaggggaagg atgttcttct
acttgtccac aatttgcccc agaatcttgc tggctacatt 300tggtacaaag
ggcaaatgac atacctctac cattacatta catcatatgt agtagacggt
360caaagaatta tatatgggcc tgcatacagt ggaagagaaa gagtatattc
caatgcatcc 420ctgctgatcc agaatgtcac gcaggaggat gcaggatcct
acaccttaca catcataaag 480cgacgcgatg ggactggagg agtaactgga
catttcacct tcaccttaca cctggagact 540cccaagccct ccatctccag
cagcaactta aatcccaggg aggccatgga ggctgtgatc 600ttaacctgtg
atcctgcgac tccagccgca agctaccagt ggtggatgaa tggtcagagc
660ctccctatga ctcacaggtt gcagctgtcc aaaaccaaca ggaccctctt
tatatttggt 720gtcacaaagt atattgcagg accctatgaa tgtgaaatac
ggaacccagt gagtgccagc 780cgcagtgacc cagtcaccct gaatctcctc
catggtccag acctccccag catttaccct 840tcattcacct attaccgttc
aggagaaaac ctctacttgt cctgcttcgc cgagtctaac 900ccacgggcac
aatattcttg gacaattaat gggaagtttc agctatcagg acaaaagctc
960tctatccccc aaataactac aaagcatagt gggctctatg cttgctctgt
tcgtaactca 1020gccactggca aggaaagctc caaatccatc acagtcaaag
tctctgactg gatattaccc 1080tgaattctac tagttcctcc aattccattt
tctcccatgg aatcacgaag agcaagaccc 1140actctgttcc agaagcccta
taagctggag gtggacaact cgatgtaaat ttcatgggaa 1200aacccttgta
cctgacatgt gagccactca gaactcacca aaatgttcga caccataaca
1260acagctactc aaactgtaaa ccaggataac aagttgatga cttcacactg
tggacagttt 1320ttccaaagat gtcagaacaa gactccccat catgataagg
ctcccacccc tcttaaccgt 1380ccttgctcat gcctgcctct ttcacttggc
aggataatgc agtcattaga atttcacatg 1440tagtagcttc tgagggtaac
aacagagtgt cagatatgtc atctcaacct caaactttta 1500cgtaacatct
caggggaaat gtggctctct ccatcttgca tacagggctc ccaatagaaa
1560tgaacacaga gatattgcct gtgtgtttgc agagaagatg gtttctataa
agagtaggaa 1620agctgaaatt atagtagagt ctcctttaaa tgcacattgt
gtggatggct ctcaccattt 1680cctaagagat acagtgtaaa acgtgacagt
aatactgatt ctagcagaat aaaacatgta 1740ccacatttgc taaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 178029326PRTHomo sapiens 29Met Gly Pro Leu
Ser Ala Pro Pro Cys Thr Gln Arg Ile Thr Trp Lys 1 5 10 15 Gly Val
Leu Leu Thr Ala Ser Leu Leu Asn Phe Trp Asn Pro Pro Thr 20 25 30
Thr Ala Gln Val Thr Ile Glu Ala Gln Pro Pro Lys Val Ser Glu Gly 35
40 45 Lys Asp Val Leu Leu Leu Val His Asn Leu Pro Gln Asn Leu Ala
Gly 50 55 60 Tyr Ile Trp Tyr Lys Gly Gln Met Thr Tyr Leu Tyr His
Tyr Ile Thr 65 70 75 80 Ser Tyr Val Val Asp Gly Gln Arg Ile Ile Tyr
Gly Pro Ala Tyr Ser 85 90 95 Gly Arg Glu Arg Val Tyr Ser Asn Ala
Ser Leu Leu Ile Gln Asn Val 100 105 110 Thr Gln Glu Asp Ala Gly Ser
Tyr Thr Leu His Ile Ile Lys Arg Arg 115 120 125 Asp Gly Thr Gly Gly
Val Thr Gly His Phe Thr Phe Thr Leu His Leu 130 135 140 Glu Thr Pro
Lys Pro Ser Ile Ser Ser Ser Asn Leu Asn Pro Arg Glu 145 150 155 160
Ala Met Glu Ala Val Ile Leu Thr Cys Asp Pro Ala Thr Pro Ala Ala 165
170 175 Ser Tyr Gln Trp Trp Met Asn Gly Gln Ser Leu Pro Met Thr His
Arg 180 185 190 Leu Gln Leu Ser Lys Thr Asn Arg Thr Leu Phe Ile Phe
Gly Val Thr 195 200 205 Lys Tyr Ile Ala Gly Pro Tyr Glu Cys Glu Ile
Arg Asn Pro Val Ser 210 215 220 Ala Ser Arg Ser Asp Pro Val Thr Leu
Asn Leu Leu His Gly Pro Asp 225 230 235 240 Leu Pro Ser Ile Tyr Pro
Ser Phe Thr Tyr Tyr Arg Ser Gly Glu Asn 245 250 255 Leu Tyr Leu Ser
Cys Phe Ala Glu Ser Asn Pro Arg Ala Gln Tyr Ser 260 265 270 Trp Thr
Ile Asn Gly Lys Phe Gln Leu Ser Gly Gln Lys Leu Ser Ile 275 280 285
Pro Gln Ile Thr Thr Lys His Ser Gly Leu Tyr Ala Cys Ser Val Arg 290
295 300 Asn Ser Ala Thr Gly Lys Glu Ser Ser Lys Ser Ile Thr Val Lys
Val 305 310 315 320 Ser Asp Trp Ile Leu Pro 325 301989DNAHomo
sapiens 30attcgggcct aggctcatct ccacagagga gaacacgcag ggagcagaga
ccatggggcc 60cctctcagcc cctccctgca cacagcatat aacctggaaa gggctcctgc
tcacagcatc 120acttttaaac ttctggaacc cgcccaccac agcccaagtc
acgattgaag cccagccacc 180aaaagtttct gaggggaagg atgttcttct
acttgtccac aatttgcccc agaatcttac 240tggctacatc tggtacaaag
gacaaatcag ggacctctac cattatgtta catcatatgt 300agtagacggt
caaataatta aatatgggcc tgcatacagt ggacgagaaa cagtatattc
360caatgcatcc ctgctgatcc agaatgtcac ccaggaagac acaggatcct
acactttaca 420catcataaag cgaggtgatg ggactggagg agtaactgga
cgtttcacct tcaccttata 480cctggagact cccaaaccct ccatctccag
cagcaatttc aaccccaggg aggccacgga 540ggctgtgatc ttaacctgtg
atcctgagac tccagatgca agctacctgt ggtggatgaa 600tggtcagagc
ctccctatga ctcacagctt gcagctgtct gaaaccaaca ggaccctcta
660cctatttggt gtcacaaact atactgcagg accctatgaa tgtgaaatac
ggaacccagt 720gagtgccagc cgcagtgacc cagtcaccct gaatctcctc
ccgaagctgc ccaagcccta 780catcaccatc aataacttaa accccaggga
gaataaggat gtctcaacct tcacctgtga 840acctaagagt gagaactaca
cctacatttg gtggctaaat ggtcagagcc tcccggtcag 900tcccagggta
aagcgacgca ttgaaaacag gatcctcatt ctacccagtg tcacgagaaa
960tgaaacagga ccctatcaat gtgaaatacg ggaccgatat ggtggcatcc
gcagtgaccc 1020agtcaccctg aatgtcctct atggtccaga cctccccaga
atttaccctt cgttcaccta 1080ttaccattca ggacaaaacc tctacttgtc
ctgctttgcg gactctaacc caccggcaca 1140gtattcttgg acaattaatg
ggaagtttca gctatcagga caaaagcttt ctatccccca 1200gattactaca
aagcatagcg ggctctatgc ttgctctgtt cgtaactcag ccactggcaa
1260ggaaagctcc aaatccgtga cagtcagagt ctctgactgg acattaccct
gaattctact 1320agttcctcca attccatctt ctcccatgga acctcaaaga
gcaagaccca ctctgttcca 1380gaagccctat aagtcagagt tggacaactc
aatgtaaatt tcatgggaaa atccttgtac 1440ctgatgtctg agccactcag
aactcaccaa aatgttcaac accataacaa cagctgctca 1500aactgtaaac
aaggaaaaca agttgatgac ttcacactgt ggacagtttt tcccaagatg
1560tcagaataag actccccatc atgatgaggc tctcacccct cttagctgtc
cttgcttgtg 1620cctgcctctt tcacttggca ggataatgca gtcattagaa
