U.S. patent application number 14/043109 was filed with the patent office on 2014-07-03 for genetic products differentially expressed in tumors and the use thereof.
The applicant listed for this patent is Michael Koslowski, Ugur Sahin, Ozlem Tureci. Invention is credited to Michael Koslowski, Ugur Sahin, Ozlem Tureci.
Application Number | 20140186338 14/043109 |
Document ID | / |
Family ID | 32240310 |
Filed Date | 2014-07-03 |
United States Patent
Application |
20140186338 |
Kind Code |
A1 |
Sahin; Ugur ; et
al. |
July 3, 2014 |
Genetic Products Differentially Expressed In Tumors And The Use
Thereof
Abstract
The present technology relates to the identification of genetic
products expressed in association with tumors and to coding nucleic
acids for the expressed products. An embodiment of the present
technology also relates to the therapy and diagnosis of disease in
which the genetic products are aberrantly expressed in association
with tumors, proteins, polypeptides and peptides which are
expressed in association with tumors, and to the nucleic acids
coding for the polypeptides, peptides and proteins.
Inventors: |
Sahin; Ugur; (Mainz, DE)
; Tureci; Ozlem; (Mainz, DE) ; Koslowski;
Michael; (Mainz, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sahin; Ugur
Tureci; Ozlem
Koslowski; Michael |
Mainz
Mainz
Mainz |
|
DE
DE
DE |
|
|
Family ID: |
32240310 |
Appl. No.: |
14/043109 |
Filed: |
October 1, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12423153 |
Apr 14, 2009 |
8586047 |
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14043109 |
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12326997 |
Dec 3, 2008 |
8088588 |
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12423153 |
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10537002 |
May 20, 2005 |
7527933 |
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PCT/EP03/13091 |
Nov 21, 2003 |
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12326997 |
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Current U.S.
Class: |
424/133.1 ;
424/139.1; 424/178.1; 530/387.3; 530/387.9; 530/391.7 |
Current CPC
Class: |
A61K 2039/505 20130101;
A61P 35/00 20180101; A61K 47/549 20170801; A61P 17/00 20180101;
C07K 2317/30 20130101; A61P 13/12 20180101; C07K 2317/34 20130101;
C07K 16/30 20130101; C07K 16/18 20130101; G01N 33/57484 20130101;
A61P 13/08 20180101; C07K 16/3023 20130101; A61P 11/00 20180101;
A61P 1/18 20180101; C07K 14/47 20130101; A61P 43/00 20180101; C07K
16/3076 20130101; C07K 16/3046 20130101; A61P 1/04 20180101; A61P
15/00 20180101 |
Class at
Publication: |
424/133.1 ;
530/387.9; 530/387.3; 530/391.7; 424/139.1; 424/178.1 |
International
Class: |
C07K 16/30 20060101
C07K016/30; A61K 47/48 20060101 A61K047/48 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 22, 2002 |
DE |
102 54 601.0 |
Claims
1.-27. (canceled)
28. A purified antibody or an antigen binding fragment thereof that
is capable of specifically binding to a tumor-associated antigen
that is expressed or abnormally expressed on a cell, said
tumor-associated antigen being selected from the group consisting
of: (a) a tumor-associated antigen comprising the amino acid
sequence of SEQ ID NO: 16; and (b) a tumor-associated antigen
encoded by a nucleic acid comprising SEQ ID NO: 7, wherein said
purified antibody binds to a deglycosylated form of the
tumor-associated antigen.
29. The purified antibody of claim 28 that is a monoclonal,
chimeric, or humanized antibody.
30. A conjugate between the purified antibody or an antigen binding
fragment thereof of claim 28 and at least one therapeutic
agent.
31. The conjugate of claim 30, wherein said therapeutic agent is a
toxin.
32. The conjugate of claim 30, wherein said therapeutic agent is
selected from the group consisting essentially of
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
hydrochloride, thioguanine, vinblastine sulfate, and vincristine
sulfate.
33. A pharmaceutical composition, comprising the purified antibody
or antigen binding fragment thereof of claim 28 and a
pharmaceutically compatible carrier.
34. The pharmaceutical composition of claim 33, wherein the
purified antibody causes induction of cell death, reduction in cell
growth, cell membrane damage, or secretion of cytokines.
35. The pharmaceutical composition of claim 34, wherein said
purified antibody is a complement-activating antibody, a monoclonal
antibody, a chimeric antibody, or a humanized antibody.
36. The pharmaceutical composition of claim 34, wherein said
purified antibody is coupled to a therapeutic agent.
37. The pharmaceutical composition of claim 36, wherein said
therapeutic agent is a toxin.
38. The pharmaceutical composition of claim 36, wherein said
therapeutic agent is selected from the group consisting essentially
of 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
hydrochloride, thioguanine, vinblastine sulfate and vincristine
sulfate.
39. A method of treating a cancer disease characterized by
expression or abnormal expression of a tumor-associated antigen,
comprising administering to a patient having said cancer disease an
effective amount of the pharmaceutical composition of claim 33.
40. The method of claim 39, wherein the cancer disease is selected
from the group consisting of esophageal cancer, stomach cancer,
gastric carcinoma, liver cancer, and ear, nose and throat (ENT)
cancer.
41. The method of claim 39, wherein said antibody or an antigen
binding fragment thereof is coupled to a therapeutic agent.
42. The method of claim 41, wherein said therapeutic agent is a
toxin.
43. The method of claim 42, wherein said therapeutic agent is
selected from the group consisting essentially of
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
hydrochloride, thioguanine, vinblastine sulfate, and vincristine
sulfate.
44. The method of claim 39, wherein said purified antibody is a
complement-activating antibody, a monoclonal antibody, a chimeric
antibody, or a humanized antibody.
45. The method of claim 39, wherein said purified antibody causes
induction of cell death, reduction in cell growth, cell membrane
damage, or secretion of cytokines.
Description
[0001] This application is a divisional application of U.S. patent
application Ser. No. 12/423,153, which was filed on Apr. 14, 2009,
as a continuation application of U.S. patent application Ser. No.
12/326,997, now U.S. Pat. No. 8,088,588, which was filed on Dec. 3,
2008 and is a continuation application of U.S. patent application
Ser. No. 10/537,002, now U.S. Pat. No. 7,527,933, which was filed
on May 20, 2005, which is a National Stage Entry of PCT/EP03/13091,
which was filed on Nov. 21, 2003 and claimed priority to German
Patent Application Number 102 54 601.0, which was filed on Nov. 22,
2002. The contents of U.S. patent application Ser. Nos. 12/423,153,
10/537,002, and 12/326,997, international patent application number
PCT/EP03/13091, and German Patent Application Number 102 54 601.0
are incorporated herein by reference in their entireties.
BACKGROUND OF THE INVENTION
[0002] 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.
[0003] In recent years a multiplicity of antigens have been defined
in various neoplasias by these approaches. However, the probes
utilized for antigen identification in the classical methods
illustrated above 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.
BRIEF SUMMARY OF THE INVENTION
[0004] It was the object of the present technology to provide
target structures for a diagnosis and therapy cancers.
[0005] According to the present technology, this object is achieved
by the subject matter of the claims.
[0006] According to the present technology, a strategy for
identifying and providing antigens expressed in association with a
tumor and the nucleic acids coding therefor was pursued. This
strategy is based on the fact that particular genes which are
expressed in an organ specific manner, e.g. exclusively in colon,
lung or kidney tissue, are reactivated also in tumor cells of the
respective organs and moreover in tumor cells of other tissues in
an ectopic and forbidden manner. First, data mining produces a list
as complete as possible of all known organ-specific genes which are
then evaluated for their aberrant activation in different tumors by
expression analyses by means of specific RT-PCR. Data mining is a
known method of identifying tumor-associated genes. In the
conventional strategies, however, transcriptoms of normal tissue
libraries are usually subtracted electronically from tumor tissue
libraries, with the assumption that the remaining genes are
tumor-specific (Schmitt et al., Nucleic Acids Res. 27:4251-60,
1999; Vasmatzis et al., Proc. Natl. Acad. Sci. USA. 95:300-4, 1998;
Scheurle et al., Cancer Res. 60:4037-43, 2000).
[0007] The concept of the present technology, which has proved much
more successful, however, is based on utilizing data mining for
electronically extracting all organ-specific genes and then
evaluating said genes for expression in tumors.
[0008] The present technology thus relates in one aspect to a
strategy for identifying tissue-specific genes differentially
expressed in tumors. Said strategy combines data mining of public
sequence libraries ("in silico") with subsequent evaluating
laboratory-experimental ("wet bench") studies.
[0009] According to the present technology, a combined strategy
based on two different bioinformatic scripts enabled new tumor
genes to be identified. These have previously been classified as
being purely organ-specific. The finding that these genes are
aberrantly activated in tumor cells allows them to be assigned a
substantially new quality with functional implications. According
to the present technology, these tumor-associated genes and the
genetic products encoded thereby were identified and provided
independently of an immunogenic action.
[0010] The tumor-associated antigens identified according to the
present technology 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: 1-8, 41-44, 51-59, 84, 117, and
119, 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 present
technology has an amino acid sequence encoded by a nucleic acid
which is selected from the group consisting of SEQ ID NOs: 1-8,
41-44, 51-59, 84, 117, and 119. In a further preferred embodiment,
a tumor-associated antigen identified according to the present
technology comprises an amino acid sequence selected from the group
consisting of SEQ ID NOs: 9-19, 45-48, 60-66, 85, 90-97, 100-102,
105, 106, 111-116, 118, 120, 123, 124, and 135-137, a part or
derivative thereof.
[0011] The present technology generally relates to the use of
tumor-associated antigens identified according to the present
technology or of parts or derivatives thereof, of nucleic acids
coding therefor or of nucleic acids directed against said coding
nucleic acids or of antibodies directed against the
tumor-associated antigens identified according to the present
technology or parts or derivatives thereof for therapy and
diagnosis. This utilization may relate to individual but also to
combinations of two or more of these antigens, functional
fragments, nucleic acids, antibodies, etc., in one embodiment also
in combination with other tumor-associated genes and antigens for
diagnosis, therapy and progress control.
[0012] Preferred diseases for a therapy and/or diagnosis are those
in which one or more of the tumor-associated antigens identified
according to the present technology are selectively expressed or
abnormally expressed.
[0013] The present technology also relates to nucleic acids and
genetic products which are expressed in association with a tumor
cell.
[0014] Furthermore, the present technology relates to genetic
products, i.e. nucleic acids and proteins or peptides, which are
produced by altered splicing (splice variants) of known genes or
altered translation using alternative open reading frames. In this
aspect the present technology relates to nucleic acids which
comprise a nucleic acid sequence selected from the group consisting
of sequences according to SEQ ID NOs: 3-5 of the sequence listing.
Moreover, in this aspect, the present technology relates to
proteins or peptides which comprise an amino acid sequence selected
from the group consisting of the sequences according to SEQ ID NOs:
10 and 12-14 of the sequence listing. The splice variants of the
present technology can be used according to the present technology
as targets for diagnosis and therapy of tumor diseases.
[0015] In particular, the present technology relates to the amino
acid sequence according to SEQ ID NO: 10 of the sequence listing
which is encoded by an alternative open reading frame identified
according to the present technology and differs from the previously
described protein sequence (SEQ ID NO: 9) in additional 85 amino
acids at the N terminus of the protein.
[0016] Very different mechanisms may cause splice variants to be
produced, for example [0017] utilization of variable transcription
initiation sites [0018] utilization of additional exons [0019]
complete or incomplete splicing out of single or two or more exons,
[0020] splice regulator sequences altered via mutation (deletion or
generation of new donor/acceptor sequences), [0021] incomplete
elimination of intron sequences.
[0022] 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, for example in blood, serum, bone marrow,
sputum, bronchial lavage, bodily secretions and tissue biopsies,
may be carried out according to the present technology, for
example, after extraction of nucleic acids by PCR amplification
with splice variant-specific oligonucleotides. In particular, pairs
of primers are suitable as oligonucleotides at least one of which
binds to the region of the splice variant which is tumor-associated
under stringent conditions. According to the present technology,
oligonucleotides described for this purpose in the examples are
suitable, in particular oligonucleotides which have or comprise a
sequence selected from SEQ ID NOs: 34-36, 39, 40, and 107-110 of
the sequence listing. According to the present technology, 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 present technology 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. In this aspect, the present technology
relates, in particular, to peptides which have or comprise a
sequence selected from SEQ ID NOs: 17-19, 111-115, 120, and 137 of
the sequence listing and specific antibodies which are directed
thereto. Suitable for immunization are particularly the amino acids
whose epitopes 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. In addition to diagnostic
usability, splice variants having new or altered epitopes are
attractive targets for immunotherapy. The epitopes of the present
technology may be utilized for targeting therapeutically active
monoclonal antibodies or T lymphocytes. 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 (immunization of animals, panning strategies for
isolation of recombinant antibodies) with utilization of
polypeptides which include these epitopes. Alternatively, it is
possible to utilize for immunization nucleic acids coding for
oligo- or polypeptides 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 oligo- or polypeptides which contain
the splice variant-specific epitopes or nucleic acids coding for
said oligo- or polypeptides. Oligo- or polypeptides which contain
the splice variant-specific epitopes or nucleic acids coding for
said polypeptides may also be used as pharmaceutically active
substances in active immunotherapy (vaccination, vaccine
therapy).
[0023] The present technology also describes proteins which differ
in nature and degree of their secondary modifications in normal and
tumor tissue (for example Durand & Seta, 2000; Clin. Chem. 46:
795-805; Hakomori, 1996; Cancer Res. 56: 5309-18).
[0024] The analysis of protein modifications can be done in Western
blots. In particular, glycosylations which as a rule have a size of
several kDa result in a higher overall mass of the target protein
which can be separated in an SDS-PAGE. For the detection of
specific O- and N-glycosidic bonds protein lysates are incubated
with O- or N-glycosylases (according to the instructions of the
respective manufactures, for example, PNgase, endoglycosidase F,
endoglycosidase H, Roche Diagnostics) prior to denaturation using
SDS. Thereafter, a Western blot is performed. If the size of target
protein is reduced a specific glycosylation can be detected in this
manner following incubation with a glycosidase and thus, also the
tumor specificity of a modification can be analyzed. Protein
regions which are differentially glycosylated in tumor cells and
healthy cells are of particular interest. Such differences in
glycosylation, however, have hitherto only been described for a few
cell surface proteins (for example, Muc1).
[0025] According to the present technology, it was possible to
detect a differential glycosylation for Claudin-18 in tumors.
Gastrointestinal carcinomas, pancreas carcinomas, esophagus tumors,
prostate tumors as well as lung tumors have a form of Claudin-18
which is glycosylated at a lower level. Glycosylation in healthy
tissues masks protein epitopes of Claudin-18 which are not covered
on tumor cells due to lacking glycosylation. Correspondingly it is
possible according to the present technology to select ligands and
antibodies which bind to these domains. Such ligands and antibodies
according to the present technology do not bind to Claudin-18 on
healthy cells since here the epitopes are covered due to
glycosylation.
[0026] As has been described above for protein epitopes which are
derived from tumor-associated splice variants it is thus possible
to use the differential glycosylation to distinguish normal cells
and tumor cells with diagnostic as well as therapeutic
intention.
[0027] In one aspect, the present technology relates to a
pharmaceutical composition comprising an agent which recognizes the
tumor-associated antigen identified according to the present
technology and which is preferably selective for cells which have
expression or abnormal expression of a tumor-associated antigen
identified according to the present technology. 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 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
further embodiment, the agent comprises two or more agents which
each selectively recognize different tumor-associated antigens, at
least one of which is a tumor-associated antigen identified
according to the present technology. Recognition needs not be
accompanied directly with inhibition of activity or expression of
the antigen. In this aspect of the present technology, the antigen
selectively limited to tumors preferably serves as a label for
recruiting effector mechanisms to this specific location. In a
preferred embodiment, the agent is a cytotoxic T lymphocyte which
recognizes the antigen on an HLA molecule and lyses the cells
labeled in this way. In a further embodiment, the agent is an
antibody which binds selectively to the tumor-associated antigen
and thus recruits natural or artificial effector mechanisms to said
cell. In a further embodiment, the agent is a T helper lymphocyte
which enhances effector functions of other cells specifically
recognizing said antigen.
[0028] In one aspect, the present technology relates to a
pharmaceutical composition comprising an agent which inhibits
expression or activity of a tumor-associated antigen identified
according to the present technology. In a preferred 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 an antibody which binds
selectively to the tumor-associated antigen. In a further
embodiment, the agent comprises two or more agents which each
selectively inhibit expression or activity of different
tumor-associated antigens, at least one of which is a
tumor-associated antigen identified according to the present
technology.
[0029] The present technology furthermore relates to a
pharmaceutical composition which comprises an agent which, when
administered, selectively increases the amount of complexes between
an HLA molecule and a peptide epitope from the tumor-associated
antigen identified according to the present technology. In one
embodiment, the agent 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. In one embodiment,
the agent comprises two or more agents which each selectively
increase the amount of complexes between MHC molecules and peptide
epitopes of different tumor-associated antigens, at least one of
which is a tumor-associated antigen identified according to the
present technology.
[0030] The present technology 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 present technology or a part
thereof, (ii) a nucleic acid which codes for a tumor-associated
antigen identified according to the present technology or for a
part thereof, (iii) an antibody which binds to a tumor-associated
antigen identified according to the present technology 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 present technology, (v) a host
cell which expresses a tumor-associated antigen identified
according to the present technology or a part thereof, and (vi)
isolated complexes between a tumor-associated antigen identified
according to the present technology or a part thereof and an HLA
molecule.
[0031] A nucleic acid coding for a tumor-associated antigen
identified according to the present technology or for a part
thereof may be present in the pharmaceutical composition in an
expression vector and functionally linked to a promoter.
[0032] A host cell present in a pharmaceutical composition of the
present technology may secrete the tumor-associated antigen or the
part thereof, express it on the surface or may additionally express
an HLA molecule which binds to said tumor-associated antigen or
said part thereof. In one embodiment, the host cell expresses the
HLA molecule endogenously. In a further embodiment, the host cell
expresses the HLA 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.
[0033] An antibody present in a pharmaceutical composition of the
present technology may be a monoclonal antibody. In further
embodiments, the antibody is a chimeric or humanized antibody, a
fragment of a natural antibody or a synthetic antibody, all of
which may be produced by combinatory techniques. The antibody may
be coupled to a therapeutically or diagnostically useful agent.
[0034] An antisense nucleic acid present in a pharmaceutical
composition of the present technology 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 present technology.
[0035] In further embodiments, a tumor-associated antigen, provided
by a pharmaceutical composition of the present technology either
directly or via expression of a nucleic acid, or a part thereof
binds to MHC molecules on the surface of cells, said binding
preferably causing a cytolytic response and/or inducing cytokine
release.
[0036] A pharmaceutical composition of the present technology may
comprise a pharmaceutically compatible carrier and/or an adjuvant.
The adjuvant may be selected from saponin, GM-CSF, CpG nucleotides,
RNA, a cytokine or a chemokine. A pharmaceutical composition of the
present technology is preferably used for the treatment of a
disease characterized by selective expression or abnormal
expression of a tumor-associated antigen. In a preferred
embodiment, the disease is cancer.
[0037] The present technology furthermore relates to methods of
treating or diagnosing a disease characterized by expression or
abnormal expression of one of more tumor-associated antigens. In
one embodiment, the treatment comprises administering a
pharmaceutical composition of the present technology.
[0038] In one aspect, the present technology relates to a method of
diagnosing a disease characterized by expression or abnormal
expression of a tumor-associated antigen identified according to
the present technology. The method comprises detection of (i) a
nucleic acid which codes for the tumor-associated antigen or of a
part thereof and/or (ii) detection of the tumor-associated antigen
or of a part thereof, and/or (iii) detection of an antibody to the
tumor-associated antigen or to a part thereof and/or (iv) detection
of cytotoxic or T helper lymphocytes which are specific for the
tumor-associated antigen or for a part thereof in a biological
sample isolated from a patient. In particular embodiments,
detection comprises (i) contacting the biological sample with an
agent which binds specifically to the nucleic acid coding for the
tumor-associated antigen or to the part thereof, to said
tumor-associated antigen or said part thereof, to the antibody or
to cytotoxic or T helper lymphocytes specific for the
tumor-associated antigen or parts thereof, and (ii) detecting the
formation 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 cytotoxic or T helper lymphocytes. In one
embodiment, the disease is characterized by expression or abnormal
expression of two or more different tumor-associated antigens and
detection comprises detection of two or more nucleic acids coding
for said two or more different tumor-associated antigens or of
parts thereof, detection of two or more different tumor-associated
antigens or of parts thereof, detection of two or more antibodies
binding to said two or more different tumor-associated antigens or
to parts thereof or detection of two or more cytotoxic or T helper
lymphocytes specific for said two or more different
tumor-associated antigens. In a further embodiment, the biological
sample isolated from the patient is compared to a comparable normal
biological sample.
[0039] In a further aspect, the present technology relates to a
method for determining regression, course or onset of a disease
characterized by expression or abnormal expression of a
tumor-associated antigen identified according to the present
technology, which method comprises monitoring a sample from a
patient who has said disease or is suspected of falling ill with
said disease, with respect to one or more parameters selected from
the group consisting of (i) the amount of nucleic acid which codes
for the tumor-associated antigen or of a part thereof, (ii) the
amount of the tumor-associated antigen or a part thereof, (iii) the
amount of antibodies which bind to the tumor-associated antigen or
to a part thereof, and (iv) the amount of cytolytic T cells or T
helper cells which are specific for a complex between the
tumor-associated antigen or a part thereof and an MHC molecule. The
method preferably comprises determining the parameter(s) in a first
sample at a first point in time and in a further sample at a second
point in time and in which the course of the disease is determined
by comparing the two samples. In particular embodiments, the
disease is characterized by expression or abnormal expression of
two or more different tumor-associated antigens and monitoring
comprises monitoring (i) the amount of two or more nucleic acids
which code for said two or more different tumor-associated antigens
or of parts thereof, and/or (ii) the amount of said two or more
different tumor-associated antigens or of parts thereof, and/or
(iii) the amount of two or more antibodies which bind to said two
or more different tumor-associated antigens or to parts thereof,
and/or (iv) the amount of two or more cytolytic T cells or of T
helper cells which are specific for complexes between said two or
more different tumor-associated antigens or of parts thereof and
MHC molecules.
[0040] According to the present technology, detection of a nucleic
acid or of a part thereof or monitoring the amount of a nucleic
acid or of a part thereof may be carried out using a 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. In one embodiment, the
polynucleotide probe comprises a sequence of 6-50, in particular
10-30, 15-30 and 20-30, contiguous nucleotides of said nucleic
acid.
[0041] In particular embodiments, the tumor-associated antigen to
be detected or the part thereof is present intracellularly or on
the cell surface. According to the present technology, detection of
a tumor-associated antigen or of a part thereof or monitoring the
amount 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.
[0042] In further embodiments, the tumor-associated antigen to be
detected or the part thereof is present in a complex with an MHC
molecule, in particular an HLA molecule.
[0043] According to the present technology, detection of an
antibody or monitoring the amount of antibodies may be carried out
using a protein or peptide binding specifically to said
antibody.
[0044] According to the present technology, detection of cytolytic
T cells or of T helper cells or monitoring the amount of cytolytic
T cells or of T helper cells which are specific for complexes
between an antigen or a part thereof and MHC molecules may be
carried out using a cell presenting the complex between said
antigen or said part thereof and an MHC molecule.
[0045] The polynucleotide probe, the antibody, the protein or
peptide or the cell, which is used for detection or monitoring, is
preferably labeled in a detectable manner. In particular
embodiments, the detectable marker is a radioactive marker or an
enzymic marker. T lymphocytes may additionally be detected by
detecting their proliferation, their cytokine production, and their
cytotoxic activity triggered by specific stimulation with the
complex of MHC and tumor-associated antigen or parts thereof. T
lymphocytes may also be detected via a recombinant MHC molecule or
else a complex of two or more MHC molecules which are loaded with
the particular immunogenic fragment of one or more of the
tumor-associated antigens and which can identify the specific T
lymphocytes by contacting the specific T cell receptor.
[0046] In a further aspect, the present technology relates to a
method of treating, diagnosing or monitoring a disease
characterized by expression or abnormal expression of a
tumor-associated antigen identified according to the present
technology, 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.
[0047] The present technology also 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 present technology, which method comprises (i) removing a
sample containing immunoreactive cells from said patient, (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. The
present technology likewise relates to cloning the T cell receptor
of cytolytic T cells against the tumor-associated antigen. Said
receptor may be transferred to other T cells which thus receive the
desired specificity and, as under (iii), may be introduced into the
patient.
[0048] In one embodiment, the host cell endogenously expresses an
HLA molecule. In a further embodiment, the host cell recombinantly
expresses an HLA molecule and/or the tumor-associated antigen or
the part thereof. 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.
[0049] In a further aspect, the present technology relates to a
method of treating a patient having a disease characterized by
expression or abnormal expression of a tumor-associated antigen,
which method comprises (i) identifying a nucleic acid which codes
for a tumor-associated antigen identified according to the present
technology and which is expressed by cells associated with said
disease, (ii) transfecting a host cell with said nucleic acid or a
part thereof, (iii) culturing the transfected host cell for
expression of said nucleic acid (this is not obligatory when a high
rate of transfection is obtained), and (iv) introducing the host
cells or an extract thereof into the patient in an amount suitable
for increasing the immune response to the patient's cells
associated with the disease. The method may further comprise
identifying an MHC molecule presenting the tumor-associated antigen
or a part thereof, with the host cell expressing the identified MHC
molecule and presenting said tumor-associated antigen or a part
thereof. The immune response may comprise a B cell response or a T
cell response. Furthermore, a T cell response may comprise
production of cytolytic T cells and/or T helper cells which are
specific for the host cells presenting the tumor-associated antigen
or a part thereof or specific for cells of the patient which
express said tumor-associated antigen or a part thereof.
[0050] The present technology also relates to a method of treating
a disease characterized by expression or abnormal expression of a
tumor-associated antigen identified according to the present
technology, 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.
[0051] Preferably, the host cells used according to the present
technology are nonproliferative or are rendered nonproliferative. A
disease characterized by expression or abnormal expression of a
tumor-associated antigen is in particular cancer.
[0052] The present technology 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: 3-5, 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
present technology 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: 10 and
12-14, a part or derivative thereof.
[0053] In a further aspect, the present technology relates to
promoter sequences of nucleic acids of the present technology.
These sequences may be functionally linked to another gene,
preferably in an expression vector, and thus ensure selective
expression of said gene in appropriate cells.
[0054] In a further aspect, the present technology relates to a
recombinant nucleic acid molecule, in particular DNA or RNA
molecule, which comprises a nucleic acid of the present
technology.
[0055] The present technology also relates to host cells which
contain a nucleic acid of the present technology or a recombinant
nucleic acid molecule comprising a nucleic acid of the present
technology.
[0056] The host cell may also comprise a nucleic acid coding for a
HLA molecule. In one embodiment, the host cell endogenously
expresses the HLA molecule. In a further embodiment, the host cell
recombinantly expresses the HLA molecule and/or the nucleic acid of
the present technology 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.
[0057] In a further embodiment, the present technology relates to
oligonucleotides which hybridize with a nucleic acid identified
according to the present technology and which may be used as
genetic probes or as "antisense" molecules. Nucleic acid molecules
in the form of oligonucleotide primers or competent samples, which
hybridize with a nucleic acid identified according to the present
technology or parts thereof, may be used for finding nucleic acids
which are homologous to said nucleic acid identified according to
the present technology. PCR amplification, Southern and Northern
hybridization may be employed for finding homologous nucleic acids.
Hybridization may be carried out under low stringency, more
preferably under medium stringency and most preferably under high
stringency conditions. The term "stringent conditions" according to
the present technology refers to conditions which allow specific
hybridization between polynucleotides.
[0058] In a further aspect, the present technology relates to a
protein, polypeptide 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: 3-5, 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 present technology relates to a protein
or polypeptide or peptide which comprises an amino acid sequence
selected from the group consisting of SEQ ID NOs: 10 and 12-14, a
part or derivative thereof.
[0059] In a further aspect, the present technology relates to an
immunogenic fragment of a tumor-associated antigen identified
according to the present technology. Said fragment preferably binds
to a human HLA receptor or to a human antibody. A fragment of the
present technology 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.
[0060] In this aspect the present technology relates, in
particular, to a peptide which has or comprises a sequence selected
from the group consisting of SEQ ID NOs: 17-19, 90-97, 100-102,
105, 106, 111-116, 120, 123, 124, and 135-137, a part or derivative
thereof.
[0061] In a further aspect, the present technology relates to an
agent which binds to a tumor-associated antigen identified
according to the present technology or to a part thereof. In a
preferred embodiment, the agent is an antibody. In further
embodiments, the antibody is a chimeric, a humanized antibody or an
antibody produced by combinatory techniques or is a fragment of an
antibody. Furthermore, the present technology relates to an
antibody which binds selectively to a complex of (i) a
tumor-associated antigen identified according to the present
technology or a part thereof and (ii) an MHC molecule to which said
tumor-associated antigen identified according to the present
technology or said part thereof binds, with said antibody not
binding to (i) or (ii) alone. An antibody of the present technology
may be a monoclonal antibody. In further embodiments, the antibody
is a chimeric or humanized antibody or a fragment of a natural
antibody.
[0062] In particular, the present technology 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: 17-19, 90-97, 100-102, 105, 106, 111-116,
120, 123, 124, and 135-137, a part or derivative thereof.
[0063] The present technology furthermore relates to a conjugate
between an agent of the present technology which binds to a
tumor-associated antigen identified according to the present
technology or to a part thereof or an antibody of the present
technology and a therapeutic or diagnostic agent. In one
embodiment, the therapeutic or diagnostic agent is a toxin.
[0064] In a further aspect, the present technology relates to a kit
for detecting expression or abnormal expression of a
tumor-associated antigen identified according to the present
technology, which kit comprises agents for detection (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 a complex between the tumor-associated antigen or a
part thereof and 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
[0065] According to the present technology, genes are described
which are expressed in tumor cells selectively or aberrantly and
which are tumor-associated antigens.
[0066] According to the present technology, these genes and/or
their genetic products and/or their derivatives and/or parts are
preferred target structures for therapeutic approaches.
Conceptionally, said therapeutic approaches may aim at inhibiting
the activity of the selectively expressed tumor-associated genetic
product. This is useful, if said aberrant respective selective
expression is functionally important in tumor pathogenecity and if
its ligation is accompanied by selective damage of the
corresponding cells. Other therapeutic concepts contemplate
tumor-associated antigens as labels which recruit effector
mechanisms having cell-damaging potential selectively to tumor
cells. Here, the function of the target molecule itself and its
role in tumor development are totally irrelevant.
[0067] "Derivative" of a nucleic acid means according to the
present technology that single or multiple 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.
[0068] According to the present technology, a nucleic acid is
preferably deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
Nucleic acids comprise according to the present technology genomic
DNA, cDNA, mRNA, recombinantly produced and chemically synthesized
molecules. According to the present technology, a nucleic acid may
be present as a single-stranded or double-stranded and linear or
covalently circularly closed molecule.
[0069] The nucleic acids described according to the present
technology have preferably been isolated. The term "isolated
nucleic acid" means according to the present technology 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.
[0070] 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% SOS, 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.
[0071] According to the present technology, 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.
[0072] Nucleic acids coding for tumor-associated antigens may,
according to the present technology, 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.
[0073] The term "expression control sequence" or "regulatory
sequence" comprises according to the present technology promoters,
enhancers and other control elements which regulate expression of a
gene. In particular embodiments of the present technology, 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.
[0074] Thus, on the one hand, the tumor-associated antigens
illustrated herein may be combined with any expression control
sequences and promoters. On the other hand, however, the promoters
of the tumor-associated genetic products illustrated herein may,
according to the present technology, be combined with any other
genes. This allows the selective activity of these promoters to be
utilized.
[0075] According to the present technology, a nucleic acid may
furthermore be present in combination with another nucleic acid
which codes for a polypeptide controlling secretion of the protein
or polypeptide encoded by said nucleic acid from a host cell.
According to the present technology, a nucleic acid may also be
present in combination with another nucleic acid which codes for a
polypeptide causing the encoded protein or polypeptide 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".
[0076] In a preferred embodiment, a recombinant DNA molecule is
according to the present technology 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 of the
present technology. 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 or viral genomes.
[0077] The nucleic acids coding for a tumor-associated antigen
identified according to the present technology 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.
[0078] According to the present technology, 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 present technology 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.
[0079] 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.
[0080] According to the present technology, 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).
[0081] In those cases of the present technology in which an HLA
molecule presents a tumor-associated antigen or a part thereof, an
expression vector may also comprise a nucleic acid sequence coding
for said HLA molecule. The nucleic acid sequence coding for the HLA
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 HLA
molecule, both nucleic acids coding therefor are transfected into
the cell either on the same expression vector or on different
expression vectors. If the cell already expresses the HLA molecule,
only the nucleic acid sequence coding for the tumor-associated
antigen or the part thereof can be transfected into the cell.
[0082] The present technology 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.
[0083] "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 present technology to an
oligonucleotide which is an oligoribonucleotide,
oligodeoxyribonucleotide, modified oligoribonucleotide or modified
oligodeoxyribonucleotide 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 present technology, 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.
[0084] Another possibility is the use of ribozymes for inactivating
a nucleic acid. Antisense oligonucleotides preferred according to
the present technology 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.
[0085] 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.
[0086] In one embodiment, an oligonucleotide of the present
technology 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.
[0087] In preferred embodiments, an oligonucleotide of the present
technology 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
present technology, 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.
[0088] 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.
[0089] Preferably, the proteins and polypeptides described
according to the present technology have been isolated. The terms
"isolated protein" or "isolated polypeptide" mean that the protein
or polypeptide has been separated from its natural environment. An
isolated protein or polypeptide may be in an essentially purified
state. The term "essentially purified" means that the protein or
polypeptide is essentially free of other substances with which it
is associated in nature or in vivo.
[0090] Such proteins and polypeptides may be used, for example, in
producing antibodies and in an immunological or diagnostic assay or
as therapeutics. Proteins and polypeptides described according to
the present technology 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.
[0091] For the purposes of the present technology, "derivatives" of
a protein or polypeptide or of an amino acid sequence comprise
amino acid insertion variants, amino acid deletion variants and/or
amino acid substitution variants.
[0092] 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
polypeptides. Preference is given 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:
[0093] 1. small aliphatic, nonpolar or slightly polar residues:
Ala, Ser, Thr (Pro, Gly)
[0094] 2. negatively charged residues and their amides: Asn, Asp,
Glu, Gln
[0095] 3. positively charged residues: H is, Arg, Lys
[0096] 4. large aliphatic, nonpolar residues: Met, Leu, Ile, Val
(Cys)
[0097] 5. large aromatic residues: Phe, Tyr, Trp.
[0098] 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.
[0099] 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. Techniques for
introducing substitution mutations at predetermined sites into DNA
which has a known or partially known sequence are well known and
comprise M13 mutagenesis, for example. The manipulation of DNA
sequences for preparing proteins having substitutions, insertions
or deletions, is described in detail in Sambrook et al. (1989), for
example.
[0100] According to the present technology, "derivatives" of
proteins, polypeptides or peptides also comprise single or multiple
substitutions, deletions and/or additions of any molecules
associated with the enzyme, such as carbohydrates, lipids and/or
proteins, polypeptides or peptides. The term "derivative" also
extends to all functional chemical equivalents of said proteins,
polypeptides or peptides.
[0101] According to the present technology, a part or fragment of a
tumor-associated antigen has a functional property of the
polypeptide from which it has been derived. Such functional
properties comprise the interaction with antibodies, the
interaction with other polypeptides or proteins, the selective
binding of nucleic acids and an enzymatic activity. A particular
property is the ability to form a complex with HLA and, where
appropriate, generate an immune response. This immune response may
be based on stimulating cytotoxic or T helper cells. A part or
fragment of a tumor-associated antigen of the present technology
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.
[0102] A part or a fragment of a nucleic acid coding for a
tumor-associated antigen relates according to the present
technology 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.
[0103] The isolation and identification of genes coding for
tumor-associated antigens also make possible the diagnosis of a
disease characterized by expression of one or more tumor-associated
antigens. These methods comprise determining one or more nucleic
acids which code for a tumor-associated antigen and/or determining
the encoded tumor-associated antigens and/or peptides derived
therefrom. The nucleic acids may be determined in the conventional
manner, including by polymerase chain reaction or hybridization
with a labeled probe. Tumor-associated antigens or peptides derived
therefrom may be determined by screening patient antisera with
respect to recognizing the antigen and/or the peptides. They may
also be determined by screening T cells of the patient for
specificities for the corresponding tumor-associated antigen.
[0104] The present technology also enables proteins binding to
tumor-associated antigens described herein to be isolated,
including antibodies and cellular binding partners of said
tumor-associated antigens.
[0105] According to the present technology, particular embodiments
ought to involve providing "dominant negative" polypeptides derived
from tumor-associated antigens. A dominant negative polypeptide is
an inactive protein variant which, by way of interacting with the
cellular machinery, displaces an active protein from its
interaction with the cellular machinery or which competes with the
active protein, thereby reducing the effect of said active protein.
For example, a dominant negative receptor which binds to a ligand
but does not generate any signal as response to binding to the
ligand can reduce the biological effect of said ligand. Similarly,
a dominant negative catalytically inactive kinase which usually
interacts with target proteins but does not phosphorylate said
target proteins may reduce phosphorylation of said target proteins
as response to a cellular signal. Similarly, a dominant negative
transcription factor which binds to a promoter site in the control
region of a gene but does not increase transcription of said gene
may reduce the effect of a normal transcription factor by occupying
promoter binding sites, without increasing transcription.
[0106] The result of expression of a dominant negative polypeptide
in a cell is a reduction in the function of active proteins. The
skilled worker may prepare dominant negative variants of a protein,
for example, by conventional mutagenesis methods and by evaluating
the dominant negative effect of the variant polypeptide.
[0107] The present technology also comprises substances such as
polypeptides which bind to tumor-associated antigens. Such binding
substances may be used, for example, in screening assays for
detecting tumor-associated antigens and complexes of
tumor-associated antigens with their binding partners and in the
purification of said tumor-associated antigens and of complexes
thereof with their binding partners. Such substances may also be
used for inhibiting the activity of tumor-associated dominant
antigens, for example by binding to such antigens.
[0108] The present technology therefore comprises binding
substances such as, for example, antibodies or antibody fragments,
which are capable of selectively binding to tumor-associated
antigens. Antibodies comprise polyclonal and monoclonal antibodies
which are produced in the conventional manner.
[0109] Such antibodies can recognize proteins in the native and/or
denaturated state (Anderson et al., J. Immunol. 143: 1899-1904,
1989; Gardsvoll, J. Immunol. Methods 234: 107-116, 2000; Kayyem et
al., Eur. J. Biochem. 208: 1-8, 1992; Spiller et al., J. Immunol.
Methods 224: 51-60, 1999).
[0110] 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: 30 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.
[0111] 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).
[0112] 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.
[0113] 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.
[0114] 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.
[0115] This is utilized in the so called "SLAM" technology, wherein
B cells from whole blood are isolated and the cells are monocloned.
Then, the supernatant of the single B cells is analyzed with
respect to its antibody specificity. In contrast to the hybridoma
technology the variable region of the antibody gene is amplified
using single cell PCR and cloned into a suitable vector. In this
way, the provision of monoclonal antibodies is accelerated (de
Wildt et al., J. Immunol. Methods 207: 61-67, 1997).
[0116] 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.
[0117] The present technology also provides 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 present technology
also comprises "single-chain" antibodies.
[0118] The present technology also comprises polypeptides which
bind specifically to tumor-associated antigens. Polypeptide 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.
[0119] Phage display may be particularly effective in identifying
binding peptides of the present technology. In this connection, for
example, a phage library is prepared (using, for example, the M13,
fd or lambda phages) which presents inserts of from 4 to about 80
amino acid residues in length. Phages are then selected which carry
inserts which bind to the tumor-associated antigen. This process
may be repeated via two or more cycles of a reselection of phages
binding to the tumor-associated antigen. Repeated rounds result in
a concentration of phages carrying particular sequences. An
analysis of DNA sequences may be carried out in order to identify
the sequences of the expressed polypeptides. The smallest linear
portion of the sequence binding to the tumor-associated antigen may
be determined. The "two-hybrid system" of yeast may also be used
for identifying polypeptides which bind to a tumor-associated
antigen. Tumor-associated antigens described according to the
present technology or fragments thereof may be used for screening
peptide libraries, including phage-display libraries, in order to
identify and select peptide binding partners of the
tumor-associated antigens. Such molecules may be used, for example,
for screening assays, purification protocols, for interference with
the function of the tumor-associated antigen and for other purposes
known to the skilled worker.
[0120] The antibodies described above and other binding molecules
may be used, for example, for identifying tissue which expresses a
tumor-associated antigen. 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. 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. According to the present technology, the term
"therapeutically useful substance" means any therapeutic molecule
which, as desired, is selectively guided to a cell which expresses
one or more tumor-associated antigens, including 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.
[0121] The term "patient" means according to the present technology
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.
[0122] According to the present technology, the term "disease"
refers to any pathological state in which tumor-associated antigens
are expressed or abnormally expressed. "Abnormal expression" means
according to the present technology 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 present
technology comprises leukemias, seminomas, melanomas, teratomas,
gliomas, kidney cancer, adrenal cancer, thyroid cancer, intestinal
cancer, liver cancer, colon cancer, stomach 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.
[0123] According to the present technology, a biological sample may
be a tissue sample 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, for use in the various methods
described herein.
[0124] According to the present technology, 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.
[0125] 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 T 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.
[0126] In another method of selecting antigen-specific cytotoxic T
lymphocytes, fluorogenic tetramers of MHC class I molecule/peptide
complexes are used for detecting specific clones of cytotoxic T
lymphocytes (Altman et al., Science 274:94-96, 1996; Dunbar et al.,
Curr. Biol. 8:413-416, 1998). Soluble MHC class I molecules are
folded in vitro in the presence of .beta..sub.2 microglobulin and a
peptide antigen binding to said class I molecule. The MHC/peptide
complexes are purified and then labeled with biotin. Tetramers are
formed by mixing the biotinylated peptide-MHC complexes with
labeled avidin (e.g. phycoerythrin) in a molar ratio of 4:1.
Tetramers are then contacted with cytotoxic T lymphocytes such as
peripheral blood or lymph nodes. The tetramers bind to cytotoxic T
lymphocytes which recognize the peptide antigen/MHC class I
complex. Cells which are bound to the tetramers may be sorted by
fluorescence-controlled cell sorting to isolate reactive cytotoxic
T lymphocytes. The isolated cytotoxic T lymphocytes may then be
propagated in vitro.
[0127] In a therapeutic method 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), 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.
[0128] Often, of the T cell repertoire of a patient, only T cells
with low affinity for a specific complex of this kind can be
propagated, since those with high affinity have been extinguished
due to development of tolerance. An alternative here may be a
transfer of the T cell receptor itself. For this too, cells
presenting the desired complex (e.g. dendritic cells) are combined
with cytotoxic T lymphocytes of healthy individuals or another
species (e.g. mouse). This results in propagation of specific
cytotoxic T lymphocytes with high affinity if the T lymphocytes are
derived from a donor organism which had no previous contact with
the specific complex. The high affinity T cell receptor of these
propagated specific T lymphocytes is cloned. If the high affinity T
cell receptors have been cloned from another species they can be
humanized to a different extent. Such T cell receptors are then
transduced via gene transfer, for example using retroviral vectors,
into T cells of patients, as desired. Adoptive transfer is then
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).
[0129] The therapeutic aspects above start out from the fact that
at least some of the abnormal cells of the patient present a
complex of a tumor-associated antigen and an HLA molecule. Such
cells may be identified in a manner known per se. As soon as cells
presenting the complex have been identified, they may be combined
with a sample from the patient, which contains cytotoxic T
lymphocytes. If the cytotoxic T lymphocytes lyse the cells
presenting the complex, it can be assumed that a tumor-associated
antigen is presented.
[0130] Adoptive transfer is not the only form of therapy which can
be applied according to the present technology. 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 HLA molecule). Various cell
types may be used. Furthermore, it is possible to use vectors which
carry one or both of the genes of interest. Particular preference
is given to viral or bacterial vectors. For example, nucleic acids
coding for a tumor-associated antigen or for a part thereof may be
functionally linked to promoter and enhancer sequences which
control expression of said tumor-associated antigen or a fragment
thereof in particular tissues or cell types. The nucleic acid may
be incorporated into an expression vector. Expression vectors may
be nonmodified extrachromosomal nucleic acids, plasmids or viral
genomes into which exogenous nucleic acids may be inserted. Nucleic
acids coding for a tumor-associated antigen may also be inserted
into a retroviral genome, thereby enabling the nucleic acid to be
integrated into the genome of the target tissue or target cell. In
these systems, a microorganism such as vaccinia virus, pox virus,
Herpes simplex virus, retrovirus or adenovirus carries the gene of
interest and de facto "infects" host cells. 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.
[0131] 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 HLA 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 HLA 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). It may also be carried out in combination with
reagents which facilitate uptake into dendritic cells. Preferred
tumor-associated antigens comprise those which react with allogenic
cancer antisera or with T cells of many cancer patients. Of
particular interest, however, are those against which no
spontaneous immune responses pre-exist. Evidently, it is possible
to induce against these immune responses which can lyse tumors
(Keogh et al., J. Immunol. 167:787-96, 2001; Appella et al., Biomed
Pept Proteins Nucleic Acids 1:177-84, 1995; Wentworth et al., Mol
Immunol. 32:603-12, 1995).
[0132] The pharmaceutical compositions described according to the
present technology may also be used as vaccines for immunization.
According to the present technology, the terms "immunization" or
"vaccination" mean 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.
[0133] As part of the composition for an immunization, 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.
[0134] 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.
[0135] 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. Examples of such costimulating molecules
are B7-1 and B7-2 (CD80 and CD86, respectively) which are expressed
on dendritic cells (DC) and interact with the CD28 molecule
expressed on the T cells. This interaction provides a costimulation
(signal 2) for an antigen/MHC/TCR-stimulated (signal 1) T cell,
thereby enhancing propagation of said T cell and the effector
function. B7 also interacts with CTLA4 (CD152) on T cells, and
studies involving CTLA4 and B7 ligands demonstrate that B7-CTLA4
interaction can enhance antitumor immunity and CTL propagation
(Zheng, P. et al., Proc. Natl. Acad. Sci. USA 95(11):6284-6289
(1998)).
[0136] B7 is typically not expressed on tumor cells so that these
are not effective antigen-presenting cells (APCs) for T cells.
Induction of B7 expression would enable tumor cells to stimulate
more effectively propagation of cytotoxic T lymphocytes and an
effector function. Costimulation by a combination of B7/IL-6/IL-12
revealed induction of IFN-gamma and Th1-cytokine profile in a T
cell population, resulting in further enhanced T cell activity
(Gajewski et al., J. Immunol. 154:5637-5648 (1995)).
[0137] A complete activation of cytotoxic T lymphocytes and a
complete effector function require an involvement of T helper cells
via interaction between the CD40 ligand on said T helper cells and
the CD40 molecule expressed by dendritic cells (Ridge et al.,
Nature 393:474 (1998), Bennett et al., Nature 393:478. (1998),
Schonberger et al., Nature 393:480 (1998)). The mechanism of this
costimulating signal probably relates to the increase in B7
production and associated IL-6/IL-12 production by said dendritic
cells (antigen-presenting Cells). CD40-CD40L interaction thus
complements the interaction of signal 1 (antigen/MHC-TCR) and
signal 2 (B7-CD28).
[0138] The use of anti-CD40 antibodies for stimulating dendritic
cells would be expected to directly enhance a response to tumor
antigens which are usually outside the range of an inflammatory
response or which are presented by nonprofessional
antigen-presenting cells (tumor cells). In these situations, T
helper and B7-costimulating signals are not provided. This
mechanism could be used in connection with therapies based on
antigen-pulsed dendritic cells.
[0139] The present technology also provides for administration of
nucleic acids, polypeptides or peptides. Polypeptides 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 present technology
also provides for administering nucleic acids in vivo by using
vectors such as viruses and target-controlled liposomes.
[0140] In a preferred embodiment, a 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).
[0141] Various methods may be used in order to introduce according
to the present technology nucleic acids into cells in vitro or in
vivo. Methods of this kind comprise transfection of nucleic acid
CaPO.sub.4 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.
[0142] The therapeutic compositions of the present technology 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, CpG and cytokines and, where appropriate, other
therapeutically active compounds.
[0143] The therapeutically active compounds of the present
technology 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.
[0144] The compositions of the present technology 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 relates to inhibition of the course of the disease. This
comprises slowing down the progress of the disease and, in
particular, interrupting 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.
[0145] An effective amount of a composition of the present
technology 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.
[0146] The pharmaceutical compositions of the present technology
are preferably sterile and contain an effective amount of the
therapeutically active substance to generate the desired reaction
or the desired effect.
[0147] The doses administered of the compositions of the present
technology 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.
[0148] 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.
[0149] The pharmaceutical compositions of the present technology
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 present technology. 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.
[0150] A pharmaceutical composition of the present technology may
comprise a pharmaceutically compatible carrier. According to the
present technology, 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 present
technology are usually such that no interaction occurs which
substantially impairs the desired pharmaceutical efficacy.
[0151] The pharmaceutical compositions of the present technology
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.
[0152] The pharmaceutical compositions may, where appropriate, also
contain suitable preservatives such as benzalkonium chloride,
chlorobutanol, paraben and thimerosal.
[0153] 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 present technology may be in the
form of capsules, tablets, lozenges, suspensions, syrups, elixir or
in the form of an emulsion, for example.
[0154] 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.
[0155] The present technology 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
present technology are accessible to the skilled worker.
BRIEF DESCRIPTION OF THE DRAWINGS
[0156] FIG. 1. GPR35 mRNA expression in colon carcinoma
biopsies
[0157] RT-PCR investigations with DNA-free RNA show GER35
expression in most of the colon carcinoma biopsies. By contrast,
there is no detectable expression in normal tissues. (1-Breast,
2-lung, 3-lymph nodes, 4-thymus, 5-colon, 6-15 colon carcinoma,
16-neg. control).
[0158] FIG. 2. Quantitative PCR analysis of GUCY2C mRNA expression
in normal and tumor tissues
[0159] Real-time PCR investigation with GUCY2C-specific primers
(SEQ ID NO: 22-23) shows selective mRNA expression in normal ileum,
colon, and in all colon carcinoma biopsies. Distinct quantities of
GUCY2C transcripts were also detected in a colon carcinoma
metastasis in the liver.
[0160] FIG. 3. Identification of tumor-specific GUCY2C splice
variants
[0161] PCR products from normal colon tissues and colon carcinomas
were cloned, and clones from both groups were checked by
restriction analysis (EcoR I) and sequenced.
[0162] FIG. 4. Selective SCGB3A expression in normal lung and lung
carcinoma
[0163] RT-PCR analysis with gene-specific SCGB3A2 primers (SEQ ID
NO: 37, 38) shows cDNA amplification exclusively in normal lung
(lane 8, 14-15) and in lung carcinoma biopsies (lane 16-24).
(1-Liver-N, 2-PBMC-N, 3-lymph node-N, 4-stomach-N, 5-testis-N,
6-breast-N, 7-kidney-N, 8-lung-N, 9-thymus-N, 10-ovary-N,
11-adrenal-N, 12-spleen-N, 14-15-lung-N, 16-24-lung carcinoma,
25-negative control).
[0164] FIG. 5. Claudin-18A2.1 expression in stomach, esophagus,
stomach carcinoma and pancreatic carcinoma
[0165] RT-PCR analysis with claudin-18A2.1-specific primers (SEQ ID
NO: 39, 40) showed according to the present technology pronounced
claudin-18A2.1 expression in 8/10 stomach carcinoma biopsies and in
3/6 pancreatic carcinoma biopsies. Distinct expression was also
detected in stomach and normal esophageal tissue. In contrast
thereto, no expression was detected in the ovary and in ovarian
carcinoma.
[0166] FIG. 6. SLC13A1 expression in the kidney and renal cell
carcinoma
[0167] RT-PCR analysis with SLC13A1-specific primers (SEQ ID NO:
49, 50) showed expression in 7/8 renal cell carcinoma samples.
Otherwise, transcripts within normal tissues were detected
exclusively in the kidney. (1-2kidney, 3-10-renal cell carcinoma,
11-breast, 12-lung, 13-liver, 14-colon, 15-lymph nodes, 16-spleen,
17-esophagus, 18-thymus, 19-thyroid, 20-PBMCs, 21-ovary,
22-testis).
[0168] FIG. 7. CLCA1 expression in colon, colon carcinoma and
stomach carcinoma
[0169] RT-PCR investigations with CLCA1-specific primers. (SEQ ID
NO: 67, 68) confirmed selective expression in the colon and showed
high expression in (3/7) investigated colon carcinoma and (1/3)
investigated stomach carcinoma samples. The other normal tissues
(NT) showed no or only very weak expression.
[0170] FIG. 8. FLJ21477 expression in the colon and colon
carcinoma
[0171] RT-PCR investigations with FLJ21477-specific primers (SEQ ID
NO: 69, 70) showed selective expression in the colon and
additionally various levels of expression in (7/12) investigated
colon carcinoma samples. The other normal tissues (NT) showed no
expression.
[0172] FIG. 9. FLJ20694 expression in the colon and colon
carcinoma
[0173] RT-PCR investigations with FLJ20694-specific primers (SEQ ID
NO: 71, 72) showed selective expression in the colon and
additionally various levels of expression in (5/9) investigated
colon carcinoma samples. The other normal tissues (NT) showed no
expression.
[0174] FIG. 10. von Ebner expression in stomach, lung and lung
carcinoma
[0175] RT-PCR investigations with von Ebner-specific primers (SEQ
ID NO: 73, 74) showed selective expression in the stomach, in the
lung and in (5/10) investigated lung carcinoma samples. The other
normal tissues (NT) showed no expression.
[0176] FIG. 11. Plunc expression in thymus, lung and lung
carcinoma
[0177] RT-PCR investigations with Plunc-specific primers (SEQ ID
NO: 75, 76) showed selective expression in the thymus, in the lung
and in (6/10) investigated lung carcinoma samples. The other normal
tissues showed no expression.
[0178] FIG. 12. SLC26A9 expression in lung, lung carcinoma and
thyroid
[0179] RT-PCR investigations with SLC26A9-specific primers (SEQ ID
NO: 77, 78) showed selective expression in the lung and in all
(13/13) investigated lung carcinoma samples. The other normal
tissues (NT) showed no expression with the exception of the
thyroid.
[0180] FIG. 13. THC1005163 expression in stomach, ovary, lung and
lung carcinoma
[0181] RT-PCR investigations with a THC1005163-specific primer (SEQ
ID NO: 79) and a nonspecific oligo dT tag primer showed expression
in stomach, ovary, lung and in (5/9) lung carcinoma biopsies. The
other normal tissues (NT) showed no expression.
[0182] FIG. 14. LOC134288 expression in kidney and renal cell
carcinoma
[0183] RT-PCR investigations with LOC134288-specific primers (SEQ
ID NO: 80, 81) showed selective expression in the kidney and in
(5/8) investigated renal cell carcinoma biopsies.
[0184] FIG. 15. THC943866 expression in kidney and renal cell
carcinoma
[0185] RT-PCR investigations with THC943866-specific primers (SEQ
ID NO: 82, 83) showed selective expression in the kidney and in
(4/8) investigated renal cell carcinoma biopsies.
[0186] FIG. 16. FLJ21458 expression in colon and colon
carcinoma
[0187] RT-PCR investigations with FLJ21458-specific primers (SEQ ID
NO: 86, 87) showed selective expression in the colon and in (7/10)
investigated colon carcinoma biopsies. (1-2-colon, 3.-liver,
4-PBMCs, 5-spleen, 6-prostate, 7-kidney, 8-ovary, 9-skin, 10-ileum,
11-lung, 12-testis, 13-22 colon carcinoma, 23-neg. control).
[0188] FIG. 17. Cellular localization of GPR35
[0189] Immunofluorescence for detecting the cellular localization
of GPR35 after transfection of a plasmid that expresses a GPR35-GFP
fusion protein. The arrows identify the membrane-associated
fluorescence of the fluorescent GFP.
[0190] FIG. 18. Quantitative expression of GPR35
[0191] A. Quantitative RT-PCR with GPR35-specific primers (SEQ ID
NO: 88, 89) show selective expression in the intestine, in colon
tumor samples and in metastases from intestinal tumors. The
following normal tissues were analyzed: liver, lung, lymph nodes,
stomach, spleen, adrenal, kidney, esophagus, ovary, testis, thymus,
skin, breast, pancreas, lymphocytes, activated lymphocytes,
prostate, thyroid, fallopian tube, endometrium, cerebellum,
brain.
[0192] B. Prevalence of GPR35 in colon tumors and metastases
thereof. GPR35 is expressed both in the tumor and in metastases in
more than 90% of the cases.
[0193] FIG. 19. Quantitative expression of GUCY2C
[0194] Quantitative RT-PCR with GUCY2C-specific primers (SEQ ID NO:
98, 99) show high and selective expression in normal colonic and
gastric tissue (A) and GUCY2C-specific expression in colonic and
gastric tumor samples (B). GUCY2C is detectable in 11/12 colon
carcinomas and in 7/10 stomach carcinomas.
[0195] FIG. 20. Quantitative expression of SCGB3A2
[0196] Quantitative RT-PCR with SCGB3A2-specific primers (SEQ ID
NO: 103, 104) show selective expression in lung samples and lung
tumor samples. 19/20 lung tumor samples are SCGB3A2-positive, and
SCGB3A2 is overexpressed by a factor of at least 10 in more than
50% of the samples. The following normal tissues were analyzed:
liver, lung, lymph nodes, stomach, spleen, adrenal, kidney,
esophagus, ovary, testis, thymus, skin, breast, pancreas,
lymphocytes, activated lymphocytes, prostate, thyroid, fallopian
tube, endometrium, cerebellum, brain.
[0197] FIG. 21. Immunofluorescence with SCGB3A2-specific
antibodies
[0198] COS7 cells were transfected with a plasmid which codes for
an SCGB3A2-GFP fusion protein. A. Detection of the transfected
fusion protein with an SCGB3A2-specific rabbit antiserum
(immunization with SEQ ID NO: 105). B. Detection of the transfected
fusion protein by GFP fluorescence. C. Superimposition of the two
fluorescences from A and B. The yellow color is produced at the
points where the two fluorescences are superimposed and thus
demonstrates the specificity of the SCGB3A2 antiserum.
[0199] FIG. 22. Diagrammatic depiction of claudin-18 splice
variants
[0200] The two claudin-18 splice variants A1 and A2 differ in the N
terminus and show different potential glycosylation sites.
[0201] FIG. 23. Quantitative expression of claudin-18, variant
A1
[0202] Claudin-A1 is highly activated in a large number of tumor
tissues. Particularly strong expression is found in gastric tumors,
lung tumors, pancreatic carcinomas and esophageal carcinomas.
[0203] FIG. 24. Quantitative expression of claudin-18, variant
A2
[0204] Variant A2 is, like variant A1, activated in many
tumors.
[0205] FIG. 25. Use of claudin-18A2-specific antibodies
(extracellular domain)
[0206] (Top) Staining of claudin-18A2-positive gastric carcinoma
cells (SNU-16) with an antibody which was produced by immunization
with a peptide (SEQ ID NO: 17). Membrane staining appears
particularly strongly in the cell/cell interaction regions.
A-preimmune, MeOH; B-immune serum MeOH, 5 .mu.g/ml;
[0207] (Below) Demonstration of the specificity of the antibody by
colocalization analysis in claudin-18A2 GFP-transfected 293T cells.
A-Claudin-18A2 GFP; B-anti-claudin-A2; C-superimposition.
[0208] FIG. 26. Use of claudin-18A2-specific antibodies
(extracellular domain)
[0209] Membrane staining of claudin-18A2-positive gastric carcinoma
cells (SNU-16) with an antibody which was produced by immunization
with a peptide (SEQ ID NO: 113, N-terminally located extracellular
domain). A monoclonal antibody which is directed against E-cadherin
was used for counterstaining. A-antibody; B-counterstaining;
C-superimposition.
[0210] FIG. 27. Use of antibodies against the C-terminal
extracellular domain of claudin-18
[0211] (Left, top and below) Membrane staining of
claudin-18A2-positive gastric carcinoma cells (SNU-16) with an
antibody which was produced by immunization with a peptide (SEQ ID
NO: 116, C-terminally located extra-cellular domain). A monoclonal
antibody which is directed against E-cadherin was used for
counter-staining (right top, below).
[0212] FIG. 28. Use of claudin-18A1-specific antibodies
[0213] (Top) Weak to absent staining of gastric carcinoma cells
(SNU-16; claudin18A2 positive) with an antibody which was produced
by immunization with a claudin-18A1-specific peptide (SEQ ID NO:
115). A-anti-E-cadherin; B-anti-claudin-18A1;
C-superimposition.
[0214] (Below) Demonstration of the specificity of the antibody by
colocalization analysis in claudin-18A1-GFP-transfected 293T cells.
A-GFP-claudin-18A1; B-anti-claudin-18A1; C-superimposition.
[0215] FIG. 29. Detection of claudin-18A2 in a Western blot.
[0216] Western blotting with lysates from various healthy tissues
with a claudin-18A2-specific antibody directed against the epitope
with SEQ ID NO: 17. 1-Stomach; 2-testis; 3-skin; 4-breast; 5-liver;
6-colon; 7-lung; 8-kidney; 9-lymph nodes.
[0217] FIG. 30. Claudin-18A2 Western blotting with samples from
stomach and stomach tumors
[0218] Lysates from stomach and stomach tumors were blotted and
tested using a claudin-18A2-specific antibody against the epitope
having SEQ ID NO: 17. Stomach tumors show a less glycosylated form
of claudin-18A2. PNGase F treatment of stomach lysates leads to the
formation of the low-glycosylated form.
[0219] Left: 1-stomach No #A; 2-stomach Tu #A; 3-stomach No #B;
4-stomach Tu #B
[0220] Right: 1-stomach No #A; 2-stomach No #B; 3-stomach No
#B+PNGase F; 4-stomach Tu #C; 5-stomach Tu #D; 6-stomach Tu
#D+PNGase F
[0221] FIG. 31. Expression of claudin-18 in lung tumors
[0222] Low-glycosylated claudin-18A2 variants were detected in lung
tumors in accordance with FIG. 30. 1-Stomach No; 2-stomach Tu;
3-9-lung Tu.
[0223] FIG. 32. Immunohistochemlcal analysis of claudin-18 using
claudin-18A2-specific antibodies in stomach tumor tissue
[0224] FIG. 33. Indirect immunofluorescence of stomach-specific
Snu16 cells with a claudin-18-specific polyclonal antiserum
[0225] A. Staining with a preimmune serum generated before the
immunization; B. Staining with the claudin-18-specific serum.
[0226] FIG. 34. Quantitative expression of SLC13A1
[0227] Quantitative RT-PCR with SLC13A1-specific primers (SEQ ID
NO: 121, 122) show high and selective expression in normal kidney
tissue (A) and SLC13A1-specific expression in renal cell carcinomas
(B). SLC13A1 transcription is detectable in 5/8 renal cell
carcinomas.
[0228] FIG. 35. Cellular localization of SLC13A1
[0229] Immunofluorescence to demonstrate the cellular localization
of SLC13A1 after transfection of a plasmid which provides an
SLC13A1-GFP fusion protein. The membrane-associated fluorescence of
the SLC13A1 fusion protein is to be seen clearly (as ring around
the transfected cell).
[0230] FIG. 36. Quantitative expression of CLCA1
[0231] Quantitative RT-PCR with CLCA1-specific primers (SEQ ID NO:
125, 126) show high and selective expression in normal colonic
tissue and stomach tissue (A) and CLCA1-specific expression in
colonic and gastric tumor samples (8). CLCA1 is detectable in 6/12
colon carcinomas and in 7/10 stomach carcinomas.
[0232] FIG. 37. Quantitative expression of FLJ21477
[0233] Quantitative RT-PCR with FLJ21477-specific primers (SEQ ID
NO: 127, 128) show high and selective expression in normal colonic
and gastric tissue and weak expression in thymus, esophagus and
brain (A) and the FLJ21477-specific expression in colonic tumor
samples (B). FLJ21477 is detectable in 11/12 colon carcinomas.
[0234] FIG. 38. Quantitative expression of FLJ20694
[0235] Quantitative RT-PCR with FLJ20694-specific primers (SEQ ID
NO: 129, 130) show high and selective expression in normal colonic
and gastric tissue (A) and FLJ20694-specific overexpression in
colonic and gastric tumor samples (B). FLJ20694 is detectable in
11/12 colon carcinomas and in 7/10 stomach carcinomas.
[0236] FIG. 39. Quantitative expression of FLJ21458
[0237] Quantitative RT-PCR with FLJ21458-specific primers (SEQ ID
NO: 133, 134) show selective expression in testis, gastric and
intestinal tissue. In addition, FLJ21458-specific transcripts were
detectable in 20/20 colonic tumors and in 7/11 colonic metastases.
The following normal tissues were analyzed: liver, lung, lymph
nodes, spleen, adrenal, kidney, esophagus, ovary, testis, thymus,
skin, breast, pancreas, lymphocytes, activated lymphocytes,
prostate, thyroid, fallopian tube, endometrium, cerebellum,
brain.
[0238] FIG. 40. immunofluorescence with FLJ21458-specific
antibodies
[0239] (Top) 293 cells were transfected with a plasmid which codes
for an FLJ21458-GFP fusion protein. A: detection of the transfected
fusion protein with an FLJ21458-specific rabbit antiserum
(immunization with SEQ ID NO: 136). B: detection of the transfected
fusion protein by GFP fluorescence. C: superimposition of the two
fluorescences from A and B. The yellow color is produced at the
points where the two fluorescences are superimposed and thus
demonstrates the specificity of the FLJ21458 antiserum.
[0240] (Below) Analysis of Snu16 cells which endogenously
synthesize FLJ21458. A: protein detection using an
FLJ21458-specific rabbit antiserum (immunization with SEQ ID NO:
136). B: detection of the membrane protein E-cadherin. C:
superimposition of the two fluorescences from A and B. The yellow
color is produced at the points where the two fluorescences are
superimposed, and demonstrates the membrane localization of
FLJ21458.
[0241] FIG. 41. Sequences
[0242] The sequences to which reference is made herein are
shown.
EXAMPLES
Material and Methods
[0243] The terms "in silico", "electronic" and "virtual cloning"
refer solely to the utilization of methods based on databases,
which may also be used to simulate laboratory experimental
processes.
[0244] Unless expressly defined otherwise, all other terms and
expressions are used so as to be understood by the skilled worker.
The techniques and methods mentioned 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.
[0245] Datamining-Based Strategy for Determining New
Tumor-Associated Genes
[0246] Two in silico strategies, namely GenBank keyword search and
the cDNAxProfiler, were combined. Utilizing the NCBI ENTREZ Search
and Retrieval System (www.ncbi.nlm.nih.gov/Entrez), a GenBank
search was carried out for candidate genes annotated as being
specifically expressed in specific tissues (Wheeler et al., Nucleic
Acids Research 28:10-14, 2000).
[0247] Carrying out queries with keywords such as "colon-specific
gene", "stomach-specific gene" or "kidney-specific gene", candidate
genes (GOI, genes of interest) were extracted from the databases.
The search was restricted to part of the total information of these
databases by using the limits "homo sapiens", for the organism, and
"mRNA", for the type of molecule.
[0248] The list of the GOI found was curated by determining
different names for the same sequence and eliminating such
redundancies.
[0249] All candidate genes obtained by the keyword search were in
turn studied with respect to their tissue distribution by the
"electronic Northern" (eNorthen) method. The eNorthern is based on
aligning the sequence of a GOI with an EST (expressed sequence tag)
database (Adams et al., Science 252:1651, 1991)
(www.ncbi.nlm.nih.gov/BLAST). The tissue origin of each EST which
is found to be homologous to the inserted GOI can be determined and
in this way the sum of all ESTs produces a preliminary assessment
of the tissue distribution of the GOI. Further studies were carried
out only with those GOI which had no homologies to EST from non
organ-specific normal tissues. This evaluation also took into
account that the public domain contains wrongly annotated cDNA
libraries (Scheurle et al., Cancer Res. 60:4037-4043, 2000)
(www.fau.edu/cmbb/publications/cancergenes6.htm).
[0250] The second datamining method utilized was the cDNA xProfiler
of the NCBI Cancer Genome Anatomy Project
(http://cgap.nci.nih.gov/Tissues/xProfiler) (Hillier et al., Genome
Research 6:807-828, 1996; Pennisi, Science 276:1023-1024, 1997).
This allows pools of transcriptomes deposited in databases to be
related to one another by logical operators. We have defined a pool
A to which all expression libraries prepared for example from colon
were assigned, excluding mixed libraries. All cDNA libraries
prepared from normal tissues other than colon were assigned to pool
B. Generally, all cDNA libraries were utilized independently of
underlying preparation methods, but only those with a size >1000
were admitted. Pool B was digitally subtracted from pool A by means
of the BUT NOT operator. The set of GOI found in this manner was
also subjected to eNorthern studies and validated by a literature
research.
[0251] This combined datamining includes all of the about 13 000
full-length genes in the public domain and predicts out of these
genes having potential organ-specific expression.
[0252] All other genes were first evaluated in normal tissues by
means of specific RT-PCR. All GOI which had proved to be expressed
in non-organ specific normal tissues had to be regarded as
false-positives and were excluded from further studies. The
remaining ones were studied in a large panel of a wide variety of
tumor tissues. The antigens depicted below proved here to be
activated in tumor cells.
[0253] RNA Extraction, Preparation of Poly-d(T) Primed cDNA and
Conventional RT-PCR Analysis
[0254] 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.
[0255] First strand cDNA synthesis from 2-4 .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
(sense CGTGAGCGCTTCGAGATGTTCCG (SEQ ID NO. 138), antisense
CCTAACCAGCTGCCCAACTGTAG (SEQ ID NO. 139), hybridization temperature
67.degree. C.
[0256] 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 0.3 mM 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 (1 min at 94.degree. C., 1 min at the
particular hybridization temperature, 2 min at 72.degree. C. and
final elongation at 72.degree. C. for 6 min). 20 .mu.l of this
reaction were fractionated and analyzed on an ethidium
bromide-stained agarose gel.
[0257] The following primers were used for expression analysis of
the corresponding antigens at the hybridization temperature
indicated.
TABLE-US-00001 GPR35 (65.degree. C.) (SEQ ID NO. 20) Sense:
5'-AGGTACATGAGCATCAGCCTG-3' (SEQ ID NO. 21) Antisense:
5'-GCAGCAGTTGGCATCTGAGAG-3' GUCY2C (62.degree. C.) (SEQ ID NO. 22)
Sense: 5'-GCAATAGACATTGCCAAGATG-3' (SEQ ID NO. 23) Antisense:
5'-AACGCTGTTGATTCTCCACAG-3' SCGB3A2 (66.degree. C.) (SEQ ID NO. 37)
Sense: 5'-CAGCCTTTGTAGTTACTCTGC-3' (SEQ ID NO. 38) Antisense:
5'-TGTCACACCAAGTGTGATAGC-3' Claudin18A2 (68.degree. C.) (SEQ ID NO.
39) Sense1: 5'-GGTTCGTGGTTTCACTGATTGGGATTGC-3' (SEQ ID NO. 40)
Antisense1: 5'-CGGCTTTGTAGTTGGTTTCTTCTGGTG-3' (SEQ ID NO. 107)
Sense2: 5'-TGTTTTCAACTACCAGGGGC-3' (SEQ ID NO. 108) Antisense2:
5'-TGTTGGCTTTGGCAGAGTCC-3' Claudin18A1 (64.degree. C.) (SEQ ID NO.
109) Sense: 5'-GAGGCAGAGTTCAGGCTTCACCGA-3' (SEQ ID NO. 110)
Antisense: 5'-TGTTGGCTTTGGCAGAGTCC-3' SLC13A1-(64.degree. C.) (SEQ
ID NO. 50) Sense: 5'-CAGATGGTTGTGAGGAGTCTG-3' (SEQ ID NO. 49)
Antisense: 5'-CCAGCTTTAACCATGTCAATG-3' CLCA1 (62.degree. C.) (SEQ
ID NO. 67) Sense: 5'-ACACGAATGGTAGATACAGTG-3' (SEQ ID NO. 68)
Antisense: 5'-ATACTTGTGAGCTGTTCCATG-3' FLJ21477 (68.degree. C.)
(SEQ ID NO. 69) Sense: 5'-ACTGTTACCTTGCATGGACTG-3' (SEQ ID NO. 70)
Antisense: 5'-CAATGAGAACACATGGACATG-3' FLJ20694 (64.degree. C.)
(SEQ ID NO. 140) Sense: 5'-CCATGAAAGCTCCATGTCTA-3' (SEQ ID NO. 72)
Antisense: 5'-AGAGATGGCACATATTCTGTC-3' Ebner (70.degree. C.) (SEQ
ID NO. 73) Sense: 5'-ATCGGCTGAAGTCAAGCATCG-3' (SEQ ID NO. 74)
Antisense: 5'-TGGTCAGTGAGGACTCAGCTG-3' Plunc (55.degree. C.) (SEQ
ID NO. 75) Sense: 5'-TTTCTCTGCTTGATGCACTTG-3' (SEQ ID NO. 76)
Antisense: 5'-GTGAGCACTGGGAAGCAGCTC-3' SLC26A9 (67.degree. C.) (SEQ
ID NO. 141) Sense: 5'-GGCAAATGCTAGAGACGTGA-3' (SEQ ID NO. 78)
Antisense: 5'-AGGTGTCCTTCAGCTGCCAAG-3' THC1005163 (60.degree. C.)