tttcacatgt agtataggag 1680cttctgaggg taacaacaga gtgtcagata
tgtcatctca acctcaaact tttacataac 1740atctcaggag gaaatgtggc
tctctccatc ttgcatacag ggctcccaat agaaatgaac 1800acagagatat
tgcctgtgtg tttgcagaga agatggtttc tataaagagt aggaaagctg
1860aaattatagt agagtcccct ttaaatgcac attgtgtgga tggctctcac
catttcctaa 1920gagatacatt gtaaaacgtg acagtaagac tgattctagc
agaataaaac atgtactaca 1980tttgctaaa 198931419PRTHomo sapiens 31Met
Gly Pro Leu Ser Ala Pro Pro Cys Thr Gln His Ile Thr Trp Lys 1 5 10
15 Gly Leu Leu Leu Thr Ala Ser Leu Leu Asn Phe Trp Asn Pro Pro Thr
20 25 30 Thr Ala Gln Val Thr Ile Glu Ala Gln Pro Pro Lys Val Ser
Glu Gly 35 40 45 Lys Asp Val Leu Leu Leu Val His Asn Leu Pro Gln
Asn Leu Thr Gly 50 55 60 Tyr Ile Trp Tyr Lys Gly Gln Ile Arg Asp
Leu Tyr His Tyr Val Thr 65 70 75 80 Ser Tyr Val Val Asp Gly Gln Ile
Ile Lys Tyr Gly Pro Ala Tyr Ser 85 90 95 Gly Arg Glu Thr Val Tyr
Ser Asn Ala Ser Leu Leu Ile Gln Asn Val 100 105 110 Thr Gln Glu Asp
Thr Gly Ser Tyr Thr Leu His Ile Ile Lys Arg Gly 115 120 125 Asp Gly
Thr Gly Gly Val Thr Gly Arg Phe Thr Phe Thr Leu Tyr Leu 130 135 140
Glu Thr Pro Lys Pro Ser Ile Ser Ser Ser Asn Phe Asn Pro Arg Glu 145
150 155 160 Ala Thr Glu Ala Val Ile Leu Thr Cys Asp Pro Glu Thr Pro
Asp Ala 165 170 175 Ser Tyr Leu Trp Trp Met Asn Gly Gln Ser Leu Pro
Met Thr His Ser 180 185 190 Leu Gln Leu Ser Glu Thr Asn Arg Thr Leu
Tyr Leu Phe Gly Val Thr 195 200 205 Asn Tyr Thr Ala Gly Pro Tyr Glu
Cys Glu Ile Arg Asn Pro Val Ser 210 215 220 Ala Ser Arg Ser Asp Pro
Val Thr Leu Asn Leu Leu Pro Lys Leu Pro 225 230 235 240 Lys Pro Tyr
Ile Thr Ile Asn Asn Leu Asn Pro Arg Glu Asn Lys Asp 245 250 255 Val
Ser Thr Phe Thr Cys Glu Pro Lys Ser Glu Asn Tyr Thr Tyr Ile 260 265
270 Trp Trp Leu Asn Gly Gln Ser Leu Pro Val Ser Pro Arg Val Lys Arg
275 280 285 Arg Ile Glu Asn Arg Ile Leu Ile Leu Pro Ser Val Thr Arg
Asn Glu 290 295 300 Thr Gly Pro Tyr Gln Cys Glu Ile Arg Asp Arg Tyr
Gly Gly Ile Arg 305 310 315 320 Ser Asp Pro Val Thr Leu Asn Val Leu
Tyr Gly Pro Asp Leu Pro Arg 325 330 335 Ile Tyr Pro Ser Phe Thr Tyr
Tyr His Ser Gly Gln Asn Leu Tyr Leu 340 345 350 Ser Cys Phe Ala Asp
Ser Asn Pro Pro Ala Gln Tyr Ser Trp Thr Ile 355 360 365 Asn Gly Lys
Phe Gln Leu Ser Gly Gln Lys Leu Ser Ile Pro Gln Ile 370 375 380 Thr
Thr Lys His Ser Gly Leu Tyr Ala Cys Ser Val Arg Asn Ser Ala 385 390
395 400 Thr Gly Lys Glu Ser Ser Lys Ser Val Thr Val Arg Val Ser Asp
Trp 405 410 415 Thr Leu Pro 3221DNAArtificial SequenceDescription
of artificial sequence Oligonucleotide 32taccctcaga agctcctata c
213323DNAArtificial SequenceDescription of artificial sequence
Oligonucleotide 33cgtgagcgct tcgagatgtt ccg 233423DNAArtificial
SequenceDescription of artificial sequence Oligonucleotide
34cctaaccagc tgcccaactg tag 23352881DNAHomo sapiens 35acagaagtgc
tagaagccag tgctcgtgaa ctaaggagaa aaagaacaga caagggaaca 60gcctggacat
ggcatcagag atccacatga caggcccaat gtgcctcatt gagaacacta
120atgggcgact gatggcgaat ccagaagctc tgaagatcct ttctgccatt
acacagccta 180tggtggtggt ggcaattgtg ggcctctacc gcacaggcaa
atcctacctg atgaacaagc 240tggctggaaa gaaaaagggc ttctctctgg
gctccacggt gcagtctcac actaaaggaa 300tctggatgtg gtgtgtgccc
caccccaaga agccaggcca catcctagtt ctgctggaca 360ccgagggtct
gggagatgta gagaagggtg acaaccagaa tgactcctgg atcttcgccc
420tggccgtcct cctgagcagc accttcgtgt acaatagcat aggaaccatc
aaccagcagg 480ctatggacca actgtactat gtgacagagc tgacacatag
aatccgatca aaatcctcac 540ctgatgagaa tgagaatgag gttgaggatt
cagctgactt tgtgagcttc ttcccagact 600ttgtgtggac actgagagat
ttctccctgg acttggaagc agatggacaa cccctcacac 660cagatgagta
cctgacatac tccctgaagc tgaagaaagg taccagtcaa aaagatgaaa
720cttttaacct gcccagactc tgtatccgga aattcttccc aaagaaaaaa
tgctttgtct 780ttgatcggcc cgttcaccgc aggaagcttg cccagctcga
gaaactacaa gatgaagagc 840tggaccccga atttgtgcaa caagtagcag
acttctgttc ctacatcttt agtaattcca 900aaactaaaac tctttcagga
ggcatccagg tcaacgggcc tcgtctagag agcctggtgc 960tgacctacgt
caatgccatc agcagtgggg atctgccgtg catggagaac gcagtcctgg
1020ccttggccca gatagagaac tcagctgcag tgcaaaaggc tattgcccac
tatgaacagc 1080agatgggcca gaaggtgcag ctgcccacag aaagcctcca
ggagctgctg gacctgcaca 1140gggacagtga gagagaggcc attgaagtct
tcatcaggag ttccttcaaa gatgtggacc 1200atctatttca aaaggagtta
gcggcccagc tagaaaaaaa gcgggatgac ttttgtaaac 1260agaatcagga
agcatcatca gatcgttgct caggtttact tcaggtcatt ttcagtcctc
1320tagaagaaga agtgaaggcg ggaatttatt cgaaaccagg gggctatcgt
ctctttgttc 1380agaagctaca agacctgaag aaaaagtact atgaggaacc
gaggaagggg atacaggctg 1440aagagattct gcagacatac ttgaaatcca
aggagtctat gactgatgca attctccaga 1500cagaccagac tctcacagaa
aaagaaaagg agattgaagt ggaacgtgtg aaagctgagt 1560ctgcacaggc
ttcagcaaaa atgttgcagg aaatgcaaag aaagaatgag cagatgatgg
1620aacagaagga gaggagttat caggaacact tgaaacaact gactgagaag
atggagaacg 1680acagggtcca gttgctgaaa gagcaagaga ggaccctcgc
tcttaaactt caggaacagg 1740agcaactact aaaagaggga tttcaaaaag
aaagcagaat aatgaaaaat gagatacagg 1800atctccagac gaaaatgaga
cgacgaaagg catgtaccat aagctaaaga ccagagcctt 1860cctgtcaccc
ctaaccaagg cataattgaa acaattttag aatttggaac aagcgtcact
1920acatttgata ataattagat cttgcatcat aacaccaaaa gtttataaag
gcatgtggta 1980caatgatcaa aatcatgttt tttcttaaaa aaaaaaaaaa
gactgtaaat tgtgcaacaa 2040agatgcattt acctctgtat caactcagga
aatctcataa gctggtacca ctcaggagaa 2100gtttattctt ccagatgacc
agcagtagac aaatggatac tgagcagagt cttaggtaaa 2160agtcttggga
aatatttggg cattggtctg gccaagtcta caatgtccca atatcaagga
2220caaccaccct agcttcttag tgaagacaat gtacagttat ccattagatc