(SEQ ID NO. 79) Sense: 5'-GTTAAGTGCTCTCTGGATTTG-3' LOC134288
(64.degree. C.) (SEQ ID NO. 80) Sense: 5'-ATCCTGATTGCTGTGTGCAAG-3'
(SEQ ID NO. 81) Antisense: 5'-CTCTTCTAGCTGGTCAACATC-3' THC943866
(59.degree. C.) (SEQ ID NO. 82) Sense: 5'-CCAGCAACAACTTACGTGGTC-3'
(SEQ ID NO. 83) Antisense: 5'-CCTTTATTCACCCAATCACTC-3' FLJ21458
(62.degree. C.) (SEQ ID NO. 86) Sense: 5'-ATTCATGGTTCCAGCAGGGAC-3'
(SEQ ID NO. 87) Antisense: 5'-GGGAGACAAAGTCACGTACTC-3'.
[0258] Preparation of Random Hexamer-Primed cDNA and Quantitative
Real-Time PCR
[0259] 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 AA-C.sub.r 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 using the high capacity cDNA
Archive Kit (PE Biosystems, USA) with use of hexamer primers in
accordance with the manufacturer's instructions. Each 5 .mu.l
portions of the diluted cDNA were employed in a total volume of 25
.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.
[0260] Cloning and Sequence Analysis
[0261] 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.
[0262] Western Blotting
[0263] Cells from cell culture (endogenous expression of the target
gene or synthesis of the target protein after transfection of an
expression vector which encodes the target protein) or tissue
samples which might contain the target protein are lysed in a 1%
SDS solution. The SDS denatures the proteins present in the lysate.
The lysates of an experimental mixture are fractionated according
to size by electrophoresis on 8-15% denaturing polyacrylamide gels
(containing 1% SDS) depending on the expected protein size (SDS
polyacrylamide gel electrophoresis, SDS-PAGE). The proteins are
then transferred by the semi-dry electroblotting method (Biorad) to
nitrocellulose membrane (Schleicher & Schull) on which the
desired protein can be detected. For this purpose, the membrane is
initially blocked (e.g. with milk powder) and then incubated with
the specific antibody in a dilution of 1:20-1:200 (depending on the
specificity of the antibody) for 60 minutes. After a washing step,
the membrane is incubated with a second antibody coupled to a
marker (e.g. enzymes such as peroxidase or alkaline phosphatase)
which recognizes the first antibody. After a further washing step,
subsequently the target protein is visualized in a color or
chemiluminescence reaction, on the membrane by means of an enzyme
reaction (e.g. ECL, Amersham Bioscience). The result is documented
by photographing with a suitable camera.
[0264] Analysis of protein modifications usually takes place by
Western blotting. Glycosilations, which usually have a size of
several kDa, lead to a larger total mass of the target protein,
which can be fractionated in the SDS-PAGE. To detect specific 0-
and N-glycosidic linkages, protein lysates from tissues or cells
are incubated before denaturation by SDS with 0- or N-glycosidases
(in accordance with their respective manufacturer's instructions,
e.g. PNgase, endoglycosidase F, endoglycosidase H, Roche
Diagnostics). This is followed by Western blotting as described
above. Thus, if there is a reduction in the size of a target
protein after incubation with a glycosidase it is possible to
detect a specific glycosilation and, in this way, also analyse the
tumor specificity of a modification. The exact position of the
glycosilated amino acid can be predicted with algorithms and
prediction programs.
[0265] Immunofluorescence
[0266] Cells of established cell lines which either synthesize the
target protein endogenously (detection of the RNA in RT-PCR or of
the protein by Western blotting) or else have been transfected with
plasmid DNA before the IF are used. A wide variety of methods (e.g.
electroporation, liposome-based transfection, calcium phosphate
precipitation) are well established for transfecting cell lines
with DNA (e.g. Lemoine et al. Methods Mol. Biol. 1997; 75: 441-7).
The transfected plasmid may in the immunofluorescence encode the
unmodified protein or else couple various amino acid markers to the
target protein. The most important markers are, for example, the
fluorescing "green fluorescent protein" (GFP) in its various
differentially fluorescing forms and short peptide sequences of
6-12 amino acids for which high-affinity and specific antibodies
are available. Cells which synthesize the target protein are fixed
with paraformaldehyde, saponin or methanol. The cells can then if
required be permeabilized by incubation with detergents (e.g. 0.2%
Triton X-100). After the fixation/permeabilization, the cells are
incubated with a primary antibody which is directed against the
target protein or against one of the coupled markers. After a
washing step, the mixture is incubated with a second antibody
coupled to a fluorescent marker (e.g. fluorescin, Texas Red, Dako)
which binds to the first antibody. The cells labeled in this way
are then covered with a layer of glycerol and analysed with the aid
of a fluorescence microscope according to the manufacturer's
instructions. Specific fluorescence emissions are achieved in this
case by specific excitation depending on the substances employed.
The analysis normally allows reliable localization of the target
protein, the antibody quality and the target protein being
confirmed in double stainings to stain in addition to the target
protein also the coupled amino acid markers or other marker
proteins whose localization has been described in the literature.
GFP and its derivatives represents a special case that can be
directly excited and itself fluoresces, so that no antibodies are
necessary for the detection.
[0267] Immunohistochemistry
[0268] IHC serves specifically for (1) being able to estimate the
amount of target protein in tumor and normal tissues, (2) analysing
how many cells in the tumor and healthy tissue synthesize the
target gene, and/or (3) defining the cell type in a tissue (tumor,
healthy cells) in which the target protein is detectable. Different
protocols must be used depending on the individual antibody (e.g.
"Diagnostic Immunohistochemistry by David J., MD Dabbs ISBN:
0443065667" or in "Microscopy, Immunohistochemistry, and Antigen
Retrieval Methods: For Light and Electron Microscopy ISBN:
0306467704").
[0269] Immunohistochemistry (1HC) on specific tissue samples serves
to detect protein in the corresponding tissue. The aim of this
method is to identify the localization of a protein in a
functionally intact tissue aggregate. IHC serves specifically for
(1) being able to estimate the amount of target protein in tumor
and normal tissues, (2) analysing how many cells in tumor and
healthy tissue synthesize the target gene, and (3) defining the
cell type in a tissue (tumor, healthy cells) in which the target
protein is detectable. Alternatively, the amounts of protein of a
target gene can be quantified by tissue immunofluorescence using a
digital camera and suitable software (e.g. Tillvision,
Till-photonics, Germany). The technology has frequently been
published, and details of staining and microscopy can therefore be
found for example in "Diagnostic Immunohistochemistry" by David J.,
MD Dabbs ISBN: 0443065667 or "Microscopy, Immunohistochemistry, and
Antigen Retrieval Methods: For Light and Electron Microscopy" ISBN:
0306467704. It should be noted that, because of the properties of
antibodies, different protocols have to be used (an example is
described below) in order to obtain a valid result.
[0270] Ordinarily, histologically defined tumor tissues and, as
reference, comparable healthy tissues are employed in the IHC. It
is moreover possible to use as positive and negative controls cell
lines in which the presence of the target gene is known thrbugh
RT-PCR analyses. A background control must always be included.
[0271] Fixed tissue (e.g. fixation with aldehyde-containing
substances, formaldehyde, paraformaldehyde or in alcoholic
solutions) or shock-frozen tissue pieces with a thickness of 1-10
.mu.m are applied to a glass-support. Paraffin-embedded samples are
deparaffinated for example with xylene. The samples are washed with
TBS-T and blocked in serum. This is followed by incubation with the
first antibody (dilution: 1:2 to 1:2000) for 1-18 hours, with
affinity-purified antibodies normally being used. A washing step is
followed by incubation with a second antibody which is coupled to
an alkaline phosphatase (alternative: for example peroxidase), and.
is directed against the first antibody, for about 30-60 minutes.
This is followed by color reaction using color substrates which are
converted by the bound enzymes (cf. for example, Shi et al., J.
Histochem. CytOchem. 39: 741-748, 1991; Shin et al., Lab. Invest.
64: 693-702, 1991). To demonstrate the antibody specificity, the
reaction can be blocked by previous addition of the immunogen.
[0272] Immunization
[0273] (See also 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). The process for
preparing antibodies is described briefly below, and details can be
found in the cited publications. Firstly, animals (e.g. rabbits)
are immunized by a first injection of the desired target protein.
The animal's immune response to the immunogen can be enhanced by a
second or third immunization within a defined period (about 2-4
weeks after the preceding immunization). Again after various
defined periods (first bleeding after 4 weeks, then about every 2
weeks with a total of up to 5 samplings), blood is taken from the
animals, and an immune serum is obtained therefrom.
[0274] The animals are usually immunized by one of four
well-established methods, with other methods also being available.
It is moreover possible to immunize with peptides which are
specific for the target protein, with the complete protein or with
extracellular partial sequences of a protein which can be
identified experimentally or via prediction programs. [0275] (1) In
the first case, peptides (length: 8-12 amino acids) conjugated to
KLH (keyhole limpet hemocyanin) are synthesized by a standardized
in vitro method, and these peptides are used for the immunization.
Usually, 3 immunizations are carried out with a concentration of
5-1000 .mu.g/immunization. The immunization can also be carried out
as service from service providers. [0276] (2) Alternatively, the
immunization can be carried out with recombinant proteins. For this
purpose, the cloned DNA of the target gene is cloned into an
expression vector, and the target protein is synthesized in analogy
to the conditions of the particular manufacturer (e.g. Roche
Diagnostics, Invitrogen, Clontech, Qiagen) for example cell-free in
vitro, in bacteria (e.g. E. coli), in yeast (e.g. S. pombe), in
insect cells or in mammalian cells. After synthesis in one of the
systems, the target protein is purified, the purification in this
case usually taking place by standardized chromatographic methods.
It is also possible in this connection to use for the immunization
proteins which have a molecular anchor as aid for purification
(e.g. His tag, Qiagen; FLAG tag, Roche Diagnostics; Gst fusion
proteins). A large number of protocols is to be found for example
in the "Current Protocols in Molecular Biology", John Wiley &
Sons Ltd., Wiley Interscience. [0277] (3) If a cell line which
synthesizes the desired protein endogenously is available, this
cell line can also be used to produce the specific antiserum. In
this case, the immunization takes place in 1-3 injections in each
case with about 1-5.times.10.sup.7 cells. [0278] (4) The
immunization can also take place by injection of DNA (DNA
immunization). For this purpose, the target gene is initially
cloned into an expression vector so that the target sequence is
under the control of a strong eukaryotic promoter (e.g. CMV
promoter). Subsequently, 5-100 .mu.g of DNA are transferred as
immunogen using a "gene gun" into capillary regions with a strong
blood flow in an organism (e.g. mouse, rabbit). The transferred DNA
is taken up by the animal's cells, the target gene is expressed,
and the animal finally develops an immune response to the target
gene (Jung et al., Mol Cells 12:41-49, 2001; Kasinrerk et al.,
Hybrid Hybridomics 21:287-293, 2002).
[0279] Quality Control of the Polyclonal Serum or Antibody
[0280] Assays based on cell culture with subsequent Western
blotting are most suitable for demonstrating specificity (various
variations are described for example in "Current Protocols in
Protein Chemistry", John Wiley & Sons Ltd., Wiley
InterScience). For the demonstration, cells are transfected with a
cDNA, which is under the control of a strong eukaryotic promoter
(e.g. cytomegalovirus promoter), for the target protein. A wide
variety of methods (e.g. electroporation, liposome-based
transfection, calcium phosphate precipitation) are well established
for transfecting cell lines with DNA (e.g. Lemoine et al., Methods
Mol. Biol. 75:441-7, 1997). It is also possible alternatively to
use cell lines which express the target gene endogenously
(demonstration by target gene-specific RT-PCR). As control, in the
ideal case homologous genes are also transfected in the experiment,
in order to be able to demonstrate in the following Western blot
the specificity of the analysed antibody.
[0281] In the subsequent Western blot, cells from cell culture or
tissue samples which might contain the target protein are lysed in
a 1% SDS solution, and the proteins are denatured thereby. The
lysates are fractionated according to size by electrophoresis on
8-15% denaturing polyacrylamide gels (contain 1% SDS) (SDS
polyacrylamide gel electrophoresis, SDS-PAGE). The proteins are
then transferred by one of a plurality of blotting methods (e.g.
semi-dry electroblot; Biorad) to a specific membrane (e.g.
nitrocellulose, Schleicher & Schull). The desired protein can
be visualized on this membrane. For this purpose, the membrane is
first incubated with the antibody which recognizes the target
protein (dilution about 1:20-1:200, depending on the specificity of
the antibody) for 60 minutes. After a washing step, the membrane is
incubated with a second antibody which is coupled to a marker (e.g.
enzymes such as peroxidase or alkaline phosphatase) and which
recognizes the first antibody. It is then possible in a color or
chemiluminescent reaction to visualize the target protein on the
membrane (e.g. ECL, Amersham Bioscience). An antibody with a high
specificity for the target protein should in the ideal case
recognize only the desired protein itself.
[0282] Various methods are used to confirm the membrane
localization of the target protein identified in the in silica
approach. An important and well-established method using the
antibodies described above is immuno-fluorescence (IF). Cells of
established cell lines which either synthesize the target protein
(detection of the RNA in an RT-PCR or of the protein in a Western
blot) or else have been transfected with plasmid DNA are used for
this. A wide variety of methods (e.g. electroporation,
liposome-based transfection, calcium phosphate precipitation) are
well established for transfection of cell lines with DNA (e.g.
Lemoine et al., Methods Mol. Biol. 75:441-7, 1997). The plasmid
transfected into the cells can in the immunofluorescence encode the
unmodified protein or else couple various amino acid markers to the
target protein. The principal markers are, for example, the
fluorescent "green fluorescent protein" (GFP) in its various
differentially fluorescent forms, short peptide sequences of 6-12
amino acids for which high-affinity and specific antibodies are
available, or the short amino acid sequence Cys-Cys-X-X-Cys-Cys
which can bind via its cysteine specific fluorescent substances
(Invitrogen). Cells which synthesize the target protein are fixed
for example with paraformaldehyde or methanol. The cells can then,
if required, be permeabilized by incubation with detergents (e.g.
0.2% Triton X-100). The cells are then incubated with a primary
antibody which is directed against the target protein or against
one of the coupled markers. After a washing step, the mixture is
incubated with a second antibody which is coupled to a fluorescent
marker (e.g. fluorescin, Texas Red, Dako) and which binds to the,
first antibody. The cells labeled in this way are then covered with
a layer of glycerol and analysed with the aid of a fluorescence
microscope according to the manufacturer's instructions. Specific
fluorescence emissions are achieved in this case by specific
excitation depending on the substances employed. The analysis
usually permits reliable localization of the target protein, the
antibody quality and the target protein being confirmed in double
stainings to stain in addition to the target protein also the
coupled amino acid markers or other marker proteins whose
localization has already been described in the literature. GFP and
its derivatives represents a special case, being excitable directly
and themselves fluorescing. The membrane permeability, which can be
controlled through the use of detergents, permits demonstration in
the immunofluorescence of whether an immunogenic epitope is located
inside or outside the cell. The prediction of the selected proteins
can thus be supported experimentally. An alternative possibility is
to detect extracellular domains by means of flow cytometry. For
this purpose, cells are fixed under non-permeabilizing conditions
(e.g. with PBS/Na azide/2%. FCS/5 mM EDTA) and analysed in a flow
cytometer in accordance with the manufacturer's instructions. Only
extracellular epitopes can be recognized by the antibody to be
analysed in this method. A difference from immunofluorescence is
that it is possible to distinguish between dead and living cells by
use of, for example, propidium iodide or Trypan blue, and thus
avoid false-positive results.
[0283] Affinity Purification
[0284] Purification of the polyclonal sera took place in the case
of the peptide antibodies entirely, or in the case of the
antibodies against recombinant proteins in part, as service by the
contracted companies. For this purpose; in both cases, the
appropriate peptide or recombinant protein was covalently bonded to
a matrix, and the latter was, after the coupling, equilibrated with
a native buffer (PBS: phosphate buffered saline) and then incubated
with the crude serum. After a further PBS washing step, the
antibody was eluted with 100 mM glycine, pH 2.7, and the eluate was
immediately neutralized in 2M TRIS, pH 8. The antibodies purified
in this way could then be employed for specific detection of the
target proteins both by Western blotting and by
immunofluorescence.
[0285] Preparation of EGFP Transfectants
[0286] For the immunofluorescence microscopy of heterologously
expressed tumor-associated antigens, the complete ORF of the
antigens was cloned in pEGFP-Cl and pEGFP-N3 vectors (Clontech).
CHO and NIH3T3 cells cultivated on slides were transfected with the
appropriate plasmid constructs using Fugene transfection reagent
(Roche) in accordance with the manufacturer's instructions and,
after 12-24 h, analysed by immunofluorescence microscopy.
Example 1
Identification of GPR35 as Diagnostic and Therapeutic Cancer
Target
[0287] GPR35 (SEQ ID NO: 1) and its translation product (SEQ ID NO:
9) have been described as putative G protein-coupled receptor. The
sequence is published in Genbank under accession No. AF089087. This
transcript codes for a protein of 309 amino acids with a molecular
weight of 34 kDa. It was predicted that GPR35 belongs to the
superfamily of G protein-coupled receptors with 7 transmembrane
domains (O'Dowd et al., Genomics 47:310-13, 1998). In order to
confirm the predicted localization of GPR35 in the cell, the
protein was fused to eGFP as reporter molecule and, after
transfection of the appropriate plasmid, expressed heterologously
in 293 cells. The localization was then analysed in a fluorescence
microscope. It was confirmed according to the present technology
that GPR35 is an integral transmembrane molecule (FIG. 17).
Investigation to date on human GPR35 (see, inter alia, Horikawa Y,
Oda N, Cox N J, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner T
H, Mashima H, Schwarz P E, del Bosque-Plata L, Horikawa Y, Oda Y,
Yoshiuchi I, Colilla S, Polonsky K S, Wei S, Concannon P, Iwasaki
N, Schulze J, Baler L J, Bogardus C, Groop L, Boerwinkle E, Hanis C
L, Bell G I Nat Genet. 2000 October; 26(2):163-75) suggested that
GPR35 is activated in many healthy tissues. The reading frame of
the gene comprises a single exon. According to the present
technology, a gene-specific primer pair (SEQ ID NO: 20, 21). for
GPR35 was used in RT-PCR analyses to amplify cDNA in the colon and
in colon carcinoma (13/26). By contrast, no significant expression
is detectable in other normal tissues. Because of the particular
fact that GPR35 consists of a single exon, genomic DNA impurities
cannot be detected with intron-spanning primers. In order to
preclude genomic contamination of the RNA samples, therefore, all
RNAs were treated with DNAse. GPR35 transcripts were detected
according to the present technology only in the colon, in the
rectum, in the testis and in colon carcinomas using DNA-free
RNA_
TABLE-US-00002 TABLE 1 GPR35 expression in normal tissues Normal
tissue Expression Brain - Cerebellum - Myocardium - Skeletal muscle
- Rectum ++ Stomach - Colon ++ Pancreas - Kidney - Testis - Thymus
- Mammary glands - Ovary - Uterus n.d. Skin - Lung - Thyroid -
Lymph nodes - Spleen - PBMC - Adrenal - Esophagus - Small intestine
+ Prostate - (nd = not determined)
[0288] The selective and high expression of GPR35 transcripts in
normal colonic tissue and in colon carcinoma biopsies (FIG. 1) was
not previously known and can be utilized according to the present
technology for molecular diagnostic methods such as RT-PCR for
detecting disseminating tumor cells in the serum and bone marrow
and for detecting disseminating tumor cells in the serum and bone
marrow and for detecting metastases in other tissues. Quantitative
RT-PCR with specific primers (SEQ ID NO: 88 and 89) also confirms
that GPR35 is a highly selective intestine-specific differentiation
antigen which is also contained in intestinal tumors and in
intestinal tumor metastases. In some intestinal tumors, it is in
fact overexpressed by one log compared with normal intestine (FIG.
18). Antibodies were produced by immunizing rabbits for detecting
GPR35 protein. The following peptides were used to propagate these
antibodies:
TABLE-US-00003 SEQ ID NO: 90 GSSDLTWPPAIKLGC (AA 9-23) SEQ ID NO:
91: DRYVAVRHPLRARGLR (AA 112-127) SEQ ID NO: 92: VAPRAKAHKSQDSLC (C
terminus) SEQ ID NO: 93 CFRSTRHNFNSMR (extracell. domain 2)
Stainings with these antibodies for example in a Western blot
confirm the expression in tumors. All 4 extracellular domains of
GPR35 (position of the predicted extracellular domains in the
sequence of SEQ ID NO: 9 AA 1-22 (SEQ ID NO: 94); AA 81-94 (SEQ ID
NO: 95); AA 156-176 (SEQ. ID NO: 96); AA 280-309 (SEQ ID NO: 97))
can be used according to the present technology as target
structures of monoclonal antibodies. These antibodies bind
specifically to the cell surface of tumor cells and can be used
both for diagnostic and for therapeutic methods. Overexpression of
GPR35 in tumors provides additional support for such a use. In
addition, the sequences coding for proteins can be used according
to the present technology as vaccine (RNA, DNA, peptide, protein)
for inducing tumor-specific immune responses (T-cell and
B-cell-mediated immune responses). In addition, it has surprisingly
been found that a further start codon exists 5' in front of the
generally known start codon and expresses an N-terminally extended
protein.
[0289] It has thus been found according to the present technology
that GPR35, a protein which was previously described as expressed
ubiquitously, is tumor-associated overexpressed, selectively in
gastrointestinal tumors, especially in tumors of the colon. GPR35
is therefore suitable in particular as molecular target structure
for the diagnosis and treatment of these tumors. Investigation to
date of human GPR35, cf., for example, Horikawa Y, Oda N, Cox N J,
Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner T H, Mashima H,
Schwarz P E, del Bosque-Plata L, Horikawa Y, Oda Y, Yoshiuchi I,
Colilla S, Polonsky K S, Wei S, Concannon P, Iwasaki N, Schulze J,
Baier L J, Bogardus C, Groop L, Boerwinkle E, Hanis C L, Bell G I
Nat Genet. 2000 October; 26(2):163-75 suggested that GPR35 is
activated in many healthy tissues. By contrast, the investigations
according to the present technology showed that GPR35 is
surprisingly not significantly detectable in most normal tissues
and, in contrast thereto, is highly activated in primary and
metastatic colon tumors. In addition, besides the described GPR35
sequence, according to the present technology a novel translation
variant which makes use of an alternative start codon has been
found (SEQ ID NO: 10):
[0290] GPR35 is a member of the group of G-coupled receptors
(GPCR), a very large protein family whose structure and function
has been very well investigated. GPCR are outstandingly suitable as
target structures for the development of pharmaceutically active
substances, because the methods necessary therefor (e.g. receptor
expression, purification, ligand screening, mutagenizing,
functional inhibition, selection of agonistic and antagonistic
ligands, radiolabeling of ligands) is very well developed and
described in detail, cf., for example, "G Protein-Coupled
Receptors" by Tatsuya Haga, Gabriel Berstein and Gabriel Bernstein
ISBN: 0849333849 and in "Identification and Expression of G-Protein
Coupled Receptors Receptor Biochemistry and Methodology" by Kevin
R. Lynch ASIN: 0471183105. Realization according to the present
technology that GPR35 is undetectable in most healthy tissues but
undergoes tumor-associated expression on the cell surface, enables
it to be used as tumor-associated target structure for example for
pharmaceutically active ligands, especially in conjugation for
example with radioactive molecules as pharmaceutical substances. It
is possible in a particular embodiment to use radiolabeled ligands
which bind to GPR35 for detecting tumor cells or for treating colon
tumors in vivo.
Example 2
Identification of GUCY2C in Hepatic and Ovarian Tumors and Novel
GUCY2C Splice Variants as Diagnostic and Therapeutic Cancer
Targets
[0291] Guanylate cyclase 2C (SEQ ID NO: 2; translation product: SEQ
ID NO: 11) --a type I transmembrane protein--belongs to the family
of natriuretic peptide receptors. The sequence is published in
Genbank under the accession number NM.sub.--004963. Binding of the
peptides guanylin and uroguanylin or else heat-stable enterotoxins
(STa) increases the intracellular cGMP concentration, thus inducing
signal transduction processes inside the cell. Recent
investigations indicate that expression of GUCY2C also extends to
extraintestinal regions such as, for example, primary and
metastatic adenocarcinomas of the stomach and of the esophagus
(Park et al., Cancer Epidemiol Biomarkers Prev. 11: 739-44, 2002).
A splice variant of GUCYC which is found both in normal and
transformed tissue of the intestine comprises a 142 bp deletion in
exon 1, thus preventing translation of a GUCY2C-like product
(Pearlman et al., Dig. Dis. Sci. 45:298-05, 2000). The only splice
variant described to date leads to no translation product.
[0292] The aim according to the present technology was to identify
tumor-associated splice variants for GUCY2C which can be utilized
both for diagnosis and for therapy. RT-PCR investigations with a
GUCY2C-specific primer pair (SEQ ID NO: 22, 23, 98, 99) show
pronounced expression of GUCY2C transcripts in normal colon and
stomach, and weak expression in liver, testis, ovary, thymus,
spleen, brain and lung (tab. 2, FIG. 19). Expression in colon and
stomach was at least 50 times higher than in all other normal
tissues. Marked GUCY2C transcript levels were detected in colon
carcinoma and stomach carcinoma (tab. 2). These results were
specified by a quantitative PCR analysis and showed pronounced
GUCY2C expression in normal colon, ileum, and in almost all colon
carcinoma samples investigated (FIG. 2, 19B). A massive
overexpression was detectable in some colon carcinoma samples. In
addition, expression is found in 7/10 stomach tumors. We also
surprisingly found that the gene is activated in many other
previously undescribed tumors, inter alia ovarian, breast, liver
and prostate tumors (FIG. 19B, tab. 2).
TABLE-US-00004 TABLE 2 GUC2C expression in normal and tumor tissues
Normal tissues Expression Tumor type Expression Brain + Colon +++
Cerebellum carcinoma Myocardium Pancreatic - Skeletal - carcinoma
muscle Esophageal - Myocardium carcinoma Stomach +++ Stomach +++
Colon +++ carcinoma Pancreas - Bronchial - Kidney - carcinoma Liver
+ Mammary -+ Testis ++ carcinoma Thymus + Ovarian + Breast -
carcinoma Ovary + Endometrial Uterus + carci Skin ENT tumors Lung +
Renal cell Thyroid carcinoma Lymph nodes - Prostate + Spleen +
carcinoma PBMC - Liver + Prostate - carcinoma
[0293] The following primer pairs were used to detect splice
variants in colonic tissue and colon carcinoma tissue:
TABLE-US-00005 (SEQ ID NO: 24, 29) GUCY2C-118 s/GUCY2C-498 as; (SEQ
ID NO: 25, 30) GUCY2C-621 s/GUCY2C-1140 as; (SEQ ID NO: 26, 31)
GUCY2C-1450 s/GUCY2C-1790 as; (SEQ ID NO: 27, 32) GUCY2C-1993
s/GUCY2C-2366 as; (SEQ ID NO: 28, 33) GUCY2C-2717 s/GUCY2C-3200 as;
(SEQ ID NO: 24, 30) GUCY2C-118 s/GUCY2C-1140 as; (SEQ ID NO: 25,
31) GUCY2C-621 s/GUCY2C-1790 as; (SEQ ID NO: 26, 32) GUCY2C-1450
s/GUCY2C-2366 as; (SEQ ID NO: 27, 33) GUCY2C-1993 s/GUCY2C-3200
as.
[0294] On investigation of splice variants in colon carcinoma
tissue, three previously unknown forms were identified according to
the present technology. [0295] a) A deletion of exon 3 (SEQ ID NO:
3) which leads to a variant of GUCY2C which is only 111 amino acids
long and in which the asparagine at position 111 is replaced by a
proline. [0296] b) A deletion of exon 6 (SEQ ID NO: 4) which
results in an expression product 258 amino acids long. This would
generate a C-terminal neoepitope comprising 13 amino acids. [0297]
c) A variant in which the nucleotides at positions 1606-1614, and
the corresponding amino acids L(536), L(537) and Q(538), are
deleted (SEQ ID NO:5).
[0298] The splice variants according to the present technology with
deletions respectively in exon 3 and exon 6 (SEQ ID NO: 3, 4) are
distinguished in particular by the translation products (SEQ ID NO:
12, 13) having no transmembrane domain. The result in the case of
exon 6 deletion is a C-terminal neoepitope of 13 amino acids which
shows no homology whatsoever with previously known proteins. This
neoepitope is thus predestined to be a target structure for
immunotherapy. The splice variant of the present technology with
base deletions at positions 1606-1614 (SEQ ID NO: 5) and its
translation product (SEQ ID NO: 14) likewise comprises a
neoepitope. Antibodies for detecting GUCY2C protein were produced
by immunizing rabbits. The following peptides were used to
propagate these antibodies:
TABLE-US-00006 SEQ ID NO: 100: HNGSYEISVLMMGNS (AA 31-45) SEQ ID
NO: 101: NLPTPPTVENQQRLA (AA 1009-1023)
Such antibodies can in principle be used for diagnostic and
therapeutic purposes.
[0299] In particular, the extracellular domain of GUCY2C (position
of the predicted extracellular domain from the sequence of SEQ ID
NO: 11: AA 454-1073 (SEQ ID NO: 102)) can be used according to the
present technology as target structure of monoclonal antibodies.
However, the structural prediction is somewhat ambiguous and not
yet verified experimentally, so that an alternative membrane
orientation is also conceivable. In this case, amino acids 1-431
would be outside the cell and be suitable as starting point for
monoclonal antibodies. These antibodies bind specifically to the
cell surface of tumor cells and can be used both for diagnostic and
for therapeutic methods. Overexpression of GUCY2C, especially in
the colon tumors, provides additional support for such a use.
Sequences coding for proteins can moreover be used according to the
present technology as vaccine (RNA, DNA, peptides, protein) for
inducing tumor-specific immune responses (T-cell- and
B-cell-mediated immune responses).
[0300] It is moreover possible in accordance with the cellular
function of the GUCY2C molecule to develop according to the present
technology substances, especially small molecules, which modulate
the function of the enzyme on tumor cells. The product of the
enzymic reaction, cGMP, is a known cellular signal molecule with a
wide variety of functions (Tremblay et al. Mol Cell Biochem 230,
31).
Example 3
Identification of SCGB3A2 as Diagnostic and Therapeutic Cancer
Target
[0301] SCGB3A2 (SEQ ID NO: 6) (translation product: SEQ ID NO: 15)
belongs to the secretoglobin gene family. The sequence is published
in GenBank under accession number NM.sub.--054023. SCGB3A2 (UGRP1)
is a homodimeric secretory protein with a size of 17 kDa, which is
expressed exclusively in the lung and in the spiracles (Niimi et
al., Am J Hum Genet 70:718-25, 2002). RT PCR investigations with a
primer pair (SEQ ID NO: 37, 38) confirmed selective expression in
normal lung tissue. Lung- and trachea-specific genes, e.g. for
surfactant proteins, are highly downregulated in malignant tumors
during dedifferentiation and are normally undetectable in lung
tumors. It was surprisingly found that SCGB3A2 is active in primary
and metastatic lung tumors. The investigations according to the
present technology showed that SCGB3A2 is strongly and frequently
expressed in bronchial carcinomas (FIG. 4). All the other 23 normal
tissues tested, apart from lung and trachea, show no expression
(cf. FIG. 20).
[0302] This was additionally confirmed in a specific quantitative
RT-PCR (SEQ ID NO: 103, 104) (FIG. 20) which additionally shows
overexpression by at least one log in more than 50% of bronchial
carcinomas. The selective and high expression of SCGB3A2 in normal
lung tissue and in lung carcinoma biopsies can be used according to
the present technology for molecular diagnostic methods such as
RT-PCR for detecting disseminating tumor cells in blood and bone
marrow, sputum, bronchial aspirate or lavage and for detecting
metastases in other tissues, e.g. in local lymph nodes. In the
healthy lung, SCGB3A2 is secreted by specialized cells exclusively
into the bronchi. Accordingly, it is not to be expected that
SCGB3A2 protein will be detectable in body fluids outside the
respiratory tract in healthy individuals. By contrast, in
particular metastatic tumor cells secrete their protein products
directly into the bloodstream. One aspect of the present technology
therefore relates to detection of SCGB3A2 products in serum or
plasma of patients via a specific antibody assay as diagnostic
finding for lung tumors.
[0303] Antibodies for detecting SCGB3A2 protein were produced by
immunizing rabbits. The following peptides were used to propagate
these antibodies:
TABLE-US-00007 SEQ ID NO: 105: LINKVPLPVDKLAPL SEQ ID NO: 106:
SEAVKKLLEALSHLV
An SCGB3A2-specific reaction was detectable in immunofluorescence
(FIG. 21). As expected for a secreted protein, the distribution of
SCGB3A2 in the cell was assignable to the endoplasmic reticulum and
secretion granules (FIG. 21A). To check the specificity, the cells
were transfected in parallel with a plasmid that synthesizes an
SCGB3A2-GFP fusion protein. Protein detection took place in this
case via the autofluorescent GFP (green fluorescent protein) (FIG.
21B). Superimposition of the two fluorescence diagrams shows
unambiguously that the immune serum specifically recognizes SCGB3A2
protein (FIG. 21C). Such antibodies can be used according to the
present technology for example in the form of immunoassays for
diagnostic and therapeutic purposes.
Example 4
Identification of Claudin-18A1 and Claudin-10 18A2 Splice Variants
as Diagnostic and Therapeutic Cancer Targets
[0304] The claudin-18 gene codes for a surface membrane molecule
having 4 transmembrane domains and intracellular N terminus and C
terminus. Niimi and colleagues (Mol. Cell. Biol. 21:7380-90, 2001)
describe two splice variants of the murine and human claudin-18
which have been described as expressed selectively in lung tissue.
(claudin-18A1) and in stomach tissue (claudin-18A2), respectively.
These variants differ in the N terminus (FIG. 22).
[0305] It was investigated according to the present technology how
far the splice variants claudin-18A2 (SEQ ID NO: 7) and
claudin-18A1 (SEQ ID NO: 117), and their respective translation
products (SEQ ID NO: 16 and 118), can be used as markers or
therapeutic target structures for tumors. A quantitative PCR able
to distinguish between the two variants was established by
selecting A1-specific (SEQ ID NO: 109 110) and A2-specific (SEQ ID
NO: 107 & 108) primer pairs. The A2 splice variant was
additionally tested with a second primer pair in a conventional PCR
(SEQ ID NO: 39 & 40). The A1 variant is described to be active
only in normal lung. However, it was surprisingly found according
to the present technology that the A1 variant is also active in the
gastric mucosa. Stomach and lung are the only normal tissues
showing significant activation. All other normal tissues are
negative for claudin-A1. On investigating tumors, it was
surprisingly found that claudin-A1 is highly activated in a large
number of tumor tissues. Particularly strong expression is to be
found in stomach tumors, lung tumors, pancreatic carcinomas,
esophageal carcinomas (FIG. 23), ENT tumors and prostate
carcinomas. The claudin-A1 expression levels in ENT, prostate,
pancreatic and esophageal tumors are 100-10 000 higher than the
levels in the corresponding normal tissues. The oligonucleotides
used to investigate the claudin-A2 splice variant specifically
enable this transcript to be amplified (SEQ ID NO: 39 & 40 and
107 & 108). Investigation revealed that the A2 splice variant
is expressed in none of the more than 20 normal tissues
investigated apart from gastric mucosa and to a small extent also
testis tissue. We have found that the A2 variant is also, like the
A1 variant, activated in many tumors (depicted by way of example in
FIG. 24). These include stomach tumors (8/10), pancreatic tumors
(6/6), esophageal carcinomas (5/10) and liver carcinomas. A1 though
no activation of claudin-18A2 is detectable in healthy lung, it was
surprisingly found that some lung tumors express the A2.1 splice
variant.
TABLE-US-00008 TABLE 3A Expression of claudin-18A2 in normal and
tumor tissues. Normal tissues Expression Tumor type Expression
Brain - Colon - Cerebellum - carcinoma Myocardium - Pancreatic ++
Skeletal - carcinoma muscle Esophageal ++ Endometrium - carcinoma
Stomach +++ Gastric +++ Colon - carcinoma Pancreas - Bronchial ++
Kidney - carcinoma Liver - Breast - Testis + carcinoma Thymus -
Ovarian - Breast - carcinoma Ovary - Endometrial n.i. Uterus -
carcinoma Skin - ENT tumors ++ Lung - Renal cell - Thyroid -
carcinoma Lymph nodes - Prostate - Spleen - carcinoma PBMC -
Esophagus -
TABLE-US-00009 TABLE 3B Expression of claudin-18A1 in normal and
tumor tissues. Normal tissues Expression Tumor type Expression
Brain - Colon - Cerebellum - carcinoma Myocardium - Pancreatic ++
Skeletal - carcinoma muscle Esophageal ++ Endometrium - carcinoma
Stomach +++ Gastric +++ Colon - carcinoma Pancreas - Bronchial ++
Kidney - carcinoma Liver - Breast + Testis + carcinoma Thymus -
Ovarian n.i. Breast - carcinoma Ovary - Endometrial n.i. Uterus -
carcinoma Skin - ENT tumors ++ Lung +++ Renal cell - Thyroid -
carcinoma Lymph nodes - Prostate ++ Spleen - carcinoma PBMC -
Esophagus -
[0306] Conventional PCR as independent control investigation also
confirmed the results of the quantitative PCR. The oligonucleotides
(SEQ ID NO: 39, 40) used for this permit specific amplification of
the A2 splice variant. It was shown according to the present
technology that 8/10 gastric carcinomas and half of the tested
pancreatic carcinomas showed strong expression of this splice
variant (FIG. 5). By contrast, expression is not detectable in
other tissues by conventional PCR. In particular, there is no
expression in lung, liver, blood, lymph nodes, breast tissue and
kidney tissue (tab. 3).
[0307] The splice variants thus represent according to the present
technology highly specific molecular markers for tumors of the
upper gastrointestinal tract as well as lung tumors, ENT tumors,
prostate carcinomas and metastases thereof. These molecular markers
can be used according to the present technology for detecting tumor
cells. Detection of the tumors is possible according to the present
technology with the oligonucleotides described (SEQ ID NO: 39, 40,
107-110). Particularly suitable oligonucleotides are primer pairs
of which at least one binds under stringent conditions to a segment
of the transcript which is 180 base pairs long and is specific for
one (SEQ ID NO: 8) or the other splice variant (SEQ ID NO:
119).
[0308] In order to confirm these data at the protein level,
claudin-specific antibodies and immune sera were generated by
immunizing animals. The plasma membrane localization of claudin-18
and the protein topology was confirmed by analysis of the
transmembrane domains with bioinformatic tools (TMHMM, TMPRED) and
immunofluorescence investigations of cells which expressed
claudin-18 fusion proteins tagged with enhanced GFP. Claudin-18 has
two extracellular domains. The N-terminal extracellular domain
differs in sequence in the two splice variants (SEQ ID NO: 111 for
A1 and SEQ ID NO: 112 for A2). The C-terminal extracellular domain
is identical for both variants (SEQ ID NO: 137). To date, no
antibodies which bind to the extracellular domains of claudin-18
have yet been described. According to the present technology,
peptide epitopes which are located extracellularly and are specific
for variant A1 or A2 or occur in both variants were selected for
the immunization. Both variants of claudin-18 have no conventional
glycosylation motifs and the glycosylation of the protein was
therefore not to be expected. Nevertheless, account was taken in
the selection of the epitopes that epitopes which comprise
asparagine, serine, threonine are potentially glycosylated in rare
cases even without conventional glycosylation sites. Glycosylation
of an epitope may prevent the binding of an antibody specific for
this epitope. Inter alia, epitopes were selected according to the
present technology so that the antibodies generated thereby permit
the glycosylation status of the antigen to be distinguished. The
following peptides, inter alia, were selected for producing
antibodies for the immunization: SEQ ID NO: 17: DQWSTQDLYN
(N-terminal extracellular domain, A2-specific, binding independent
of glycosylation) SEQ ID NO: 18: NNPVTAVFNYQ (N-terminal
extracellular domain, A2-specific, binding mainly to unglycosylated
form, N37) SEQ ID NO: 113: STQDLYNNPVTAVF (N-terminal extracellular
domain, A2-specific, binding only to non-glycosylated form, N37)
SEQ ID NO: 114: DMWSTQDLYDNP (N-terminal extracellular domain,
A1-specific) SEQ ID NO: 115: CRPYFTILGLPA (N-terminal extracellular
domain, mainly specific for A1) SEQ ID NO: 116: TNFWMSTANMYTG
(C-terminal extracellular domain, recognizes both A1 and A2).
[0309] The data for the A2-specific antibody produced by
immunization with SEQ ID NO: 17 are shown by way of example. The
specific antibody can be utilized under various fixation conditions
for immunofluorescence investigations. With comparative stainings
of RT-PCR-positive and negative cell lines, in an amount which is
readily detectable, the corresponding protein can be specifically
detected in the gastric carcinoma cell lines typed as positive
(FIG. 25). The endogenous protein is membrane-located and forms
relatively large focal aggregates on the membrane. This antibody
was additionally employed for protein detection in Western
blotting. As expected, protein is detected only in stomach and in
no other normal tissue, not even lung (FIG. 29). The comparative
staining of stomach tumors and adjacent normal stomach tissue from
patients surprisingly revealed that claudin-18 A2 has a smaller
mass weight in all stomach tumors in which this protein is detected
(FIG. 30, left). It was found according to the present technology
in a series of experiments that a band also appears at this level
when lysate of normal stomach tissue is treated with the
deglycosylating agent PNGase F (FIG. 30, right). Whereas
exclusively the glycosylated form of the A2 variant is detectable
in all normal stomach tissues, A2 is detectable as such in more
than 60% of the investigated gastric carcinomas, in particular
exclusively in the deglycosylated form. A1 though the A2 variant of
claudin-18 is not detected in normal lung even at the protein
level, it is to be found in bronchial carcinomas, as also
previously in the quantitative RT-PCR. Once again, only the
deglycosylated variant is present (FIG. 31). Antibodies which
recognize the extracellular domain of the claudin-18-A2 splice
variant have been produced according to the present technology. In
addition, antibodies which selectively recognize the N-terminal
domain of the claudin-18-A1 splice variant (FIG. 28) and antibodies
which bind to both variants in the region of the C-terminal
extracellular domain (FIG. 27) have been produced. It is possible
according to the present technology to use such antibodies for
diagnostic purposes, e.g. immunohistology (FIG. 32), but also for
therapeutic purposes as explained above. A further important aspect
relates to differentially glycosylated domains of claudin-18.
Antibodies which exclusively bind to non-glycosylated epitopes have
been produced according to the present technology. Claudin-18
itself is a highly selective differentiating antigen for stomach
tissue (A2) and for the lung and stomach (A1). Since it is
evidently affected by changes in the glycosylation machinery in
tumors, a particular, deglycosylated, variant of A2 is produced in
tumors. This can be utilized diagnostically and therapeutically.
Immune sera such as the one described here (against peptide of SEQ
ID NO:17) can be utilized diagnostically for example in Western
blotting. Antibodies which are entirely unable to bind to the
glycosylated epitope as obtained for example by immunization with
peptide of SEQ ID NO:113 (FIG. 26), can distinguish tumor tissue
from normal tissue in the binding. It is possible in particular to
employ such antibodies therapeutically because they are highly
selective. The produced antibodies can be used directly also for
producing chimeric or humanized recombinant antibodies. This can
also take place directly with antibodies obtained from rabbits
(concerning this, see J Biol Chem. 2000 May 5; 275(18):13668-76 by
Rader C, Ritter G, Nathan S, Elia M, Gout I, Jungbluth A A, Cohen L
S, Welt S, Old L J, Barbas C F 3rd. "The rabbit antibody repertoire
as a novel source for the generation of therapeutic human
antibodies"). For this purpose, lymphocytes from the immunized
animals were preserved. The amino acids 1-47 (SEQ ID NO: 19 and
120) also represent particularly good epitopes for
immunotherapeutic methods such as vaccines and the adoptive
transfer of antigen-specific T lymphocytes.
Example 5
Identification of SLC13A1 as Diagnostic and Therapeutic Cancer
Target
[0310] SLC13A1 belongs to the family of sodium sulfate
cotransporters. The human gene is, in contrast to the mouse homolog
of this gene, selectively expressed in the kidney (Lee et al.,
Genomics 70:354-63). SLC13A1 codes for a protein of 595 amino acids
and comprises 13 putative transmembrane domains. Alternative
splicing results in 4 different transcripts (SEQ ID NO: 41-44) and
its corresponding translation products (SEQ ID NO: 45-48). It was
investigated whether SLC13A1 can be used as marker for kidney
tumors. Oligonucleotides (SEQ ID NO: 49, 50) which enable specific
amplification of SLC13A1 were used for this purpose.
TABLE-US-00010 TABLE 4 Expression of SLC13A1 in normal and tumor
tissues Normal tissues Expression Tumor type Expression Brain -
Colon nd Cerebellum nd carcinoma Myocardium nd Pancreatic nd
Skeletal nd carcinoma muscle Esophageal nd Mycardium - carcinoma
Stomach - Gastric nd Colon - carcinoma Pancreas nd Bronchial nd
Kidney +++ carcinoma Liver - Breast nd Testis + carcinoma Thymus -
Ovarian nd Breast - carcinoma Ovary - Endometrial nd Uterus nd
carcinoma Skin nd ENT tumors nd Lung - Renal cell +++ Thyroid -
carcinoma Lymph nodes - Prostate nd Spleen - carcinoma PBMC -
Sigmoid - Esophagus -
[0311] RT-PCR investigations with an SLC13A1-specific primer pair
(SEQ ID NO: 49, 50) confirmed virtually selective expression in the
kidney, and showed according to the present technology a high
expression in virtually all (7/8) investigated renal cell carcinoma
biopsies (tab. 4, FIG. 6). Quantitative RT-PCR with specific
primers (SEQ ID NO: 121, 122) also confirmed these data (FIG. 34).
Weak signals were detectable in the following normal tissues:
colon, stomach, testis, breast, liver and brain. Expression in
renal carcinomas was, however, at least 100 times higher than in
all other normal tissues.
[0312] In order to analyse the subcellular localization of SLC13A1
in the cell, the protein was fused to eGFP as reporter molecule
and, after transfection of the appropriate plasmid, expressed
heterologously in 293 cells. The localization was then analysed
under the fluorescence microscope. Our data impressively confirmed
that SLC13A1 is an integral transmembrane molecule (FIG. 35).
[0313] Antibodies for detecting the SLC13A1 protein were produced
by immunizing rabbits. The peptides of SEQ ID NO: 123 and 124 were
used for propagating these antibodies. Such antibodies can in
principle be used for diagnostic and therapeutic purposes. The
SLC13A1 protein has 13 transmembrane domains and 7 extracellular
regions. These extracellular domains of SLC13A1 in particular can
be used according to the present technology as target structures
for monoclonal antibodies. SLC13A1 is involved as channel protein
in the transport of ions. The extracellular domains of SLC13A1 in
the healthy kidney are directed polarically in the direction of the
urinary tract (luminally). However, high molecular weight
monoclonal antibodies employed therapeutically are not excreted
into the urinary tract, so that no binding to SLC13A1 takes place
in the healthy kidney. By contrast, the polarity of SLC13A1 is
abolished in tumor cells, and the protein is available for antibody
targeting directly via the bloodstream. The pronounced expression
and high incidence of SLC13A1 in renal cell carcinomas make this
protein according to the present technology a highly interesting
diagnostic and therapeutic marker. This includes according to the
present technology the detection of disseminated tumor cells in
serum, bone marrow, urine, and detection of metastases in other
organs by means of RT-PCR. It is additionally possible to use the
extracellular domains of SLC13A1 according to the present
technology as target structure for immunodiagnosis and therapy by
means of monoclonal antibodies. SLC13A1 can moreover be employed
according to the present technology as vaccine (RNA, DNA, protein,
peptides) for inducing tumor-specific immune responses (T and B
cell-mediated immune responses). This includes according to the
present technology also the development of so-called small
compounds which modulate the biological activity of SLC13A1 and can
be employed for the therapy of renal tumors.
Example 6
Identification of CLCA1 as Diagnostic and Therapeutic Cancer
Target
[0314] CLCA1 (SEQ ID NO: 51; translation product: SEQ ID NO: 60)
belongs to the family of Ca.sup.++-activated Cl.sup.- channels. The
sequence is published in Genbank under the accession No. NM 001285.
CLCA1 is exclusively expressed in the intestinal crypt epithelium
and in the goblet cells (Gruber et al., Genomics 54:200-14, 1998).
It was investigated whether CLCA1 can be used as marker for colonic
and gastric carcinoma. Oligonucleotides (SEQ ID NO: 67, 68) which
enable specific amplification of CLCA1 were used for this purpose.
RT-ECR investigations with this primer set confirmed selective
expression in the colon, and showed according to the present
technology high expression in (3/7) investigated colonic and (1/3)
investigated gastric carcinoma samples (FIG. 7). The other normal
tissues showed no or only very weak expression. This was
additionally confirmed with a specific quantitative RT-PCR (SEQ ID
NO: 125, 126), in which case no expression could be detected in the
normal tissues analyzed (FIG. 36). Of the tumor samples
investigated in this experiment, 6/12 colonic carcinoma samples and
5/10 gastric carcinoma samples were positive for CLCA1. Overall,
expression of the gene in tumors appears to be dysregulated.
Besides samples with very strong expression, CLCA1 was markedly
downregulated in other samples.
[0315] The protein is predicted to have 4 transmembrane domains
with a total of 2 extracellular regions. These extracellular
domains of CLCA1 in particular can be used according to the present
technology as target structures for monoclonal antibodies.
[0316] The pronounced expression and high incidence of CLCA1 in
gastric and colonic carcinomas make this protein according to the
present technology an interesting diagnostic and therapeutic
marker. This includes according to the present technology the
detection of disseminated tumor cells in serum, bone marrow, urine,
and detection of metastases in other organs by means of RT-PCR. It
is additionally possible to use the extracellular domains of CLCA1
according to the present technology as target structure for
immunodiagnosis and therapy by means of monoclonal antibodies.
CLCA1 can moreover be employed according to the present technology
as vaccine (RNA, DNA, protein, peptides) for inducing
tumor-specific immune responses (T and B cell-mediated immune
responses). This includes according to the present technology also
the development of so-called small compounds which modulate the
biological activity as transport proteins of CLCA1 and can be
employed for the therapy of gastrointestinal tumors.
Example 7
Identification of FLJ21477 as Diagnostic and Therapeutic Cancer
Target
[0317] FLJ21477 (SEQ ID NO: 52) and its predicted translation
product (SEQ ID NO: 61) was published as hypothetical protein in
Genbank under the accession No. NM.sub.--025153. It is an integral
membrane protein having ATPase activity and 4 transmembrane
domains, which is accordingly suitable for therapy with specific
antibodies. RT-PCR investigations with FLJ21477-specific primers
(SEQ ID NO: 69, 70) showed selective expression in the colon, and
additionally various levels of expression in (7/12) investigated
colonic carcinoma samples (FIG. 8). The other normal tissues showed
no expression. This was confirmed additionally by a specific
quantitative RT-PCR (SEQ ID NO: 127, 128). FLJ21477-specific
expression was detectable both in colon (FIG. 37A) and in 11/12 of
colonic carcinomas. Besides the expression in colon tissue,
expression was additionally detectable in stomach tissue. In
addition, under the conditions of the quantitative RT-PCR, the
expression detectable in brain, thymus and esophagus was distinctly
weaker compared with colon and stomach (FIG. 37A). It was moreover
additionally possible to detect FLJ21477-specific expression in the
following tumor samples: stomach, pancreas, esophagus and liver.
The protein is predicted to have 4 transmembrane domains with a
total of 2 extracellular regions. These extracellular domains of
FLJ21477 in particular can be used according to the present
technology as' target structures for monoclonal antibodies.
[0318] The expression and the high incidence of FLJ21477 for
gastric and colonic carcinomas make this protein according to the
present technology a valuable diagnostic and therapeutic marker.
This includes according to the present technology 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 FLJ21477 can be used
according to the present technology as target structure for
immunodiagnosis and therapy by means of monoclonal antibodies. In
addition, FLJ21477 can be employed according to the present
technology as vaccine (RNA, DNA, protein, peptides) for inducing
tumor-specific immune responses (T and B cell-mediated immune
responses).
Example 8
Identification of FLJ20694 as Diagnostic and Therapeutic Cancer
Target
[0319] FLJ20694 (SEQ ID NO: 53) and its translation product (SEQ ID
NO: 62) were published as hypothetical protein in Genbank under
accession No. NM.sub.--017928. This protein is an integral
transmembrane molecule (transmembrane domain AA 33-54), very
probably with thioredoxin function. RT-PCR investigations with
FLJ20694-specific primers (SEQ ID NO: 71, 72) showed selective
expression in the colon, and additionally various levels of
expression in (5/9) investigated colonic carcinoma samples (FIG.
9). The other normal tissues showed no expression. This was
additionally confirmed by a specific quantitative RT-PCR (SEQ ID
NO: 129, 130) (FIG. 38). FLJ29694 expression was undetectable in
any other normal tissue apart from colon and stomach (not analysed
in the first experiment).
[0320] The protein is predicted to have one transmembrane domain
with an extracellular region. These extracellular domains of
FLJ20694 in particular can be used according to the present
technology as target structures for monoclonal antibodies.
[0321] In addition, FLJ20694 can be employed according to the
present technology as vaccine (RNA, DNA, protein, peptides) for
inducing tumor-specific immune responses (T and B cell-mediated
immune responses). This includes according to the present
technology also the development of so-called small compounds which
modulate the biological activity of FLJ20694 and can be employed
for the therapy of gastrointestinal tumors.
Example 9
Identification of Von Ebner's Protein (c20orfll4) as Diagnostic and
Therapeutic Cancer Target
[0322] von Ebner's protein (SEQ ID NO:54) and its translation
product (SEQ ID NO: 63) were published as Plunc-related protein of
the upper airways and of the nasopharyngeal epithelium in Genbank
under the accession No. AF364078. It was investigated according to
the present technology whether von Ebner's protein can be used as
marker of lung carcinoma. Oligonucleotides (SEQ ID NO: 73, 74)
which enable specific amplification of Ebner's protein were used
for this purpose. RT-PCR investigations with this primer set showed
selective expression in the lung and in (5/10) investigated lung
carcinoma samples (FIG. 10). In the group of normal tissues there
was also expression in the stomach. The other normal tissues showed
no expression.
Example 10
Identification of Plunc as Diagnostic and Therapeutic Cancer
Target
[0323] Plunc (SEQ ID NO: 55) and its translation product (SEQ ID
NO: 64) were published in Genbank under the accession No. NM
016583. Human Plunc codes for a protein of 256 amino acids and
shows 72% homology with the murine Plunc protein (Single and
Single, Biochem Biophys Acta 1493:363-7, 2000). Expression of Plunc
is confined to the trachea, the upper airways, nasopharyngeal
epithelium and salivary gland.
[0324] It was investigated according to the present technology
whether Plunc can be used as marker of lung carcinoma.
Oligonucleotides (SEQ ID NO: 75, 76) which enable specific
amplification of Plunc were used for this purpose.
[0325] RT-PCR investigations with this primer set showed selective
expression in the thymus, in the lung and in (6/10) investigated
lung carcinoma samples (FIG. 11). Other normal tissues showed no
expression.
Example 11
Identification of SLC26A9 as Diagnostic and Therapeutic Cancer
Target
[0326] SLC26A9 (SEQ ID NO: 56) and its translation product (SEQ ID
NO: 65) were published in Genbank under the accession No.
NM.sub.--134325. SLC26A9 belongs to the family of anion exchangers.
Expression of SLC26A9 is confined to the bronchiolar and alveolar
epithelium of the lung (Lohi et al., J Biol Chem 277:14246-54,
2002).
[0327] It was investigated whether SLC26A9 can be used as marker of
lung carcinoma. Oligonucleotides (SEQ ID NO: 77, 78) which enable
specific amplification of SLC26A9 were used for this purpose.
RT-PCR investigations with SLC26A9-specific primers (SEQ ID NO: 77,
78) showed selective expression in the lung and in all (13/13)
investigated lung carcinoma samples (FIG. 12). The other normal
tissues showed no expression, with the exception of the thyroid. It
was possible in quantitative RT-PCR experiments with the primers of
SEQ ID NO: 131 and 132 firstly to confirm these results, and to
obtain additional information. It was possible in pooled samples of
4-5 tumor tissues to detect high expression levels for
SLC26A9-specific RNA in lung, colon, pancreas and stomach tumors.
SLC26A9 is member of a family of transmembrane anion transporters.
In the healthy lung, the protein is luminally directed in the
direction of the airways and thus not directly available to IgG
antibodies from the blood. By contrast, the polarity of the protein
is abolished in tumors. It is therefore possible according to the
present technology to address SLC26A9 as therapeutic target using
monoclonal antibodies in the defined tumors, inter alia lung
tumors, gastric carcinomas, pancreatic carcinomas. The pronounced,
high expression and high incidence of SLC26A9 for lung, stomach,
pancreatic and esophageal carcinomas make this protein according to
the present technology an excellent diagnostic and therapeutic
marker. This includes according to the present technology the
detection of disseminated tumor cells in serum, bone marrow and
urine, and detection of metastases in other organs by means of
RT-PCR. In addition, the extracellular domains of SLC26A9 can be
used according to the present technology as target structure for
immunodiagnosis and therapy by means of monoclonal antibodies. It
is additionally possible to employ SLC26A9 according to the present
technology as vaccine (RNA, DNA, protein, peptides) for inducing
tumor-specific immune responses (T and B cell-mediated immune
responses). This includes according to the present technology also
the development of so-called small compounds which modulate the
biological activity of SLC26A9 and can be employed for the therapy
of lung tumors and gastrointestinal tumors.
Example 12
Identification of THC1005163 as Diagnostic and Therapeutic Cancer
Target
[0328] THC1005163 (SEQ ID NO: 57) is a gene fragment from the TIGR
gene index. The gene is defined only in the 3' region, while an ORF
is lacking. RT-PCR investigations took place with a
THC1005163-specific primer (SEQ ID NO: 79) and an oligo dT.sub.18
primer which had a specific tag of 21 specific bases at the 5' end.
This tag was examined using database search programs for homology
with known sequences. This specific primer was initially employed
in the cDNA synthesis in order to preclude genomic DNA
contaminations. RT-PCR investigations with this primer set showed
expression in the stomach, ovary, lung and in (5/9) lung carcinoma
biopsies (FIG. 13). Other normal tissues showed no expression.
Example 13
Identification of LOC134288 as Diagnostic and Therapeutic Cancer
Target
[0329] LOC134288 (SEQ ID NO: 58) and its predicted translation
product (SEQ ID NO: 66) were published in Genbank under accession
No. XM.sub.--059703.
[0330] It was investigated according to the present technology
whether LOC134288 can be used as marker of renal cell carcinoma.
Oligonucleotides (SEQ ID NO: 80, 81) which enable specific
amplification of LOC134288 were used for this purpose. RT-PCR
investigations showed selective expression in the kidney and in
(5/8) investigated renal cell carcinoma biopsies (FIG. 14).
Example 14
Identification of THC943866 as Diagnostic and Therapeutic Cancer
Target
[0331] THC 943866 (SEQ ID NO: 59) is a gene fragment from the TIGR
gene index. It was investigated whether THC943866 can be used as
marker of renal cell carcinoma. Oligonucleotides (SEQ ID NO: 82,
83) which enable specific amplification of THC943866 were used for
this purpose.
[0332] RT-PCR investigations with THC943866-specific primers (SEQ
ID NO: 82, 83) showed selective expression in the kidney and in
(4/8) investigated renal cell carcinoma biopsies (FIG. 15).
Example 15
Identification of FLJ21458 as Diagnostic and Therapeutic Cancer
Target
[0333] FLJ21458 (SEQ ID NO: 84) and its predicted translation
product (SEQ ID NO: 85) were published in Genbank under the
accession No. NM.sub.--034850. Sequence analyses revealed that the
protein represents a new member of the butyrophillin family.
Structural analyses revealed that it represents a type 1
transmembrane protein with an extracellular immunoglobulin domain.
Oligonucleotides (SEQ ID NO: 86, 87) which enable specific
amplification of FLJ21458 were used for investigating expression.
RT-PCR investigations with FLJ21458-specific primers (SEQ ID NO:
86, 87) showed selective expression in colon and in (7/10)
investigated colonic carcinoma biopsies (FIG. 16, tab. 5).
Quantitative RT-PCR with specific primers (SEQ ID NO: 133, 134)
confirmed this selective expression profile (FIG. 39). It was
additionally possible in the experiment to detect FLJ21458
gastrointestinal-specifically in the colon, and in stomach, in the
rectum and cecum and in testis. 7/11 colon metastasis samples were
also positive in the quantitative PCR. FLJ21458-specific expression
was extended to other tumors, and a protein-specific expression was
detectable in stomach, pancreas and liver tumors (tab. 5).
Antibodies for detecting FLJ21458 protein were produced by
immunizing rabbits. The following peptides were used to propagate
these antibodies:
TABLE-US-00011 SEQ ID NO: 135: QWQVFGPDKPVQAL SEQ ID NO: 136:
AKWKGPQGQDLSTDS
An FLJ21458-specific reaction was detectable in immuno-fluorescence
(FIG. 40). To check the specificity of the antibodies, 293 cells
were transfected with a plasmid that codes for an FLJ21458-GFP
fusion protein. Specificity was demonstrated on the one hand by
colocalization investigations using the FLJ21458-specific antibody,
and on the other hand via the auto-fluorescent GFP. Superimposition
of the two fluorescent diagrams showed unambiguously that the
immune serum specifically recognises FLJ21458 protein (FIG. 40a).
Owing to the overexpression of the protein, the resultant cell
staining was diffuse and did not allow unambiguous protein
localization. For this reason, a further immunofluorescence
experiment was carried out with the stomach tumor-specific cell
line Snu16 which expresses FLJ21458 endogenously (FIG. 41B). The
cells were stained with the FLJ21458-specific antiserum and with
another antibody which recognizes the membrane protein E-cadherin.
The FLJ21458-specific antibody stains the cell membranes at least
weakly and is thus evidence that FLF21458 is localized in the cell
membrane.
[0334] Bioinformatic investigations showed that the protein encoded
by FLJ21458 represents a cell surface molecule and has an
immunoglobulin supermolecule domain. Selective expression of this
surface molecule makes it a good target for developing diagnostic
methods for the detection of tumor cells and therapeutic methods
for the elimination of tumor cells.
[0335] The pronounced expression and high incidence of FLJ21458 for
gastric and colonic carcinomas make this protein according to the
present technology a highly interesting diagnostic and therapeutic
marker. This includes according to the present technology the
detection of disseminated tumor cells in serum, bone marrow and
urine, and the detection of metastases in other organs by means of
RT-PCR. It is additionally possible to employ the extracellular
domains of FLJ21458 according to the present technology as target
structure for immuno-diagnosis and therapy by means of monoclonal
antibodies. It is additionally possible to employ FLJ21458
according to the present technology as vaccine (RNA, DNA, protein,
peptides) for inducing tumor-specific immune responses (T and B
cell-mediated immune responses). This includes according to the
present technology also the development of so-called small
compounds which modulate the biological activity of FLJ21458 and
can be employed for the therapy of gastrointestinal tumors.