aagactacac 2280ggtctatgag caataatgtg atttctggac attgcccatg
tataatcctc actgatgatt 2340tcaagctaaa gcaaaccacc ttatacagag
atctagaatc tctttatgtt ctccagagga 2400aggtggaaga aaccatgggc
aggagtagga attgagtgat aaacaattgg gctaatgaag 2460aaaacttctc
ttattgttca gttcatccag attataactt caatgggaca ctttagacca
2520ttagacaatt gacactggat taaacaaatt cacataatgc caaatacaca
atgtatttat 2580agcaacgtat aatttgcaaa gatggacttt aaaagatgct
gtgtaactaa actgaaataa 2640ttcaattact tattatttag aatgttaaag
cttatgatag tcttttctaa ttcttaacac 2700tcatacttga aatctttccg
agtttcccca gaagagaata tgggattttt tttgacattt 2760ttgacccatt
taataatgct cttgtgttta cctagtatat gtagactttg tcttatgtgt
2820caaaagtcct aggaaagtgg ttgatgtttc ttatagcaat taaaaattat
ttttgaactg 2880a 288136592PRTHomo sapiens 36Met Ala Ser Glu Ile His
Met Thr Gly Pro Met Cys Leu Ile Glu Asn 1 5 10 15 Thr Asn Gly Arg
Leu Met Ala Asn Pro Glu Ala Leu Lys Ile Leu Ser 20 25 30 Ala Ile
Thr Gln Pro Met Val Val Val Ala Ile Val Gly Leu Tyr Arg 35 40 45
Thr Gly Lys Ser Tyr Leu Met Asn Lys Leu Ala Gly Lys Lys Lys Gly 50
55 60 Phe Ser Leu Gly Ser Thr Val Gln Ser His Thr Lys Gly Ile Trp
Met 65 70 75 80 Trp Cys Val Pro His Pro Lys Lys Pro Gly His Ile Leu
Val Leu Leu 85 90 95 Asp Thr Glu Gly Leu Gly Asp Val Glu Lys Gly
Asp Asn Gln Asn Asp 100 105 110 Ser Trp Ile Phe Ala Leu Ala Val Leu
Leu Ser Ser Thr Phe Val Tyr 115 120 125 Asn Ser Ile Gly Thr Ile Asn
Gln Gln Ala Met Asp Gln Leu Tyr Tyr 130 135 140 Val Thr Glu Leu Thr
His Arg Ile Arg Ser Lys Ser Ser Pro Asp Glu 145 150 155 160 Asn Glu
Asn Glu Val Glu Asp Ser Ala Asp Phe Val Ser Phe Phe Pro 165 170 175
Asp Phe Val Trp Thr Leu Arg Asp Phe Ser Leu Asp Leu Glu Ala Asp 180
185 190 Gly Gln Pro Leu Thr Pro Asp Glu Tyr Leu Thr Tyr Ser Leu Lys
Leu 195 200 205 Lys Lys Gly Thr Ser Gln Lys Asp Glu Thr Phe Asn Leu
Pro Arg Leu 210 215 220 Cys Ile Arg Lys Phe Phe Pro Lys Lys Lys Cys
Phe Val Phe Asp Arg 225 230 235 240 Pro Val His Arg Arg Lys Leu Ala
Gln Leu Glu Lys Leu Gln Asp Glu 245 250 255 Glu Leu Asp Pro Glu Phe
Val Gln Gln Val Ala Asp Phe Cys Ser Tyr 260 265 270 Ile Phe Ser Asn
Ser Lys Thr Lys Thr Leu Ser Gly Gly Ile Gln Val 275 280 285 Asn Gly
Pro Arg Leu Glu Ser Leu Val Leu Thr Tyr Val Asn Ala Ile 290 295 300
Ser Ser Gly Asp Leu Pro Cys Met Glu Asn Ala Val Leu Ala Leu Ala 305
310 315 320 Gln Ile Glu Asn Ser Ala Ala Val Gln Lys Ala Ile Ala His
Tyr Glu 325 330 335 Gln Gln Met Gly Gln Lys Val Gln Leu Pro Thr Glu
Ser Leu Gln Glu 340 345 350 Leu Leu Asp Leu His Arg Asp Ser Glu Arg
Glu Ala Ile Glu Val Phe 355
360 365 Ile Arg Ser Ser Phe Lys Asp Val Asp His Leu Phe Gln Lys Glu
Leu 370 375 380 Ala Ala Gln Leu Glu Lys Lys Arg Asp Asp Phe Cys Lys
Gln Asn Gln 385 390 395 400 Glu Ala Ser Ser Asp Arg Cys Ser Gly Leu
Leu Gln Val Ile Phe Ser 405 410 415 Pro Leu Glu Glu Glu Val Lys Ala
Gly Ile Tyr Ser Lys Pro Gly Gly 420 425 430 Tyr Arg Leu Phe Val Gln
Lys Leu Gln Asp Leu Lys Lys Lys Tyr Tyr 435 440 445 Glu Glu Pro Arg
Lys Gly Ile Gln Ala Glu Glu Ile Leu Gln Thr Tyr 450 455 460 Leu Lys
Ser Lys Glu Ser Met Thr Asp Ala Ile Leu Gln Thr Asp Gln 465 470 475
480 Thr Leu Thr Glu Lys Glu Lys Glu Ile Glu Val Glu Arg Val Lys Ala
485 490 495 Glu Ser Ala Gln Ala Ser Ala Lys Met Leu Gln Glu Met Gln
Arg Lys 500 505 510 Asn Glu Gln Met Met Glu Gln Lys Glu Arg Ser Tyr
Gln Glu His Leu 515 520 525 Lys Gln Leu Thr Glu Lys Met Glu Asn Asp
Arg Val Gln Leu Leu Lys 530 535 540 Glu Gln Glu Arg Thr Leu Ala Leu
Lys Leu Gln Glu Gln Glu Gln Leu 545 550 555 560 Leu Lys Glu Gly Phe
Gln Lys Glu Ser Arg Ile Met Lys Asn Glu Ile 565 570 575 Gln Asp Leu
Gln Thr Lys Met Arg Arg Arg Lys Ala Cys Thr Ile Ser 580 585 590
3718DNAArtificial SequenceDescription of artificial sequence
Oligonucleotide 37aggagattga agtggaac 183816DNAArtificial
SequenceDescription of artificial sequence Oligonucleotide
38ttgctcctgt tcctga 16392380DNAHomo sapiens 39ctccaggctg tggaaccttt
gttctttcac tctttgcaat aaatcttgct gctgctcact 60ctttgggtcc acactgcctt
tatgagctgt aacactcact gggaatgtct gcagcttcac 120tcctgaagcc
agcgagacca cgaacccacc aggaggaaca aacaactcca gacgcgcagc
180cttaagagct gtaacactca ccgcgaaggt ctgcagcttc actcctgagc
cagccagacc 240acgaacccac cagaaggaag aaactccaaa cacatccgaa
catcagaagg agcaaactcc 300tgacacgcca cctttaagaa ccgtgacact
caacgctagg gtccgcggct tcattcttga 360agtcagtgag accaagaacc
caccaattcc ggacacgcta attgttgtag atcatcactt 420caaggtgccc
atatctttct agtggaaaaa ttattctggc ctccgctgca tacaaatcag
480gcaaccagaa ttctacatat ataaggcaaa gtaacatcct agacatggct
ttagagatcc 540acatgtcaga ccccatgtgc ctcatcgaga actttaatga
gcagctgaag gttaatcagg 600aagctttgga gatcctgtct gccattacgc
aacctgtagt tgtggtagcg attgtgggcc 660tctatcgcac tggcaaatcc
tacctgatga acaagctggc tgggaagaac aagggcttct 720ctgttgcatc
tacggtgcag tctcacacca agggaatttg gatatggtgt gtgcctcatc
780ccaactggcc aaatcacaca ttagttctgc ttgacaccga gggcctggga
gatgtagaga 840aggctgacaa caagaatgat atccagatct ttgcactggc
actcttactg agcagcacct 900ttgtgtacaa tactgtgaac aaaattgatc
agggtgctat cgacctactg cacaatgtga 960cagaactgac agatctgctc
aaggcaagaa actcacccga ccttgacagg gttgaagatc 1020ctgctgactc
tgcgagcttc ttcccagact tagtgtggac tctgagagat ttctgcttag
1080gcctggaaat agatgggcaa cttgtcacac cagatgaata cctggagaat
tccctaaggc 1140caaagcaagg tagtgatcaa agagttcaaa atttcaattt
gccccgtctg tgtatacaga 1200agttctttcc aaaaaagaaa tgctttatct
ttgacttacc tgctcaccaa aaaaagcttg 1260cccaacttga aacactgcct
gatgatgagc tagagcctga atttgtgcaa caagtgacag 1320aattctgttc
ctacatcttt agccattcta tgaccaagac tcttccaggt ggcatcatgg
1380tcaatggatc tcgtctaaag aacctggtgc tgacctatgt caatgccatc
agcagtgggg 1440atctgccttg catagagaat gcagtcctgg ccttggctca
gagagagaac tcagctgcag 1500tgcaaaaggc cattgcccac tatgaccagc
aaatgggcca gaaagtgcag ctgcccatgg 1560aaaccctcca ggagctgctg
gacctgcaca ggaccagtga gagggaggcc attgaagtct 1620tcatgaaaaa
ctctttcaag gatgtagacc aaagtttcca gaaagaattg gagactctac
1680tagatgcaaa acagaatgac atttgtaaac ggaacctgga agcatcctcg
gattattgct 1740cggctttact taaggatatt tttggtcctc tagaagaagc
agtgaagcag ggaatttatt 1800ctaagccagg aggccataat ctcttcattc
agaaaacaga agaactgaag gcaaagtact 1860atcgggagcc tcggaaagga
atacaggctg aagaagttct gcagaaatat ttaaagtcca 1920aggagtctgt
gagtcatgca atattacaga ctgaccaggc tctcacagag acggaaaaaa
1980agaagaaaga ggcacaagtg aaagcagaag ctgaaaaggc tgaagcgcaa
aggttggcgg 2040cgattcaaag gcagaacgag caaatgatgc aggagaggga
gagactccat caggaacaag 2100tgagacaaat ggagatagcc aaacaaaatt
ggctggcaga gcaacagaaa atgcaggaac 2160aacagatgca ggaacaggct
gcacagctca gcacaacatt ccaagctcaa aatagaagcc 2220ttctcagtga
gctccagcac gcccagagga ctgttaataa cgatgatcca tgtgttttac
2280tctaaagtgc taaatatggg agtttccttt ttttactctt tgtcactgat
gacacaacag 2340aaaagaaact gtagaccttg ggacaatcaa catttaaata
238040586PRTHomo sapiens 40Met Ala Leu Glu Ile His Met Ser Asp Pro
Met Cys Leu Ile Glu Asn 1 5 10 15 Phe Asn Glu Gln Leu Lys Val Asn
Gln Glu Ala Leu Glu Ile Leu Ser 20 25 30 Ala Ile Thr Gln Pro Val
Val Val Val Ala Ile Val Gly Leu Tyr Arg 35 40 45 Thr Gly Lys Ser
Tyr Leu Met Asn Lys Leu Ala Gly Lys Asn Lys Gly 50 55 60 Phe Ser
Val Ala Ser Thr Val Gln Ser His Thr Lys Gly Ile Trp Ile 65 70 75 80
Trp Cys Val Pro His Pro Asn Trp Pro Asn His Thr Leu Val Leu Leu 85
90 95 Asp Thr Glu Gly Leu Gly Asp Val Glu Lys Ala Asp Asn Lys Asn
Asp 100 105 110 Ile Gln Ile Phe Ala Leu Ala Leu Leu Leu Ser Ser Thr
Phe Val Tyr 115 120 125 Asn Thr Val Asn Lys Ile Asp Gln Gly Ala Ile
Asp Leu Leu His Asn 130 135 140 Val Thr Glu Leu Thr Asp Leu Leu Lys
Ala Arg Asn Ser Pro Asp Leu 145 150 155 160 Asp Arg Val Glu Asp Pro
Ala Asp Ser Ala Ser Phe Phe Pro Asp Leu 165 170 175 Val Trp Thr Leu
Arg Asp Phe Cys Leu Gly Leu Glu Ile Asp Gly Gln 180 185 190 Leu Val
Thr Pro Asp Glu Tyr Leu Glu Asn Ser Leu Arg Pro Lys Gln 195 200 205
Gly Ser Asp Gln Arg Val Gln Asn Phe Asn Leu Pro Arg Leu Cys Ile 210
215 220 Gln Lys Phe Phe Pro Lys Lys Lys Cys Phe Ile Phe Asp Leu Pro
Ala 225 230 235 240 His Gln Lys Lys Leu Ala Gln Leu Glu Thr Leu Pro
Asp Asp Glu Leu 245 250 255 Glu Pro Glu Phe Val Gln Gln Val Thr Glu
Phe Cys Ser Tyr Ile Phe 260 265 270 Ser His Ser Met Thr Lys Thr Leu
Pro Gly Gly Ile Met Val Asn Gly 275 280 285 Ser Arg Leu Lys Asn Leu
Val Leu Thr Tyr Val Asn Ala Ile Ser Ser 290 295 300 Gly Asp Leu Pro
Cys Ile Glu Asn Ala Val Leu Ala Leu Ala Gln Arg 305 310 315 320 Glu
Asn Ser Ala Ala Val Gln Lys Ala Ile Ala His Tyr Asp Gln Gln 325 330
335 Met Gly Gln Lys Val Gln Leu Pro Met Glu Thr Leu Gln Glu Leu Leu
340 345 350 Asp Leu His Arg Thr Ser Glu Arg Glu Ala Ile Glu Val Phe
Met Lys 355 360 365 Asn Ser Phe Lys Asp Val Asp Gln Ser Phe Gln Lys
Glu Leu Glu Thr 370 375 380 Leu Leu Asp Ala Lys Gln Asn Asp Ile Cys
Lys Arg Asn Leu Glu Ala 385 390 395 400 Ser Ser Asp Tyr Cys Ser Ala
Leu Leu Lys Asp Ile Phe Gly Pro Leu 405 410 415 Glu Glu Ala Val Lys
Gln Gly Ile Tyr Ser Lys Pro Gly Gly His Asn 420 425 430 Leu Phe Ile
Gln Lys Thr Glu Glu Leu Lys Ala Lys Tyr Tyr Arg Glu 435 440 445 Pro
Arg Lys Gly Ile Gln Ala Glu Glu Val Leu Gln Lys Tyr Leu Lys 450 455
460 Ser Lys Glu Ser Val Ser His Ala Ile Leu Gln Thr Asp Gln Ala Leu
465 470 475 480 Thr Glu Thr Glu Lys Lys Lys Lys Glu Ala Gln Val Lys
Ala Glu Ala 485 490 495 Glu Lys Ala Glu Ala Gln Arg Leu Ala Ala Ile
Gln Arg Gln Asn Glu 500 505 510 Gln Met Met Gln Glu Arg Glu Arg Leu
His Gln Glu Gln Val Arg Gln 515 520 525 Met Glu Ile Ala Lys Gln Asn
Trp Leu Ala Glu Gln Gln Lys Met Gln 530 535 540 Glu Gln Gln Met Gln
Glu Gln Ala Ala Gln Leu Ser Thr Thr Phe Gln 545 550 555 560 Ala Gln
Asn Arg Ser Leu Leu Ser Glu Leu Gln His Ala Gln Arg Thr 565 570 575
Val Asn Asn Asp Asp Pro Cys Val Leu Leu 580 585 411051DNAHomo
sapiens 41ggaccgccgc ctggttaaag gcgcttattt cccaggcagc cgctgcagtc
gccacacctt 60tgcccctgct gcgatgaccc tgtcgccact tctgctgttc ctgccaccgc
tgctgctgct 120gctggacgtc cccacggcgg cggtgcaggc gtcccctctg
caagcgttag acttctttgg 180gaatgggcca ccagttaact acaagacagg
caatctatac ctgcgggggc ccctgaagaa 240gtccaatgca ccgcttgtca
atgtgaccct ctactatgaa gcactgtgcg gtggctgccg 300agccttcctg
atccgggagc tcttcccaac atggctgttg gtcatggaga tcctcaatgt
360cacgctggtg ccctacggaa acgcacagga acaaaatgtc agtggcaggt
gggagttcaa 420gtgccagcat ggagaagagg agtgcaaatt caacaaggtg
gaggcctgcg tgttggatga 480acttgacatg gagctagcct tcctgaccat
tgtctgcatg gaagagtttg aggacatgga 540gagaagtctg ccactatgcc
tgcagctcta cgccccaggg ctgtcgccag acactatcat 600ggagtgtgca
atgggggacc gcggcatgca gctcatgcac gccaacgccc agcggacaga
660tgctctccag ccaccacacg agtatgtgcc ctgggtcacc gtcaatggga
aacccttgga 720agatcagacc cagctcctta cccttgtctg ccagttgtac
cagggcaaga agccggatgt 780ctgcccttcc tcaaccagct ccctcaggag
tgtttgcttc aagtgatggc cggtgagctg 840cggagagctc atggaaggcg
agtgggaacc cggctgcctg cctttttttc tgatccagac 900cctcggcacc
tgctacttac caactggaaa attttatgca tcccatgaag cccagataca
960caaaattcca ccccatgatc aagaatcctg ctccactaag aatggtgcta
aagtaaaact 1020agtttaataa gcaaaaaaaa aaaaaaaaaa a 105142250PRTHomo