TABLE-US-00012 TABLE 5 FLJ21458 expression in normal and tumor
tissues Normal tissues Expression Tumor type Expression Brain -
Colonic 7/10 Cerebellum - carcinoma Myocardium nd Pancreatic 5/6
Skeletel - carcinoma muscle Esophageal nd Mycardium - carcinoma
Stomach ++ Gastric 8/10 Colon +++ carcinoma Pancreas - Bronchial nd
Kidney - carcinoma Liver - Breast nd Testis ++ carcinoma Thymus nd
Ovarian nd Breast nd carcinoma Ovary - Endometrial nd Uterus -
carcinoma Skin - ENT tumors nd Lung - Renal cell nd Thyroid nd
carcinoma Lymph nodes - Prostate nd Spleen - carcinoma PBMC -
Colonic 7/11 Adrenal nd metastases Esophagus - Liver 5/8 Small
intestine - carcinoma Prostate -
Sequence CWU 1
1
14111875DNAHomo Sapiens 1caggccagag tcccagctgt cctggactct
gctgtgggga agggctgatg caggtgtgga 60gtcaaatgtg ggtgcctcct gcagccgggt
gccaggaggg gtggaggggc caccctgggc 120tttgtccggg agcctggtct
tcccgtcctt gggctgacag gtgctgctgc ctctgagccc 180tccctgctaa
gagctgtgtg ctgggtaagg ctggtggccc tttgggctcc ctgtccagga
240tttgtgctct ggagggtagg gcttgctggg ctggggactg gaggggaacg
tggagctcct 300tctgcctcct ttcctgcccc atgacagcag gcagatccca
ggagagaaga gctcaggaga 360tgggaagagg atctgtccag gggttagacc
tcaagggtga cttggagttc tttacggcac 420ccatgctttc tttgaggagt
tttgtgtttg tgggtgtggg gtcggggctc acctcctccc 480acatccctgc
ccagaggtgg gcagagtggg ggcagtgcct tgctccccct gctcgctctc
540tgctgacctc cggctccctg tgctgcccca ggaccatgaa tggcacctac
aacacctgtg 600gctccagcga cctcacctgg cccccagcga tcaagctggg
cttctacgcc tacttgggcg 660tcctgctggt gctaggcctg ctgctcaaca
gcctggcgct ctgggtgttc tgctgccgca 720tgcagcagtg gacggagacc
cgcatctaca tgaccaacct ggcggtggcc gacctctgcc 780tgctgtgcac
cttgcccttc gtgctgcact ccctgcgaga cacctcagac acgccgctgt
840gccagctctc ccagggcatc tacctgacca acaggtacat gagcatcagc
ctggtcacgg 900ccatcgccgt ggaccgctat gtggccgtgc ggcacccgct
gcgtgcccgc gggctgcggt 960cccccaggca ggctgcggcc gtgtgcgcgg
tcctctgggt gctggtcatc ggctccctgg 1020tggctcgctg gctcctgggg
attcaggagg gcggcttctg cttcaggagc acccggcaca 1080atttcaactc
catggcgttc ccgctgctgg gattctacct gcccctggcc gtggtggtct
1140tctgctccct gaaggtggtg actgccctgg cccagaggcc acccaccgac
gtggggcagg 1200cagaggccac ccgcaaggct gcccgcatgg tctgggccaa
cctcctggtg ttcgtggtct 1260gcttcctgcc cctgcacgtg gggctgacag
tgcgcctcgc agtgggctgg aacgcctgtg 1320ccctcctgga gacgatccgt
cgcgccctgt acataaccag caagctctca gatgccaact 1380gctgcctgga
cgccatctgc tactactaca tggccaagga gttccaggag gcgtctgcac
1440tggccgtggc tcccagtgct aaggcccaca aaagccagga ctctctgtgc
gtgaccctcg 1500cctaagaggc gtgctgtggg cgctgtgggc caggtctcgg
gggctccggg aggtgctgcc 1560tgccagggga agctggaacc agtagcaagg
agcccgggat cagccctgaa ctcactgtgt 1620attctcttgg agccttgggt
gggcagggac ggcccaggta cctgctctct tgggaagaga 1680gagggacagg
gacaagggca agaggactga ggccagagca aggccaatgt cagagacccc
1740cgggatgggg cctcacactt gccaccccca gaaccagctc acctggccag
agtgggttcc 1800tgctggccag ggtgcagcct tgatgacacc tgccgctgcc
cctcggggct ggaataaaac 1860tccccaccca gagtc 187523222DNAHomo Sapiens
2atgaagacgt tgctgttgga cttggctttg tggtcactgc tcttccagcc cgggtggctg
60tcctttagtt cccaggtgag tcagaactgc cacaatggca gctatgaaat cagcgtcctg
120atgatgggca actcagcctt tgcagagccc ctgaaaaact tggaagatgc
ggtgaatgag 180gggctggaaa tagtgagagg acgtctgcaa aatgctggcc
taaatgtgac tgtgaacgct 240actttcatgt attcggatgg tctgattcat
aactcaggcg actgccggag tagcacctgt 300gaaggcctcg acctactcag
gaaaatttca aatgcacaac ggatgggctg tgtcctcata 360gggccctcat
gtacatactc caccttccag atgtaccttg acacagaatt gagctacccc
420atgatctcag ctggaagttt tggattgtca tgtgactata aagaaacctt
aaccaggctg 480atgtctccag ctagaaagtt gatgtacttc ttggttaact
tttggaaaac caacgatctg 540cccttcaaaa cttattcctg gagcacttcg
tatgtttaca agaatggtac agaaactgag 600gactgtttct ggtaccttaa
tgctctggag gctagcgttt cctatttctc ccacgaactc 660ggctttaagg
tggtgttaag acaagataag gagtttcagg atatcttaat ggaccacaac
720aggaaaagca atgtgattat tatgtgtggt ggtccagagt tcctctacaa
gctgaagggt 780gaccgagcag tggctgaaga cattgtcatt attctagtgg
atcttttcaa tgaccagtac 840ttggaggaca atgtcacagc ccctgactat
atgaaaaatg tccttgttct gacgctgtct 900cctgggaatt cccttctaaa
tagctctttc tccaggaatc tatcaccaac aaaacgagac 960tttgctcttg
cctatttgaa tggaatcctg ctctttggac atatgctgaa gatatttctt
1020gaaaatggag aaaatattac cacccccaaa tttgctcatg ctttcaggaa
tctcactttt 1080gaagggtatg acggtccagt gaccttggat gactgggggg
atgttgacag taccatggtg 1140cttctgtata cctctgtgga caccaagaaa
tacaaggttc ttttgaccta tgatacccac 1200gtaaataaga cctatcctgt
ggatatgagc cccacattca cttggaagaa ctctaaactt 1260cctaatgata
ttacaggccg gggccctcag atcctgatga ttgcagtctt caccctcact
1320ggagctgtgg tgctgctcct gctcgtcgct ctcctgatgc tcagaaaata
tagaaaagat 1380tatgaacttc gtcagaaaaa atggtcccac attcctcctg
aaaatatctt tcctctggag 1440accaatgaga ccaatcatgt tagcctcaag
atcgatgatg acaaaagacg agatacaatc 1500cagagactac gacagtgcaa
atacgacaaa aagcgagtga ttctcaaaga tctcaagcac 1560aatgatggta
atttcactga aaaacagaag atagaattga acaagttgct tcagattgac
1620tattacaacc tgaccaagtt ctacggcaca gtgaaacttg ataccatgat
cttcggggtg 1680atagaatact gtgagagagg atccctccgg gaagttttaa
atgacacaat ttcctaccct 1740gatggcacat tcatggattg ggagtttaag
atctctgtct tgtatgacat tgctaaggga 1800atgtcatatc tgcactccag
taagacagaa gtccatggtc gtctgaaatc taccaactgc 1860gtagtggaca
gtagaatggt ggtgaagatc actgattttg gctgcaattc cattttacct
1920ccaaaaaagg acctgtggac agctccagag cacctccgcc aagccaacat
ctctcagaaa 1980ggagatgtgt acagctatgg gatcatcgca caggagatca
ttctgcggaa agaaaccttc 2040tacactttga gctgtcggga ccggaatgag
aagattttca gagtggaaaa ttccaatgga 2100atgaaaccct tccgcccaga
tttattcttg gaaacagcag aggaaaaaga gctagaagtg 2160tacctacttg
taaaaaactg ttgggaggaa gatccagaaa agagaccaga tttcaaaaaa
2220attgagacta cacttgccaa gatatttgga ctttttcatg accaaaaaaa
tgaaagctat 2280atggatacct tgatccgacg tctacagcta tattctcgaa
acctggaaca tctggtagag 2340gaaaggacac agctgtacaa ggcagagagg
gacagggctg acagacttaa ctttatgttg 2400cttccaaggc tagtggtaaa
gtctctgaag gagaaaggct ttgtggagcc ggaactatat 2460gaggaagtta
caatctactt cagtgacatt gtaggtttca ctactatctg caaatacagc
2520acccccatgg aagtggtgga catgcttaat gacatctata agagttttga
ccacattgtt 2580gatcatcatg atgtctacaa ggtggaaacc atcggtgatg
cgtacatggt ggctagtggt 2640ttgcctaaga gaaatggcaa tcggcatgca
atagacattg ccaagatggc cttggaaatc 2700ctcagcttca tggggacctt
tgagctggag catcttcctg gcctcccaat atggattcgc 2760attggagttc
actctggtcc ctgtgctgct ggagttgtgg gaatcaagat gcctcgttat
2820tgtctatttg gagatacggt caacacagcc tctaggatgg aatccactgg
cctccctttg 2880agaattcacg tgagtggctc caccatagcc atcctgaaga
gaactgagtg ccagttcctt 2940tatgaagtga gaggagaaac atacttaaag
ggaagaggaa atgagactac ctactggctg 3000actgggatga aggaccagaa
attcaacctg ccaacccctc ctactgtgga gaatcaacag 3060cgtttgcaag
cagaattttc agacatgatt gccaactctt tacagaaaag acaggcagca
3120gggataagaa gccaaaaacc cagacgggta gccagctata aaaaaggcac
tctggaatac 3180ttgcagctga ataccacaga caaggagagc acctattttt aa
32223336DNAHomo Sapiens 3atgaagacgt tgctgttgga cttggctttg
tggtcactgc tcttccagcc cgggtggctg 60tcctttagtt cccaggtgag tcagaactgc
cacaatggca gctatgaaat cagcgtcctg 120atgatgggca actcagcctt
tgcagagccc ctgaaaaact tggaagatgc ggtgaatgag 180gggctggaaa
tagtgagagg acgtctgcaa aatgctggcc taaatgtgac tgtgaacgct
240actttcatgt attcggatgg tctgattcat aactcaggcg actgccggag
tagcacctgt 300gaaggcctcg acctactcag gaaaatttca ccttga
3364777DNAHomo Sapiens 4atgaagacgt tgctgttgga cttggctttg tggtcactgc
tcttccagcc cgggtggctg 60tcctttagtt cccaggtgag tcagaactgc cacaatggca
gctatgaaat cagcgtcctg 120atgatgggca actcagcctt tgcagagccc
ctgaaaaact tggaagatgc ggtgaatgag 180gggctggaaa tagtgagagg
acgtctgcaa aatgctggcc taaatgtgac tgtgaacgct 240actttcatgt
attcggatgg tctgattcat aactcaggcg actgccggag tagcacctgt
300gaaggcctcg acctactcag gaaaatttca aatgcacaac ggatgggctg
tgtcctcata 360gggccctcat gtacatactc caccttccag atgtaccttg
acacagaatt gagctacccc 420atgatctcag ctggaagttt tggattgtca
tgtgactata aagaaacctt aaccaggctg 480atgtctccag ctagaaagtt
gatgtacttc ttggttaact tttggaaaac caacgatctg 540cccttcaaaa
cttattcctg gagcacttcg tatgtttaca agaatggtac agaaactgag
600gactgtttct ggtaccttaa tgctctggag gctagcgttt cctatttctc
ccacgaactc 660ggctttaagg tggtgttaag acaagataag gagtttcagg
atatcttaat ggaccacaac 720aggaaaagca atgtgaccag tacttggagg
acaatgtcac agcccctgac tatatga 77753213DNAHomo Sapiens 5atgaagacgt
tgctgttgga cttggctttg tggtcactgc tcttccagcc cgggtggctg 60tcctttagtt
cccaggtgag tcagaactgc cacaatggca gctatgaaat cagcgtcctg
120atgatgggca actcagcctt tgcagagccc ctgaaaaact tggaagatgc
ggtgaatgag 180gggctggaaa tagtgagagg acgtctgcaa aatgctggcc
taaatgtgac tgtgaacgct 240actttcatgt attcggatgg tctgattcat
aactcaggcg actgccggag tagcacctgt 300gaaggcctcg acctactcag
gaaaatttca aatgcacaac ggatgggctg tgtcctcata 360gggccctcat
gtacatactc caccttccag atgtaccttg acacagaatt gagctacccc
420atgatctcag ctggaagttt tggattgtca tgtgactata aagaaacctt
aaccaggctg 480atgtctccag ctagaaagtt gatgtacttc ttggttaact
tttggaaaac caacgatctg 540cccttcaaaa cttattcctg gagcacttcg
tatgtttaca agaatggtac agaaactgag 600gactgtttct ggtaccttaa
tgctctggag gctagcgttt cctatttctc ccacgaactc 660ggctttaagg
tggtgttaag acaagataag gagtttcagg atatcttaat ggaccacaac
720aggaaaagca atgtgattat tatgtgtggt ggtccagagt tcctctacaa
gctgaagggt 780gaccgagcag tggctgaaga cattgtcatt attctagtgg
atcttttcaa tgaccagtac 840ttggaggaca atgtcacagc ccctgactat
atgaaaaatg tccttgttct gacgctgtct 900cctgggaatt cccttctaaa
tagctctttc tccaggaatc tatcaccaac aaaacgagac 960tttgctcttg
cctatttgaa tggaatcctg ctctttggac atatgctgaa gatatttctt
1020gaaaatggag aaaatattac cacccccaaa tttgctcatg ctttcaggaa
tctcactttt 1080gaagggtatg acggtccagt gaccttggat gactgggggg
atgttgacag taccatggtg 1140cttctgtata cctctgtgga caccaagaaa
tacaaggttc ttttgaccta tgatacccac 1200gtaaataaga cctatcctgt
ggatatgagc cccacattca cttggaagaa ctctaaactt 1260cctaatgata
ttacaggccg gggccctcag atcctgatga ttgcagtctt caccctcact
1320ggagctgtgg tgctgctcct gctcgtcgct ctcctgatgc tcagaaaata
tagaaaagat 1380tatgaacttc gtcagaaaaa atggtcccac attcctcctg
aaaatatctt tcctctggag 1440accaatgaga ccaatcatgt tagcctcaag
atcgatgatg acaaaagacg agatacaatc 1500cagagactac gacagtgcaa
atacgacaaa aagcgagtga ttctcaaaga tctcaagcac 1560aatgatggta
atttcactga aaaacagaag atagaattga acaagattga ctattacaac
1620ctgaccaagt tctacggcac agtgaaactt gataccatga tcttcggggt
gatagaatac 1680tgtgagagag gatccctccg ggaagtttta aatgacacaa
tttcctaccc tgatggcaca 1740ttcatggatt gggagtttaa gatctctgtc
ttgtatgaca ttgctaaggg aatgtcatat 1800ctgcactcca gtaagacaga
agtccatggt cgtctgaaat ctaccaactg cgtagtggac 1860agtagaatgg
tggtgaagat cactgatttt ggctgcaatt ccattttacc tccaaaaaag
1920gacctgtgga cagctccaga gcacctccgc caagccaaca tctctcagaa
aggagatgtg 1980tacagctatg ggatcatcgc acaggagatc attctgcgga
aagaaacctt ctacactttg 2040agctgtcggg accggaatga gaagattttc
agagtggaaa attccaatgg aatgaaaccc 2100ttccgcccag atttattctt
ggaaacagca gaggaaaaag agctagaagt gtacctactt 2160gtaaaaaact
gttgggagga agatccagaa aagagaccag atttcaaaaa aattgagact
2220acacttgcca agatatttgg actttttcat gaccaaaaaa atgaaagcta
tatggatacc 2280ttgatccgac gtctacagct atattctcga aacctggaac
atctggtaga ggaaaggaca 2340cagctgtaca aggcagagag ggacagggct
gacagactta actttatgtt gcttccaagg 2400ctagtggtaa agtctctgaa
ggagaaaggc tttgtggagc cggaactata tgaggaagtt 2460acaatctact
tcagtgacat tgtaggtttc actactatct gcaaatacag cacccccatg
2520gaagtggtgg acatgcttaa tgacatctat aagagttttg accacattgt
tgatcatcat 2580gatgtctaca aggtggaaac catcggtgat gcgtacatgg
tggctagtgg tttgcctaag 2640agaaatggca atcggcatgc aatagacatt
gccaagatgg ccttggaaat cctcagcttc 2700atggggacct ttgagctgga
gcatcttcct ggcctcccaa tatggattcg cattggagtt 2760cactctggtc
cctgtgctgc tggagttgtg ggaatcaaga tgcctcgtta ttgtctattt
2820ggagatacgg tcaacacagc ctctaggatg gaatccactg gcctcccttt
gagaattcac 2880gtgagtggct ccaccatagc catcctgaag agaactgagt
gccagttcct ttatgaagtg 2940agaggagaaa catacttaaa gggaagagga
aatgagacta cctactggct gactgggatg 3000aaggaccaga aattcaacct
gccaacccct cctactgtgg agaatcaaca gcgtttgcaa 3060gcagaatttt
cagacatgat tgccaactct ttacagaaaa gacaggcagc agggataaga
3120agccaaaaac ccagacgggt agccagctat aaaaaaggca ctctggaata
cttgcagctg 3180aataccacag acaaggagag cacctatttt taa 32136550DNAHomo
Sapiens 6ggggacactt tgtatggcaa gtggaaccac tggcttggtg gattttgcta
gatttttctg 60atttttaaac tcctgaaaaa tatcccagat aactgtcatg aagctggtaa
ctatcttcct 120gctggtgacc atcagccttt gtagttactc tgctactgcc
ttcctcatca acaaagtgcc 180ccttcctgtt gacaagttgg cacctttacc
tctggacaac attcttccct ttatggatcc 240attaaagctt cttctgaaaa
ctctgggcat ttctgttgag caccttgtgg aggggctaag 300gaagtgtgta
aatgagctgg gaccagaggc ttctgaagct gtgaagaaac tgctggaggc
360gctatcacac ttggtgtgac atcaagataa agagcggagg tggatgggga
tggaagatga 420tgctcctatc ctccctgcct gaaacctgtt ctaccaatta
tagatcaaat gccctaaaat 480gtagtgaccc gtgaaaagga caaataaagc
aatgaatact aaaaaaaaaa aaaaaaaaaa 540aaaaaaaaaa 5507786DNAHomo
Sapiens 7atggccgtga ctgcctgtca gggcttgggg ttcgtggttt cactgattgg
gattgcgggc 60atcattgctg ccacctgcat ggaccagtgg agcacccaag acttgtacaa
caaccccgta 120acagctgttt tcaactacca ggggctgtgg cgctcctgtg
tccgagagag ctctggcttc 180accgagtgcc ggggctactt caccctgctg
gggctgccag ccatgctgca ggcagtgcga 240gccctgatga tcgtaggcat
cgtcctgggt gccattggcc tcctggtatc catctttgcc 300ctgaaatgca
tccgcattgg cagcatggag gactctgcca aagccaacat gacactgacc
360tccgggatca tgttcattgt ctcaggtctt tgtgcaattg ctggagtgtc
tgtgtttgcc 420aacatgctgg tgactaactt ctggatgtcc acagctaaca
tgtacaccgg catgggtggg 480atggtgcaga ctgttcagac caggtacaca
tttggtgcgg ctctgttcgt gggctgggtc 540gctggaggcc tcacactaat
tgggggtgtg atgatgtgca tcgcctgccg gggcctggca 600ccagaagaaa
ccaactacaa agccgtttct tatcatgcct caggccacag tgttgcctac
660aagcctggag gcttcaaggc cagcactggc tttgggtcca acaccaaaaa
caagaagata 720tacgatggag gtgcccgcac agaggacgag gtacaatctt
atccttccaa gcacgactat 780gtgtaa 7868180DNAHomo Sapiens 8tgcgccacca
tggccgtgac tgcctgtcag ggcttggggt tcgtggtttc actgattggg 60attgcgggca
tcattgctgc cacctgcatg gaccagtgga gcacccaaga cttgtacaac
120aaccccgtaa cagctgtttt caactaccag gggctgtggc gctcctgtgt
ccgagagagc 1809309PRTHomo Sapiens 9Met Asn Gly Thr Tyr Asn Thr Cys
Gly Ser Ser Asp Leu Thr Trp Pro 1 5 10 15 Pro Ala Ile Lys Leu Gly
Phe Tyr Ala Tyr Leu Gly Val Leu Leu Val 20 25 30 Leu Gly Leu Leu
Leu Asn Ser Leu Ala Leu Trp Val Phe Cys Cys Arg 35 40 45 Met Gln
Gln Trp Thr Glu Thr Arg Ile Tyr Met Thr Asn Leu Ala Val 50 55 60
Ala Asp Leu Cys Leu Leu Cys Thr Leu Pro Phe Val Leu His Ser Leu 65
70 75 80 Arg Asp Thr Ser Asp Thr Pro Leu Cys Gln Leu Ser Gln Gly
Ile Tyr 85 90 95 Leu Thr Asn Arg Tyr Met Ser Ile Ser Leu Val Thr
Ala Ile Ala Val 100 105 110 Asp Arg Tyr Val Ala Val Arg His Pro Leu
Arg Ala Arg Gly Leu Arg 115 120 125 Ser Pro Arg Gln Ala Ala Ala Val
Cys Ala Val Leu Trp Val Leu Val 130 135 140 Ile Gly Ser Leu Val Ala
Arg Trp Leu Leu Gly Ile Gln Glu Gly Gly 145 150 155 160 Phe Cys Phe
Arg Ser Thr Arg His Asn Phe Asn Ser Met Arg Phe Pro 165 170 175 Leu
Leu Gly Phe Tyr Leu Pro Leu Ala Val Val Val Phe Cys Ser Leu 180 185
190 Lys Val Val Thr Ala Leu Ala Gln Arg Pro Pro Thr Asp Val Gly Gln
195 200 205 Ala Glu Ala Thr Arg Lys Ala Ala Arg Met Val Trp Ala Asn
Leu Leu 210 215 220 Val Phe Val Val Cys Phe Leu Pro Leu His Val Gly
Leu Thr Val Arg 225 230 235 240 Leu Ala Val Gly Trp Asn Ala Cys Ala
Leu Leu Glu Thr Ile Arg Arg 245 250 255 Ala Leu Tyr Ile Thr Ser Lys
Leu Ser Asp Ala Asn Cys Cys Leu Asp 260 265 270 Ala Ile Cys Tyr Tyr
Tyr Met Ala Lys Glu Phe Gln Glu Ala Ser Ala 275 280 285 Leu Ala Val
Ala Pro Arg Ala Lys Ala His Lys Ser Gln Asp Ser Leu 290 295 300 Cys
Val Thr Leu Ala 305 10394PRTHomo Sapiens 10Met Thr Ala Gly Arg Ser
Gln Glu Arg Arg Ala Gln Glu Met Gly Arg 1 5 10 15 Gly Ser Val Gln
Gly Leu Asp Leu Lys Gly Asp Leu Glu Phe Phe Thr 20 25 30 Ala Pro
Met Leu Ser Leu Arg Ser Phe Val Phe Val Gly Val Gly Ser 35 40 45
Gly Leu Thr Ser Ser His Ile Pro Ala Gln Arg Trp Ala Glu Trp Gly 50
55 60 Gln Cys Leu Ala Pro Pro Ala Arg Ser Leu Leu Thr Ser Gly Ser
Leu 65 70 75 80 Cys Cys Pro Arg Thr Met Asn Gly Thr Tyr Asn Thr Cys
Gly Ser Ser 85 90 95 Asp Leu Thr Trp Pro Pro Ala Ile Lys Leu Gly
Phe Tyr Ala Tyr Leu 100 105 110 Gly Val Leu Leu Val Leu Gly Leu Leu
Leu Asn Ser Leu Ala Leu Trp 115 120 125 Val Phe Cys Cys Arg Met Gln
Gln Trp Thr Glu Thr Arg Ile Tyr Met 130 135 140 Thr Asn Leu Ala Val
Ala Asp Leu Cys Leu Leu Cys Thr Leu Pro Phe 145 150 155 160 Val Leu
His Ser Leu Arg Asp Thr Ser Asp Thr Pro Leu Cys Gln Leu 165 170 175
Ser Gln Gly Ile Tyr Leu Thr Asn Arg Tyr Met Ser Ile Ser Leu Val 180
185 190 Thr Ala Ile Ala Val Asp Arg Tyr Val Ala Val Arg His Pro Leu
Arg 195 200 205 Ala Arg Gly Leu Arg Ser Pro Arg Gln Ala Ala Ala Val
Cys Ala Val 210 215 220 Leu Trp Val Leu Val Ile Gly Ser Leu Val Ala
Arg Trp Leu Leu Gly 225 230 235 240 Ile Gln Glu Gly Gly Phe Cys Phe
Arg Ser Thr Arg His Asn Phe Asn 245 250 255 Ser Met Ala
Phe Pro Leu Leu Gly Phe Tyr Leu Pro Leu Ala Val Val 260 265 270 Val
Phe Cys Ser Leu Lys Val Val Thr Ala Leu Ala Gln Arg Pro Pro 275 280
285 Thr Asp Val Gly Gln Ala Glu Ala Thr Arg Lys Ala Ala Arg Met Val
290 295 300 Trp Ala Asn Leu Leu Val Phe Val Val Cys Phe Leu Pro Leu
His Val 305 310 315 320 Gly Leu Thr Val Arg Leu Ala Val Gly Trp Asn
Ala Cys Ala Leu Leu 325 330 335 Glu Thr Ile Arg Arg Ala Leu Tyr Ile
Thr Ser Lys Leu Ser Asp Ala 340 345 350 Asn Cys Cys Leu Asp Ala Ile
Cys Tyr Tyr Tyr Met Ala Lys Glu Phe 355 360 365 Gln Glu Ala Ser Ala
Leu Ala Val Ala Pro Ser Ala Lys Ala His Lys 370 375 380 Ser Gln Asp
Ser Leu Cys Val Thr Leu Ala 385 390 111073PRTHomo Sapiens 11Met Lys
Thr Leu Leu Leu Asp Leu Ala Leu Trp Ser Leu Leu Phe Gln 1 5 10 15
Pro Gly Trp Leu Ser Phe Ser Ser Gln Val Ser Gln Asn Cys His Asn 20
25 30 Gly Ser Tyr Glu Ile Ser Val Leu Met Met Gly Asn Ser Ala Phe
Ala 35 40 45 Glu Pro Leu Lys Asn Leu Glu Asp Ala Val Asn Glu Gly
Leu Glu Ile 50 55 60 Val Arg Gly Arg Leu Gln Asn Ala Gly Leu Asn
Val Thr Val Asn Ala 65 70 75 80 Thr Phe Met Tyr Ser Asp Gly Leu Ile
His Asn Ser Gly Asp Cys Arg 85 90 95 Ser Ser Thr Cys Glu Gly Leu
Asp Leu Leu Arg Lys Ile Ser Asn Ala 100 105 110 Gln Arg Met Gly Cys
Val Leu Ile Gly Pro Ser Cys Thr Tyr Ser Thr 115 120 125 Phe Gln Met
Tyr Leu Asp Thr Glu Leu Ser Tyr Pro Met Ile Ser Ala 130 135 140 Gly
Ser Phe Gly Leu Ser Cys Asp Tyr Lys Glu Thr Leu Thr Arg Leu 145 150
155 160 Met Ser Pro Ala Arg Lys Leu Met Tyr Phe Leu Val Asn Phe Trp
Lys 165 170 175 Thr Asn Asp Leu Pro Phe Lys Thr Tyr Ser Trp Ser Thr
Ser Tyr Val 180 185 190 Tyr Lys Asn Gly Thr Glu Thr Glu Asp Cys Phe
Trp Tyr Leu Asn Ala 195 200 205 Leu Glu Ala Ser Val Ser Tyr Phe Ser
His Glu Leu Gly Phe Lys Val 210 215 220 Val Leu Arg Gln Asp Lys Glu
Phe Gln Asp Ile Leu Met Asp His Asn 225 230 235 240 Arg Lys Ser Asn
Val Ile Ile Met Cys Gly Gly Pro Glu Phe Leu Tyr 245 250 255 Lys Leu
Lys Gly Asp Arg Ala Val Ala Glu Asp Ile Val Ile Ile Leu 260 265 270
Val Asp Leu Phe Asn Asp Gln Tyr Leu Glu Asp Asn Val Thr Ala Pro 275
280 285 Asp Tyr Met Lys Asn Val Leu Val Leu Thr Leu Ser Pro Gly Asn
Ser 290 295 300 Leu Leu Asn Ser Ser Phe Ser Arg Asn Leu Ser Pro Thr
Lys Arg Asp 305 310 315 320 Phe Ala Leu Ala Tyr Leu Asn Gly Ile Leu
Leu Phe Gly His Met Leu 325 330 335 Lys Ile Phe Leu Glu Asn Gly Glu
Asn Ile Thr Thr Pro Lys Phe Ala 340 345 350 His Ala Phe Arg Asn Leu
Thr Phe Glu Gly Tyr Asp Gly Pro Val Thr 355 360 365 Leu Asp Asp Trp
Gly Asp Val Asp Ser Thr Met Val Leu Leu Tyr Thr 370 375 380 Ser Val
Asp Thr Lys Lys Tyr Lys Val Leu Leu Thr Tyr Asp Thr His 385 390 395
400 Val Asn Lys Thr Tyr Pro Val Asp Met Ser Pro Thr Phe Thr Trp Lys
405 410 415 Asn Ser Lys Leu Pro Asn Asp Ile Thr Gly Arg Gly Pro Gln
Ile Leu 420 425 430 Met Ile Ala Val Phe Thr Leu Thr Gly Ala Val Val
Leu Leu Leu Leu 435 440 445 Val Ala Leu Leu Met Leu Arg Lys Tyr Arg
Lys Asp Tyr Glu Leu Arg 450 455 460 Gln Lys Lys Trp Ser His Ile Pro
Pro Glu Asn Ile Phe Pro Leu Glu 465 470 475 480 Thr Asn Glu Thr Asn
His Val Ser Leu Lys Ile Asp Asp Asp Lys Arg 485 490 495 Arg Asp Thr
Ile Gln Arg Leu Arg Gln Cys Lys Tyr Asp Lys Lys Arg 500 505 510 Val
Ile Leu Lys Asp Leu Lys His Asn Asp Gly Asn Phe Thr Glu Lys 515 520
525 Gln Lys Ile Glu Leu Asn Lys Leu Leu Gln Ile Asp Tyr Tyr Asn Leu
530 535 540 Thr Lys Phe Tyr Gly Thr Val Lys Leu Asp Thr Met Ile Phe
Gly Val 545 550 555 560 Ile Glu Tyr Cys Glu Arg Gly Ser Leu Arg Glu
Val Leu Asn Asp Thr 565 570 575 Ile Ser Tyr Pro Asp Gly Thr Phe Met
Asp Trp Glu Phe Lys Ile Ser 580 585 590 Val Leu Tyr Asp Ile Ala Lys
Gly Met Ser Tyr Leu His Ser Ser Lys 595 600 605 Thr Glu Val His Gly
Arg Leu Lys Ser Thr Asn Cys Val Val Asp Ser 610 615 620 Arg Met Val
Val Lys Ile Thr Asp Phe Gly Cys Asn Ser Ile Leu Pro 625 630 635 640
Pro Lys Lys Asp Leu Trp Thr Ala Pro Glu His Leu Arg Gln Ala Asn 645
650 655 Ile Ser Gln Lys Gly Asp Val Tyr Ser Tyr Gly Ile Ile Ala Gln
Glu 660 665 670 Ile Ile Leu Arg Lys Glu Thr Phe Tyr Thr Leu Ser Cys
Arg Asp Arg 675 680 685 Asn Glu Lys Ile Phe Arg Val Glu Asn Ser Asn
Gly Met Lys Pro Phe 690 695 700 Arg Pro Asp Leu Phe Leu Glu Thr Ala
Glu Glu Lys Glu Leu Glu Val 705 710 715 720 Tyr Leu Leu Val Lys Asn
Cys Trp Glu Glu Asp Pro Glu Lys Arg Pro 725 730 735 Asp Phe Lys Lys
Ile Glu Thr Thr Leu Ala Lys Ile Phe Gly Leu Phe 740 745 750 His Asp
Gln Lys Asn Glu Ser Tyr Met Asp Thr Leu Ile Arg Arg Leu 755 760 765
Gln Leu Tyr Ser Arg Asn Leu Glu His Leu Val Glu Glu Arg Thr Gln 770
775 780 Leu Tyr Lys Ala Glu Arg Asp Arg Ala Asp Arg Leu Asn Phe Met
Leu 785 790 795 800 Leu Pro Arg Leu Val Val Lys Ser Leu Lys Glu Lys
Gly Phe Val Glu 805 810 815 Pro Glu Leu Tyr Glu Glu Val Thr Ile Tyr
Phe Ser Asp Ile Val Gly 820 825 830 Phe Thr Thr Ile Cys Lys Tyr Ser
Thr Pro Met Glu Val Val Asp Met 835 840 845 Leu Asn Asp Ile Tyr Lys
Ser Phe Asp His Ile Val Asp His His Asp 850 855 860 Val Tyr Lys Val
Glu Thr Ile Gly Asp Ala Tyr Met Val Ala Ser Gly 865 870 875 880 Leu
Pro Lys Arg Asn Gly Asn Arg His Ala Ile Asp Ile Ala Lys Met 885 890
895 Ala Leu Glu Ile Leu Ser Phe Met Gly Thr Phe Glu Leu Glu His Leu
900 905 910 Pro Gly Leu Pro Ile Trp Ile Arg Ile Gly Val His Ser Gly
Pro Cys 915 920 925 Ala Ala Gly Val Val Gly Ile Lys Met Pro Arg Tyr
Cys Leu Phe Gly 930 935 940 Asp Thr Val Asn Thr Ala Ser Arg Met Glu
Ser Thr Gly Leu Pro Leu 945 950 955 960 Arg Ile His Val Ser Gly Ser
Thr Ile Ala Ile Leu Lys Arg Thr Glu 965 970 975 Cys Gln Phe Leu Tyr
Glu Val Arg Gly Glu Thr Tyr Leu Lys Gly Arg 980 985 990 Gly Asn Glu
Thr Thr Tyr Trp Leu Thr Gly Met Lys Asp Gln Lys Phe 995 1000 1005
Asn Leu Pro Thr Pro Pro Thr Val Glu Asn Gln Gln Arg Leu Gln 1010
1015 1020 Ala Glu Phe Ser Asp Met Ile Ala Asn Ser Leu Gln Lys Arg
Gln 1025 1030 1035 Ala Ala Gly Ile Arg Ser Gln Lys Pro Arg Arg Val
Ala Ser Tyr 1040 1045 1050 Lys Lys Gly Thr Leu Glu Tyr Leu Gln Leu
Asn Thr Thr Asp Lys 1055 1060 1065 Glu Ser Thr Tyr Phe 1070
12111PRTHomo Sapiens 12Met Lys Thr Leu Leu Leu Asp Leu Ala Leu Trp
Ser Leu Leu Phe Gln 1 5 10 15 Pro Gly Trp Leu Ser Phe Ser Ser Gln
Val Ser Gln Asn Cys His Asn 20 25 30 Gly Ser Tyr Glu Ile Ser Val
Leu Met Met Gly Asn Ser Ala Phe Ala 35 40 45 Glu Pro Leu Lys Asn
Leu Glu Asp Ala Val Asn Glu Gly Leu Glu Ile 50 55 60 Val Arg Gly
Arg Leu Gln Asn Ala Gly Leu Asn Val Thr Val Asn Ala 65 70 75 80 Thr
Phe Met Tyr Ser Asp Gly Leu Ile His Asn Ser Gly Asp Cys Arg 85 90
95 Ser Ser Thr Cys Glu Gly Leu Asp Leu Leu Arg Lys Ile Ser Pro 100
105 110 13258PRTHomo Sapiens 13Met Lys Thr Leu Leu Leu Asp Leu Ala
Leu Trp Ser Leu Leu Phe Gln 1 5 10 15 Pro Gly Trp Leu Ser Phe Ser
Ser Gln Val Ser Gln Asn Cys His Asn 20 25 30 Gly Ser Tyr Glu Ile
Ser Val Leu Met Met Gly Asn Ser Ala Phe Ala 35 40 45 Glu Pro Leu
Lys Asn Leu Glu Asp Ala Val Asn Glu Gly Leu Glu Ile 50 55 60 Val
Arg Gly Arg Leu Gln Asn Ala Gly Leu Asn Val Thr Val Asn Ala 65 70
75 80 Thr Phe Met Tyr Ser Asp Gly Leu Ile His Asn Ser Gly Asp Cys
Arg 85 90 95 Ser Ser Thr Cys Glu Gly Leu Asp Leu Leu Arg Lys Ile
Ser Asn Ala 100 105 110 Gln Arg Met Gly Cys Val Leu Ile Gly Pro Ser
Cys Thr Tyr Ser Thr 115 120 125 Phe Gln Met Tyr Leu Asp Thr Glu Leu
Ser Tyr Pro Met Ile Ser Ala 130 135 140 Gly Ser Phe Gly Leu Ser Cys
Asp Tyr Lys Glu Thr Leu Thr Arg Leu 145 150 155 160 Met Ser Pro Ala
Arg Lys Leu Met Tyr Phe Leu Val Asn Phe Trp Lys 165 170 175 Thr Asn
Asp Leu Pro Phe Lys Thr Tyr Ser Trp Ser Thr Ser Tyr Val 180 185 190
Tyr Lys Asn Gly Thr Glu Thr Glu Asp Cys Phe Trp Tyr Leu Asn Ala 195
200 205 Leu Glu Ala Ser Val Ser Tyr Phe Ser His Glu Leu Gly Phe Lys
Val 210 215 220 Val Leu Arg Gln Asp Lys Glu Phe Gln Asp Ile Leu Met
Asp His Asn 225 230 235 240 Arg Lys Ser Asn Val Thr Ser Thr Trp Arg
Thr Met Ser Gln Pro Leu 245 250 255 Thr Ile 141070PRTHomo Sapiens
14Met Lys Thr Leu Leu Leu Asp Leu Ala Leu Trp Ser Leu Leu Phe Gln 1
5 10 15 Pro Gly Trp Leu Ser Phe Ser Ser Gln Val Ser Gln Asn Cys His
Asn 20 25 30 Gly Ser Tyr Glu Ile Ser Val Leu Met Met Gly Asn Ser
Ala Phe Ala 35 40 45 Glu Pro Leu Lys Asn Leu Glu Asp Ala Val Asn
Glu Gly Leu Glu Ile 50 55 60 Val Arg Gly Arg Leu Gln Asn Ala Gly
Leu Asn Val Thr Val Asn Ala 65 70 75 80 Thr Phe Met Tyr Ser Asp Gly
Leu Ile His Asn Ser Gly Asp Cys Arg 85 90 95 Ser Ser Thr Cys Glu
Gly Leu Asp Leu Leu Arg Lys Ile Ser Asn Ala 100 105 110 Gln Arg Met
Gly Cys Val Leu Ile Gly Pro Ser Cys Thr Tyr Ser Thr 115 120 125 Phe
Gln Met Tyr Leu Asp Thr Glu Leu Ser Tyr Pro Met Ile Ser Ala 130 135
140 Gly Ser Phe Gly Leu Ser Cys Asp Tyr Lys Glu Thr Leu Thr Arg Leu
145 150 155 160 Met Ser Pro Ala Arg Lys Leu Met Tyr Phe Leu Val Asn
Phe Trp Lys 165 170 175 Thr Asn Asp Leu Pro Phe Lys Thr Tyr Ser Trp
Ser Thr Ser Tyr Val 180 185 190 Tyr Lys Asn Gly Thr Glu Thr Glu Asp
Cys Phe Trp Tyr Leu Asn Ala 195 200 205 Leu Glu Ala Ser Val Ser Tyr
Phe Ser His Glu Leu Gly Phe Lys Val 210 215 220 Val Leu Arg Gln Asp
Lys Glu Phe Gln Asp Ile Leu Met Asp His Asn 225 230 235 240 Arg Lys
Ser Asn Val Ile Ile Met Cys Gly Gly Pro Glu Phe Leu Tyr 245 250 255
Lys Leu Lys Gly Asp Arg Ala Val Ala Glu Asp Ile Val Ile Ile Leu 260
265 270 Val Asp Leu Phe Asn Asp Gln Tyr Leu Glu Asp Asn Val Thr Ala