sapiens 42Met Thr Leu Ser Pro Leu Leu Leu Phe Leu Pro Pro Leu Leu
Leu Leu 1 5 10 15 Leu Asp Val Pro Thr Ala Ala Val Gln Ala Ser Pro
Leu Gln Ala Leu 20 25 30 Asp Phe Phe Gly Asn Gly Pro Pro Val Asn
Tyr Lys Thr Gly Asn Leu 35 40 45 Tyr Leu Arg Gly Pro Leu Lys Lys
Ser Asn Ala Pro Leu Val Asn Val 50 55 60 Thr Leu Tyr Tyr Glu Ala
Leu Cys Gly Gly Cys Arg Ala Phe Leu Ile 65 70 75 80 Arg Glu Leu Phe
Pro Thr Trp Leu Leu Val Met Glu Ile Leu Asn Val 85 90 95 Thr Leu
Val Pro Tyr Gly Asn Ala Gln Glu Gln Asn Val Ser Gly Arg 100 105 110
Trp Glu Phe Lys Cys Gln His Gly Glu Glu Glu Cys Lys Phe Asn Lys 115
120 125 Val Glu Ala Cys Val Leu Asp Glu Leu Asp Met Glu Leu Ala Phe
Leu 130 135 140 Thr Ile Val Cys Met Glu Glu Phe Glu Asp Met Glu Arg
Ser Leu Pro 145 150 155 160 Leu Cys Leu Gln Leu Tyr Ala Pro Gly Leu
Ser Pro Asp Thr Ile Met 165 170 175 Glu Cys Ala Met Gly Asp Arg Gly
Met Gln Leu Met His Ala Asn Ala 180 185 190 Gln Arg Thr Asp Ala Leu
Gln Pro Pro His Glu Tyr Val Pro Trp Val 195 200 205 Thr Val Asn Gly
Lys Pro Leu Glu Asp Gln Thr Gln Leu Leu Thr Leu 210 215 220 Val Cys
Gln Leu Tyr Gln Gly Lys Lys Pro Asp Val Cys Pro Ser Ser 225 230 235
240 Thr Ser Ser Leu Arg Ser Val Cys Phe Lys 245 250
4317DNAArtificial SequenceDescription of artificial sequence
Oligonucleotide 43ctacaagaca ggcaatc 174417DNAArtificial
SequenceDescription of artificial sequence Oligonucleotide
44ttcatccaac acgcagg 1745875DNAHomo sapiens 45ggttgctaag gagtgggtgc
ctcagaatca ggctgcaatg ggcattgtct gtgcacaatg 60ttcctttatt ctgctgctgt
ccataataag ggctcgtcca cctcccttcc tcttctgccc 120attgagcagt
caaagaactg aaagtcctta taagcctgtg cacctgggcc tgggccctac
180agataaggta gctgctattg ctatggcccg catcattgac ctggtgccct
gggacgatgg 240ctccacacat gtgtatgcct ccccggccat cctgcttccc
atggagcggc agcgcaacca 300gctggcgggc gtgaagcagc agctctacca
cccagccctg cccaccctgc gccacatgga 360cagggacacc gtcaaggcct
gccttcctga tgagcactgc cagtccacca cctactgccg 420caaagatgaa
tttgacaacg cccattttac actccttggg gtccccaaca aacccctgca
480gtgtttggac atcaccgcca caggccagaa gcttcgcaac aggtaccacg
agggaaagct 540ggcgcccatc gcgccaggca tcaaccgagt ggactggccc
tgcttcacgc gcgccatcga 600ggactggtcc cacttcgtgt cctcggccgg
ggagttcaag ctgccttgcc tgaggaagcg 660agcggagggt ctcagcggct
acgcggtgcg gtacttgaag cccgacgtga cccagacctg 720gcggtactgc
ctcaaccaga accccagcct ggaccgctac ggacagaagc ccctgccttt
780cgactccctg aacactttcc gaagcttcgg ctccagctac agtcgtgtca
actacctgac 840cccctggcat taatctctgg aaaggaggct gactc
87546271PRTHomo sapiens 46Met Gly Ile Val Cys Ala Gln Cys Ser Phe
Ile Leu Leu Leu Ser Ile 1 5 10 15 Ile Arg Ala Arg Pro Pro Pro Phe
Leu Phe Cys Pro Leu Ser Ser Gln 20 25 30 Arg Thr Glu Ser Pro Tyr
Lys Pro Val His Leu Gly Leu Gly Pro Thr 35 40 45 Asp Lys Val Ala
Ala Ile Ala Met Ala Arg Ile Ile Asp Leu Val Pro 50 55 60 Trp Asp
Asp Gly Ser Thr His Val Tyr Ala Ser Pro Ala Ile Leu Leu 65 70 75 80
Pro Met Glu Arg Gln Arg Asn Gln Leu Ala Gly Val Lys Gln Gln Leu 85
90 95 Tyr His Pro Ala Leu Pro Thr Leu Arg His Met Asp Arg Asp Thr
Val 100 105 110 Lys Ala Cys Leu Pro Asp Glu His Cys Gln Ser Thr Thr
Tyr Cys Arg 115 120 125 Lys Asp Glu Phe Asp Asn Ala His Phe Thr Leu
Leu Gly Val Pro Asn 130 135 140 Lys Pro Leu Gln Cys Leu Asp Ile Thr
Ala Thr Gly Gln Lys Leu Arg 145 150 155 160 Asn Arg Tyr His Glu Gly
Lys Leu Ala Pro Ile Ala Pro Gly Ile Asn 165 170 175 Arg Val Asp Trp
Pro Cys Phe Thr Arg Ala Ile Glu Asp Trp Ser His 180 185 190 Phe Val
Ser Ser Ala Gly Glu Phe Lys Leu Pro Cys Leu Arg Lys Arg 195 200 205
Ala Glu Gly Leu Ser Gly Tyr Ala Val Arg Tyr Leu Lys Pro Asp Val 210
215 220 Thr Gln Thr Trp Arg Tyr Cys Leu Asn Gln Asn Pro Ser Leu Asp
Arg 225 230 235 240 Tyr Gly Gln Lys Pro Leu Pro Phe Asp Ser Leu Asn
Thr Phe Arg Ser 245 250 255 Phe Gly Ser Ser Tyr Ser Arg Val Asn Tyr
Leu Thr Pro Trp His 260 265 270 4722DNAArtificial
SequenceDescription of artificial sequence Oligonucleotide
47tccaccacct actgccgcaa ag 224820DNAArtificial SequenceDescription
of artificial sequence Oligonucleotide 48accctccgct cgcttcctca
2049956DNAHomo sapiens 49ggttgctaag gagtgggtgc ctcagaatca
ggctgcaatg ggcattgtct gtgcacaatg 60ttcctttatt ctgctgctgt ccataataag
ggctcgtcca cctcccttcc tcttctgccc 120attgagcagt caaagaactg
aaagtcctta taagcctgtg cacctgggcc tgggccctac 180agataaggta
gctgctattg ctatggcccg catcattgac ctggtgccct gggacgatgg
240ctccacacat gtgtatgcct ccccggccat cctgcttccc atggagcggc
agcgcaacca 300gctggcgggc gtgaagcagc agctctacca cccagccctg
cccaccctgc gccacatgga 360cagggacacc gtcaaggcct gccttcctga
tgagcactgc cagtccacca cctactgccg 420caaagatgaa tttgacaacg
cccattttac actccttggg gtccccaaca aacccctgca 480gtgtttggac
atcaccgcca caggccagaa gcttcgcaac aggtaccacg agggaaagct
540ggcgcccatc gcgccaggca tcaaccgagt ggactggccc tgcttcacgc
gcgccatcga 600ggactggtcc cacttcgtgt cctcggccgg ggagttcaag
ctgccttgcc tgaggaagcg 660agcggagggt ctcagcggct acgcggtgcg
gtacttgaag cccgacgtga cccagacctg 720gcggcaggaa gccctgtgtg
gggcgtgggc agggcgtccg tgctcccccg actcaccgcg 780cccctatccc
tgctgcccgc ctcagtactg cctcaaccag aaccccagcc tggaccgcta
840cggacagaag cccctgcctt tcgactccct gaacactttc cgaagcttcg
gctccagcta 900cagtcgtgtc aactacctga ccccctggca ttaatctctg
gaaaggaggc tgactc 95650298PRTHomo sapiens 50Met Gly Ile
Val Cys Ala Gln Cys Ser Phe Ile Leu Leu Leu Ser Ile 1 5 10 15 Ile
Arg Ala Arg Pro Pro Pro Phe Leu Phe Cys Pro Leu Ser Ser Gln 20 25
30 Arg Thr Glu Ser Pro Tyr Lys Pro Val His Leu Gly Leu Gly Pro Thr
35 40 45 Asp Lys Val Ala Ala Ile Ala Met Ala Arg Ile Ile Asp Leu
Val Pro 50 55 60 Trp Asp Asp Gly Ser Thr His Val Tyr Ala Ser Pro
Ala Ile Leu Leu 65 70 75 80 Pro Met Glu Arg Gln Arg Asn Gln Leu Ala
Gly Val Lys Gln Gln Leu 85 90 95 Tyr His Pro Ala Leu Pro Thr Leu
Arg His Met Asp Arg Asp Thr Val 100 105 110 Lys Ala Cys Leu Pro Asp
Glu His Cys Gln Ser Thr Thr Tyr Cys Arg 115 120 125 Lys Asp Glu Phe
Asp Asn Ala His Phe Thr Leu Leu Gly Val Pro Asn 130 135 140 Lys Pro
Leu Gln Cys Leu Asp Ile Thr Ala Thr Gly Gln Lys Leu Arg 145 150 155
160 Asn Arg Tyr His Glu Gly Lys Leu Ala Pro Ile Ala Pro Gly Ile Asn
165 170 175 Arg Val Asp Trp Pro Cys Phe Thr Arg Ala Ile Glu Asp Trp
Ser His 180 185 190 Phe Val Ser Ser Ala Gly Glu Phe Lys Leu Pro Cys
Leu Arg Lys Arg 195 200 205 Ala Glu Gly Leu Ser Gly Tyr Ala Val Arg
Tyr Leu Lys Pro Asp Val 210 215 220 Thr Gln Thr Trp Arg Gln Glu Ala
Leu Cys Gly Ala Trp Ala Gly Arg 225 230 235 240 Pro Cys Ser Pro Asp
Ser Pro Arg Pro Tyr Pro Cys Cys Pro Pro Gln 245 250 255 Tyr Cys Leu
Asn Gln Asn Pro Ser Leu Asp Arg Tyr Gly Gln Lys Pro 260 265 270 Leu
Pro Phe Asp Ser Leu Asn Thr Phe Arg Ser Phe Gly Ser Ser Tyr 275 280
285 Ser Arg Val Asn Tyr Leu Thr Pro Trp His 290 295 5116PRTHomo
sapiens 51Cys Ser Lys Asp Ser Arg Gly Ile Ala Asp Pro Asn Gln Ser
Ala Lys 1 5 10 15 5213PRTHomo sapiens 52Cys Glu Glu Asp Ser Lys Ile
Lys Gly Ile His Asp Glu 1 5 10 5312PRTHomo sapiens 53Cys Lys Leu
Arg Asn Arg Ile Trp Gly Met Val Asn 1 5 10 5464PRTHomo sapiens
54Arg Tyr Arg Lys Cys Ile Lys Leu Lys Gln Val Gln Ser Pro Pro Thr 1
5 10 15 Glu Thr Leu Gly Val Glu Asn Lys Gly Tyr Phe Gly Asp Glu Gln
Gln 20 25 30 Ile Arg Thr Glu Pro Ile Leu Pro Glu Ile His Phe Leu
Asn Lys Pro 35 40 45 Ala Ser Lys Asp Ser Arg Gly Ile Ala Asp Pro
Asn Gln Ser Ala Lys 50 55 60 55667PRTHomo sapiens 55Met Leu Pro Gly
Cys Ile Phe Leu Met Ile Leu Leu Ile Pro Gln Val 1 5 10 15 Lys Glu
Lys Phe Ile Leu Gly Val Glu Gly Gln Gln Leu Val Arg Pro 20 25 30
Lys Lys Leu Pro Leu Ile Gln Lys Arg Asp Thr Gly His Thr His Asp 35
40 45 Asp Asp Ile Leu Lys Thr Tyr Glu Glu Glu Leu Leu Tyr Glu Ile
Lys 50 55 60 Leu Asn Arg Lys Thr Leu Val Leu His Leu Leu Arg Ser
Arg Glu Phe 65 70 75 80 Leu Gly Ser Asn Tyr Ser Glu Thr Phe Tyr Ser
Met Lys Gly Glu Ala 85 90 95 Phe Thr Arg His Pro Gln Ile Met Asp
His Cys Phe Tyr Gln Gly Ser 100 105 110 Ile Val His Glu Tyr Asp Ser
Ala Ala Ser Ile Ser Thr Cys Asn Gly 115 120 125 Leu Arg Gly Phe Phe
Arg Ile Asn Asp Gln Arg Tyr Leu Ile Glu Pro 130 135 140 Val Lys Tyr
Ser Asp Glu Gly Glu His Leu Val Phe Lys Tyr Asn Leu 145 150 155 160
Arg Val Pro Tyr Gly Ala Asn Tyr Ser Cys Thr Glu Leu Asn Phe Thr 165
170 175 Arg Lys Thr Val Pro Gly Asp Asn Glu Ser Glu Glu Asp Ser Lys
Ile 180 185 190 Lys Gly Ile His Asp Glu Lys Tyr Val Glu Leu Phe Ile
Val Ala Asp 195 200 205 Asp Thr Val Tyr Arg Arg Asn Gly His Pro His
Asn Lys Leu Arg Asn 210 215 220 Arg Ile Trp Gly Met Val Asn Phe Val
Asn Met Ile Tyr Lys Thr Leu 225 230 235 240 Asn Ile His Val Thr Leu
Val Gly Ile Glu Ile Trp Thr His Glu Asp 245 250 255 Lys Ile Glu Leu
Tyr Ser Asn Ile Glu Thr Thr Leu Leu Arg Phe Ser 260 265 270 Phe Trp
Gln Glu Lys Ile Leu Lys Thr Arg Lys Asp Phe Asp His Val 275 280 285
Val Leu Leu Ser Gly Lys Trp Leu Tyr Ser His Val Gln Gly Ile Ser 290
295 300 Tyr Pro Gly Gly Met Cys Leu Pro Tyr Tyr Ser Thr Ser Ile Ile
Lys 305 310 315 320 Asp Leu Leu Pro Asp Thr Asn Ile Ile Ala Asn Arg
Met Ala His Gln 325 330 335 Leu Gly His Asn Leu Gly Met Gln His Asp
Glu Phe Pro Cys Thr Cys 340 345 350 Pro Ser Gly Lys Cys Val Met Asp
Ser Asp Gly Ser Ile Pro Ala Leu 355 360 365 Lys Phe Ser Lys Cys Ser
Gln Asn Gln Tyr His Gln Tyr Leu Lys Asp 370 375 380 Tyr Lys Pro Thr
Cys Met Leu Asn Ile Pro Phe Pro Tyr Asn Phe His 385 390 395 400 Asp
Phe Gln Phe Cys Gly Asn Lys Lys Leu Asp Glu Gly Glu Glu Cys 405 410
415 Asp Cys Gly Pro Ala Gln Glu Cys Thr Asn Pro Cys Cys Asp Ala His
420 425 430 Thr Cys Val Leu Lys Pro Gly Phe Thr Cys Ala Glu Gly Glu
Cys Cys 435 440 445 Glu Ser Cys Gln Ile Lys Lys Ala Gly Ser Ile Cys
Arg Pro Ala Lys 450 455 460 Asp Glu Cys Asp Phe Pro Glu Met Cys Thr
Gly His Ser Pro Ala Cys 465 470 475 480 Pro Lys Asp Gln Phe Arg Val
Asn Gly Phe Pro Cys Lys Asn Ser Glu 485 490 495 Gly Tyr Cys Phe Met
Gly Lys Cys Pro Thr Arg Glu Asp Gln Cys Ser 500 505 510 Glu Leu Phe
Asp Asp Asp Ala Ile Glu Ser His Asp Ile Cys Tyr Lys 515 520 525 Met
Asn Thr Lys Gly Asn Lys Phe Gly Tyr Cys Lys Asn Lys Glu Asn 530 535
540 Arg Phe Leu Pro Cys Glu Glu Lys Asp Val Arg Cys Gly Lys Ile Tyr
545 550 555 560 Cys Thr Gly Gly Glu Leu Ser Ser Leu Leu Gly Glu Asp
Lys Thr Tyr 565 570 575 His Leu Lys Asp Pro Gln Lys Asn Ala Thr Val
Lys Cys Lys Thr Ile 580 585 590 Phe Leu Tyr His Asp Ser Thr Asp Ile
Gly Leu Val Ala Ser Gly Thr 595 600 605 Lys Cys Gly Glu Gly Met Val
Cys Asn Asn Gly Glu Cys Leu Asn Met 610 615 620 Glu Lys Val Tyr Ile
Ser Thr Asn Cys Pro Ser Gln Cys Asn Glu Asn 625 630 635 640 Pro Val
Asp Gly His Gly Leu Gln Cys His Cys Glu Glu Gly Gln Ala 645 650 655
Pro Val Ala Cys Glu Glu Thr Leu His Val Thr 660 665 5639PRTHomo
sapiens 56Ile Ser Thr Asn Cys Pro Ser Gln Cys Asn Glu Asn Pro Val
Asp Gly 1 5 10 15 His Gly Leu Gln Cys His Cys Glu Glu Gly Gln Ala
Pro Val Ala Cys 20 25 30 Glu Glu Thr Leu His Val Thr 35 5762PRTHomo
sapiens 57Ile Ser Thr Asn Cys Pro Ser Gln Cys Asn Glu Asn Pro Val
Asp Gly 1 5 10 15 His Gly Leu Gln Cys His Cys Glu Glu Gly Gln Ala
Pro Val Ala Cys 20 25 30 Glu Glu Thr Leu His Val Thr Asn Ile Thr