Pro 275 280 285 Asp Tyr Met Lys Asn Val Leu Val Leu Thr Leu Ser Pro
Gly Asn Ser 290 295 300 Leu Leu Asn Ser Ser Phe Ser Arg Asn Leu Ser
Pro Thr Lys Arg Asp 305 310 315 320 Phe Ala Leu Ala Tyr Leu Asn Gly
Ile Leu Leu Phe Gly His Met Leu 325 330 335 Lys Ile Phe Leu Glu Asn
Gly Glu Asn Ile Thr Thr Pro Lys Phe Ala 340 345 350 His Ala Phe Arg
Asn Leu Thr Phe Glu Gly Tyr Asp Gly Pro Val Thr 355 360 365 Leu Asp
Asp Trp Gly Asp Val Asp Ser Thr Met Val Leu Leu Tyr Thr 370 375 380
Ser Val Asp Thr Lys Lys Tyr Lys Val Leu Leu Thr Tyr Asp Thr His 385
390 395 400 Val Asn Lys Thr Tyr Pro Val Asp Met Ser Pro Thr Phe Thr
Trp Lys 405 410 415 Asn Ser Lys Leu Pro Asn Asp Ile Thr Gly Arg Gly
Pro Gln Ile Leu 420 425 430 Met Ile Ala Val Phe Thr Leu Thr Gly Ala
Val Val Leu Leu Leu Leu 435 440 445 Val Ala Leu Leu Met Leu Arg Lys
Tyr Arg Lys Asp Tyr Glu Leu Arg 450 455 460 Gln Lys Lys Trp Ser His
Ile Pro Pro Glu Asn Ile Phe Pro Leu Glu 465 470 475 480 Thr Asn Glu
Thr Asn His Val Ser Leu Lys Ile Asp Asp Asp Lys Arg 485 490 495 Arg
Asp Thr Ile Gln Arg Leu Arg Gln Cys Lys Tyr Asp Lys Lys Arg 500 505
510 Val Ile Leu Lys Asp Leu Lys His Asn Asp Gly Asn Phe Thr Glu Lys
515 520 525 Gln Lys Ile Glu Leu Asn Lys Ile Asp Tyr Tyr Asn Leu Thr
Lys Phe 530 535 540 Tyr Gly Thr Val Lys Leu Asp Thr Met Ile Phe Gly
Val Ile Glu Tyr 545 550 555 560 Cys Glu Arg Gly Ser Leu Arg Glu Val
Leu Asn Asp Thr Ile Ser Tyr 565 570 575 Pro Asp Gly Thr Phe Met Asp
Trp Glu Phe Lys Ile Ser Val Leu Tyr 580 585 590 Asp Ile Ala Lys Gly
Met Ser Tyr Leu His Ser Ser Lys Thr Glu Val 595 600 605 His Gly Arg
Leu Lys Ser Thr Asn Cys Val Val Asp Ser Arg Met Val 610 615 620 Val
Lys Ile Thr Asp Phe Gly Cys Asn Ser Ile Leu Pro Pro Lys Lys 625 630
635 640 Asp Leu Trp Thr Ala Pro Glu His Leu Arg Gln Ala Asn Ile Ser
Gln 645 650 655 Lys Gly Asp Val Tyr Ser Tyr Gly Ile Ile Ala Gln Glu
Ile Ile Leu 660 665 670 Arg Lys Glu Thr Phe Tyr Thr Leu Ser Cys Arg
Asp Arg Asn Glu Lys 675 680 685 Ile Phe Arg Val Glu Asn Ser Asn Gly
Met Lys Pro Phe Arg Pro Asp 690 695 700 Leu Phe Leu Glu Thr Ala Glu
Glu Lys Glu Leu Glu Val Tyr Leu Leu 705
710 715 720 Val Lys Asn Cys Trp Glu Glu Asp Pro Glu Lys Arg Pro Asp
Phe Lys 725 730 735 Lys Ile Glu Thr Thr Leu Ala Lys Ile Phe Gly Leu
Phe His Asp Gln 740 745 750 Lys Asn Glu Ser Tyr Met Asp Thr Leu Ile
Arg Arg Leu Gln Leu Tyr 755 760 765 Ser Arg Asn Leu Glu His Leu Val
Glu Glu Arg Thr Gln Leu Tyr Lys 770 775 780 Ala Glu Arg Asp Arg Ala
Asp Arg Leu Asn Phe Met Leu Leu Pro Arg 785 790 795 800 Leu Val Val
Lys Ser Leu Lys Glu Lys Gly Phe Val Glu Pro Glu Leu 805 810 815 Tyr
Glu Glu Val Thr Ile Tyr Phe Ser Asp Ile Val Gly Phe Thr Thr 820 825
830 Ile Cys Lys Tyr Ser Thr Pro Met Glu Val Val Asp Met Leu Asn Asp
835 840 845 Ile Tyr Lys Ser Phe Asp His Ile Val Asp His His Asp Val
Tyr Lys 850 855 860 Val Glu Thr Ile Gly Asp Ala Tyr Met Val Ala Ser
Gly Leu Pro Lys 865 870 875 880 Arg Asn Gly Asn Arg His Ala Ile Asp
Ile Ala Lys Met Ala Leu Glu 885 890 895 Ile Leu Ser Phe Met Gly Thr
Phe Glu Leu Glu His Leu Pro Gly Leu 900 905 910 Pro Ile Trp Ile Arg
Ile Gly Val His Ser Gly Pro Cys Ala Ala Gly 915 920 925 Val Val Gly
Ile Lys Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr Val 930 935 940 Asn
Thr Ala Ser Arg Met Glu Ser Thr Gly Leu Pro Leu Arg Ile His 945 950
955 960 Val Ser Gly Ser Thr Ile Ala Ile Leu Lys Arg Thr Glu Cys Gln
Phe 965 970 975 Leu Tyr Glu Val Arg Gly Glu Thr Tyr Leu Lys Gly Arg
Gly Asn Glu 980 985 990 Thr Thr Tyr Trp Leu Thr Gly Met Lys Asp Gln
Lys Phe Asn Leu Pro 995 1000 1005 Thr Pro Pro Thr Val Glu Asn Gln
Gln Arg Leu Gln Ala Glu Phe 1010 1015 1020 Ser Asp Met Ile Ala Asn
Ser Leu Gln Lys Arg Gln Ala Ala Gly 1025 1030 1035 Ile Arg Ser Gln
Lys Pro Arg Arg Val Ala Ser Tyr Lys Lys Gly 1040 1045 1050 Thr Leu
Glu Tyr Leu Gln Leu Asn Thr Thr Asp Lys Glu Ser Thr 1055 1060 1065
Tyr Phe 1070 1593PRTHomo Sapiens 15Met Lys Leu Val Thr Ile Phe Leu
Leu Val Thr Ile Ser Leu Cys Ser 1 5 10 15 Tyr Ser Ala Thr Ala Lys
Leu Ile Asn Lys Cys Pro Leu Pro Val Asp 20 25 30 Lys Leu Ala Pro
Leu Pro Leu Asp Asn Ile Leu Pro Phe Met Asp Pro 35 40 45 Leu Lys
Leu Leu Leu Lys Thr Leu Gly Ile Ser Val Glu His Leu Val 50 55 60
Glu Gly Leu Arg Lys Cys Val Asn Glu Leu Gly Pro Glu Ala Ser Glu 65
70 75 80 Ala Val Lys Lys Leu Leu Glu Ala Leu Ser His Leu Val 85 90
16261PRTHomo Sapiens 16Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe
Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr
Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro
Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys
Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe
Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala
Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90
95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser
100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile
Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala
Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met
Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr
Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala
Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala
Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val
Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215
220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile
225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser
Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 1710PRTHomo Sapiens
17Asp Gln Trp Ser Thr Gln Asp Leu Tyr Asn 1 5 10 1811PRTHomo
Sapiens 18Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln 1 5 10
1947PRTHomo Sapiens 19Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe
Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr
Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro
Val Thr Ala Val Phe Asn Tyr Gln 35 40 45 2021DNAArtificial
SequenceOligonucleotide 20aggtacatga gcatcagcct g
212121DNAArtificial SequenceOligonucleotide 21gcagcagttg gcatctgaga
g 212221DNAArtificial SequenceOligonucleotide 22gcaatagaca
ttgccaagat g 212321DNAArtificial SequenceOligonucleotide
23aacgctgttg attctccaca g 212433DNAArtificial
SequenceOligonucleotide 24ggatcctcct ttagttccca ggtgagtcag aac
332521DNAArtificial SequenceOligonucleotide 25tgctctggag gctagcgttt
c 212621DNAArtificial SequenceOligonucleotide 26accaatcatg
ttagcctcaa g 212721DNAArtificial SequenceOligonucleotide
27agctatggga tcatcgcaca g 212821DNAArtificial
SequenceOligonucleotide 28cctttgagct ggagcatctt c
212921DNAArtificial SequenceOligonucleotide 29ctttctagct ggagacatca
g 213021DNAArtificial SequenceOligonucleotide 30caccatggta
ctgtcaacat c 213121DNAArtificial SequenceOligonucleotide
31atgtcataca agacagagat c 213221DNAArtificial
SequenceOligonucleotide 32tctgccttgt acagctgtgt c
213321DNAArtificial SequenceOligonucleotide 33tctgtggtat tcagctgcaa
g 213422DNAArtificial SequenceOligonucleotide 34tactcaggaa
aatttcacct tg 223527DNAArtificial SequenceOligonucleotide
35gaccacaaca ggaaaagcaa tgtgacc 273622DNAArtificial
SequenceOligonucleotide 36gatagaattg aacaagattg ac
223721DNAArtificial SequenceOligonucleotide 37cagcctttgt agttactctg
c 213821DNAArtificial SequenceOligonucleotide 38tgtcacacca
agtgtgatag c 213928DNAArtificial SequenceOligonucleotide
39ggttcgtggt ttcactgatt gggattgc 284027DNAArtificial
SequenceOligonucleotide 40cggctttgta gttggtttct tctggtg
27413814DNAHomo Sapiens 41ctattgaagc cacctgctca ggacaatgaa
attcttcagt tacattctgg tttatcgccg 60atttctcttc gtggttttca ctgtgttggt
tttactacct ctgcccatcg tcctccacac 120caaggaagca gaatgtgcct
acacactctt tgtggtcgcc acattttggc tcacagaagc 180attgcctctg
tcggtaacag ctttgctacc tagtttaatg ttacccatgt ttgggatcat
240gccttctaag aaggtggcat ctgcttattt caaggatttt cacttactgc
taattggagt 300tatctgttta gcaacatcca tagaaaaatg gaatttgcac
aagagaattg ctctgaaaat 360ggtgatgatg gttggtgtaa atcctgcatg
gctgacgctg gggttcatga gcagcactgc 420ctttttgtct atgtggctca
gcaacacctc gacggctgcc atggtgatgc ccattgcgga 480ggctgtagtg
cagcagatca tcaatgcaga agcagaggtc gaggccactc agatgactta
540cttcaacgga tcaaccaacc acggactaga aattgatgaa agtgttaatg
gacatgaaat 600aaatgagagg aaagagaaaa caaaaccagt tccaggatac
aataatgata cagggaaaat 660ttcaagcaag gtggagttgg aaaagaactc
aggcatgaga accaaatatc gaacaaagaa 720gggccacgtg acacgtaaac
ttacgtgttt gtgcattgcc tactcttcta ccattggtgg 780actgacaaca
atcactggta cctccaccaa cttgatcttt gcagagtatt tcaatacacg
840ctatcctgac tgtcgttgcc tcaactttgg atcatggttt acgttttcct
tcccagctgc 900ccttatcatt ctactcttat cctggatctg gcttcagtgg
cttttcctag gattcaattt 960taaggagatg ttcaaatgtg gcaaaaccaa
aacagtccaa caaaaagctt gtgctgaggt 1020gattaagcaa gaataccaaa
agcttgggcc aataaggtat caagaaattg tgaccttggt 1080cctcttcatt
ataatggctc tgctatggtt tagtcgagac cccggatttg ttcctggttg
1140gtctgcactt ttttcagagt accctggttt tgctacagat tcaactgttg
ctttacttat 1200agggctgcta ttctttctta tcccagctaa gacactgact
aaaactacac ctacaggaga 1260aattgttgct tttgattact ctccactgat
tacttggaaa gaattccagt cattcatgcc 1320ctgggatata gccattcttg
ttggtggagg gtttgccctg gcagatggtt gtgaggagtc 1380tggattatct
aagtggatag gaaataaatt atctcctctg ggttcattac cagcatggct
1440aataattctg atatcttctt tgatggtgac atctttaact gaggtagcca
gcaatccagc 1500taccattaca ctctttctcc caatattatc tccattggcc
gaagccattc atgtgaaccc 1560tctttatatt ctgatacctt ctactctgtg
tacttcattt gcattcctcc taccagtagc 1620aaatccaccc aatgctattg
tcttttcata tggtcatctg aaagtcattg acatggttaa 1680agctggactt
ggtgtcaaca ttgttggtgt tgctgtggtt atgcttggca tatgtacttg
1740gattgtaccc atgtttgacc tctacactta cccttcgtgg gctcctgcta
tgagtaatga 1800gaccatgcca taataagcac aaaatttctg actatcttgc
ggtaatttct ggaagacatt 1860aatgattgac tgtaaaatgt ggctctaaat
aactaatgac acacatttaa atcagttatg 1920gtgtagctgc tgcaattccc
gtgaataccc gaaacctgct ggtataactc agagtccata 1980tttgttattg
cagtgcaact aaagagcatc tatgtgcctt catcaagaag cccatgtttt
2040gagattttgc tcatgaacca tctgcaactt gcttcatcat aagaataatt
tataacttga 2100ccttcaaaga gattagagca tttgtttcat cttacagttg
gagttcaatg taacatttta 2160aatgcaattt attatttcag aaatttccca
tgaaactaaa aatagaaaat aagatataca 2220agttaattcg gtacttggat
aaatcatttc tgcattgttg ttccagagaa tttgctgaga 2280aatcaaagcc
atggtcatct ggtgatgaag agaaaaggtt aatctaaatg atatgtgcat
2340ttcctcattt aaaaaatcca attggattat tcttaatata tacatgtaat
atgaaaattg 2400agattgaagc actaattcca aaattatggc tgaatatact
aaataacaga aaagttacag 2460ataagaattt atttctactg aactctatag
ttagtgtaat ataattcata tttttatgat 2520attggcacac tgagaaattc
attttgtaga gctatggata aggcttgcta tgatttgcac 2580tattagtaca
gtatagttag aaaggaaagc tgaacactat aaaactatta acatattttc
2640gtatatgagt aacaactttg cttaagtgtt tatcttagtt cagaaataca
taatgtcata 2700tgttaaaaat aaagagatgt agaaatctaa atgaattatc
actgtgtata cagacagaaa 2760aatcacataa ctctggtgtg ttaacattgc
aatgaaaaaa tgaaaaaaag aaggaaaaaa 2820gaataagaat gaaaactgct
gacgtattac aaaacagaaa aataaatgat ttaaaatcaa 2880atcaaaaaga
aaaaaactaa acatttaaac aaaaatggga taagaatagt cttctagaag
2940tgaggatgcg taaaagaatg agtttccaat taccctgatg tgacaattac
acattgtaga 3000caggtagcaa aatatcacat acacccccaa aatatgtaca
aatattatat atcaataaat 3060aaatttttaa agagtaagtg ctattggcat
tccaaaattc agctaaagga aaaatgatca 3120aaaacaaagt aaggtgcaca
gttagcaaaa gatgcagatg ttatatcaca gcaattctca 3180tgctaaaaat
acaacaaaag acaaagcaaa aaataaacct ttgctttttt tttttttttt
3240tttttttttt gagacggagt ctcgctctgt cgcccaggct ggagtgcagt
ggcgggatct 3300cggctcactg caagctccgc ctcccaggtt cacgccattc
tcctgcctca gccaaacctt 3360tgctattttt aatcttcgtt ggcactttcc
agctgttact gaccttgtca ttttttgttc 3420aaataagatt atttacaaac
ttattcttga aactaaatat agtaaagagg gtttttaaaa 3480taatatttaa
catacgaatt attaattggc catgttcatt atttatctat gtttattaat
3540gggccaatgc aaaaaatcat tttttcaaag aaaaatttgt ccatgtaaag
cttaaattat 3600aatattgctg ctttgtataa ctcttctatg tttattctat
tcatttgttc ctttccctac 3660catattttac acatgtattt ataatctgta
gtatttatta catttctgct tttttctagt 3720cattcaattt atcactgctg
aattgcatca gatcatggat gcatttttat tatgaaaaaa 3780taaaatgact
tttcaaatta aaaaaaaaaa aaaa 381442734DNAHomo Sapiens 42caggacaatg
aaattcttca gttacattct ggtttatcgc cgatttctct tcgtggtttt 60cactgtgttg
gttttactac ctctgcccat cgtcctccac accaaggaag cagaatgtgc
120ctacacactc tttgtggtcg ccacattttg gctcacagaa gcattgcctc
tgtcggtaac 180agctttgcta cctagtttaa tgttacccat gtttgggatc
atgccttcta agaaggtggc 240atctgcttat ttcaaggatt ttcacttact
gctaattgga gttatctgtt tagcaacatc 300catagaaaaa tggaatttgc
acaagagaat tgctctgaaa atggtgatga tggttggtgt 360aaatcctgca
tggctgacgc tggggttcat gagcagcact gcctttttgt ctatgtggct
420cagcaacacc tcgacggctg ccatggtgat gcccattgcg gaggctgtag
tgcagcagat 480catcaatgca gaagcagagg tcgaggccac tcagatgact
tacttcaacg gatcaaccaa 540ccacggacta gaaattgatg aaagtgttaa
tggacatgaa ataaatgaga ggaaagagaa 600aacaaaacca gttccaggat
acaataatga tacagggaaa atttcaagca aggtggagtt 660ggaaaagact
gtttaactac tgaaatgaag ctattctcct gactaaacat aactgaaaaa
720ccattcatta aatg 73443539DNAHomo Sapiens 43gccactcaga tgacttactt
caacggatca accaaccacg gactagaaat tgatgaaagt 60gttaatggac atgaaataaa
tgagaggaaa gagaaaacaa aaccagttcc aggatacaat 120aatgatacag
ggaaaatttc aagcaaggtg gagttggaaa agcactggaa acttgcagtt
180caagatggct ccccatctcc ctctgtccat tctgtatcgc agctagctgc
tcaaggaaag 240gagaaagtgg aaggcatatg tacttagaaa ttattctatt
actttcctgg atttaagagt 300attcagattt tctatttcaa catcaaacaa
ttgcattttt aaaaagaaat ttatgtgttc 360catgtcaaat ttagtagtgt
gtggttgttt ataatatttt cttatatcta cttaatttct 420atagtattta
tagttatatg tctttatttc taacattttt cttgtgcttt taaagattat
480ttaaagatta tttttaaata atctttattt catttaaata aaatatttta tttaagtct
53944556DNAHomo Sapiens 44cacggactag aaattgatga aagtgttaat
ggacatgaaa taaatgagag gaaagagaaa 60acaaaaccag ttccaggata caataatgat
acagggaaaa tttcaagcaa ggtggagttg 120gaaaagaact caggcatgag
aaccaaatat cgaacaaaga agggccacgt gacacgtaaa 180cttacgtgtt
tgtgcattgc ctactcttct accattggtg gactgacaac aatcactggt
240acctccacca acttgatctt tgcagagtat ttcaatacat tccatccaca
cagaagagga 300gatcgtacaa ggcatgtaca ccaggaggca gaaatttgag
gcatatcttg gaactctgtc 360taccacatcc tgaacatcac acagtttcca
ctcttgttgc cttcaatcct gagaatgcat 420ccaggagcca ttctgtttta
tgtcaattac taattagatc atgtcacgtt actaacttac 480tacgttccaa
ttagtcctta ttgcatttgt aataaaatcc gcatactttc ggactggcta
540caaggttata catgat 55645595PRTHomo Sapiens 45Met Lys Phe Phe Ser
Tyr Ile Leu Val Tyr Arg Arg Phe Leu Phe Val 1 5 10 15 Val Phe Thr
Val Leu Val Leu Leu Pro Leu Pro Ile Val Leu His Thr 20 25 30 Lys
Glu Ala Glu Cys Ala Tyr Thr Leu Phe Val Val Ala Thr Phe Trp 35 40
45 Leu Thr Glu Ala Leu Pro Leu Ser Val Thr Ala Leu Leu Pro Ser Leu
50 55 60 Met Leu Pro Met Phe Gly Ile Met Pro Ser Lys Lys Val Ala
Ser Ala 65 70 75 80 Tyr Phe Lys Asp Phe His Leu Leu Leu Ile Gly Val
Ile Cys Leu Ala 85 90 95 Thr Ser Ile Glu Lys Trp Asn Leu His Lys
Arg Ile Ala Leu Lys Met 100 105 110 Val Met Met Val Gly Val Asn Pro
Ala Trp Leu Thr Leu Gly Phe Met 115 120 125 Ser Ser Thr Ala Phe Leu
Ser Met Trp Leu Ser Asn Thr Ser Thr Ala 130 135 140 Ala Met Val Met
Pro Ile Ala Glu Ala Val Val Gln Gln Ile Ile Asn 145 150 155 160 Ala
Glu Ala Glu Val Glu Ala Thr Gln Met Thr Tyr Phe Asn Gly Ser 165 170
175 Thr Asn His Gly Leu Glu Ile Asp Glu Ser Val Asn Gly His Glu Ile
180 185 190 Asn Glu Arg Lys Glu Lys Thr Lys Pro Val Pro Gly Tyr Asn
Asn Asp 195 200 205 Thr Gly Lys Ile Ser Ser Lys Val Glu Leu Glu Lys
Asn Ser Gly Met 210 215 220 Arg Thr Lys Tyr Arg Thr Lys Lys Gly His
Val Thr Arg Lys Leu Thr 225 230 235 240 Cys Leu Cys Ile Ala Tyr Ser
Ser Thr Ile Gly Gly Leu Thr Thr Ile 245 250 255 Thr Gly Thr Ser Thr
Asn Leu Ile Phe Ala Glu Tyr Phe Asn Thr Arg 260 265 270 Tyr Pro Asp
Cys Arg Cys Leu Asn Phe Gly Ser Trp Phe Thr Phe Ser 275 280 285
Phe Pro Ala Ala Leu Ile Ile Leu Leu Leu Ser Trp Ile Trp Leu Gln 290
295 300 Trp Leu Phe Leu Gly Phe Asn Phe Lys Glu Met Phe Lys Cys Gly
Lys 305 310 315 320 Thr Lys Thr Val Gln Gln Lys Ala Cys Ala Glu Val
Ile Lys Gln Glu 325 330 335 Tyr Gln Lys Leu Gly Pro Ile Arg Tyr Gln
Glu Ile Val Thr Leu Val 340 345 350 Leu Phe Ile Ile Met Ala Leu Leu
Trp Phe Ser Arg Asp Pro Gly Phe 355 360 365 Val Pro Gly Trp Ser Ala
Leu Phe Ser Glu Tyr Pro Gly Phe Ala Thr 370 375 380 Asp Ser Thr Val
Ala Leu Leu Ile Gly Leu Leu Phe Phe Leu Ile Pro 385 390 395 400 Ala
Lys Thr Leu Thr Lys Thr Thr Pro Thr Gly Glu Ile Val Ala Phe 405 410
415 Asp Tyr Ser Pro Leu Ile Thr Trp Lys Glu Phe Gln Ser Phe Met Pro
420 425 430 Trp Asp Ile Ala Ile Leu Val Gly Gly Gly Phe Ala Leu Ala
Asp Gly 435 440 445 Cys Glu Glu Ser Gly Leu Ser Lys Trp Ile Gly Asn
Lys Leu Ser Pro 450 455 460 Leu Gly Ser Leu Pro Ala Trp Leu Ile Ile
Leu Ile Ser Ser Leu Met 465 470 475 480 Val Thr Ser Leu Thr Glu Val
Ala Ser Asn Pro Ala Thr Ile Thr Leu 485 490 495 Phe Leu Pro Ile Leu
Ser Pro Leu Ala Glu Ala Ile His Val Asn Pro 500 505 510 Leu Tyr Ile
Leu Ile Pro Ser Thr Leu Cys Thr Ser Phe Ala Phe Leu 515 520 525 Leu
Pro Val Ala Asn Pro Pro Asn Ala Ile Val Phe Ser Tyr Gly His 530 535
540 Leu Lys Val Ile Asp Met Val Lys Ala Gly Leu Gly Val Asn Ile Val
545 550 555 560 Gly Val Ala Val Val Met Leu Gly Ile Cys Thr Trp Ile
Val Pro Met 565 570 575 Phe Asp Leu Tyr Thr Tyr Pro Ser Trp Ala Pro
Ala Met Ser Asn Glu 580 585 590 Thr Met Pro 595 46224PRTHomo
Sapiens 46Arg Thr Met Lys Phe Phe Ser Tyr Ile Leu Val Tyr Arg Arg
Phe Leu 1 5 10 15 Phe Val Val Phe Thr Val Leu Val Leu Leu Pro Leu
Pro Ile Val Leu 20 25 30 His Thr Lys Glu Ala Glu Cys Ala Tyr Thr
Leu Phe Val Val Ala Thr 35 40 45 Phe Trp Leu Thr Glu Ala Leu Pro
Leu Ser Val Thr Ala Leu Leu Pro 50 55 60 Ser Leu Met Leu Pro Met
Phe Gly Ile Met Pro Ser Lys Lys Val Ala 65 70 75 80 Ser Ala Tyr Phe
Lys Asp Phe His Leu Leu Leu Ile Gly Val Ile Cys 85 90 95 Leu Ala
Thr Ser Ile Glu Lys Trp Asn Leu His Lys Arg Ile Ala Leu 100 105 110
Lys Met Val Met Met Val Gly Val Asn Pro Ala Trp Leu Thr Leu Gly 115
120 125 Phe Met Ser Ser Thr Ala Phe Leu Ser Met Trp Leu Ser Asn Thr
Ser 130 135 140 Thr Ala Ala Met Val Met Pro Ile Ala Glu Ala Val Val
Gln Gln Ile 145 150 155 160 Ile Asn Ala Glu Ala Glu Val Glu Ala Thr
Gln Met Thr Tyr Phe Asn 165 170 175 Gly Ser Thr Asn His Gly Leu Glu
Ile Asp Glu Ser Val Asn Gly His 180 185 190 Glu Ile Asn Glu Arg Lys
Glu Lys Thr Lys Pro Val Pro Gly Tyr Asn 195 200 205 Asn Asp Thr Gly
Lys Ile Ser Ser Lys Val Glu Leu Glu Lys Thr Val 210 215 220
4788PRTHomo Sapiens 47Ala Thr Gln Met Thr Tyr Phe Asn Gly Ser Thr
Asn His Gly Leu Glu 1 5 10 15 Ile Asp Glu Ser Val Asn Gly His Glu
Ile Asn Glu Arg Lys Glu Lys 20 25 30 Thr Lys Pro Val Pro Gly Tyr
Asn Asn Asp Thr Gly Lys Ile Ser Ser 35 40 45 Lys Val Glu Leu Glu
Lys His Trp Lys Leu Ala Val Gln Asp Gly Ser 50 55 60 Pro Ser Pro
Ser Val His Ser Val Ser Gln Leu Ala Ala Gln Gly Lys 65 70 75 80 Glu
Lys Val Glu Gly Ile Cys Thr 85 48112PRTHomo Sapiens 48His Gly Leu
Glu Ile Asp Glu Ser Val Asn Gly His Glu Ile Asn Glu 1 5 10 15 Arg
Lys Glu Lys Thr Lys Pro Val Pro Gly Tyr Asn Asn Asp Thr Gly 20 25
30 Lys Ile Ser Ser Lys Val Glu Leu Glu Lys Asn Ser Gly Met Arg Thr
35 40 45 Lys Tyr Arg Thr Lys Lys Gly His Val Thr Arg Lys Leu Thr
Cys Leu 50 55 60 Cys Ile Ala Tyr Ser Ser Thr Ile Gly Gly Leu Thr
Thr Ile Thr Gly 65 70 75 80 Thr Ser Thr Asn Leu Ile Phe Ala Glu Tyr
Phe Asn Thr Phe His Pro 85 90 95 His Arg Arg Gly Asp Arg Thr Arg
His Val His Gln Glu Ala Glu Ile 100 105 110 4921DNAArtificial
SequenceOligonucleotide 49ccagctttaa ccatgtcaat g
215021DNAArtificial SequenceOligonucleotide 50cagatggttg tgaggagtct
g 21513311DNAHomo Sapiens 51tgctaatgct tttggtacaa atggatgtgg
aatataattg aatattttct tgtttaaggg 60gagcatgaag aggtgttgag gttatgtcaa
gcatctggca cagctgaagg cagatggaaa 120tatttacaag tacgcaattt
gagactaaga tattgttatc attctcctat tgaagacaag 180agcaatagta
aaacacatca ggtcaggggg ttaaagacct gtgataaacc acttccgata
240agttggaaac gtgtgtctat attttcatat ctgtatatat ataatggtaa
agaaagacac 300cttcgtaacc cgcattttcc aaagagagga atcacaggga
gatgtacagc aatggggcca 360tttaagagtt ctgtgttcat cttgattctt
caccttctag aaggggccct gagtaattca 420ctcattcagc tgaacaacaa
tggctatgaa ggcattgtcg ttgcaatcga ccccaatgtg 480ccagaagatg
aaacactcat tcaacaaata aaggacatgg tgacccaggc atctctgtat
540ctgtttgaag ctacaggaaa gcgattttat ttcaaaaatg ttgccatttt
gattcctgaa 600acatggaaga caaaggctga ctatgtgaga ccaaaacttg
agacctacaa aaatgctgat 660gttctggttg ctgagtctac tcctccaggt
aatgatgaac cctacactga gcagatgggc 720aactgtggag agaagggtga
aaggatccac ctcactcctg atttcattgc aggaaaaaag 780ttagctgaat
atggaccaca aggtaaggca tttgtccatg agtgggctca tctacgatgg
840ggagtatttg acgagtacaa taatgatgag aaattctact tatccaatgg
aagaatacaa 900gcagtaagat gttcagcagg tattactggt acaaatgtag
taaagaagtg tcagggaggc 960agctgttaca ccaaaagatg cacattcaat
aaagttacag gactctatga aaaaggatgt 1020gagtttgttc tccaatcccg
ccagacggag aaggcttcta taatgtttgc acaacatgtt 1080gattctatag
ttgaattctg tacagaacaa aaccacaaca aagaagctcc aaacaagcaa
1140aatcaaaaat gcaatctccg aagcacatgg gaagtgatcc gtgattctga
ggactttaag 1200aaaaccactc ctatgacaac acagccacca aatcccacct
tctcattgct gcagattgga 1260caaagaattg tgtgtttagt ccttgacaaa
tctggaagca tggcgactgg taaccgcctc 1320aatcgactga atcaagcagg
ccagcttttc ctgctgcaga cagttgagct ggggtcctgg 1380gttgggatgg
tgacatttga cagtgctgcc catgtacaaa gtgaactcat acagataaac
1440agtggcagtg acagggacac actcgccaaa agattacctg cagcagcttc
aggagggacg 1500tccatctgca gcgggcttcg atcggcattt actgtgatta
ggaagaaata tccaactgat 1560ggatctgaaa ttgtgctgct gacggatggg
gaagacaaca ctataagtgg gtgctttaac 1620gaggtcaaac aaagtggtgc
catcatccac acagtcgctt tggggccctc tgcagctcaa 1680gaactagagg
agctgtccaa aatgacagga ggtttacaga catatgcttc agatcaagtt
1740cagaacaatg gcctcattga tgcttttggg gccctttcat caggaaatgg
agctgtctct 1800cagcgctcca tccagcttga gagtaaggga ttaaccctcc
agaacagcca gtggatgaat 1860ggcacagtga tcgtggacag caccgtggga
aaggacactt tgtttcttat cacctggaca 1920acgcagcctc cccaaatcct
tctctgggat cccagtggac agaagcaagg tggctttgta 1980gtggacaaaa
acaccaaaat ggcctacctc caaatcccag gcattgctaa ggttggcact
2040tggaaataca gtctgcaagc aagctcacaa accttgaccc tgactgtcac
gtcccgtgcg 2100tccaatgcta ccctgcctcc aattacagtg acttccaaaa
cgaacaagga caccagcaaa 2160ttccccagcc ctctggtagt ttatgcaaat
attcgccaag gagcctcccc aattctcagg 2220gccagtgtca cagccctgat
tgaatcagtg aatggaaaaa cagttacctt ggaactactg 2280gataatggag
caggtgctga tgctactaag gatgacggtg tctactcaag gtatttcaca
2340acttatgaca cgaatggtag atacagtgta aaagtgcggg ctctgggagg
agttaacgca 2400gccagacgga gagtgatacc ccagcagagt ggagcactgt
acatacctgg ctggattgag 2460aatgatgaaa tacaatggaa tccaccaaga
cctgaaatta ataaggatga tgttcaacac 2520aagcaagtgt gtttcagcag
aacatcctcg ggaggctcat ttgtggcttc tgatgtccca 2580aatgctccca
tacctgatct cttcccacct ggccaaatca ccgacctgaa ggcggaaatt
2640cacgggggca gtctcattaa tctgacttgg acagctcctg gggatgatta
tgaccatgga 2700acagctcaca agtatatcat tcgaataagt acaagtattc
ttgatctcag agacaagttc 2760aatgaatctc ttcaagtgaa tactactgct
ctcatcccaa aggaagccaa ctctgaggaa 2820gtctttttgt ttaaaccaga
aaacattact tttgaaaatg gcacagatct tttcattgct 2880attcaggctg
ttgataaggt cgatctgaaa tcagaaatat ccaacattgc acgagtatct
2940ttgtttattc ctccacagac tccgccagag acacctagtc ctgatgaaac
gtctgctcct 3000tgtcctaata ttcatatcaa cagcaccatt cctggcattc
acattttaaa aattatgtgg 3060aagtggatag gagaactgca gctgtcaata
gcctagggct gaatttttgt cagataaata 3120aaataaatca ttcatccttt
ttttgattat aaaattttct aaaatgtatt ttagacttcc 3180tgtagggggc
gatatactaa atgtatatag tacatttata ctaaatgtat tcctgtaggg
3240ggcgatatac taaatgtatt ttagacttcc tgtagggggc gataaaataa
aatgctaaac 3300aactgggtaa a 3311523067DNAHomo Sapiens 52aattaaatta
tgagaattaa aaagacaaca ttgagcagag atgaaaaagg aagggaggaa 60aaggtggaaa
agaaaagaag acaagaagcg agtagtggtc tctaacttgc tctttgaagg
120atggtctcac aaagagaacc ccaacagaca tcatcgtggg aatcaaatca
agaccagcaa 180gtacaccgtg ttgtccttcg tccccaaaaa catttttgag
cagctacacc ggtttgccaa 240tctctatttt gtgggcattg cggttctgaa
ttttatccct gtggtcaatg ctttccagcc 300tgaggtgagc atgataccaa
tctgtgttat cctggcagtc actgccatca aggacgcttg 360ggaagacctc
cggaggtaca aatcggataa agtcatcaat aaccgagagt gcctcatcta
420cagcagaaaa gagcagacct atgtgcagaa gtgctggaag gatgtgcgtg
tgggagactt 480catccaaatg aaatgcaatg agattgtccc agcagacata
ctcctccttt tttcctctga 540ccccaatggg atatgccatc tggaaactgc
cagcttggat ggagagacaa acctcaagca 600aagacgtgtc gtgaagggct
tctcacagca ggaggtacag ttcgaaccag agcttttcca 660caataccatc
gtgtgtgaga aacccaacaa ccacctcaac aaatttaagg gttatatgga
720gcatcctgac cagaccagga ctggctttgg ctgtgagagt cttctgcttc
gaggctgcac 780catcagaaac accgagatgg ctgttggcat tgtcatctat
gcaggccatg agacgaaagc 840catgctgaac aacagtggcc cccggtacaa
acgcagcaag attgagcggc gcatgaatat 900agacatcttc ttctgcattg
ggatcctcat cctcatgtgc cttattggag ctgtaggtca 960cagcatctgg
aatgggacct ttgaagaaca ccctcccttc gatgtgccag atgccaatgg
1020cagcttcctt cccagtgccc ttgggggctt ctacatgttc ctcacaatga
tcatcctgct 1080ccaggtgctg atccccatct ctttgtatgt ctccattgag
ctggtgaagc tcgggcaagt 1140gttcttcttg agcaatgacc ttgacctgta
tgatgaagag accgatttat ccattcaatg 1200tcgagccctc aacatcgcag
aggacttggg ccagatccag tacatcttct ccgataagac 1260ggggaccctg
acagagaaca agatggtgtt ccgacgttgc accatcatgg gcagcgagta
1320ttctcaccaa gaaaatggta tagaagctcc caagggctcc atccctcttt
ctaaaaggaa 1380ataccctgct ctcctaagaa acgaggagat aaaagacatt
ctcctggctc tcttagaggc 1440tgtgtggcat ttccacaagt tgcttcctgt
atccctgtgg tcttccttgt cacagatcag 1500ggctgttcca attacttgta
aactttcatt tgtttacaaa ggttagaagt tatcccatat 1560gtggttcccc
ttcagctgat ctttgtctgg tgccagacaa agcactttat gagacgagtt
1620ttttatctgt cagcaatgga ttggagacat ttcccaattg tgtgccagtc
acacaaccaa 1680ggcttaggaa tttctcaggc caccttacct gacatgtcag
ggcaggtctg tgtctaggtg 1740catggtcaga tttaatacat ccagaagatg
tcttctattc taacagatct cttagcttgt 1800cactgaggca aagttttgat
ttaggagata gggctataaa atgcctggac tgttaccttg 1860catggactga
atatgactca taaaactgat ctgattcctt cagccatcat ctgcccaact
1920tggttcccct ccccaccccc ccacaacaca cacacacact ttctaagaaa
agaaaagaaa 1980ttcttttttt tcaatacttt aagttctggg atacatgtgc
agaatgtgca ggtttgttac 2040ataggtatac atgtgtcatg gtggtttgca