Ile Leu Val Val Val Leu 35 40 45 Val Leu Val Ile Val Gly Ile Gly
Val Leu Ile Leu Leu Val 50 55 60 5812PRTHomo sapiens 58Ala Pro Gln
Lys Ser Pro Trp Leu Thr Lys Pro Cys 1 5 10 5914PRTHomo sapiens
59Cys Pro Leu Gln Pro Ser His Phe Leu Asp Ile Ser Glu Asp 1 5 10
6012PRTHomo sapiens 60Cys Ile Tyr Ser Thr Glu Ile His Tyr Ser Ser
Lys 1 5 10 61500DNAHomo sapiens 61gcagcctcac tgtcctgtct tgaacatcct
ccaccaaagt gtgaacacag gtggcatggg 60cttcgtaacg aataagagcg ccttcaaagc
aggagattcc ctgtacctgc gaagggcctt 120cgtgaacaac cttggagagg
aaaggagaac caggattcag atccaaagca tccagaaggc 180tttagacatc
cagatcaggg agattgatag agaaaaagca gccctgaaga gatttttggt
240aaagcttcac aagacaactg gctattttcc tcaaaagcca ttgtggtgac
tgatgctgtg 300ccaccatagg ggacgagttc atctgaaaca tccaatacag
acagaacaca cctgggtctc 360cttctgtatc tctctgtgac ccaagaaagc
agtagaagtg gctactggga caagccaaaa 420aagaaaaaaa aggactagat
aattgaggac acaaatggac tgcctctaaa ataataaaca 480aaacctccta
caaaagagag 500622256DNAHomo sapiens 62atgcggctga tgggccccct
gagcctgaga gtagggatga ccctggaagc ggcgctagtg 60ctgatccgca ggtccccgcg
ctcccgccag agccgcgacc cggcttgtgc cacccagctt 120cggggcctgc
agggagcgct gcttcttctg agcctgagtc tgggaccctc tttgcagtgt
180ggctgtgggg gcgctggtcc catcacggcc tttttcataa agcacatggt
gccaactgtt 240agtggatcta ccatcctgga ggatgatagc cctcttctaa
cagctccact aggcagtgcc 300ccagtaggga ctctgtgtag gcgccctcac
cccacatttc ccttccacac tgccctagca 360gaggttctcc atgagggcgc
tgcccctgca gcaaacttct gcctggacat ccaggaaccg 420ccccagtggt
cagctgccgc gctgttgcta ggcaacagcg tgcgagctca gatcagcgtg
480gggtggagga gaagtggagt ttggaagttc aggggcacag gggcacaggc
ccacgactgc 540agcgggatgg accagtactg catcctgggc cgcatcgggg
agggcgccca cggcatcgtc 600ttcaaggcca agcacgtgga gccgagggtg
ggctggcagt gtctgccttc tatcctgcag 660actggcgaga tagttgccct
caagaaggtg gccctaaggc ggttggagga cggcttccct 720aaccaggccc
tgcgggagat taaggctctg caggagatgg aggacaatca gtatgtggta
780caactgaagg ctgtgttccc acacggtgga ggctttgtgc tggcctttga
gttcatgctg 840tcggatctgg ccgaggtggt gcgccatgcc cagaggccac
tagcccaggc acaggtcaag 900agctacctgc agatgctgct caagggtgtc
gccttctgcc atgccaacaa cattgtacat 960cgggacctga aacctgccaa
cctgctcatc agcgcctcag gccagctcaa gatagcggac 1020tttggcctgg
ctcgagtctt ttccccagac ggcagccgcc tctacacaca ccaggtggcc
1080accaggtggt accgagcccc cgagctcctg tatggtgccc gccagtatga
ccagggcgtc 1140gatctgtggt ctgtgggctg catcatgggg gagctgttga
atgggtcccc ccttttcccg 1200ggcaagaacg atattgaaca gctttgctat
gtgcttcgca tcttgggcac cccaaaccct 1260caagtctggc cggagctcac
tgagctgccg gactacaaca agatctcctt taaggagcag 1320gtgcccatgc
ccctggagga ggtgctgcct gacgtctctc cccaggcatt ggatctgctg
1380ggtcaattcc ttctctaccc tcctcaccag cgcatcgcag cttccaagct
gtgggagccc 1440caggagagag gcaaggccga ctttggggtc aagcaaagct
tcctggtgga gggcaggttt 1500aagcatgaga tgtttgagga ggtgttcgct
gaagagagaa atgctcccct gcctgcccat 1560ccatctgagc tgccgattcc
tcagcgtcta gggggacctg cccccaaggc ccatccaggg 1620cccccccaca
tccatgactt ccacgtggac cggcctcttg aggagtcgct gttgaaccca
1680gagctgattc ggcccttcat cctggagggc ccttgcgagg gttggtctcg
aggcagaggt 1740catgttccca gccaagagta tgagaacatc cagtcgagca
gaggagattc atggcctgtg 1800ctcgtttcac tgacctgcat gttctggaag
gagccaggac agcagcctac cccgcaagct 1860gcccagaagc cccggatggc
ccctcagcca gctgaaacca ccgtccactg ggaagatggc 1920tacttggagg
cctctggagg ggagggggtt gttcacgaag gcccttcgca ggtacaggga
1980atctcctgct ttgaaggcgc tcttattcgt tacgaagccc atgccacctg
tgttcacact 2040ttggtggagg atgttcaaga caggacacat ctactggccc
ctcctaaagc ctttgcctgg 2100ctgctcctgc aagagcatgc acaaagcaca
gcctccactg ccttgcctgg gaacactttg 2160gctcccccag cacagccaat
gctcaactca acaggacaaa gctgcaggcc cagtcccaaa 2220ccgccagggt
gtgagaatat agctcaggag tattga 225663751PRTHomo sapiens 63Met Arg Leu
Met Gly Pro Leu Ser Leu Arg Val Gly Met Thr Leu Glu 1 5 10 15 Ala
Ala Leu Val Leu Ile Arg Arg Ser Pro Arg Ser Arg Gln Ser Arg 20 25
30 Asp Pro Ala Cys Ala Thr Gln Leu Arg Gly Leu Gln Gly Ala Leu Leu
35 40 45 Leu Leu Ser Leu Ser Leu Gly Pro Ser Leu Gln Cys Gly Cys
Gly Gly 50 55 60 Ala Gly Pro Ile Thr Ala Phe Phe Ile Lys His Met
Val Pro Thr Val 65 70 75 80 Ser Gly Ser Thr Ile Leu Glu Asp Asp Ser
Pro Leu Leu Thr Ala Pro 85 90 95 Leu Gly Ser Ala Pro Val Gly Thr
Leu Cys Arg Arg Pro His Pro Thr 100 105 110 Phe Pro Phe His Thr Ala
Leu Ala Glu Val Leu His Glu Gly Ala Ala 115 120 125 Pro Ala Ala Asn
Phe Cys Leu Asp Ile Gln Glu Pro Pro Gln Trp Ser 130 135 140 Ala Ala
Ala Leu Leu Leu Gly Asn Ser Val Arg Ala Gln Ile Ser Val 145 150 155
160 Gly Trp Arg Arg Ser Gly Val Trp Lys Phe Arg Gly Thr Gly Ala Gln
165 170 175 Ala His Asp Cys Ser Gly Met Asp Gln Tyr Cys Ile Leu Gly
Arg Ile 180 185 190 Gly Glu Gly Ala His Gly Ile Val Phe Lys Ala Lys
His Val Glu Pro 195 200 205 Arg Val Gly Trp Gln Cys Leu Pro Ser Ile
Leu Gln Thr Gly Glu Ile 210 215 220 Val Ala Leu Lys Lys Val Ala Leu
Arg Arg Leu Glu Asp Gly Phe Pro 225 230 235 240 Asn Gln Ala Leu Arg
Glu Ile Lys Ala Leu Gln Glu Met Glu Asp Asn 245 250 255 Gln Tyr Val
Val Gln Leu Lys Ala Val Phe Pro His Gly Gly Gly Phe 260 265 270 Val
Leu Ala Phe Glu Phe Met Leu Ser Asp Leu Ala Glu Val Val Arg 275 280
285 His Ala Gln Arg Pro Leu Ala Gln Ala Gln Val Lys Ser Tyr Leu Gln
290 295 300 Met Leu Leu Lys Gly Val Ala Phe Cys His Ala Asn Asn Ile
Val His 305 310 315 320 Arg Asp Leu Lys Pro Ala Asn Leu Leu Ile Ser
Ala Ser Gly Gln Leu 325 330 335 Lys Ile Ala Asp Phe Gly Leu Ala Arg
Val Phe Ser Pro Asp Gly Ser 340 345 350 Arg Leu Tyr Thr His Gln Val
Ala Thr Arg Trp Tyr Arg Ala Pro Glu 355 360 365 Leu Leu Tyr Gly Ala
Arg Gln Tyr Asp Gln Gly Val Asp Leu Trp Ser 370 375 380 Val Gly Cys
Ile Met Gly Glu Leu Leu Asn Gly Ser Pro Leu Phe Pro 385 390 395 400
Gly Lys Asn Asp Ile Glu Gln Leu Cys Tyr Val Leu Arg Ile Leu Gly 405
410 415 Thr Pro Asn Pro Gln Val Trp Pro Glu Leu Thr Glu Leu Pro Asp
Tyr 420 425 430 Asn Lys Ile Ser Phe Lys Glu Gln Val Pro Met Pro Leu
Glu Glu Val 435 440 445 Leu Pro Asp Val Ser Pro Gln Ala Leu Asp Leu
Leu Gly Gln Phe Leu 450 455 460 Leu Tyr Pro Pro His Gln Arg Ile Ala
Ala Ser Lys Leu Trp Glu Pro 465 470 475 480 Gln Glu Arg Gly Lys Ala
Asp Phe Gly Val Lys Gln Ser Phe Leu Val 485 490 495 Glu Gly Arg Phe
Lys His Glu Met Phe Glu Glu Val Phe Ala Glu Glu 500 505 510 Arg Asn
Ala Pro Leu Pro Ala His Pro Ser Glu Leu Pro Ile Pro Gln 515 520 525
Arg Leu Gly Gly Pro Ala Pro Lys Ala His Pro Gly Pro Pro His Ile 530
535 540 His Asp Phe His Val Asp Arg Pro Leu Glu Glu Ser Leu Leu Asn
Pro 545 550 555 560 Glu Leu Ile Arg Pro Phe Ile Leu Glu Gly Pro Cys
Glu Gly Trp Ser 565 570 575 Arg Gly Arg Gly His Val Pro Ser Gln Glu
Tyr Glu Asn Ile Gln Ser 580 585 590 Ser Arg Gly Asp Ser Trp Pro Val
Leu Val Ser Leu Thr Cys Met Phe 595 600 605 Trp Lys Glu Pro Gly Gln
Gln Pro Thr Pro Gln Ala Ala Gln Lys Pro 610 615 620 Arg Met Ala Pro
Gln Pro Ala Glu
Thr Thr Val His Trp Glu Asp Gly 625 630 635 640 Tyr Leu Glu Ala Ser
Gly Gly Glu Gly Val Val His Glu Gly Pro Ser 645 650 655 Gln Val Gln
Gly Ile Ser Cys Phe Glu Gly Ala Leu Ile Arg Tyr Glu 660 665 670 Ala
His Ala Thr Cys Val His Thr Leu Val Glu Asp Val Gln Asp Arg 675 680
685 Thr His Leu Leu Ala Pro Pro Lys Ala Phe Ala Trp Leu Leu Leu Gln
690 695 700 Glu His Ala Gln Ser Thr Ala Ser Thr Ala Leu Pro Gly Asn
Thr Leu 705 710 715 720 Ala Pro Pro Ala Gln Pro Met Leu Asn Ser Thr
Gly Gln Ser Cys Arg 725 730 735 Pro Ser Pro Lys Pro Pro Gly Cys Glu
Asn Ile Ala Gln Glu Tyr 740 745 750 64455DNAHomo sapiens
64tcagaccgac agtcaggcag aagcagggcc cccggtctgt ctaggcttca ggatgcctaa
60gaagctctcg gtgggcagcc agtgctccag aaccaggggg ttgttgtggg caggtctctg
120gggaggcctg aggggccaca cggtgtcctg ggtgtggggt ctgcatctac
atacgactca 180taggcacctg ccattggggc gatttgtgat ggtttaggga
aacagggtct gcccttggat 240gggctggcct acagtcaggg gccttccttg
tctgtcttac tttccccgct ctgccagagt 300gaacccaagg atgccgccag
cctcacccca gtgggtggca gttacgccgg catggaactc 360agactcaccg
gcacgcatga cttccaaccg tccacacatc tttgcaccca cagtatctgc
420ttcagagaat gtattctaaa taataaaaat cataa 45565746DNAHomo sapiens
65cccactttgc tggatgaagc tctgtgaact cttcctggtt cttagtactg cctaattcat
60gaatccttct ttgctcaaat gaactcaaag ttaatttaaa gtttttattc taacatgaga
120ctctttctcc catgagactt tgcatgaggg agaacagtcg ctaattaaca
gccagttctt 180gaaatcacgc agtcctgaaa tccagctgat gctgctcagc
agagaagcag atgtttggag 240gttaaaacaa agtcacaagt ctgacccaca
ctcaaaggga gaagattatg caagagcaaa 300agcacgagat ggaaaccatg
gggccatctt atagtctgtc tgccggagaa agatgaacac 360aaatcctcgt
gcagaaaagt gtgacactaa aagaaccaac ctgatactac tatccttaga
420aaagcctgca tacaaggttg gctgtcatct gggaatgtag gtgatcaaga
gttctctcca 480ctgacataaa ctttgtttaa ataataagga tgactcattg
tgactagact atgcaaataa 540tgggatgtgt gttaaacatc tcctttcctt
ctgggagtct ggaattttgg tgtatgctaa 600acagtgggtg cctgcaagac
cagcctcaaa taaaaaccct gggcactgag tctctaaaaa 660gactcccagt
aggcaatatt tcacacgtgt tctcacaaac ttactgctgg aggaattatg
720cacgccttgc atgactctat tggaaa 74666782DNAHomo sapiens
66gcacgagaga tgttataata attttcaaaa gaaggtaaaa tcatgactca tatggatata
60aaaatgaaca ccttgttatg gattgaactg tgtgccccta aaagatatgt tgaagcctga
120actcccagta cctgagaatg tgaccatgtt tggaaatagt gtctttgcag
gtttaatcaa 180gttaagatga ggtcattaga tgggacctaa tttaatatgg
ctgatgccct tataagaaga 240gggaattttg gacacagaca tacaaggagg
acgccatgtg aagacacagt tggaaaagca 300gggaagagat ggccttgtgt
ataaggaggc agagactgga gttatgctgc cacaagccaa 360ggaacaactg
gggctacctg agctggaaga ggcgaggaag gattctcccc tagagggttc
420tgagggagcg tggacctgcc aacaacttgg tttcagattt ctagtctcca
gaactatgca 480gtaataaatt tctgttgttt taagccaccc agtttgtggc
actttgttac gtcagccctg 540tgaaacaaac aaacaaacaa acaaaaaaca
agttaaatat tttatttaac tcataatatg 600gaaataggtg aggtgttaca
accaattcaa aaaagaagtg aaaaataagc ccaaatgtat 660atagagtaaa
ggaatgttga aatgaatgtg caaatggaag caaaactaga ttctcctaca
720atgggacaga aaagtcaatt gaacctctac tggacagaac ataatttgca
tgtgtataga 780aa 78267525DNAHomo sapiens 67gcaccaggtt tctggtaagg
ggcaattttg tgtttgcctg ttctttggga atatcacctg 60gagatagatg gattcccaga
cccaccaaat ctaggtttta gaccaaaaaa gaatgtttta 120ttattagaat
tgagtatatc tcaggtcctc tttcattact acaacacaaa ataaatctgg
180gggccaggaa cggtggctca cacctgtaat cccagagtgc taggattaca
agcaacaagc 240atgagctacc acgctcggcc tatgcatctc agtcttaaat
tttcttctag gtggatttag 300gggtttgtgc tcgtggccct aaaaatgaac
atgagaaagg cagggagtac ctgcttagtt 360gcaataggcc ttgtttaggc
taaaaataag ctcgatacct gtattttata tactgtaaag 420agcattaacc
ataacctgcc tgcaacagcc tctgtaggta gaaacaatta tggaggtgtc
480aagtaagaat aaagagctga taacatcaaa aaaaaaaaaa aaaaa 5256811PRTHomo
sapiens 68Ala Pro Gln Lys Ser Pro Trp Leu Thr Lys Pro 1 5 10
6913PRTHomo sapiens 69Pro Leu Gln Pro Ser His Phe Leu Asp Ile Ser
Glu Asp 1 5 10 7021DNAArtificial sequencesiRNA sense strand
70ccaugagagu agccagcaat t 217121DNAArtificial sequencesiRNA
antisense strand 71uugcuggcua cucucaugga g 217221DNAArtificial
sequencesiRNA sense strand 72gguucaggac aaaguccaat t
217321DNAArtificial sequencesiRNA antisense strand 73uuggacuuug
uccugaaccg g 21
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