gcacccacca acccatcatc taccttaggt 2100atttctccta atgctatccc
tcccctagcc cccaaccccc cgatgggctc cagtgtgtga 2160tgttcccctc
catgtccatg tgttctcatt gttcaattcc cacttatgag tgagaacatg
2220cagtatttgg ttttctgttc ttgtgttagt ttgctgatgg tttcctgttc
atccgtgtcc 2280ctgcaaagga catgaactca tcctttttta tggctgcata
atattccatg gtgtatatgt 2340gccacatttt ctttatccag tctatcgctg
atgggcactg gggttggttc caagtctttg 2400ctattgtgaa cagtgctgca
ataaacttac atgtgcatgt gtctttagta gaatgattta 2460taatcctttg
ggtatatacc cagtaatggg attgctggtc aaatggtatt tctggttcta
2520gatccttgag gaatctttgt cttccacaat ggttgaacta atttgtactc
ccaccaacag 2580tgtaaaagta ttcctgtttc tctacatcct cttcagcatc
tgttgtgtcc tgacatttta 2640atgatcacta ttctcactgg cgtgagatgt
tatctcattg tggttttgat ttgcatttct 2700ctaatgacca gtaatgatga
gctttttttc atatgtttgt tggctgcata aatgtcttct 2760tttgagaagt
gtctgttcat atccttcacc cattttttga agaaaacaaa ctcttaagag
2820agcagtattc attcttttga gtgtgaggga tggagaaaga gaaagatgga
gagagtatta 2880taagcagctg tatccccttt gccatggtga tagcagacca
ttcacatggg agcttctggt 2940ctctttgtaa taataataag agccacatta
ccagtactta gagtatgcta gttattttaa 3000cacattgtat cattaaatct
tcaaaacatc cctatgagtt agaaacctaa aaaaaaaaaa 3060aaaaaaa
3067532778DNAHomo Sapiens 53ctcattttga tgtctagaat caggggatcc
aggatcatca ccaaggtcat tttcccaggt 60atggaggggt ctttctgctt ctttcttgtc
atgcacagct gctgaggaag gggctgggag 120taaagacagt gaaatgggga
ggaggagtcc attcaaaccg agaaacaaag tgtttggttt 180ttcttacccc
tggtgtagaa gctaccaacc ttttccaaga aagagggcct ggcccccttc
240tcgggtctgg ctgggtgcct gctgtgcctc tctggcctcc cctccgaagg
gcaccattcc 300ctcgggtgag tactaccggc ctgcaccgtc ttccagtggg
gacagcctga gaagagagtc 360tggggcctta cttcagtacc ttccttcact
ggcctcaccc tgtgcaaatc atgccacacg 420ctgcagcctc cttttcccta
tctataaaat aaaaatgacc ctgctctatc tcactgggct 480ggcaagaaca
cactgttgtt gccttgcaga cagatgtgct gaggctgtag aaagtgcttt
540ttatttggtt gggagcttgt gcataaatgc gagaggggct gcacatctga
cggactagag 600gtgactcatg gctgaaccgg aacaggacat cggggagaag
ccagcagcca tgctgaactc 660tccacagggc cctgtgaaaa gctcttcacc
tcctctgccc tctggatcta gtgaagccta 720ttcatccttc agatgtcagc
tcaaataatc aaccttcatg gaggcctccc ttgaccccta 780acatgctttc
aaagtactgt gtatttcaca ttcatcatgc cccgacaact gtgatttccc
840atttattaat atctgtctct tctgctggcc tgcaaactcc aggagcacag
agacatcttt 900gggatttttg aacatgattt ccccagggct tagcccagtg
cctggtgcaa agcaggcttt 960caacatgttc agtggatatt gtaagaaaga
aagaaataca caaaaggcct ggcatatgca 1020aagcactcta aatattcact
cctttccctt ccctctgggt gagaaaattt ctccttataa 1080agacaccctc
ctaactgtat ctctgctaga gaactgaaga cataaagcac tctgtgccaa
1140aaatatttaa gtaaaaactt gagctaagca cagagattat aaatatttct
tccccagatt 1200acgcaccatt taaaaatact gtctcagctc cttttcatga
tttgggtggt gattaaagaa 1260aattactctt caagactgaa agtcattact
gcccttttcc tgacttgcct tttcccttga 1320gaaggggagg ataagctgca
gggcaggaag tggaagtggg gcatccttgt cctttgtctg 1380gcagacagcc
aactggtcag gtactgctcc ttctcaactc tttcctgatt cccaggtgaa
1440tataaacaag aaggcacaaa tccacacttg ccaacaacgg acccaagtga
taacaagaaa 1500cccagtgaca cctgtctagg tgaagactca gcccctatgt
gaccaggttg caaagccaaa 1560ctgaccatct gctttccatt tggactttta
gttcatactg tatcttctca ggacagttaa 1620gttggaatac aatgccactg
tcctgaaaga tggtagaatt atcctatttc tggaggagtg 1680ggggtggtgg
gtaggaatct caagagcgat ttgctcctct gcacaatagc ttctttaagg
1740acaccagggc ccccagggct atacatttcc ctgaagcttt ccagataagc
aacaaggtat 1800gagcacctgc tatgtattgc ccaagggtga tgtgtttaaa
tatccattgc atattttaaa 1860tccttggctg gcttaaagct gcaagctttc
tgtcttcagt ggatataatg ggggcataca 1920tcccagagct tgcccaacac
tccaagaaaa gaaccctcag ctaatgcaaa gtgtgtatgt 1980gcccatgaaa
gctccatgtc tacttaacat tcagttttta ggattattta tgctgtaata
2040atagatatga aaatctctga caggtatttt gtttccttta caaactgtat
ttgaatttat 2100gggtgattta gagcttgtgt ttaaagtcag aattcagaac
cccaaagaaa atgacttcat 2160tgaaattgaa ctgaagagac aagaactgag
ttaccaaaac ctactaaacg tgagttgctg 2220tgaactgggg attaaaccag
aacgagtgga gaagatcaga aagctaccaa acacactgct 2280cagaaaggac
aaagacattc gaagactgcg ggactttcag gaagtggaac tcattttaat
2340gaaaaatgga agctccagat tgacagaata tgtgccatct ctgacagaaa
ggccctgcta 2400tgatagcaaa gctgcaaaaa tgacttatta aatactccca
ggaatggccg cgcatggtgg 2460ctcaccccct gtaatcccag cactttggga
agccaaggtg ggcggatcac ctgaggtcag 2520gagttctaga ccagcctggc
caacatatag tgaaacccag tctctactaa aaaaaataca 2580aaaattagct
aggtgtggtg gcgcacacct gtagtagtcc cagctacatg ggaagctgag
2640gcaggagaat cacctgaacc caggaggcag aggttgcagt gagctgagat
tgcgccactg 2700cactccagcc tggcgacaga gcaagactct gtctctcaaa
ataaataaat aaataaataa 2760ataaataaat aaataatc 2778541646DNAHomo
Sapiens 54gcccgggaga ggagaggagc gggccgagga ctccagcgtg cccaggtctg
gcatcctgca 60cttgctgccc tctgacacct gggaagatgg ccggcccgtg gaccttcacc
cttctctgtg 120gtttgctggc agccaccttg atccaagcca ccctcagtcc
cactgcagtt ctcatcctcg 180gcccaaaagt catcaaagaa aagctgacac
aggagctgaa ggaccacaac gccaccagca 240tcctgcagca gctgccgctg
ctcagtgcca tgcgggaaaa gccagccgga ggcatccctg 300tgctgggcag
cctggtgaac accgtcctga agcacatcat ctggctgaag gtcatcacag
360ctaacatcct ccagctgcag gtgaagccct cggccaatga ccaggagctg
ctagtcaaga 420tccccctgga catggtggct ggattcaaca cgcccctggt
caagaccatc gtggagttcc 480acatgacgac tgaggcccaa gccaccatcc
gcatggacac cagtgcaagt ggccccaccc 540gcctggtcct cagtgactgt
gccaccagcc atgggagcct gcgcatccaa ctgctgcata 600agctctcctt
cctggtgaac gccttagcta agcaggtcat
gaacctccta gtgccatccc 660tgcccaatct agtgaaaaac cagctgtgtc
ccgtgatcga ggcttccttc aatggcatgt 720atgcagacct cctgcagctg
gtgaaggtgc ccatttccct cagcattgac cgtctggagt 780ttgaccttct
gtatcctgcc atcaagggtg acaccattca gctctacctg ggggccaagt
840tgttggactc acagggaaag gtgaccaagt ggttcaataa ctctgcagct
tccctgacaa 900tgcccaccct ggacaacatc ccgttcagcc tcatcgtgag
tcaggacgtg gtgaaagctg 960cagtggctgc tgtgctctct ccagaagaat
tcatggtcct gttggactct gtgcttcctg 1020agagtgccca tcggctgaag
tcaagcatcg ggctgatcaa tgaaaaggct gcagataagc 1080tgggatctac
ccagatcgtg aagatcctaa ctcaggacac tcccgagttt tttatagacc
1140aaggccatgc caaggtggcc caactgatcg tgctggaagt gtttccctcc
agtgaagccc 1200tccgcccttt gttcaccctg ggcatcgaag ccagctcgga
agctcagttt tacaccaaag 1260gtgaccaact tatactcaac ttgaataaca
tcagctctga tcggatccag ctgatgaact 1320ctgggattgg ctggttccaa
cctgatgttc tgaaaaacat catcactgag atcatccact 1380ccatcctgct
gccgaaccag aatggcaaat taagatctgg ggtcccagtg tcattggtga
1440aggccttggg attcgaggca gctgagtcct cactgaccaa ggatgccctt
gtgcttactc 1500cagcctcctt gtggaaaccc agctctcctg tctcccagtg
aagacttgga tggcagccat 1560cagggaaggc tgggtcccag ctgggagtat
gggtgtgagc tctatagacc atccctctct 1620gcaatcaata aacacttgcc tgtgat
1646551049DNAHomo Sapiens 55ggagtggggg agagagagga gaccaggaca
gctgctgaga cctctaagaa gtccagatac 60taagagcaaa gatgtttcaa actgggggcc
tcattgtctt ctacgggctg ttagcccaga 120ccatggccca gtttggaggc
ctgcccgtgc ccctggacca gaccctgccc ttgaatgtga 180atccagccct
gcccttgagt cccacaggtc ttgcaggaag cttgacaaat gccctcagca
240atggcctgct gtctgggggc ctgttgggca ttctggaaaa ccttccgctc
ctggacatcc 300tgaagcctgg aggaggtact tctggtggcc tccttggggg
actgcttgga aaagtgacgt 360cagtgattcc tggcctgaac aacatcattg
acataaaggt cactgacccc cagctgctgg 420aacttggcct tgtgcagagc
cctgatggcc accgtctcta tgtcaccatc cctctcggca 480taaagctcca
agtgaatacg cccctggtcg gtgcaagtct gttgaggctg gctgtgaagc
540tggacatcac tgcagaaatc ttagctgtga gagataagca ggagaggatc
cacctggtcc 600ttggtgactg cacccattcc cctggaagcc tgcaaatttc
tctgcttgat ggacttggcc 660ccctccccat tcaaggtctt ctggacagcc
tcacagggat cttgaataaa gtcctgcctg 720agttggttca gggcaacgtg
tgccctctgg tcaatgaggt tctcagaggc ttggacatca 780ccctggtgca
tgacattgtt aacatgctga tccacggact acagtttgtc atcaaggtct
840aagccttcca ggaaggggct ggcctctgct gagctgcttc ccagtgctca
cagatggctg 900gcccatgtgc tggaagatga cacagttgcc ttctctccga
ggaacctgcc ccctctcctt 960tcccaccagg cgtgtgtaac atcccatgtg
cctcacctaa taaaatggct cttcttctgc 1020aaaaaaaaaa aaaaaaaaaa
aaaaaaaaa 1049564815DNAHomo Sapiens 56gagcagagcc ctttcacaca
cctcaggaac acctttcggc tgcccgctcc ccagacacac 60ctgcagccct gcccagccgg
ctttgctcac ccactgcttg taaatgcccc agatatgagc 120cagcccaggc
cccgctacgt ggtagacaga gccgcatact cccttaccct cttcgacgat
180gagtttgaga agaaggaccg gacataccca gtgggagaga aacttcgcaa
tgccttcaga 240tgttcctcag ccaagatcaa agctgtggtg tttgggctgc
tgcctgtgct ctcctggctc 300cccaagtaca agattaaaga ctacatcatt
cctgacctgc tcggtggact cagcggggga 360tccatccagg tcccacaagg
catggcattt gctctgctgg ccaaccttcc tgcagtcaat 420ggcctctact
cctccttctt ccccctcctg acctacttct tcctgggggg tgttcaccag
480atggtgccag gtacctttgc cgttatcagc atcctggtgg gtaacatctg
tctgcagctg 540gccccagagt cgaaattcca ggtcttcaac aatgccacca
atgagagcta tgtggacaca 600gcagccatgg aggctgagag gctgcacgtg
tcagctacgc tagcctgcct caccgccatc 660atccagatgg gtctgggctt
catgcagttt ggctttgtgg ccatctacct ctccgagtcc 720ttcatccggg
gcttcatgac ggccgccggc ctgcagatcc tgatttcggt gctcaagtac
780atcttcggac tgaccatccc ctcctacaca ggcccagggt ccatcgtctt
taccttcatt 840gacatttgca aaaacctccc ccacaccaac atcgcctcgc
tcatcttcgc tctcatcagc 900ggtgccttcc tggtgctggt gaaggagctc
aatgctcgct acatgcacaa gattcgcttc 960cccatcccta cagagatgat
tgtggtggtg gtggcaacag ctatctccgg gggctgtaag 1020atgcccaaaa
agtatcacat gcagatcgtg ggagaaatcc aacgcgggtt ccccaccccg
1080gtgtcgcctg tggtctcaca gtggaaggac atgataggca cagccttctc
cctagccatc 1140gtgagctacg tcatcaacct ggctatgggc cggaccctgg
ccaacaagca cggctacgac 1200gtggattcga accaggagat gatcgctctc
ggctgcagca acttctttgg ctccttcttt 1260aaaattcatg tcatttgctg
tgcgctttct gtcactctgg ctgtggatgg agctggagga 1320aaatcccagg
tggccagcct gtgtgtgtct ctggtggtga tgatcaccat gctggtcctg
1380gggatctatc tgtatcctct ccctaagtct gtgctaggag ccctgatcgc
tgtcaatctc 1440aagaactccc tcaagcaact caccgacccc tactacctgt
ggaggaagag caagctggac 1500tgttgcatct gggtagtgag cttcctctcc
tccttcttcc tcagcctgcc ctatggtgtg 1560gcagtgggtg tcgccttctc
cgtcctggtc gtggtcttcc agactcagtt tcgaaatggc 1620tatgcactgg
cccaggtcat ggacactgac atttatgtga atcccaagac ctataatagg
1680gcccaggata tccaggggat taaaatcatc acgtactgct cccctctcta
ctttgccaac 1740tcagagatct tcaggcaaaa ggtcatcgcc aagacaggca
tggaccccca gaaagtatta 1800ctagccaagc aaaaatacct caagaagcag
gagaagcgga gaatgaggcc cacacaacag 1860aggaggtctc tattcatgaa
aaccaagact gtctccctgc aggagctgca gcaggacttt 1920gagaatgcgc
cccccaccga ccccaacaac aaccagaccc cggctaacgg caccagcgtg
1980tcctatatca ccttcagccc tgacagctcc tcacctgccc agagtgagcc
accagcctcc 2040gctgaggccc ccggcgagcc cagtgacatg ctggccagcg
tcccaccctt cgtcaccttc 2100cacaccctca tcctggacat gagtggagtc
agcttcgtgg acttgatggg catcaaggcc 2160ctggccaagc tgagctccac
ctatgggaag atcggcgtga aggtcttctt ggtgaacatc 2220catgcccagg
tgtacaatga cattagccat ggaggcgtct ttgaggatgg gagtctagaa
2280tgcaagcacg tctttcccag catacatgac gcagtcctct ttgcccaggc
aaatgctaga 2340gacgtgaccc caggacacaa cttccaaggg gctccagggg
atgctgagct ctccttgtac 2400gactcagagg aggacattcg cagctactgg
gacttagagc aggagatgtt cgggagcatg 2460tttcacgcag agaccctgac
cgccctgtga gggctcagcc agtcctcatg ctgcctacag 2520agtgcctggc
acttgggact tccataaagg atgagcctgg ggtcacaggg ggtgtcgggc
2580ggaggaaagt gcatccccca gagcttgggt tcctctctcc tctccccctc
tctcctccct 2640tccttccctc cccgcatctc cagagagagc ctctcagcag
caggggggtg ctacccttac 2700gggagtgaga gtctggtgag cccactcttc
acccgtcagg ccctggccgc aatggacaag 2760cctcctgctc actccacccc
acccacatct gccctgtcct tggcagctga aggacacctt 2820gacttccagc
ttttacgagt gagccaaaaa cagaaggaca agtacaactg tgctggcctg
2880ctgtacaagc ttcaaaaagt gtcccagagc ccgcacggct cggtgtcaga
tggtgtcagg 2940ctgtcacgga catagggata aacttggtta ggactctggc
ttgccttccc cagctgcctc 3000aactctgtct ctggcagctc tgcacccagg
gaccatgtgc tctccacacc caggagtcta 3060ggccttggta actatgcgcc
ccccctccat catccccaag gctgcccaaa ccaccactgc 3120tgtcagcaag
cacatcagac tctagcctgg acagtggcca ggaccgtcga gaccaccaga
3180gctacctccc cggggacagc ccactaaggt tctgcctcag cctcctgaaa
catcactgcc 3240ctcagaggct gctcccttcc cctggaggct ggctagaaac
cccaaagagg gggatgggta 3300gctggcagaa tcatctggca tcctagtaat
agataccagt tattctgcac aaaacttttg 3360ggaattcctc tttgcaccca
gagactcaga ggggaagagg gtgctagtac caacacaggg 3420aaaacggatg
ggacctgggc ccagacagtc ccccttgacc ccagggccca tcagggaaat
3480gcctcccttt ggtaaatctg ccttatcctt ctttacctgg caaagagcca
atcatgttaa 3540ctcttcctta tcagcctgtg gcccagagac acaatggggt
ccttctgtag gcaaaggtgg 3600aagtcctcca gggatccgct acatccccta
actgcatgca gatgtggaaa ggggctgatc 3660cagattgggt cttcctgcac
aggaagactc tttaacaccc ttaggacctc aggccatctt 3720ctcctatgaa
gatgaaaata ggggttaagt tttccatatg tacaaggagg tattgagagg
3780aaccctactg ttgacttgaa aataaatagg ttccatgtgt aagtgttttg
taaaatttca 3840gtggaaatgc acagaaaatc ttctggcctc tcatcactgc
ttttctcaag cttcttcagc 3900ttaacaaccc cttccctaac aggttgggct
ggcccagcct aggaaaacat ccccatttct 3960aacttcagcc agacctgcgt
tgtgtgtctg tgtgttgagt gagctggtca gctaacaagt 4020cttcttagag
ttaaaggagg gggtgctggc caagagccaa cacattcttg gcccaggagc
4080attgcttttc tgtgaattca ttatgccatc tggctgccaa tggaactcaa
aacttggaag 4140gcgaaggaca atgttatctg ggattcaccg tgcccagcac
ccgaagtgcc aaattccagg 4200aggacaagag ccttagccaa tgacaactca
ctctccccta ctccacctcc ttccaagtcc 4260agctcaggcc caggaggtgg
gagaaggtca cagagcctca ggaatttcca agtcagagtc 4320ccctttgaac
caagtatcta gatcccctga ggacttgatg aagtgatcct taacccccaa
4380gtaatcatta acccccagac cagcctcaga actgaaggag attgttgacc
cagtgacctg 4440gagttgaggc tcagggagag atctgccaca tgtctgaggg
ttgcagagcc cgctgtggag 4500gtaagattgg aaacacatga ggcagaggga
agacattgaa gaaaacatct ctgctggaat 4560atttggaaaa gaacactctt
ctggacctgg ttgaagcagg aaagatggag gcaaagtagt 4620gaaataatcc
agaatttcaa tgcttttgaa tgttcttagt gatactgacc tgtgataata
4680taattcccag ggaggactgg gaaccttatc tcttgagata tttgcataat
ttatttaatt 4740taagcctcat tctccttttg ttcattttgg taataaactg
gatttgaatt gtgaacaaaa 4800aaaaaaaaaa aaaaa 4815572572DNAHomo
Sapiens 57aatgctctaa gacctctcag cacgggcgga agaaactccc ggagagctca
cccaaaaaac 60aaggagatcc catctagatt tcttcttgct tttgactcac agctggaagt
tagaaaagcc 120tcgatttcat ctttggagag gccaaatggt cttagcctca
gtctctgtct ctaaatattc 180caccataaaa cagctgagtt atttatgaat
tagaggctat agctcacatt ttcaatcctc 240tatttctttt tttaaatata
actttctact ctgatgagag aatgtggttt taatctctct 300ctcacatttt
gatgatttag acagactccc cctcttcctc ctagtcaata aacccattga
360tgatctattt cccagcttat ccccaagaaa acttttgaaa ggaaagagta
gacccaaaga 420tgttattttc tgctgtttga attttgtctc cccaccccca
acttggctag taataaacac 480ttactgaaga agaagcaata agagaaagat
atttgtaatc tctccagccc atgatctcgg 540ttttcttaca ctgtgatctt
aaaagttacc aaaccaaagt cattttcagt ttgaggcaac 600caaacctttc
tactgctgtt gacatcttct tattacagca acaccattct aggagtttcc
660tgagctctcc actggagtcc tctttctgtc gcgggtcaga aattgtccct
agatgaatga 720gaaaattatt ttttttaatt taagtcctaa atatagttaa
aataaataat gttttagtaa 780aatgatacac tatctctgtg aaatagcctc
acccctacat gtggatagaa ggaaatgaaa 840aaataattgc tttgacattg
tctatatggt actttgtaaa gtcatgctta agtacaaatt 900ccatgaaaag
ctcactgatc ctaattcttt ccctttgagg tctctatggc tctgattgta
960catgatagta agtgtaagcc atgtaaaaag taaataatgt ctgggcacag
tggctcacgc 1020ctgtaatcct agcactttgg gaggctgagg aggaaggatc
acttgagccc agaagttcga 1080gactagcctg ggcaacatgg agaagccctg
tctctacaaa atacagagag aaaaaatcag 1140ccagtcatgg tggcatacac
ctgtagtccc agcattccgg gaggctgagg tgggaggatc 1200acttgagccc
agggaggttg gggctgcagt gagccatgat cacaccactg cactccagcc
1260aggtgacata gcgagatcct gtctaaaaaa ataaaaaata aataatggaa
cacagcaagt 1320cctaggaagt aggttaaaac taattcttta aaaaaaaaaa
aaagttgagc ctgaattaaa 1380tgtaatgttt ccaagtgaca ggtatccaca
tttgcatggt tacaagccac tgccagttgg 1440cagtagcact ttcctggcac
tgtggtcggt tttgttttgt tttgctttgt ttagagacgg 1500ggtctcactt
tccaggctgg cctcaaactc ctgcactcaa gcaattcttc taccctggcc
1560tcccaagtag ctggaattac aggtgtgcgc catcacaact agctggtggt
cagttttgtt 1620actctgagag ctgttcactt ctctgaattc acctagagtg
gttggaccat cagatgtttg 1680ggcaaaactg aaagctcttt gcaaccacac
accttccctg agcttacatc actgcccttt 1740tgagcagaaa gtctaaattc
cttccaagac agtagaattc catcccagta ccaaagccag 1800ataggccccc
taggaaactg aggtaagagc agtctctaaa aactacccac agcagcattg
1860gtgcagggga acttggccat taggttatta tttgagagga aagtcctcac
atcaatagta 1920catatgaaag tgacctccaa ggggattggt gaatactcat
aaggatcttc aggctgaaca 1980gactatgtct ggggaaagaa cggattatgc
cccattaaat aacaagttgt gttcaagagt 2040cagagcagtg agctcagagg
cccttctcac tgagacagca acatttaaac caaaccagag 2100gaagtatttg
tggaactcac tgcctcagtt tgggtaaagg atgagcagac aagtcaacta
2160aagaaaaaag aaaagcaagg aggagggttg agcaatctag agcatggagt
ttgttaagtg 2220ctctctggat ttgagttgaa gagcatccat ttgagttgaa
ggccacaggg cacaatgagc 2280tctcccttct accaccagaa agtccctggt
caggtctcag gtagtgcggt gtggctcagc 2340tgggttttta attagcgcat
tctctatcca acatttaatt gtttgaaagc ctccatatag 2400ttagattgtg
ctttgtaatt ttgttgttgt tgctctatct tattgtatat gcattgagta
2460ttaacctgaa tgttttgtta cttaaatatt aaaaacactg ttatcctaca
aaaaaaccct 2520caaaggctga aaataaagaa ggaagatgga gacaccctct
gggggtcctc tc 2572581324DNAHomo Sapiens 58ctttgcagtg gatgcccttg
gcagggtgag cccacaagga gcaatggagc agggcagcgg 60ccgcttggag gacttccctg
tcaatgtgtt ctccgtcact ccttacacac ccagcaccgc 120tgacatccag
gtgtccgatg atgacaaggc gggggccacc ttgctcttct caggcatctt
180tctgggactg gtggggatca cattcactgt catgggctgg atcaaatacc
aaggtgtctc 240ccactttgaa tggacccagc tccttgggcc cgtcctgctg
tcagttgggg tgacattcat 300cctgattgct gtgtgcaagt tcaaaatgct
ctcctgccag ttgtgcaaag aaagtgagga 360aagggtcccg gactcggaac
agacaccagg aggaccatca tttgttttca ctggcatcaa 420ccaacccatc
accttccatg gggccactgt ggtgcagtac atccctcctc cttatggttc
480tccagagcct atggggataa ataccagcta cctgcagtct gtggtgagcc
cctgcggcct 540cataacctct ggaggggcag cagccgccat gtcaagtcct
cctcaatact acaccatcta 600ccctcaagat aactctgcat ttgtggttga
tgagggctgc ctttctttca cggacggtgg 660aaatcacagg cccaatcctg
atgttgacca gctagaagag acacagctgg aagaggaggc 720ctgtgcctgc
ttctctcctc ccccttatga agaaatatac tctctccctc gctagaggct
780attctgatat aataacacaa tgctcagctc agggagcaag tgtttccgtc
attgttacct 840gacaaccgtg gtgttctatg ttgtaacctt cagaagttac
agcagcgccc aggcagcctg 900acagagatca ttcaaggggg gaaaggggaa
gtgggaggtg caatttctca gattggtaaa 960aattaggctg ggctggggaa
attctcctcc ggaacagttt caaattccct cgggtaagaa 1020atctcctgta
taaggttcag gagcaggaat ttcacttttt catccaccac cctccccctt
1080ctctgtagga aggcattggt ggctcaattt taaccccagc agccaatgga
aaaatcacga 1140cttctgagac tttgggagtt tccacagagg tgagagtcgg
gtgggaagga agcagggaag 1200agaaagcagg cccagctgga gatttcctgg
tggctgtcct tggccccaaa gcagactcac 1260taatcccaaa caactcagct
gccatctggc ctctctgagg actctgggta ccttaaagac 1320tata
132459683DNAHomo Sapiens 59caggaaagtt cgtgctgcta ggcagaggaa
ctgcagcttg ttggcaggtg aagggagcct 60gtttagctgt gtccagcaac aacttacgtg
gtcctgcttg tgttccaggt gaagcgtctg 120gccgccgagc agaggaatca
agacctgctc attctttcct cgggggatcc atccagcaat 180gacatcatct
catgctgcca caaggacccc aagtctgggc tgctggggac cagccacgct
240ccccactgct cattccttca tcctagagac attctgactc tcctccgact
gcgctgtgca 300caggcgtgac aagctctttt acatctcagt ctgcacaact
tcaggcactt agcagattga 360tatgcatcca acaaatattg attgaatatc
tgctaaatac ccagtaatgt ttcatgagtg 420attgggtgaa taaaggaatg
ctggttcctt ctggccatat taactcctgc acaatactaa 480gaaaaataaa
ttgcactagc tgtggaataa tgtgaatccc aatgtcatct attgaaatat
540tacctgacta ttaagaggta tttatttttg tatcttttct agcaaagtaa
ataaaattct 600taatacagca tatcccctta ttcacggggg gtatgttcca
agacccccgg tggatgcctg 660aaactatgga taataccaga tcc 68360914PRTHomo
Sapiens 60Met Gly Pro Phe Lys Ser Ser Val Phe Ile Leu Ile Leu His
Leu Leu 1 5 10 15 Glu Gly Ala Leu Ser Asn Ser Leu Ile Gln Leu Asn
Asn Asn Gly Tyr 20 25 30 Glu Gly Ile Val Val Ala Ile Asp Pro Asn
Val Pro Glu Asp Glu Thr 35 40 45 Leu Ile Gln Gln Ile Lys Asp Met
Val Thr Gln Ala Ser Leu Tyr Leu 50 55 60 Phe Glu Ala Thr Gly Lys
Arg Phe Tyr Phe Lys Asn Val Ala Ile Leu 65 70 75 80 Ile Pro Glu Thr
Trp Lys Thr Lys Ala Asp Tyr Val Arg Pro Lys Leu 85 90 95 Glu Thr
Tyr Lys Asn Ala Asp Val Leu Val Ala Glu Ser Thr Pro Pro 100 105 110
Gly Asn Asp Glu Pro Tyr Thr Glu Gln Met Gly Asn Cys Gly Glu Lys 115
120 125 Gly Glu Arg Ile His Leu Thr Pro Asp Phe Ile Ala Gly Lys Lys
Leu 130 135 140 Ala Glu Tyr Gly Pro Gln Gly Lys Ala Phe Val His Glu
Trp Ala His 145 150 155 160 Leu Arg Trp Gly Val Phe Asp Glu Tyr Asn
Asn Asp Glu Lys Phe Tyr 165 170 175 Leu Ser Asn Gly Arg Ile Gln Ala
Val Arg Cys Ser Ala Gly Ile Thr 180 185 190 Gly Thr Asn Val Val Lys
Lys Cys Gln Gly Gly Ser Cys Tyr Thr Lys 195 200 205 Arg Cys Thr Phe
Asn Lys Val Thr Gly Leu Tyr Glu Lys Gly Cys Glu 210 215 220 Phe Val
Leu Gln Ser Arg Gln Thr Glu Lys Ala Ser Ile Met Phe Ala 225 230 235
240 Gln His Val Asp Ser Ile Val Glu Phe Cys Thr Glu Gln Asn His Asn
245 250 255 Lys Glu Ala Pro Asn Lys Gln Asn Gln Lys Cys Asn Leu Arg
Ser Thr 260 265 270 Trp Glu Val Ile Arg Asp Ser Glu Asp Phe Lys Lys
Thr Thr Pro Met 275 280 285 Thr Thr Gln Pro Pro Asn Pro Thr Phe Ser
Leu Leu Gln Ile Gly Gln 290 295 300 Arg Ile Val Cys Leu Val Leu Asp
Lys Ser Gly Ser Met Ala Thr Gly 305 310 315 320 Asn Arg Leu Asn Arg
Leu Asn Gln Ala Gly Gln Leu Phe Leu Leu Gln 325 330 335 Thr Val Glu
Leu Gly Ser Trp Val Gly Met Val Thr Phe Asp Ser Ala 340 345 350 Ala
His Val Gln Ser Glu Leu Ile Gln Ile Asn Ser Gly Ser Asp Arg 355 360
365 Asp Thr Leu Ala Lys Arg Leu Pro Ala Ala Ala Ser Gly Gly Thr Ser
370 375 380 Ile Cys Ser Gly Leu Arg Ser Ala Phe Thr Val Ile Arg Lys
Lys Tyr 385 390 395 400 Pro Thr Asp Gly Ser Glu Ile Val Leu Leu Thr
Asp Gly Glu Asp Asn 405 410 415 Thr Ile Ser Gly Cys Phe Asn Glu Val
Lys Gln Ser Gly Ala Ile Ile 420 425 430 His Thr Val Ala Leu Gly Pro
Ser Ala Ala Gln Glu Leu Glu Glu Leu 435 440 445 Ser Lys Met Thr Gly
Gly Leu Gln Thr Tyr Ala Ser Asp Gln Val Gln 450 455 460 Asn Asn Gly
Leu Ile Asp Ala Phe Gly Ala Leu Ser Ser Gly Asn Gly 465 470 475 480
Ala Val Ser Gln Arg Ser Ile Gln Leu Glu Ser Lys Gly Leu Thr Leu 485
490 495 Gln Asn Ser Gln Trp Met Asn Gly Thr Val Ile Val Asp Ser
Thr
Val 500 505 510 Gly Lys Asp Thr Leu Phe Leu Ile Thr Trp Thr Thr Gln
Pro Pro Gln 515 520 525 Ile Leu Leu Trp Asp Pro Ser Gly Gln Lys Gln
Gly Gly Phe Val Val 530 535 540 Asp Lys Asn Thr Lys Met Ala Tyr Leu
Gln Ile Pro Gly Ile Ala Lys 545 550 555 560 Val Gly Thr Trp Lys Tyr
Ser Leu Gln Ala Ser Ser Gln Thr Leu Thr 565 570 575 Leu Thr Val Thr
Ser Arg Ala Ser Asn Ala Thr Leu Pro Pro Ile Thr 580 585 590 Val Thr
Ser Lys Thr Asn Lys Asp Thr Ser Lys Phe Pro Ser Pro Leu 595 600 605
Val Val Tyr Ala Asn Ile Arg Gln Gly Ala Ser Pro Ile Leu Arg Ala 610
615 620 Ser Val Thr Ala Leu Ile Glu Ser Val Asn Gly Lys Thr Val Thr
Leu 625 630 635 640 Glu Leu Leu Asp Asn Gly Ala Gly Ala Asp Ala Thr
Lys Asp Asp Gly 645 650 655 Val Tyr Ser Arg Tyr Phe Thr Thr Tyr Asp
Thr Asn Gly Arg Tyr Ser 660 665 670 Val Lys Val Arg Ala Leu Gly Gly
Val Asn Ala Ala Arg Arg Arg Val 675 680 685 Ile Pro Gln Gln Ser Gly
Ala Leu Tyr Ile Pro Gly Trp Ile Glu Asn 690 695 700 Asp Glu Ile Gln
Trp Asn Pro Pro Arg Pro Glu Ile Asn Lys Asp Asp 705 710 715 720 Val
Gln His Lys Gln Val Cys Phe Ser Arg Thr Ser Ser Gly Gly Ser 725 730
735 Phe Val Ala Ser Asp Val Pro Asn Ala Pro Ile Pro Asp Leu Phe Pro
740 745 750 Pro Gly Gln Ile Thr Asp Leu Lys Ala Glu Ile His Gly Gly
Ser Leu 755 760 765 Ile Asn Leu Thr Trp Thr Ala Pro Gly Asp Asp Tyr
Asp His Gly Thr 770 775 780 Ala His Lys Tyr Ile Ile Arg Ile Ser Thr
Ser Ile Leu Asp Leu Arg 785 790 795 800 Asp Lys Phe Asn Glu Ser Leu
Gln Val Asn Thr Thr Ala Leu Ile Pro 805 810 815 Lys Glu Ala Asn Ser
Glu Glu Val Phe Leu Phe Lys Pro Glu Asn Ile 820 825 830 Thr Phe Glu
Asn Gly Thr Asp Leu Phe Ile Ala Ile Gln Ala Val Asp 835 840 845 Lys
Val Asp Leu Lys Ser Glu Ile Ser Asn Ile Ala Arg Val Ser Leu 850 855
860 Phe Ile Pro Pro Gln Thr Pro Pro Glu Thr Pro Ser Pro Asp Glu Thr
865 870 875 880 Ser Ala Pro Cys Pro Asn Ile His Ile Asn Ser Thr Ile
Pro Gly Ile 885 890 895 His Ile Leu Lys Ile Met Trp Lys Trp Ile Gly
Glu Leu Gln Leu Ser 900 905 910 Ile Ala 61501PRTHomo Sapiens 61Met
Lys Lys Glu Gly Arg Lys Arg Trp Lys Arg Lys Glu Asp Lys Lys 1 5 10
15 Arg Val Val Val Ser Asn Leu Leu Phe Glu Gly Trp Ser His Lys Glu
20 25 30 Asn Pro Asn Arg His His Arg Gly Asn Gln Ile Lys Thr Ser
Lys Tyr 35 40 45 Thr Val Leu Ser Phe Val Pro Lys Asn Ile Phe Glu
Gln Leu His Arg 50 55 60 Phe Ala Asn Leu Tyr Phe Val Gly Ile Ala
Val Leu Asn Phe Ile Pro 65 70 75 80 Val Val Asn Ala Phe Gln Pro Glu
Val Ser Met Ile Pro Ile Cys Val 85 90 95 Ile Leu Ala Val Thr Ala
Ile Lys Asp Ala Trp Glu Asp Leu Arg Arg 100 105 110 Tyr Lys Ser Asp
Lys Val Ile Asn Asn Arg Glu Cys Leu Ile Tyr Ser 115 120 125 Arg Lys
Glu Gln Thr Tyr Val Gln Lys Cys Trp Lys Asp Val Arg Val 130 135 140
Gly Asp Phe Ile Gln Met Lys Cys Asn Glu Ile Val Pro Ala Asp Ile 145
150 155 160 Leu Leu Leu Phe Ser Ser Asp Pro Asn Gly Ile Cys His Leu
Glu Thr 165 170 175 Ala Ser Leu Asp Gly Glu Thr Asn Leu Lys Gln Arg
Arg Val Val Lys 180 185 190 Gly Phe Ser Gln Gln Glu Val Gln Phe Glu
Pro Glu Leu Phe His Asn 195 200 205 Thr Ile Val Cys Glu Lys Pro Asn
Asn His Leu Asn Lys Phe Lys Gly 210 215 220 Tyr Met Glu His Pro Asp
Gln Thr Arg Thr Gly Phe Gly Cys Glu Ser 225 230 235 240 Leu Leu Leu
Arg Gly Cys Thr Ile Arg Asn Thr Glu Met Ala Val Gly 245 250 255 Ile
Val Ile Tyr Ala Gly His Glu Thr Lys Ala Met Leu Asn Asn Ser 260 265
270 Gly Pro Arg Tyr Lys Arg Ser Lys Ile Glu Arg Arg Met Asn Ile Asp
275 280 285 Ile Phe Phe Cys Ile Gly Ile Leu Ile Leu Met Cys Leu Ile
Gly Ala 290 295 300 Val Gly His Ser Ile Trp Asn Gly Thr Phe Glu Glu
His Pro Pro Phe 305 310 315 320 Asp Val Pro Asp Ala Asn Gly Ser Phe
Leu Pro Ser Ala Leu Gly Gly 325 330 335 Phe Tyr Met Phe Leu Thr Met
Ile Ile Leu Leu Gln Val Leu Ile Pro 340 345 350 Ile Ser Leu Tyr Val
Ser Ile Glu Leu Val Lys Leu Gly Gln Val Phe 355 360 365 Phe Leu Ser
Asn Asp Leu Asp Leu Tyr Asp Glu Glu Thr Asp Leu Ser 370 375 380 Ile
Gln Cys Arg Ala Leu Asn Ile Ala Glu Asp Leu Gly Gln Ile Gln 385 390
395 400 Tyr Ile Phe Ser Asp Lys Thr Gly Thr Leu Thr Glu Asn Lys Met
Val 405 410 415 Phe Arg Arg Cys Thr Ile Met Gly Ser Glu Tyr Ser His
Gln Glu Asn 420 425 430 Gly Ile Glu Ala Pro Lys Gly Ser Ile Pro Leu
Ser Lys Arg Lys Tyr 435 440 445 Pro Ala Leu Leu Arg Asn Glu Glu Ile
Lys Asp Ile Leu Leu Ala Leu 450 455 460 Leu Glu Ala Val Trp His Phe
His Lys Leu Leu Pro Val Ser Leu Trp 465 470 475 480 Ser Ser Leu Ser
Gln Ile Arg Ala Val Pro Ile Thr Cys Lys Leu Ser 485 490 495 Phe Val
Tyr Lys Gly 500 62154PRTHomo Sapiens 62Met Gly Arg Arg Ser Pro Phe
Lys Pro Arg Asn Lys Val Phe Gly Phe 1 5 10 15 Ser Tyr Pro Trp Cys
Arg Ser Tyr Gln Pro Phe Pro Arg Lys Arg Ala 20 25 30 Trp Pro Pro
Ser Arg Val Trp Leu Gly Ala Cys Cys Ala Ser Leu Ala 35 40 45 Ser
Pro Pro Lys Gly Thr Ile Pro Ser Gly Glu Tyr Tyr Arg Pro Ala 50 55
60 Pro Ser Ser Ser Gly Asp Ser Leu Arg Arg Glu Ser Gly Ala Leu Leu
65 70 75 80 Gln Tyr Leu Pro Ser Leu Ala Ser Pro Cys Ala Asn His Ala
Thr Arg 85 90 95 Cys Ser Leu Leu Phe Pro Ile Tyr Lys Ile Lys Met
Thr Leu Leu Tyr 100 105 110 Leu Thr Gly Leu Ala Arg Thr His Cys Cys
Cys Leu Ala Asp Arg Cys 115 120 125 Ala Glu Ala Val Glu Ser Ala Phe
Tyr Leu Val Gly Ser Leu Cys Ile 130 135 140 Asn Ala Arg Gly Ala Ala
His Leu Thr Asp 145 150 63484PRTHomo Sapiens 63Met Ala Gly Pro Trp
Thr Phe Thr Leu Leu Cys Gly Leu Leu Ala Ala 1 5 10 15 Thr Leu Ile
Gln Ala Thr Leu Ser Pro Thr Ala Val Leu Ile Leu Gly 20 25 30 Pro
Lys Val Ile Lys Glu Lys Leu Thr Gln Glu Leu Lys Asp His Asn 35 40
45 Ala Thr Ser Ile Leu Gln Gln Leu Pro Leu Leu Ser Ala Met Arg Glu
50 55 60 Lys Pro Ala Gly Gly Ile Pro Val Leu Gly Ser Leu Val Asn
Thr Val 65 70 75 80 Leu Lys His Ile Ile Trp Leu Lys Val Ile Thr Ala
Asn Ile Leu Gln 85 90 95 Leu Gln Val Lys Pro Ser Ala Asn Asp Gln
Glu Leu Leu Val Lys Ile 100 105 110 Pro Leu Asp Met Val Ala Gly Phe
Asn Thr Pro Leu Val Lys Thr Ile 115 120 125 Val Glu Phe His Met Thr
Thr Glu Ala Gln Ala Thr Ile Arg Met Asp 130 135 140 Thr Ser Ala Ser
Gly Pro Thr Arg Leu Val Leu Ser Asp Cys Ala Thr 145 150 155 160 Ser
His Gly Ser Leu Arg Ile Gln Leu Leu His Lys Leu Ser Phe Leu 165 170
175 Val Asn Ala Leu Ala Lys Gln Val Met Asn Leu Leu Val Pro Ser Leu
180 185 190 Pro Asn Leu Val Lys Asn Gln Leu Cys Pro Val Ile Glu Ala
Ser Phe 195 200 205 Asn Gly Met Tyr Ala Asp Leu Leu Gln Leu Val Lys
Val Pro Ile Ser 210 215 220 Leu Ser Ile Asp Arg Leu Glu Phe Asp Leu
Leu Tyr Pro Ala Ile Lys 225 230 235 240 Gly Asp Thr Ile Gln Leu Tyr
Leu Gly Ala Lys Leu Leu Asp Ser Gln 245 250 255 Gly Lys Val Thr Lys
Trp Phe Asn Asn Ser Ala Ala Ser Leu Thr Met 260 265 270 Pro Thr Leu
Asp Asn Ile Pro Phe Ser Leu Ile Val Ser Gln Asp Val 275 280 285 Val
Lys Ala Ala Val Ala Ala Val Leu Ser Pro Glu Glu Phe Met Val 290 295
300 Leu Leu Asp Ser Val Leu Pro Glu Ser Ala His Arg Leu Lys Ser Ser
305 310 315 320 Ile Gly Leu Ile Asn Glu Lys Ala Ala Asp Lys Leu Gly
Ser Thr Gln 325 330 335 Ile Val Lys Ile Leu Thr Gln Asp Thr Pro Glu
Phe Phe Ile Asp Gln 340 345 350 Gly His Ala Lys Val Ala Gln Leu Ile
Val Leu Glu Val Phe Pro Ser 355 360 365 Ser Glu Ala Leu Arg Pro Leu
Phe Thr Leu Gly Ile Glu Ala Ser Ser 370 375 380 Glu Ala Gln Phe Tyr
Thr Lys Gly Asp Gln Leu Ile Leu Asn Leu Asn 385 390 395 400 Asn Ile
Ser Ser Asp Arg Ile Gln Leu Met Asn Ser Gly Ile Gly Trp 405 410 415
Phe Gln Pro Asp Val Leu Lys Asn Ile Ile Thr Glu Ile Ile His Ser 420
425 430 Ile Leu Leu Pro Asn Gln Asn Gly Lys Leu Arg Ser Gly Val Pro
Val 435 440 445 Ser Leu Val Lys Ala Leu Gly Phe Glu Ala Ala Glu Ser
Ser Leu Thr 450 455 460 Lys Asp Ala Leu Val Leu Thr Pro Ala Ser Leu
Trp Lys Pro Ser Ser 465 470 475 480 Pro Val Ser Gln 64256PRTHomo
Sapiens 64Met Phe Gln Thr Gly Gly Leu Ile Val Phe Tyr Gly Leu Leu
Ala Gln 1 5 10 15 Thr Met Ala Gln Phe Gly Gly Leu Pro Val Pro Leu
Asp Gln Thr Leu 20 25 30 Pro Leu Asn Val Asn Pro Ala Leu Pro Leu
Ser Pro Thr Gly Leu Ala 35 40 45 Gly Ser Leu Thr Asn Ala Leu Ser
Asn Gly Leu Leu Ser Gly Gly Leu 50 55 60 Leu Gly Ile Leu Glu Asn
Leu Pro Leu Leu Asp Ile Leu Lys Pro Gly 65 70 75 80 Gly Gly Thr Ser
Gly Gly Leu Leu Gly Gly Leu Leu Gly Lys Val Thr 85 90 95 Ser Val
Ile Pro Gly Leu Asn Asn Ile Ile Asp Ile Lys Val Thr Asp 100 105 110
Pro Gln Leu Leu Glu Leu Gly Leu Val Gln Ser Pro Asp Gly His Arg 115
120 125 Leu Tyr Val Thr Ile Pro Leu Gly Ile Lys Leu Gln Val Asn Thr
Pro 130 135 140 Leu Val Gly Ala Ser Leu Leu Arg Leu Ala Val Lys Leu
Asp Ile Thr 145 150 155 160 Ala Glu Ile Leu Ala Val Arg Asp Lys Gln
Glu Arg Ile His Leu Val 165 170 175 Leu Gly Asp Cys Thr His Ser Pro
Gly Ser Leu Gln Ile Ser Leu Leu 180 185 190 Asp Gly Leu Gly Pro Leu
Pro Ile Gln Gly Leu Leu Asp Ser Leu Thr 195 200 205 Gly Ile Leu Asn
Lys Val Leu Pro Glu Leu Val Gln Gly Asn Val Cys 210 215 220 Pro Leu
Val Asn Glu Val Leu Arg Gly Leu Asp Ile Thr Leu Val His 225 230 235
240 Asp Ile Val Asn Met Leu Ile His Gly Leu Gln Phe Val Ile Lys Val
245 250 255 65791PRTHomo Sapiens 65Met Ser Gln Pro Arg Pro Arg Tyr
Val Val Asp Arg Ala Ala Tyr Ser 1 5 10 15 Leu Thr Leu Phe Asp Asp
Glu Phe Glu Lys Lys Asp Arg Thr Tyr Pro 20 25 30 Val Gly Glu Lys
Leu Arg Asn Ala Phe Arg Cys Ser Ser Ala Lys Ile 35 40 45 Lys Ala
Val Val Phe Gly Leu Leu Pro Val Leu Ser Trp Leu Pro Lys 50 55 60
Tyr Lys Ile Lys Asp Tyr Ile Ile Pro Asp Leu Leu Gly Gly Leu Ser 65
70 75 80 Gly Gly Ser Ile Gln Val Pro Gln Gly Met Ala Phe Ala Leu
Leu Ala 85 90 95 Asn Leu Pro Ala Val Asn Gly Leu Tyr Ser Ser Phe
Phe Pro Leu Leu 100 105 110 Thr Tyr Phe Phe Leu Gly Gly Val His Gln
Met Val Pro Gly Thr Phe 115 120 125 Ala Val Ile Ser Ile Leu Val Gly
Asn Ile Cys Leu Gln Leu Ala Pro 130 135 140 Glu Ser Lys Phe Gln Val
Phe Asn Asn Ala Thr Asn Glu Ser Tyr Val 145 150 155 160 Asp Thr Ala
Ala Met Glu Ala Glu Arg Leu His Val Ser Ala Thr Leu 165 170 175 Ala
Cys Leu Thr Ala Ile Ile Gln Met Gly Leu Gly Phe Met Gln Phe 180 185
190 Gly Phe Val Ala Ile Tyr Leu Ser Glu Ser Phe Ile Arg Gly Phe Met
195 200 205 Thr Ala Ala Gly Leu Gln Ile Leu Ile Ser Val Leu Lys Tyr
Ile Phe 210 215 220 Gly Leu Thr Ile Pro Ser Tyr Thr Gly Pro Gly Ser
Ile Val Phe Thr 225 230 235 240 Phe Ile Asp Ile Cys Lys Asn Leu Pro
His Thr Asn Ile Ala Ser Leu 245 250 255 Ile Phe Ala Leu Ile Ser Gly
Ala Phe Leu Val Leu Val Lys Glu Leu 260 265 270 Asn Ala Arg Tyr Met
His Lys Ile Arg Phe Pro Ile Pro Thr Glu Met 275 280 285 Ile Val Val
Val Val Ala Thr Ala Ile Ser Gly Gly Cys Lys Met Pro 290 295 300 Lys
Lys Tyr His Met Gln Ile Val Gly Glu Ile Gln Arg Gly Phe Pro 305 310
315 320 Thr Pro Val Ser Pro Val Val Ser Gln Trp Lys Asp Met Ile Gly
Thr 325 330 335 Ala Phe Ser Leu Ala Ile Val Ser Tyr Val Ile Asn Leu
Ala Met Gly 340 345 350 Arg Thr Leu Ala Asn Lys His Gly Tyr Asp Val
Asp Ser Asn Gln Glu 355 360 365 Met Ile Ala Leu Gly Cys Ser Asn Phe
Phe Gly Ser Phe Phe Lys Ile 370 375 380 His Val Ile Cys Cys Ala Leu
Ser Val Thr Leu Ala Val Asp Gly Ala 385 390 395 400 Gly Gly Lys Ser
Gln Val Ala Ser Leu Cys Val Ser Leu Val Val Met 405 410 415 Ile Thr
Met Leu Val Leu Gly Ile Tyr Leu Tyr Pro Leu Pro Lys Ser 420 425 430
Val Leu Gly Ala Leu Ile Ala Val Asn Leu Lys Asn Ser Leu Lys Gln 435
440 445 Leu Thr Asp Pro Tyr Tyr Leu Trp Arg Lys Ser Lys Leu Asp Cys
Cys 450 455 460 Ile Trp Val Val Ser Phe Leu Ser Ser Phe Phe Leu Ser
Leu Pro Tyr 465 470 475 480 Gly Val Ala Val Gly Val Ala Phe Ser Val
Leu Val Val Val Phe Gln
485 490 495 Thr Gln Phe Arg Asn Gly Tyr Ala Leu Ala Gln Val Met Asp
Thr Asp 500 505 510 Ile Tyr Val Asn Pro Lys Thr Tyr Asn Arg Ala Gln
Asp Ile Gln Gly 515 520 525 Ile Lys Ile Ile Thr Tyr Cys Ser Pro Leu
Tyr Phe Ala Asn Ser Glu 530 535 540 Ile Phe Arg Gln Lys Val Ile Ala
Lys Thr Gly Met Asp Pro Gln Lys 545 550 555 560 Val Leu Leu Ala Lys
Gln Lys Tyr Leu Lys Lys Gln Glu Lys Arg Arg 565 570 575 Met Arg Pro
Thr Gln Gln Arg Arg Ser Leu Phe Met Lys Thr Lys Thr 580 585 590 Val
Ser Leu Gln Glu Leu Gln Gln Asp Phe Glu Asn Ala Pro Pro Thr 595 600
605 Asp Pro Asn Asn Asn Gln Thr Pro Ala Asn Gly Thr Ser Val Ser Tyr
610 615 620 Ile Thr Phe Ser Pro Asp Ser Ser Ser Pro Ala Gln Ser Glu
Pro Pro 625 630 635 640 Ala Ser Ala Glu Ala Pro Gly Glu Pro Ser Asp
Met Leu Ala Ser Val 645 650 655 Pro Pro Phe Val Thr Phe His Thr Leu
Ile Leu Asp Met Ser Gly Val 660 665 670 Ser Phe Val Asp Leu Met Gly
Ile Lys Ala Leu Ala Lys Leu Ser Ser 675 680 685 Thr Tyr Gly Lys Ile
Gly Val Lys Val Phe Leu Val Asn Ile His Ala 690 695 700 Gln Val Tyr
Asn Asp Ile Ser His Gly Gly Val Phe Glu Asp Gly Ser 705 710 715 720
Leu Glu Cys Lys His Val Phe Pro Ser Ile His Asp Ala Val Leu Phe 725
730 735 Ala Gln Ala Asn Ala Arg Asp Val Thr Pro Gly His Asn Phe Gln
Gly 740 745 750 Ala Pro Gly Asp Ala Glu Leu Ser Leu Tyr Asp Ser Glu
Glu Asp Ile 755 760 765 Arg Ser Tyr Trp Asp Leu Glu Gln Glu Met Phe
Gly Ser Met Phe His 770 775 780 Ala Glu Thr Leu Thr Ala Leu 785 790
66243PRTHomo Sapiens 66Met Glu Gln Gly Ser Gly Arg Leu Glu Asp Phe
Pro Val Asn Val Phe 1 5 10 15 Ser Val Thr Pro Tyr Thr Pro Ser Thr
Ala Asp Ile Gln Val Ser Asp 20 25 30 Asp Asp Lys Ala Gly Ala Thr
Leu Leu Phe Ser Gly Ile Phe Leu Gly 35 40 45 Leu Val Gly Ile Thr
Phe Thr Val Met Gly Trp Ile Lys Tyr Gln Gly 50 55 60 Val Ser His
Phe Glu Trp Thr Gln Leu Leu Gly Pro Val Leu Leu Ser 65 70 75 80 Val
Gly Val Thr Phe Ile Leu Ile Ala Val Cys Lys Phe Lys Met Leu 85 90
95 Ser Cys Gln Leu Cys Lys Glu Ser Glu Glu Arg Val Pro Asp Ser Glu
100 105 110 Gln Thr Pro Gly Gly Pro Ser Phe Val Phe Thr Gly Ile Asn
Gln Pro 115 120 125 Ile Thr Phe His Gly Ala Thr Val Val Gln Tyr Ile
Pro Pro Pro Tyr 130 135 140 Gly Ser Pro Glu Pro Met Gly Ile Asn Thr
Ser Tyr Leu Gln Ser Val 145 150 155 160 Val Ser Pro Cys Gly Leu Ile
Thr Ser Gly Gly Ala Ala Ala Ala Met 165 170 175 Ser Ser Pro Pro Gln
Tyr Tyr Thr Ile Tyr Pro Gln Asp Asn Ser Ala 180 185 190 Phe Val Val
Asp Glu Gly Cys Leu Ser Phe Thr Asp Gly Gly Asn His 195 200 205 Arg
Pro Asn Pro Asp Val Asp Gln Leu Glu Glu Thr Gln Leu Glu Glu 210 215
220 Glu Ala Cys Ala Cys Phe Ser Pro Pro Pro Tyr Glu Glu Ile Tyr Ser
225 230 235 240 Leu Pro Arg 6721DNAArtificial
SequenceOligonucleotide 67acacgaatgg tagatacagt g
216821DNAArtificial SequenceOligonucleotide 68atacttgtga gctgttccat
g 216921DNAArtificial SequenceOligonucleotide 69actgttacct
tgcatggact g 217021DNAArtificial SequenceOligonucleotide
70caatgagaac acatggacat g 217121DNAArtificial
SequenceOligonucleotide 71ccatgaaagc tccatgtcta c
217221DNAArtificial SequenceOligonucleotide 72agagatggca catattctgt
c 217321DNAArtificial SequenceOligonucleotide 73atcggctgaa
gtcaagcatc g 217421DNAArtificial SequenceOligonucleotide
74tggtcagtga ggactcagct g 217521DNAArtificial
SequenceOligonucleotide 75tttctctgct tgatgcactt g
217621DNAArtificial SequenceOligonucleotide 76gtgagcactg ggaagcagct
c 217721DNAArtificial SequenceOligonucleotide 77ggcaaatgct
agagacgtga c 217821DNAArtificial SequenceOligonucleotide
78aggtgtcctt cagctgccaa g 217921DNAArtificial
SequenceOligonucleotide 79gttaagtgct ctctggattt g
218021DNAArtificial SequenceOligonucleotide 80atcctgattg ctgtgtgcaa
g 218121DNAArtificial SequenceOligonucleotide 81ctcttctagc
tggtcaacat c 218221DNAArtificial SequenceOligonucleotide
82ccagcaacaa cttacgtggt c 218321DNAArtificial
SequenceOligonucleotide 83cctttattca cccaatcact c 21842165DNAHomo
Sapiens 84agaacagcgc agtttgccct ccgctcacgc agagcctctc cgtggcctcc
gcaccttgag 60cattaggcca gttctcctct tctctctaat ccatccgtca cctctcctgt
catccgtttc 120catgccgtga ggtccattca cagaacacat ccatggctct
catgctcagt ttggttctga 180gtctcctcaa gctgggatca gggcagtggc
aggtgtttgg gccagacaag cctgtccagg 240ccttggtggg ggaggacgca
gcattctcct gtttcctgtc tcctaagacc aatgcagagg 300ccatggaagt
gcggttcttc aggggccagt tctctagcgt ggtccacctc tacagggacg
360ggaaggacca gccatttatg cagatgccac agtatcaagg caggacaaaa
ctggtgaagg 420attctattgc ggaggggcgc atctctctga ggctggaaaa
cattactgtg ttggatgctg 480gcctctatgg gtgcaggatt agttcccagt
cttactacca gaaggccatc tgggagctac 540aggtgtcagc actgggctca
gttcctctca tttccatcac gggatatgtt gatagagaca 600tccagctact
ctgtcagtcc tcgggctggt tcccccggcc cacagcgaag tggaaaggtc
660cacaaggaca ggatttgtcc acagactcca ggacaaacag agacatgcat
ggcctgtttg 720atgtggagat ctctctgacc gtccaagaga acgccgggag
catatcctgt tccatgcggc 780atgctcatct gagccgagag gtggaatcca
gggtacagat aggagatacc tttttcgagc 840ctatatcgtg gcacctggct
accaaagtac tgggaatact ctgctgtggc ctattttttg 900gcattgttgg
actgaagatt ttcttctcca aattccagtg taagcgagag agagaagcat
960gggccggtgc cttattcatg gttccagcag ggacaggatc agagatgctc
ccacatccag 1020ctgcttctct tcttctagtc ctagcctcca ggggcccagg
cccaaaaaag gaaaatccag 1080gcggaactgg actggagaag aaagcacgga
caggcagaat tgagagacgc ccggaaacac 1140gcagtggagg tgactctgga
tccagagacg gctcacccga agctctgcgt ttctgatctg 1200aaaactgtaa
cccatagaaa agctccccag gaggtgcctc actctgagaa gagatttaca
1260aggaagagtg tggtggcttc tcagagtttc caagcaggga aacattactg
ggaggtggac 1320ggaggacaca ataaaaggtg gcgcgtggga gtgtgccggg
atgatgtgga caggaggaag 1380gagtacgtga ctttgtctcc cgatcatggg
tactgggtcc tcagactgaa tggagaacat 1440ttgtatttca cattaaatcc
ccgttttatc agcgtcttcc ccaggacccc acctacaaaa 1500ataggggtct
tcctggacta tgagtgtggg accatctcct tcttcaacat aaatgaccag
1560tcccttattt ataccctgac atgtcggttt gaaggcttat tgaggcccta
cattgagtat 1620ccgtcctata atgagcaaaa tggaactccc atagtcatct
gcccagtcac ccaggaatca 1680gagaaagagg cctcttggca aagggcctct
gcaatcccag agacaagcaa cagtgagtcc 1740tcctcacagg caaccacgcc
cttcctcccc aggggtgaaa tgtaggatga atcacatccc 1800acattcttct
ttagggatat taaggtctct ctcccagatc caaagtcccg cagcagccgg
1860ccaaggtggc ttccagatga agggggactg gcctgtccac atgggagtca
ggtgtcatgg 1920ctgccctgag ctgggaggga agaaggctga cattacattt
agtttgctct cactccatct 1980ggctaagtga tcttgaaata ccacctctca
ggtgaagaac cgtcaggaat tcccatctca 2040caggctgtgg tgtagattaa
gtagacaagg aatgtgaata atgcttagat cttattgatg 2100acagagtgta
tcctaatggt ttgttcatta tattacactt tcagtaaaaa aaaaaaaaaa 2160aaaaa
216585347PRTHomo Sapiens 85Met Ala Leu Met Leu Ser Leu Val Leu Ser
Leu Leu Lys Leu Gly Ser 1 5 10 15 Gly Gln Trp Gln Val Phe Gly Pro
Asp Lys Pro Val Gln Ala Leu Val 20 25 30 Gly Glu Asp Ala Ala Phe
Ser Cys Phe Leu Ser Pro Lys Thr Asn Ala 35 40 45 Glu Ala Met Glu
Val Arg Phe Phe Arg Gly Gln Phe Ser Ser Val Val 50 55 60 His Leu
Tyr Arg Asp Gly Lys Asp Gln Pro Phe Met Gln Met Pro Gln 65 70 75 80
Tyr Gln Gly Arg Thr Lys Leu Val Lys Asp Ser Ile Ala Glu Gly Arg 85
90 95 Ile Ser Leu Arg Leu Glu Asn Ile Thr Val Leu Asp Ala Gly Leu
Tyr 100 105 110 Gly Cys Arg Ile Ser Ser Gln Ser Tyr Tyr Gln Lys Ala
Ile Trp Glu 115 120 125 Leu Gln Val Ser Ala Leu Gly Ser Val Pro Leu
Ile Ser Ile Thr Gly 130 135 140 Tyr Val Asp Arg Asp Ile Gln Leu Leu
Cys Gln Ser Ser Gly Trp Phe 145 150 155 160 Pro Arg Pro Thr Ala Lys
Trp Lys Gly Pro Gln Gly Gln Asp Leu Ser 165 170 175 Thr Asp Ser Arg
Thr Asn Arg Asp Met His Gly Leu Phe Asp Val Glu 180 185 190 Ile Ser
Leu Thr Val Gln Glu Asn Ala Gly Ser Ile Ser Cys Ser Met 195 200 205
Arg His Ala His Leu Ser Arg Glu Val Glu Ser Arg Val Gln Ile Gly 210
215 220 Asp Thr Phe Phe Glu Pro Ile Ser Trp His Leu Ala Thr Lys Val
Leu 225 230 235 240 Gly Ile Leu Cys Cys Gly Leu Phe Phe Gly Ile Val
Gly Leu Lys Ile 245 250 255 Phe Phe Ser Lys Phe Gln Cys Lys Arg Glu
Arg Glu Ala Trp Ala Gly 260 265 270 Ala Leu Phe Met Val Pro Ala Gly
Thr Gly Ser Glu Met Leu Pro His 275 280 285 Pro Ala Ala Ser Leu Leu
Leu Val Leu Ala Ser Arg Gly Pro Gly Pro 290 295 300 Lys Lys Glu Asn
Pro Gly Gly Thr Gly Leu Glu Lys Lys Ala Arg Thr 305 310 315 320 Gly
Arg Ile Glu Arg Arg Pro Glu Thr Arg Ser Gly Gly Asp Ser Gly 325 330
335 Ser Arg Asp Gly Ser Pro Glu Ala Leu Arg Phe 340 345
8621DNAArtificial SequenceOligonucleotide 86attcatggtt ccagcaggga c
218721DNAArtificial SequenceOligonucleotide 87gggagacaaa gtcacgtact
c 218822DNAArtificial SequenceOligonucleotide 88tcctggtgtt
cgtggtctgc tt 228922DNAArtificial SequenceOligonucleotide
89gagagtcctg gcttttgtgg gc 229015PRTHomo Sapiens 90Gly Ser Ser Asp
Leu Thr Trp Pro Pro Ala Ile Lys Leu Gly Cys 1 5 10 15 9116PRTHomo
Sapiens 91Asp Arg Tyr Val Ala Val Arg His Pro Leu Arg Ala Arg Gly
Leu Arg 1 5 10 15 9215PRTHomo Sapiens 92Val Ala Pro Arg Ala Lys Ala
His Lys Ser Gln Asp Ser Leu Cys 1 5 10 15 9313PRTHomo Sapiens 93Cys
Phe Arg Ser Thr Arg His Asn Phe Asn Ser Met Arg 1 5 10 9422PRTHomo
Sapiens 94Met Asn Gly Thr Tyr Asn Thr Cys Gly Ser Ser Asp Leu Thr
Trp Pro 1 5 10 15 Pro Ala Ile Lys Leu Gly 20 9514PRTHomo Sapiens
95Arg Asp Thr Ser Asp Thr Pro Leu Cys Gln Leu Ser Gln Gly 1 5 10
9622PRTHomo Sapiens 96Gly Ile Gln Glu Gly Gly Phe Cys Phe Arg Ser
Thr Arg His Asn Phe 1 5 10 15 Asn Ser Met Arg Phe Pro 20
9730PRTHomo Sapiens 97Ala Lys Glu Phe Gln Glu Ala Ser Ala Leu Ala
Val Ala Pro Arg Ala 1 5 10 15 Lys Ala His Lys Ser Gln Asp Ser Leu
Cys Val Thr Leu Ala 20 25 30 9822DNAArtificial
SequenceOligonucleotide 98tcctgctcgt cgctctcctg at
229920DNAArtificial SequenceOligonucleotide 99tcgctttttg tcgtatttgc
2010015PRTHomo Sapiens 100His Asn Gly Ser Tyr Glu Ile Ser Val Leu
Met Met Gly Asn Ser 1 5 10 15 10115PRTHomo Sapiens 101Asn Leu Pro
Thr Pro Pro Thr Val Glu Asn Gln Gln Arg Leu Ala 1 5 10 15
102619PRTHomo Sapiens 102Arg Lys Tyr Arg Lys Asp Tyr Glu Leu Arg
Gln Lys Lys Trp Ser His 1 5 10 15 Ile Pro Pro Glu Asn Ile Phe Pro
Leu Glu Thr Asn Glu Thr Asn His 20 25 30 Val Ser Leu Lys Ile Asp
Asp Asp Lys Arg Arg Asp Thr Ile Gln Arg 35 40 45 Leu Arg Gln Cys
Lys Tyr Asp Lys Lys Arg Val Ile Leu Lys Asp Leu 50 55 60 Lys His
Asn Asp Gly Asn Phe Thr Glu Lys Gln Lys Ile Glu Leu Asn 65 70 75 80
Lys Leu Leu Gln Ile Asp Tyr Tyr Asn Leu Thr Lys Phe Tyr Gly Thr 85
90 95 Val Lys Leu Asp Thr Met Ile Phe Gly Val Ile Glu Tyr Cys Glu
Arg 100 105 110 Gly Ser Leu Arg Glu Val Leu Asn Asp Thr Ile Ser Tyr
Pro Asp Gly 115 120 125 Thr Phe Met Asp Trp Glu Phe Lys Ile Ser Val
Leu Tyr Asp Ile Ala 130 135 140 Lys Gly Met Ser Tyr Leu His Ser Ser
Lys Thr Glu Val His Gly Arg 145 150 155 160 Leu Lys Ser Thr Asn Cys
Val Val Asp Ser Arg Met Val Val Lys Ile 165 170 175 Thr Asp Phe Gly
Cys Asn Ser Ile Leu Pro Pro Lys Lys Asp Leu Trp 180 185 190 Thr Ala
Pro Glu His Leu Arg Gln Ala Asn Ile Ser Gln Lys Gly Asp 195 200 205
Val Tyr Ser Tyr Gly Ile Ile Ala Gln Glu Ile Ile Leu Arg Lys Glu 210
215 220 Thr Phe Tyr Thr Leu Ser Cys Arg Asp Arg Asn Glu Lys Ile Phe
Arg 225 230 235 240 Val Glu Asn Ser Asn Gly Met Lys Pro Phe Arg Pro
Asp Leu Phe Leu 245 250 255 Glu Thr Ala Glu Glu Lys Glu Leu Glu Val
Tyr Leu Leu Val Lys Asn 260 265 270 Cys Trp Glu Glu Asp Pro Glu Lys
Arg Pro Asp Phe Lys Lys Ile Glu 275 280 285 Thr Thr Leu Ala Lys Ile
Phe Gly Leu Phe His Asp Gln Lys Asn Glu 290 295 300 Ser Tyr Met Asp
Thr Leu Ile Arg Arg Leu Gln Leu Tyr Ser Arg Asn 305 310 315 320 Leu
Glu His Leu Val Glu Glu Arg Thr Gln Leu Tyr Lys Ala Glu Arg 325 330
335 Asp Arg Ala Asp Arg Leu Asn Phe Met Leu Leu Pro Arg Leu Val Val
340 345 350 Lys Ser Leu Lys Glu Lys Gly Phe Val Glu Pro Glu Leu Tyr
Glu Glu 355 360 365 Val Thr Ile Tyr Phe Ser Asp Ile Val Gly Phe Thr
Thr Ile Cys Lys 370 375 380 Tyr Ser Thr Pro Met Glu Val Val Asp Met
Leu Asn Asp Ile Tyr Lys 385 390 395 400 Ser Phe Asp His Ile Val Asp
His His Asp Val Tyr Lys Val Glu Thr 405 410 415 Ile Gly Asp Ala Tyr
Met Val Ala Ser Gly Leu Pro Lys Arg Asn Gly 420 425 430 Asn Arg His
Ala Ile Asp Ile Ala Lys Met Ala Leu Glu Ile Leu Ser 435 440 445 Phe
Met Gly Thr Phe Glu Leu Glu His Leu Pro Gly Leu Pro Ile Trp 450 455
460 Ile Arg Ile Gly Val His Ser Gly Pro Cys Ala Ala Gly Val Val Gly
465 470 475 480 Ile Lys Met Pro Arg Tyr Cys Leu Phe Gly Asp Thr Val
Asn Thr Ala 485 490 495 Ser Arg Met Glu Ser Thr Gly Leu Pro Leu Arg
Ile His Val Ser
Gly 500 505 510 Ser Thr Ile Ala Ile Leu Lys Arg Thr Glu Cys Gln Phe
Leu Tyr Glu 515 520 525 Val Arg Gly Glu Thr Tyr Leu Lys Gly Arg Gly
Asn Glu Thr Thr Tyr 530 535 540 Trp Leu Thr Gly Met Lys Asp Gln Lys
Phe Asn Leu Pro Thr Pro Pro 545 550 555 560 Thr Val Glu Asn Gln Gln
Arg Leu Gln Ala Glu Phe Ser Asp Met Ile 565 570 575 Ala Asn Ser Leu
Gln Lys Arg Gln Ala Ala Gly Ile Arg Ser Gln Lys 580 585 590 Pro Arg
Arg Val Ala Ser Tyr Lys Lys Gly Thr Leu Glu Tyr Leu Gln 595 600 605
Leu Asn Thr Thr Asp Lys Glu Ser Thr Tyr Phe 610 615
10320DNAArtificial SequenceOligonucleotide 103gctggtaact atcttcctgc
2010420DNAArtificial SequenceOligonucleotide 104gaagaatgtt
gtccagaggt 2010515PRTHomo Sapiens 105Leu Ile Asn Lys Val Pro Leu
Pro Val Asp Lys Leu Ala Pro Leu 1 5 10 15 10615PRTHomo Sapiens
106Ser Glu Ala Val Lys Lys Leu Leu Glu Ala Leu Ser His Leu Val 1 5
10 15 10720DNAArtificial SequenceOligonucleotide 107tgttttcaac
taccaggggc 2010820DNAArtificial SequenceOligonucleotide
108tgttggcttt ggcagagtcc 2010924DNAArtificial
SequenceOligonucleotide 109gaggcagagt tcaggcttca ccga
2411020DNAArtificial SequenceOligonucleotide 110tgttggcttt
ggcagagtcc 2011156PRTHomo Sapiens 111Thr Gly Met Asp Met Trp Ser
Thr Gln Asp Leu Tyr Asp Asn Pro Val 1 5 10 15 Thr Ser Val Phe Gln
Tyr Glu Gly Leu Trp Arg Ser Cys Val Arg Gln 20 25 30 Ser Ser Gly
Phe Thr Glu Cys Arg Pro Tyr Phe Thr Ile Leu Gly Leu 35 40 45 Pro
Ala Met Leu Gln Ala Val Arg 50 55 11253PRTHomo Sapiens 112Asp Gln
Trp Ser Thr Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val 1 5 10 15
Phe Asn Tyr Gln Gly Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly 20
25 30 Phe Thr Glu Cys Arg Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala
Met 35 40 45 Leu Gln Ala Val Arg 50 11314PRTHomo Sapiens 113Ser Thr
Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe 1 5 10 11412PRTHomo
Sapiens 114Asp Met Trp Ser Thr Gln Asp Leu Tyr Asp Asn Pro 1 5 10
11512PRTHomo Sapiens 115Cys Arg Pro Tyr Phe Thr Ile Leu Gly Leu Pro
Ala 1 5 10 11613PRTHomo Sapiens 116Thr Asn Phe Trp Met Ser Thr Ala
Asn Met Tyr Thr Gly 1 5 10 117816DNAHomo Sapiens 117gccaggatca
tgtccaccac cacatgccaa gtggtggcgt tcctcctgtc catcctgggg 60ctggccggct
gcatcgcggc caccgggatg gacatgtgga gcacccagga cctgtacgac
120aaccccgtca cctccgtgtt ccagtacgaa gggctctgga ggagctgcgt
gaggcagagt 180tcaggcttca ccgaatgcag gccctatttc accatcctgg
gacttccagc catgctgcag 240gcagtgcgag ccctgatgat cgtaggcatc
gtcctgggtg ccattggcct cctggtatcc 300atctttgccc tgaaatgcat
ccgcattggc agcatggagg actctgccaa agccaacatg 360acactgacct
ccgggatcat gttcattgtc tcaggtcttt gtgcaattgc tggagtgtct
420gtgtttgcca acatgctggt gactaacttc tggatgtcca cagctaacat
gtacaccggc 480atgggtggga tggtgcagac tgttcagacc aggtacacat
ttggtgcggc tctgttcgtg 540ggctgggtcg ctggaggcct cacactaatt
gggggtgtga tgatgtgcat cgcctgccgg 600ggcctggcac cagaagaaac
caactacaaa gccgtttctt atcatgcctc aggccacagt 660gttgcctaca
agcctggagg cttcaaggcc agcactggct ttgggtccaa caccaaaaac
720aagaagatat acgatggagg tgcccgcaca gaggacgagg tacaatctta
tccttccaag 780cacgactatg tgtaatgctc taagacctct cagcac
816118261PRTHomo Sapiens 118Met Ser Thr Thr Thr Cys Gln Val Val Ala
Phe Leu Leu Ser Ile Leu 1 5 10 15 Gly Leu Ala Gly Cys Ile Ala Ala
Thr Gly Met Asp Met Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asp Asn
Pro Val Thr Ser Val Phe Gln Tyr Glu Gly 35 40 45 Leu Trp Arg Ser
Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Pro Tyr
Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80
Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85
90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp
Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe
Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe
Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn
Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln
Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val
Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile
Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205
Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210
215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys
Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln
Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 119227DNAHomo
Sapiens 119gccaggatca tgtccaccac cacatgccaa gtggtggcgt tcctcctgtc
catcctgggg 60ctggccggct gcatcgcggc caccgggatg gacatgtgga gcacccagga
cctgtacgac 120aaccccgtca cctccgtgtt ccagtacgaa gggctctgga
ggagctgcgt gaggcagagt 180tcaggcttca ccgaatgcag gccctatttc
accatcctgg gacttcc 22712069PRTHomo Sapiens 120Met Ser Thr Thr Thr
Cys Gln Val Val Ala Phe Leu Leu Ser Ile Leu 1 5 10 15 Gly Leu Ala
Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr 20 25 30 Gln
Asp Leu Tyr Asp Asn Pro Val Thr Ser Val Phe Gln Tyr Glu Gly 35 40
45 Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg
50 55 60 Pro Tyr Phe Thr Ile 65 12120DNAArtificial
SequenceOligonucleotide 121aatgagagga aagagaaaac
2012220DNAArtificial SequenceOligonucleotide 122atggtagaag
agtaggcaat 2012315PRTHomo Sapiens 123Glu Lys Trp Asn Leu His Lys
Arg Ile Ala Leu Lys Met Val Cys 1 5 10 15 12411PRTHomo Sapiens
124Cys Leu Gly Phe Asn Phe Lys Glu Met Phe Lys 1 5 10
12523DNAArtificial SequenceOligonucleotide 125taatgatgaa ccctacactg
agc 2312620DNAArtificial SequenceOligonucleotide 126atggacaaat
gccctacctt 2012722DNAArtificial SequenceOligonucleotide
127agtgctggaa ggatgtgcgt gt 2212820DNAArtificial
SequenceOligonucleotide 128ttgaggtggt tgttgggttt
2012920DNAArtificial SequenceOligonucleotide 129agatgtgctg
aggctgtaga 2013020DNAArtificial SequenceOligonucleotide
130atgaaggttg attatttgag 2013123DNAArtificial
SequenceOligonucleotide 131agccgcatac tcccttaccc tct
2313220DNAArtificial SequenceOligonucleotide 132gcagcagccc
aaacaccaca 2013320DNAArtificial SequenceOligonucleotide
133ctgagccgag aggtggaatc 2013420DNAArtificial
SequenceOligonucleotide 134ctctctcgct tacactggaa 2013514PRTHomo
Sapiens 135Gln Trp Gln Val Phe Gly Pro Asp Lys Pro Val Gln Ala Leu
1 5 10 13615PRTHomo Sapiens 136Ala Lys Trp Lys Gly Pro Gln Gly Gln
Asp Leu Ser Thr Asp Ser 1 5 10 15 13732PRTHomo Sapiens 137Asn Met
Leu Val Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr 1 5 10 15
Gly Met Gly Gly Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly 20
25 30 13823DNAArtificial SequenceOligonucleotide 138cgtgagcgct
tcgagatgtt ccg 2313923DNAArtificial SequenceOligonucleotide
139cctaaccagc tgcccaactg tag 2314020DNAArtificial
SequenceOligonucleotide 140ccatgaaagc tccatgtcta
2014120DNAArtificial SequenceOligonucleotide 141ggcaaatgct
agagacgtga 20
* * * * *
References